WO2023155144A1

WO2023155144A1 - Methods for regulating angiogenesis

Info

Publication number: WO2023155144A1
Application number: PCT/CN2022/076866
Authority: WO
Inventors: Li Yu; Cuifang ZHANG
Original assignee: Tsinghua University
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2023-08-24

Abstract

The present disclosure provides methods for regulating angiogenesis, also provides a method for regulating angiogenesis and/or an angiogenesis related biological process, comprising regulating the formation and/or function of a migrasome generated by a monocyte and/or macrophage.

Description

METHODS FOR REGULATING ANGIOGENESIS

BACKGROUND OF THE INVENTION

As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, the vesicles are named as “migrasomes” . However, few possible roles have been identified.

Angiogenesis refers to the formation of new blood vessels, and is essential to proper embryonic development and growth, and tissue repair. Angiogenesis is also essential to many pathological conditions. Accordingly, improved compositions and methods for regulating angiogenesis and angiogenesis related diseases and disorders are required.

SUMMARY OF THE INVENTION

Using migrasomes as the signal source to guide angiogenesis may have multiple advantages. First, for many angiogenic factors which do not bind to ECM, migrasomes may provide a mechanism for creating spatial and temporal signal patterns. Secondly, multiple different kinds of angiogenic factors can be packed into the same migrasome. These factors, which may contribute to angiogenesis through different yet complementary mechanisms, can undergo synchronized release, thus ensuring the spatial and temporal coordination of diverse regulatory mechanisms for angiogenesis. Finally, the release of angiogenic factors from migrasomes may require the rupture or leakage of migrasomes. This may create a latency effect and provide more opportunity to fine-tune the formation of signal patterns.

In the first aspect, this patent application provides a method for regulating angiogenesis and/or an angiogenesis related biological process, comprising regulating the formation and/or function of a migrasome generated by a monocyte and/or macrophage.

In one embodiment, wherein said angiogenesis related biological process comprises tumor and/or wound healing.

In one embodiment, the method increases angiogenesis, and comprises promoting the formation and/or function of said migrasome.

In one embodiment, wherein said promoting the formation and/or function of said migrasome comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said monocyte, said macrophage and/or in said migrasome.

In one embodiment, wherein said promoting the formation and/or function of said migrasome comprises overexpressing the tetraspanin, the functional fragment thereof, and/or the functional variant thereof in said monocyte, said macrophage and/or in said migrasome.

In one embodiment, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.

In one embodiment, wherein said promoting the function of said migrasome comprises increasing the amount and/or function of an angiogenesis factor in said migrasome.

In one embodiment, wherein said angiogenesis factor comprises vascular endothelial growth factor (VEGF) and/or transforming growth factor (TGF) .

In one embodiment, wherein said VEGF comprises VEGFA.

In one embodiment, wherein said TGF comprises TGF-β3.

In one embodiment, the method decreases angiogenesis, and comprises inhibiting the formation and/or function of said migrasome.

In one embodiment, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting the expression and/or function of a tetraspanin in said monocyte, said macrophage and/or in said migrasome.

In one embodiment, wherein said inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for said tetraspanin in said monocyte and/or macrophage.

In one embodiment, wherein said tetraspanin comprises tetraspanin 4.

In one embodiment, wherein said inhibiting the function of said migrasome comprises decreasing the amount and/or function of an angiogenesis factor in said migrasome.

In one embodiment, wherein said decreasing the amount and/or function of said angiogenesis factor comprises knocking out or knocking down the expression of a gene encoding for said angiogenesis factor in said monocyte and/or said macrophage.

In one embodiment, wherein said decreasing the amount and/or function of said angiogenesis factor comprises treating said migrasome with an agent capable of inhibiting the function of said angiogenesis factor.

In one embodiment, wherein said agent capable of inhibiting the function of said angiogenesis factor comprises a protease, a small molecule, and/or an antibody capable of inhibiting the activity of said angiogenesis factor.

In one embodiment, wherein said VEGF comprises VEGFA.

In one embodiment, wherein said TGF comprises TGF-β3.

In the second aspect, this patent application provides a method for regulating angiogenesis and/or an angiogenesis related biological process in a subject in need thereof, comprising administering to said subject an effective amount of migrasomes generated by a monocyte and/or a macrophage.

In one embodiment, the method increases angiogenesis.

In the third aspect, this patent application provides a method for monitoring angiogenesis and/or an angiogenesis related biological process in a subject, comprising analyzing the presence, amount and/or function of a migrasome generated by a monocyte and/or a macrophage in a biological sample of said subject.

In one embodiment, wherein said biological sample comprises a body fluid sample of said subject.

In one embodiment, wherein said biological sample comprises a blood sample of said subject.

In one embodiment, wherein an increase of the amount of said migrasome indicates an increase of said angiogenesis.

In one embodiment, wherein analyzing the presence, amount and/or function of said migrasome comprises analyzing the presence and/or amount of a marker molecule of said migrasome.

In one embodiment, wherein analyzing the presence, amount and/or function of said migrasome comprises determining the presence and/or amount of Tspan4 ⁺, Integrin ⁺, Pleckstrin Homology (PH) domain ⁺, NDST1 ⁺, PIGK ⁺, CPQ ⁺, EOGT ⁺, KUL01 ⁺ and/or CD115 ⁺ vesicles in said biological sample.

In one embodiment, wherein analyzing the presence, amount and/or function of said migrasome comprises staining said biological sample with wheatgerm agglutinin (WGA) .

In one embodiment, wherein said migrasome is KUL01 ⁺ and/or CD115 ⁺.

In the fourth aspect, this patent application provides a method for regulating angiogenesis and/or an angiogenesis related biological process in a subject, comprising:

i) monitoring the angiogenesis and/or the angiogenesis related biological process in said subject according to any one of method for monitoring angiogenesis and/or an angiogenesis related biological process in a subject of the present application; and

ii) administering a regulating agent according to the result of step i) .

In one embodiment, the method is an in vitro or ex vivo method.

In one embodiment, the method is an in vivo method.

In the fifth aspect, this patent application provides an agent capable of regulating the formation and/or function of a migrasome generated by a monocyte and/or a macrophage, for use in regulating angiogenesis and/or an angiogenesis related biological process.

In one embodiment, the agent is capable of increasing the formation and/or function of said migrasome, and for use in increasing angiogenesis.

In one embodiment, the agent is capable of increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said monocyte, said macrophage and/or in said migrasome.

In one embodiment, the agent comprises a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

In one embodiment, the agent is capable of inhibiting the formation and/or function of said migrasome, and for use in decreasing angiogenesis.

In one embodiment, the agent is capable of inhibiting the expression and/or function of a tetraspanin in said monocyte, said macrophage and/or in said migrasome.

In one embodiment, the agent is capable of knocking out or knocking down the expression of a gene encoding for said tetraspanin in said monocyte and/or macrophage.

In one embodiment, wherein said tetraspanin comprises tetraspanin 4.

In the sixth aspect, this patent application provides an isolated migrasome generated by a monocyte and/or a macrophage, for use in regulating angiogenesis and/or an angiogenesis related biological process.

In the seventh aspect, this patent application provides an engineered monocyte and/or a macrophage with altered ability for regulating angiogenesis and/or an angiogenesis related biological process comparing to a corresponding unmodified monocyte and/or macrophage, said engineered monocyte and/or macrophage has been modified to alter its migrasome generation ability.

In one embodiment, the engineered monocyte and/or macrophage has increased ability for promoting angiogenesis comparing to a corresponding unmodified monocyte and/or macrophage.

In one embodiment, the engineered monocyte and/or macrophage has been modified to have increased ability for generating migrasomes.

In one embodiment, the engineered monocyte and/or macrophage has been modified to increase the amount and/or function of a tetraspanin therein.

In one embodiment, the engineered monocyte and/or macrophage has been modified to overexpress a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

In one embodiment, the engineered monocyte and/or macrophage has decreased ability for promoting angiogenesis comparing to a corresponding unmodified monocyte and/or macrophage.

In one embodiment, the engineered monocyte and/or macrophage has been modified to have decreased ability for generating migrasomes.

In one embodiment, the engineered monocyte and/or macrophage has been modified to decrease the amount and/or function of a tetraspanin therein.

In one embodiment, wherein the expression of a gene encoding for a tetraspanin has been knocked out or knocked down.

In one embodiment, wherein said tetraspanin comprises tetraspanin 4.

In the eighth aspect, this patent application provides a use of the agent of the present application, the isolated migrasome of the present application, and/or the engineered monocyte and/or a macrophage of the present application in the preparation of a regulator for angiogenesis and/or the angiogenesis related biological process.

In the ninth aspect, this patent application provides an agent capable of detecting the presence, amount and/or function of a migrasome generated by a monocyte and/or a macrophage in a biological sample of a subject, for use in monitoring angiogenesis and/or an angiogenesis related biological process in said subject.

In one embodiment, wherein said migrasome is KUL01 ⁺ and/or CD115 ⁺.

In the tenth aspect, this patent application provides a use of the agent of the present application in the preparation of an indicator for angiogenesis and/or the angiogenesis related biological process in said subject.

In the eleventh aspect, this patent application provides a composition, comprising the agent of the present application, the isolated migrasome of the present application, and/or the engineered monocyte and/or macrophage of the present application.

In one embodiment, the composition is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.

In the twelfth aspect, this patent application provides a kit, comprising the agent of the present application, the isolated migrasome of the present application, the engineered monocyte and/or macrophage of the present application, and/or the composition of the present application.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “FIG. ” , “Fig. ” and “FIG. ” herein) , of which:

FIGs. 1a-1i illustrate Detection of migrasomes in chick embryo chorioallantoic membrane (CAM) . (1a) Confocal image of WGA ^high and WGA ^low cells in CAM from a 9-day chick embryo (CAM9d) . CAM is stained by WGA (wheat germ agglutinin) and observed by spinning disk microscopy. Scale bar, 5 μm. Enlarged images of migrasomes and retraction fibers emanating from WGA ^high CAM cells are shown in the lower panels. (1b) TEM image of CAM9d. Scale bar, 1 μm. Enlarged images of individual migrasomes are shown in the right panels. (1c) Migrasomes from (1b) are quantified for their size and the number of intralumenal vesicles. 60 migrasomes are examined and quantified. (1d) Focused ion beam scanning electron microscope (FIB-SEM) analysis of CAM9d. Enlarged z-stack images of the same migrasome are shown in the lower panels to highlight the connection between the migrasome and the retraction fiber. Scale bar, 500 nm. (1e) 3D reconstruction of a migrasome (red arrowhead) . collagen fibers; retraction fiber. Scale bar, 1 μm. (1f) Migrasome formation captured by spinning disk confocal microscopy. Scale bar, 5 μm. (1g) The distribution of WGA ^high cells in CAM9d. Blood vessels are revealed by dextran staining. CAM is visualized by Dragonfly confocal z-stack imaging and presented as the maximum intensity projection. Scale bar, 15 μm. (1h) Trajectories of WGA ^high cells in CAM9d are observed by time-lapse microscopy and analyzed by NIS-Elements Viewer. Scale bar, 10 μm. (1i) Enlargement of a WGA ^high cell in E9d CAM from (1h) . Scale bar, 5 μm.

FIGs. 2a-2m illustrate that Migrasomes are generated by monocytes. (2a) Diagram showing the procedure for isolation of WGA ^high or WGA ^low cells from CAM9d. (2b) Isolated cells from WGA-stained CAM9d sorted by FACS according to WGA signal. (2c) WGA ^high and WGA ^low cells are observed by confocal microscopy. Scale bars, 10 μm. (2d) Cells from (2c) are quantified for the number of migrasomes. Data are presented as means ± SEM; n = 40 cells per group pooled from three independent experiments. P values (*P < 0.0001) are calculated using a two-tailed, unpaired t-test. (2e) TEM images of WGA ^high and WGA ^low cells. A migrasome from the WGA ^high cell is shown in the right panel. Scale bar in left panel, 10 μm; middle panel, 1 μm; right panel, 200 nm. (2f) Trajectories of cultured WGA ^high and WGA ^low cells in the same amount of time (12 h) . 20 cells are examined and quantified in each group. (2g) WGA ^high cells are subjected to single-cell RNA-seq analysis. Data are analyzed by principal component analysis (PCA) . (2h) Heat map reporting scaled expression of discriminative marker gene sets for the two cell types identified in (2g) . (2i) Cells isolated from CAM9d are stained by CD115 and sorted by FACS. (2j) The CD115-positive cells are observed by Dragonfly spinning disk confocal microscopy. Scale bar, 5 μm. (2k) CAM9d is stained by WGA and anti-CD115 or KUL01 antibody. Immunofluorescence is visualized in CAMs by confocal z-stack imaging and presented as the maximum intensity projection. Scale bar, 15 μm. (2l) WGA ^high cells are untreated or treated with GM-CSF for 72 h. Boiled yeast cells (strain BY4741) labelled with a fluorescent tracer are added. Cells are observed by spinning disk confocal microscopy. White lines indicate the outlines of WGA ^high cells. White arrows indicate the internalized yeast. Scale bar, 5 μm. (2m) Quantification of the number of monocytes from (2l) with engulfed yeast. Data are presented as mean ± SEM; n=22 from three independent experiments; *P < 0.0001.

FIGs. 3a-3h illustrate that Depletion of monocytes reduces the migrasome number in CAM and impairs angiogenesis. (3a) Vessels from CAM9d are stained by dextran and CD115. Images are taken 10 mins and 2 hours after staining. Scale bar, 50 μm. (3b) Diagram of the procedure for depleting monocytes/macrophages in chick embryos by clodronate-containing liposomes. (3c) 6-day-old commercial SPF chicken embryos are injected with 2 μl clodronate-liposomes or PBS-liposomes. 48 h later, the second microinjection of 7.5 μl clodronate-liposomes or PBS-liposomes is administered. The post-treatment CAM10d is stained by WGA488 and visualized by confocal microscopy. Scale bar, 100 μm. (3d) WGA ^high cells from (3c) are quantified. Data are presented as mean ± SEM; n = 33 from three independent experiments; *P < 0.001. (3e) After monocyte depletion, CAMs are stained by WGA to label migrasomes and imaged by spinning disk microscopy. Scale bar, 10 μm. (3f) The numbers of migrasomes from (3e) are quantified. Data are presented as mean ± SEM; n = 16 from three independent experiments; *P < 0.0001. (3g) PBS-and clodronate-treated CAMs are visualized with a stereomicroscope. Scale bar, 500 μm. (3h) The number of sprouting capillaries from (3g) is quantified. Data are presented as mean ± SEM; n = 30 from three independent experiments; *P < 0.001.

FIGs. 4a-4j illustrate that Monocyte migrasomes contain angiogenesis factors and chemokines. (4a) Diagram of the migrasome isolation procedure. (4b) Images of migrasomes purified from CAM9d. Left panel, confocal image of purified migrasomes stained by WGA, scale bar, 5 μm; right panel, TEM image of migrasomes isolated from CAM9d, scale bar, 500 nm. (4c) Western blot analysis of isolated CAM9d migrasomes with the indicated antibodies. (4d) Diagram of the procedure for TMT-labelling and quantitative mass spectrometry. (4e) Volcano plot showing the mass spectrometry-based quantification of TMT-labelled proteins. The right dots represent a migrasome: cell abundance ≥ 2, P < 0.01; the left dots represent a migrasome: cell abundance < 0.5, P < 0.01. n = 6 biologically independent experiments. P values are calculated in Excel using a two-tailed, two-sample unequal variance t-test. (4f) Data from (4e) are analyzed for the abundance of the indicated proteins. (4g) Violin plots showing the mRNA levels of indicated genes from single-cell sequencing analysis of monocyte and epithelial cells. (4h) Cell bodies and migrasomes are analyzed by western blot using anti-CXCL12 and anti-VEGFA antibodies. (4i) Monocytes isolated from CAM9d are stained with WGA and the indicated antibodies and visualized by confocal microscopy. Scale bar, 5 μm. (4j) CAM9d are stained with WGA and the indicated antibodies. CAM immunofluorescence is visualized by confocal z-stack imaging and presented as the maximum intensity projection. Scale bar, 10 μm.

FIGs. 5a-5k illustrate that Migrasomes induce capillary formation and recruitment of monocytes. (5a) Confocal and TEM images of purified migrasomes from CAM9d. Left panel, scale bar, 5 μm; right panel, scale bar, 500 nm. (5b) Migrasomes are delivered to CAM9d by mixing them with Matrigel. After 48 h, CAMs are visualized by stereomicroscopy, scale bar, 500 μm. (5c) CAMs from (5b) are quantified for newly formed capillaries. Data are presented as mean ± SEM; n =20 fields from three independent experiments; *P < 0.0001. (5d) Migrasomes are delivered to CAM9d by mixing them with low-melting-point agarose. After 48 h, CAMs are visualized by stereomicroscopy Scale bar, 500 μm. (5e) CAMs from (5d) are quantified for sprouting capillaries. Data are presented as mean ± SEM; n =20 fields from three independent experiments; *P < 0.0001. (5f) Migrasomes are delivered to CAM9d in low-melting-point agarose. After 48 h, CAMs are stained with WGA and visualized by spinning disk microscopy. Scale bar, 5 μm. The boxed areas are enlarged images. (5g) CAMs from (5f) are quantified for the number of WGA ^high cells. Data are presented as mean ± SEM; n = 25 fields from three independent experiments; *P < 0.0001. (5h) Endothelial cell tube formation assays are carried out in the absence or presence of migrasomes. Scale bar, 50 μm. (5i) Endothelial cell tubes from (5h) are quantified for mature tubes. Data are presented as mean ± SEM; n = 40 fields from three independent experiments; N. S: no significance, *P < 0.001. (5j) Diagram of the transwell assay for recruitment of WGA ^high or WGA ^low cells. (5k) Cells adhered to the underside of the transwell membrane are stained by DAPI and visualized by confocal microscopy. Scale bar, 30 μm. (5l) Statistical analysis of the migration indexes from (5k) are calculated using one-way ANOVA. Data are presented as mean ± SEM; n = 35 fields from three independent experiments; N. S: no significance, *P < 0.01.

FIGs. 6a-6p illustrate that Blocking migrasome formation impairs capillary formation. (6a) CAM8d are transfected with TSPAN4 siRNA. 72 h after transfection, CAMs are stained with WGA and visualized by spinning disk microscopy. Scale bar, 10 μm. (6b) TSPAN4 knockdown efficiency in each CAM piece is determined by RT–PCR. Data are presented as mean ± SEM; n = 15 pieces from three independent experiments; *P < 0.01. (6c) WGA ^high cells from (6a) are quantified for migrasome formation. Data are presented as mean ± SEM; n = 65 cells from three independent experiments; *P <0.0001. (6d) CAM8d are transfected with TSPAN4 siRNA. 72 h after transfection, CAMs are visualized by stereomicroscopy. Right panels show enlarged regions of interest. Scale bar, 500 μm. (6e) Quantification of the number of sprouting capillaries from (6d) . Data are presented as mean ±SEM; n = 18 fields from three independent experiments; *P < 0.0001. (6f) CAM8d are transfected with TSPAN4 siRNA. 72 h after transfection, CAMs are stained by WGA and WGA ^high cells are visualized by confocal microscopy. Scale bar, 30 μm. (6g) CAMs from (6f) are quantified for the number of WGA ^high cells. Data are presented as means ± SEM, n = 15 fields from three independent experiments, *P < 0.001. (6h) 48 h after RNAi transfection, migrasomes embedded in low-melting-point agarose are added to CAM. 48 h later, CAMs are visualized by stereomicroscopy. Scale bar, 500 μm.(6i) CAMs from (6h) are quantified for sprouting capillaries. Data are presented as means ± SEM, n = 8 fields from three independent experiments, *P < 0.0001. (6j) Diagram of the strategy for mosaic knockout of TSPAN4 in chicken embryos. (6k) Efficiency of TSPAN4 knockout (T4-KO) in CAM of 8d chicken embryos is monitored by expression of mCherry. Scale bar, 20 μm. (6l) TSPAN4 knockout efficiency in CAM of 8d chicken embryos is evaluated by flow cytometry. (6m) CAMs from (k-l) are stained for WGA and observed by spinning disk microscopy. Scale bar, 10 μm. (6n) WGA ^high cells from (6m) are quantified for migrasome formation. Data are presented as mean ± SEM; n = 45 cells from three independent experiments; *P < 0.0001 versus scramble. (6o) CAMs from (6k-6l) are visualized by stereomicroscopy. Scale bar, 500 μm. The boxed areas are enlarged in the right panels. (6p) CAMs from (6o) are quantified for capillary formation. Data are presented as mean ± SEM; n = 18 fields from three independent experiments; *P < 0.0001.

FIGs. 7a-7f illustrate that Migrasomes rescue capillary formation and monocyte recruitment defects in VEGFA-knockdown and CXCL12-knockdown CAM. (7a) Diagram showing the migrasome rescue assay after knockdown of VEGFA or CXCL12 in CAM. (7b) CAMs are transfected with the indicated RNAi. After 48 h, migrasomes embedded in low-melting-point agarose are added. After another 48 h, CAMs are visualized by stereomicroscopy. Scale bar, 1 mm. (7c) CAMs treated with VEGF siRNA (top) or CXCL12 siRNA (bottom) from (7b) are quantified for sprouting capillaries. Data are presented as mean ± SEM; n = 12 fields from three independent experiments; *P < 0.05; ns, no significant difference. (7d) CAMs from (7b) are stained with WGA and visualized by confocal microscopy. Scale bar, 50 μm. (7e) CAMs from (7d) are quantified for the number of WGA ^high cells. Data are presented as mean ± SEM; n = 22 fields from three independent experiments, respectively; *P < 0.01. (7f) . Model of the role of migrasomes from monocytes in angiogenesis during embryonic development.

FIGs. 8a-8c illustrate the movement of migrasome-generating cells. (8a) Migration of WGA ^high and WGA ^low cell is monitored by time-lapse confocal microscopy. Scale bar, 5 μm. (8b) Cells from (8a) are quantified for migration speed. Data are presented as means ± SEM; n = 21 cells per group pooled from three independent experiments. (8c) Cells from (8a) are quantified for size. Data are presented as means ± SEM; n = 21 cells per group pooled from three independent experiments. P values (*P < 0.0001) are calculated using a two-tailed, unpaired t-test.

FIGs. 9a-9d illustrate gene knock-out system and results. (9a) Diagram showing the strategy for knocking out TSPAN4 and knocking in mCherry in chick embryos. The mCherry coding sequence is inserted into TSPAN4 at the position targeted by the sgRNA. Thus, an mCherry-positive signal indicates that native TSPAN4 gene expression is silenced simultaneously. (9b) Time-lapse imaging of migration and migrasome formation by WGA ^high cells isolated from WT or TSPAN4-KO CAM8d. Cells are stained by WGA and observed by spinning disk microscopy. Scale bar, 5 μm. (9c) CAMs from (Fig. 6k, 6l) are stained for WGA and observed by spinning disk microscopy. Scale bar, 30 μm. (9d) CAMs from (Fig. 6k, 6l) are quantified for the number of WGA ^high cells. Data are presented as mean ± SEM; n = 15 cells from three independent experiments; *P < 0.0001.

FIG. 10 illustrates gene expression in monocytes. Violin plots showing the mRNA levels of TGFB3 from single-cell sequencing analysis of monocyte-like cells and epithelial cells.

FIG. 11 illustrates Immunofluorescence results of CAM. CAM9d are stained with WGA and the indicated antibodies and visualized by confocal microscopy. Scale bar, 20 μm. Immunofluorescence in CAMs is visualized by confocal z-stack imaging and presented as the maximum intensity projection.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Term

As used herein, the term “angiogenesis” generally refers to the formation, growth and/or repair of a blood vessel. For example, new blood vessels take shape from existing blood vessels by “sprouting” of endothelial cells. For example, normal angiogenesis may occur in the healthy body of a subject for healing wounds and for restoring blood flow to tissues after injury. For example, abnormal angiogenesis may provide new blood vessels that feed diseased tissues and/or destroy normal tissues, and in the case of cancer, the new vessels may allow tumor cells to escape into the circulation and lodge in other organs (tumor metastases) .

As used herein, the term “angiogenesis related biological process” generally refers to a biological progress that is affected by, mediated by, or otherwise related to angiogenesis. For example, angiogenesis may occur in normal biological process, such as tissue repairing. For example, angiogenesis may occur in abnormal biological process, such as tumor growth and/or metastases.

As used herein, the term “antibody” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, the antibody can include an immunoglobulin composed of at one or more heavy (H) chains and/or one or more light (L) chains, and include any molecule including its antigen binding portion. The term “antibody" includes monoclonal antibodies, antibodies fragment or antibody derivatives, including but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single-strand antibodies (e.g., scFv) , and antigen-binding fragments of antibodies (e.g., Fab, Fab’, VHH and (Fab) 2 fragments) .

As used herein, the term “angiogenesis factor” generally refers to an agent that promote the growth and/or repair of a blood vessel. For example, angiogenesis factor may comprise a protein inducing proliferation and/or migration of vascular endothelial cells. For example, the term “angiogenesis factor” may encompass various isoforms of the angiogenesis factor, the naturally-occurring allelic and processed forms thereof.

As used herein, the term "engineered" generally refers to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome, of a polypeptide, or of other components. The term "engineered" can refer to alterations, additions, and/or deletions of the genes, polypeptides or other components. The term "engineered cell" generally refers to a modified cell of human or non-human origin. For example, an engineered cell can refer to a cell with an added, deleted and/or altered gene, polypeptide or other components.

As used herein, the term "functional fragment" generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid.

As used herein, the term "functional variant" generally refers to a nucleic acid molecule, or a polypeptide having similar amino acid or nucleic acid sequences as the parent sequence and retain one or more properties of the parent sequence.

As used herein, the term “knock down” generally refers to a measurable reduction in the expression of a target mRNA or the corresponding protein in a genetically modified cell or organism as compared to the expression of the target mRNA or the corresponding protein in a counterpart control cell or organism that does not contain the genetic modification to reduce expression. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., siRNA, shRNA, microRNA, antisense RNA, or other RNA-mediated inhibition techniques, to knock down a target polynucleotide sequence.

As used herein, the term “knock out” generally includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence. For example, a knock-out can be achieved by altering a target polynucleotide sequence by inducing a deletion in the target polynucleotide sequence in a functional domain of the target polynucleotide sequence. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., CRISPR/Cas systems, ZFN, TALEN, TgAgo, to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

As used herein, the term “migrasome” generally refers to a membrane-bound cellular structure derived from or generated by a migrating cell. The term “migrasome” encompasses an organelle (also known as “pomegranate-like structure” or PLS) attached to a retraction fiber generated by a migrating cell. In some cases, the term “migrasome” also refers to a vesicle (e.g., an extracellular vesicle) already detached from the cell generating it. In the present disclosure, the term “migrasome” also refers to a vesicle (e.g., an artificial vesicle) with similar functions and/or compositions as such a vesicle or organelle derived from, and/or generated by migrating cells.

As used herein, the term “pharmaceutically acceptable excipient” generally refers to any material, which is inert in the sense that it substantially does not have a therapeutic and/or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties.

As used herein, the term “tetraspanin” generally refers to a membrane protein, which is also known as the transmembrane 4 superfamily (TM4SF) protein, and may have four transmembrane alpha-helices and two extracellular domains. For example, the term “tetraspanin” may encompass various isoforms of the tetraspanin, as well as the naturally-occurring allelic and processed forms thereof.

As used herein, the term “small molecule” generally refers to a naturally occurring or chemically synthesized organic or inorganic molecule. For example, small molecule may be less than about 2000 Daltons. For example, small molecule may be less than about 1000 Daltons, from about or at 1000 Daltons to about or at 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less Daltons. A small molecule as understood by those of skill in the art and used herein may be a term that evolved to differentiate traditional drugs, from the new class of drugs based on developments in genetic engineering and biotechnology, such as proteins, nucleic acids and the like. A small molecule is understood to mean any molecule that may not be a macromolecule, such as a protein or nucleic acid. A “small molecule” as used herein may include a molecule containing two or more monomeric subunits, such as a dipeptide or dinucleotide, and generally is understood to refer to molecules that may be about or at 1000 Daltons or below in molecular weight.

Unless otherwise specified, “a” , “an” , “the” and “at least one” are used interchangeably and refer to one or more than one.

In the present disclosure, the term “comprise” also encompasses “is” , “has” and “consist of” . For example, “a composition comprising X and Y” may be understood to encompass a composition that comprises at least X and Y. It shall also be understood to disclose a composition that only comprises X and Y (i.e., a composition consisting of X and Y) .

Method for regulating angiogenesis and/or an angiogenesis related biological process

For example, wherein said angiogenesis related biological process may comprise tumor and/or wound healing. For example, the method may increase angiogenesis.

Migrasome generated by a monocyte and/or macrophage

For example, the present application provides a migrasome that is generated by a monocyte and/or macrophage. For example, the migrasome may be generated when a monocyte and/or macrophage is moving, e.g., migrating. For example, when the position of monocyte and/or macrophage is changing, the migrasome may be generated. For example, the position of the edge of the monocyte and/or macrophage is changing, the migrasome may be generated.

For example, the migrasome generated by a monocyte and/or macrophage may comprise the migrasome that is generating by the monocyte and/or macrophage. For example, the migrasome generated by a monocyte and/or macrophage may comprise the migrasome that has already been generated by and detached from the monocyte and/or macrophage. For example, the migrasome generated by a monocyte and/or macrophage may have special character. For example, the migrasome generated by a monocyte and/or macrophage may comprise special marker from the monocyte and/or macrophage. For example, the migrasome generated by a monocyte and/or macrophage may comprise KUL01 ⁺ and/or CD115 ⁺ migrasome. For example, the amount of the KUL01 and/or CD115 may be higher on the migrasome generated by a monocyte and/or macrophage that the migrasome generated by a cell other than monocyte and/or macrophage.

Regulating angiogenesis and/or an angiogenesis related biological process

For example, regulating angiogenesis and/or an angiogenesis related biological process may comprise promoting angiogenesis and/or an angiogenesis related biological process or inhibiting angiogenesis and/or an angiogenesis related biological process. For example, angiogenesis related biological process may comprise tumor and/or wound healing.

Inhibiting angiogenesis and/or an angiogenesis related biological process

For example, when treating the migrasome according to the method of the present application, the degree of angiogenesis and/or an angiogenesis related biological process may be lower than the untreated migrasome or the migrasome before treating. For example, the degree of angiogenesis and/or an angiogenesis related biological process may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated migrasome or the migrasome before treating. For example, the degree of angiogenesis may be analyzed by analyzing the amount of the blood vessel. For example, the degree of angiogenesis related biological process may be analyzed by analyzing the weigh, volume and/or metastasis of the tumor.

Promoting angiogenesis and/or an angiogenesis related biological process

For example, when treating the migrasome according to the method of the present application, the degree of angiogenesis and/or an angiogenesis related biological process may be higher than the untreated migrasome or the migrasome before treating. For example, the degree of angiogenesis and/or an angiogenesis related biological process may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the untreated migrasome or the migrasome before treating. For example, the degree of angiogenesis may be analyzed by analyzing the amount of the blood vessel. For example, the degree of angiogenesis related biological process may be analyzed by analyzing the degree of wound healing.

Promoting the formation and/or function of said migrasome

For example, the method may increase angiogenesis, and may comprise promoting the formation and/or function of said migrasome.

For example, promoting the formation and/or function of migrasomes can comprise promoting the cell migration. For example, the migration ability or the migration rate of the cell may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the unmodified cell.

For example, promoting the formation and/or function of migrasomes can comprise promoting the formation of a retraction fiber. For example, the number of retraction fiber may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the number of retraction fiber derived from the unmodified cell.

For example, promoting the formation and/or function of migrasomes can comprise increasing the amount and/or function of TSPAN protein. For example, the TSPAN protein may comprise its function fragment, and/or its functional variant. For example, the TSPAN protein may comprise TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.

For example, promoting the formation and/or function of migrasomes can comprise increasing the amount of cholesterol in a cell.

Increasing the amount and/or function of an angiogenesis factor in migrasome

For example, the present application provides a method for promoting the function of migrasome, which comprises increasing the amount and/or function of an angiogenesis factor in the migrasome.

For example, when treating the migrasome according to the method of the present application, the amount and/or function of an angiogenesis factor in the treated migrasome may be higher than the untreated migrasome or the migrasome before treating. For example, the amount of an angiogenesis factor in the treated migrasome may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the untreated migrasome or the migrasome before treating. For example, the function of an angiogenesis factor may be promoting angiogenesis and/or an angiogenesis related biological process, the function of the angiogenesis factor in the treated migrasome may be 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than the untreated migrasome or the migrasome before treating. For example, amount and/or function of an angiogenesis factor in migrasome may be analyzed by western blot, Immunofluorescence and/or quantitative mass spectrometry analysis.

For example, increasing the amount and/or function of an angiogenesis factor in migrasome may comprise providing angiogenesis factor, overexpressing angiogenesis factor, and/or activating angiogenesis factor.

Increasing the expression of the angiogenesis factor (including a functional variant thereof, or a functional fragment thereof) may comprise overexpressing the angiogenesis factor. The overexpression may be achieved either by introducing an exogenous protein or an exogenous nucleic acid molecule encoding the protein, or by causing increased expression of the endogenous protein or the endogenous gene encoding for said protein. For example, such overexpression may be caused by a mutation in the regulatory region of a gene encoding for the protein. In some cases, the overexpression may be achieved by changing the function of one or more components of the transcriptional and/or translational machinery.

For example, increasing the amount and/or function of an angiogenesis factor in migrasome may comprise increasing the amount of angiogenesis factor. For example, increasing the amount and/or function of an angiogenesis factor in migrasome may comprise increasing the expression of angiogenesis factor. For example, increasing the amount and/or function of an angiogenesis factor in migrasome may comprise introducing a gene encoding for an angiogenesis factor in migrasome. For example, increasing the amount and/or function of an angiogenesis factor in migrasome may comprise activating the interaction between the angiogenesis factor and the receptor of the angiogenesis factor. For example, increasing the amount and/or function of an angiogenesis factor in migrasome may comprise muting an angiogenesis factor in migrasome.

angiogenesis factor

For example, the present application provides a method for and/or an agent for use in promoting the function of the migrasome, which comprises increasing the amount and/or function of an angiogenesis factor in the migrasome. For example, the present application provides a method for and/or an agent for use in inhibiting the function of the migrasome, which comprises decreasing the amount and/or function of an angiogenesis factor in the migrasome. For example, the angiogenesis factor may comprise vascular endothelial growth factor (VEGF) , e.g., VEGFA, and/or transforming growth factor (TGF) , e.g., TGF-β3, or functional variants thereof.

Inhibiting the formation and/or function of said migrasome

For example, the method decreases angiogenesis, and may comprise inhibiting the formation and/or function of said migrasome.

For example, inhibiting the formation and/or function of migrasomes can comprise inhibiting the cell migration. For example, the migration ability or the migration rate of the cell may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the unmodified cell.

For example, inhibiting the formation and/or function of migrasomes can comprise inhibiting the formation of a retraction fiber. For example, the number of retraction fiber may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the number of retraction fiber derived from the unmodified cell.

For example, inhibiting the formation and/or function of migrasomes can comprise decreasing the amount and/or function of TSPAN protein. For example, the TSPAN protein may comprise its function fragment, and/or its functional variant. For example, the TSPAN protein may comprise TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37. For example, wherein said tetraspanin may comprise tetraspanin 4.

For example, inhibiting the formation and/or function of migrasomes may comprise decreasing the amount of cholesterol in a cell.

For example, wherein said inhibiting the function of said migrasome may comprise decreasing the amount and/or function of an angiogenesis factor in said migrasome.

Decreasing the amount and/or function of an angiogenesis factor in migrasome

For example, the present application provides a method for inhibiting the function of migrasome, which comprises decreasing the amount and/or function of an angiogenesis factor in the migrasome.

For example, when treating the migrasome according to the method of the present application, the amount and/or function of an angiogenesis factor in the treated migrasome may be lower than the untreated migrasome or the migrasome before treating. For example, the amount of an angiogenesis factor in the treated migrasome may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated migrasome or the migrasome before treating. For example, the function of an angiogenesis factor may be promoting angiogenesis and/or an angiogenesis related biological process, the function of the angiogenesis factor in the treated migrasome may be 0.1%lower, 1%lower, 10%lower, 20%lower, 30%lower, 40%lower, 50%lower, 60%lower, 70%lower, 80%lower, 90%lower, 95%lower, 99%lower, 100%lower, 2 times lower, 3 times lower, 4 times lower, 5 times lower, 10 times lower, 50 times lower, 100 times lower, 1000 times lower, or 10000 times lower than the untreated migrasome or the migrasome before treating. For example, amount and/or function of an angiogenesis factor in migrasome may be analyzed by western blot, Immunofluorescence and/or quantitative mass spectrometry analysis.

For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise knocking out the expression of a gene encoding for angiogenesis factor, knocking down the expression of a gene encoding for angiogenesis factor, and/or treating the migrasome with an agent capable of inhibiting the function of the angiogenesis factor.

Knocking out

Knocking out refers to a genetic process in which the target protein encoding gene is made inoperative ( “knocked out” ) . When the encoding gene is knocked out, it may comprise heterozygous knock out or homozygous knock out. In the heterozygous knock out, only one of two gene copies (alleles) is knocked out, in the homozygous knock out, both copies are knocked out.

Knockouts may be accomplished through a variety of techniques. In some cases, the knockouts may be naturally occurring mutations that are screened out or identified (e.g., by DNA sequencing or other methods) .

In some cases, the knockouts are generated by homologous recombination. For example, it may involve creating a nucleic acid (e.g., DNA) construct containing the desired mutation. The construct may also comprise a drug resistance marker in place of the desired knockout gene. The construct may further contain a minimum length (e.g., 2kb or above) of homology to the target sequence. The construct may be delivered to target cells (for example, through microinjection, electroporation, or other methods, such as transfection, using a virus or a non-virus system) . This method then relies on the cell’s own repair mechanisms to recombine the nucleic acid construct into the existing DNA (e.g., the genome of the cell) . This may result in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein, if it is translated at all. The drug selection marker on the construct may be used to select for cells in which the recombination event has occurred. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection may be performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals.

In some cases, the knockouts are generated using site-specific nucleases. Various methods may be used to precisely target a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell’s repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ) , which involves directly ligating the two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene.

For example, a zinc-finger nuclease may be used to generate such knockouts. Zinc-finger nucleases comprise DNA binding domains that can precisely target a DNA sequence. Each zinc finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene.

As another example, Transcription activator-like effector nucleases (TALENs) may be used to generate such knockouts. TALENs contain a DNA binding domain and a nuclease that can cleave DNA. The DNA binding region may comprise amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene. As a further example, clustered regularly interspaced short palindromic repeats (CRISPR) system may be used to generate such knockouts. The CRISPR/Cas9 method is a method for genome editing that contains a guide RNA complexed with a Cas9 protein. The guide RNA can be engineered to match a desired DNA sequence through simple complementary base pairing. The coupled Cas9 may cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in a nonfunctional gene.

The knockout may also comprise a conditional gene knockout. A conditional gene knockout allows gene deletion in a tissue or cell when certain conditions are fulfilled, for example, in a tissue specific manner. It may be achieved by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them, and deletes the gene flanked by these sites.

Knocking down

Knocking down refers to a process by which the expression of the target protein encoding gene is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

The knocking down may be through a genetic modification or may be transient. If a DNA of an organism or cell is genetically modified, the resulting organism or cell may be referred to as a “knockdown organism” or a “knockdown cell” . If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result may be referred to as a “transient knockdown” .

In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through a variety of processes. Binding can occur either through the blocking of transcription (in the case of gene-binding) , the degradation of the mRNA transcript (e.g., by small interfering RNA (siRNA) ) or RNase-H dependent antisense, or through the blocking of either mRNA translation, pre-mRNA splicing sites, or nuclease cleavage sites used for maturation of other functional RNAs, including miRNA (e.g., by morpholino oligos or other RNase-H independent antisense) .

RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC) . The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.

For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise decreasing the amount of angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise decreasing the expression of angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise introducing CRISPR Cas9 system and/or miRNA targeting angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise inhibiting the interaction between the angiogenesis factor and the receptor of the angiogenesis factor. For example, decreasing the amount and/or function of an angiogenesis factor in migrasome may comprise binding to the angiogenesis factor.

Agent capable of inhibiting the function of angiogenesis factor

For example, the present application provides an agent for decreasing the amount and/or function of the angiogenesis factor. For example, the agent may be capable of inhibiting the function of angiogenesis factor. For example, the agent capable of inhibiting the function of angiogenesis factor may comprise an agent that may inhibit the interaction between the angiogenesis factor and the receptor of the angiogenesis factor. For example, the agent capable of inhibiting the function of angiogenesis factor may comprise an agent that may bind to the angiogenesis factor. For example, the agent capable of inhibiting the function of angiogenesis factor may comprise a protease, a small molecule, and/or an antibody. For example, the agent capable of inhibiting the function of angiogenesis factor may comprise a special inhibitor.

For example, when treating angiogenesis factor with agent capable of inhibiting the function of angiogenesis factor, the function of the angiogenesis factor may be inhibiting. For example, when treating migrasome with the agent capable of inhibiting the function of angiogenesis factor, the function of the angiogenesis factor in the migrasome may be inhibiting. For example, when treating migrasome with the agent capable of inhibiting the function of angiogenesis factor, the angiogenesis factor in the migrasome may not promote angiogenesis and/or an angiogenesis related biological process. For example, the agent capable of inhibiting the function of VEGF may be a protease, a small molecule, and/or an antibody that can bind to VEGF, e.g., VEGFA. For example, the agent capable of inhibiting the function of TGF may be a protease, a small molecule, and/or an antibody that can bind to TGF e.g., TGF-β3.

Method for monitoring angiogenesis and/or an angiogenesis related biological process

i) monitoring the angiogenesis and/or the angiogenesis related biological process in said subject according to method for regulating angiogenesis and/or an angiogenesis related biological process of the present application; and

ii) administering a regulating agent according to the result of step i) .

For example, the method may be an in vitro or ex vivo method. For example, the method may be an in vivo method.

For example, wherein said angiogenesis related biological process may comprise tumor and/or wound healing.

For example, wherein said biological sample may comprise a body fluid sample of said subject. For example, wherein said biological sample may comprise a blood sample of said subject.

For example, the biological sample may be collected and/or analyzed. For example, the biological sample may comprise but not limited to biological fluids such as sputum, blood, serum, plasma, or urine. For example, the biological sample may comprise a blood sample. For example, the blood sample may comprise whole blood, plasma, and/or serum.

For example, the biological sample may be from a human and/or an animal. For example, the biological sample may be analyzed in vivo, e.g., without removal from the human or animal, or the biological sample may be tested in vitro. For example, the biological sample may be analyzed after processing, e.g., by isolating. For example, the biological sample may be freshly taken from a human or animal, or may be processed or stored.

For example, analyzing biological sample may comprise assess a change in migrasome level in the biological sample in comparison with a reference sample. For example, the amount and/or function of a migrasome in the biological sample may be lower than in the reference sample, which may indicate that the subject is at a greater risk of angiogenesis inhibiting and/or angiogenesis related biological process inhibiting. For example, the amount and/or function of a migrasome in the biological sample may be higher than in the reference sample, which may indicate that the subject is at a greater risk of angiogenesis promoting and/or angiogenesis related biological process promoting. For example, the reference sample may be derived from the same subject, taken at a different time point or from other site of the body, and/or from another individual.

For example, wherein an increase of the amount of said migrasome may indicate an increase of said angiogenesis. For example, wherein analyzing the presence, amount and/or function of said migrasome may comprise analyzing the presence and/or amount of a marker molecule of said migrasome.

For example, wherein analyzing the presence, amount and/or function of said migrasome may comprise determining the presence and/or amount of Tspan4 ⁺, Integrin ⁺, Pleckstrin Homology (PH) domain ⁺, NDST1 ⁺, PIGK ⁺, CPQ ⁺, EOGT ⁺, KUL01 ⁺ and/or CD115 ⁺ vesicles in said biological sample.

For example, wherein analyzing the presence, amount and/or function of said migrasome may comprise staining said biological sample with wheatgerm agglutinin (WGA) .

For example, wherein said migrasome may be KUL01 ⁺ and/or CD115 ⁺. For example, the migrasome generated by a monocyte and/or macrophage may comprise KUL01 ⁺ and/or CD115 ⁺migrasome. For example, the amount of the KUL01 and/or CD115 may be higher on the migrasome generated by a monocyte and/or macrophage that the migrasome generated by a cell other than monocyte and/or macrophage.

Agent

For example, the agent may be capable of increasing the formation and/or function of said migrasome, and for use in increasing angiogenesis.

For example, the agent may be capable of increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said monocyte, said macrophage and/or in said migrasome.

For example, the agent may comprise a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

For example, wherein said tetraspanin may comprise TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.

For example, the agent may be capable of inhibiting the formation and/or function of said migrasome, and for use in decreasing angiogenesis.

For example, the agent may be capable of inhibiting the expression and/or function of a tetraspanin in said monocyte, said macrophage and/or in said migrasome.

For example, the agent may be capable of knocking out or knocking down the expression of a gene encoding for said tetraspanin in said monocyte and/or macrophage.

For example, wherein said tetraspanin may comprise tetraspanin 4.

Isolated and/or engineered migrasome generated by a monocyte and/or a macrophage

For example, this patent application provides an agent capable of isolating a migrasome generated by a monocyte and/or a macrophage, for use in regulating angiogenesis and/or an angiogenesis related biological process.

For example, this patent application provides an agent capable of modifying an engineered monocyte and/or macrophage by altering its migrasome generation ability, for use in preparing an engineered monocyte and/or a macrophage with altered ability for regulating angiogenesis and/or an angiogenesis related biological process comparing to a corresponding unmodified monocyte and/or macrophage.

For example, this patent application provides a method for preparing an engineered monocyte and/or a macrophage with altered ability for regulating angiogenesis and/or an angiogenesis related biological process comparing to a corresponding unmodified monocyte and/or macrophage, the method comprises modifying an engineered monocyte and/or macrophage by altering its migrasome generation ability.

For example, this patent application provides a use of the agent capable of modifying an engineered monocyte and/or macrophage by altering its migrasome generation ability, in the preparation of an engineered monocyte and/or a macrophage with altered ability for regulating angiogenesis and/or an angiogenesis related biological process comparing to a corresponding unmodified monocyte and/or macrophage.

For example, the monocyte and/or the macrophage may have increased ability for promoting angiogenesis comparing to a corresponding unmodified monocyte and/or macrophage.

A “corresponding unmodified cell” refers to a cell that has not been modified to alter the amount and/or function of the sphingomyelin therein, while with all the other features substantially the same as the engineered cell. In some cases, the corresponding unmodified cell is a wildtype cell (e.g., of the same cell type as the engineered cell) . In some cases, the corresponding unmodified cell may comprise one or more modifications, but the modification may be for other purposes.

For example, the monocyte and/or the macrophage may have been modified to have increased ability for generating migrasomes.

For example, the monocyte and/or the macrophage may have been modified to increase the amount and/or function of a tetraspanin therein.

For example, the monocyte and/or the macrophage may have been modified to overexpress a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

For example, the monocyte and/or the macrophage may have decreased ability for promoting angiogenesis comparing to a corresponding unmodified monocyte and/or macrophage.

For example, the monocyte and/or the macrophage may have been modified to have decreased ability for generating migrasomes.

For example, the monocyte and/or the macrophage may have been modified to decrease the amount and/or function of a tetraspanin therein.

For example, wherein the expression of a gene encoding for a tetraspanin may have been knocked out or knocked down.

For example, wherein said tetraspanin may comprise tetraspanin 4.

Medical use

In the present disclosure, an agent may be a small molecule compound, an antibody, a nucleic acid molecule, a polypeptide, or fragments thereof. In some cases, the agent may comprise one or more active components, present in a single molecule or as separate molecules. The agent may be provided in a non-active form and be converted into an active form in vitro or in vivo before, during or after administration. The agent may be a pharmaceutical agent or an agent for non-pharmaceutical use. The agent may exert the desired functions directly or indirectly via the function of additional agents, compositions, or cells.

For example, wherein said migrasome may be KUL01 ⁺ and/or CD115 ⁺.

Composition and/or kit

In the eleventh aspect, this patent application provides a composition, comprising the agent of the present application, the isolated migrasome of the present application, and/or the engineered monocyte and/or macrophage of the present application. For example, the composition may be a pharmaceutical composition and may optionally comprise a pharmaceutically acceptable excipient.

For example, the present application provides a composition comprising an agent of the present application, an isolated migrasome of the present application and/or an engineered cell of the present application. For example, the composition of the present application may be a pharmaceutical composition.

For example, the composition may be formulated with a suitable amount of a pharmaceutically acceptable excipient to provide the form for proper administration. For example, the composition may take the form of capsules, tablets, powders, solutions, or any other form suitable for administration. For example, the pharmaceutically acceptable excipient may be the excipient that is approved by a regulatory agency and/or listed in generally recognized pharmacopeia for use in subject, e.g., humans.

For example, dosages and desired concentration of the composition may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well known within the skill of an ordinary artisan. It is within the scope of the present application that different formulations may be effective.

In the twelfth aspect, this patent application provides a kit, comprising the agent of the present application, the isolated migrasome of the present application, the engineered monocyte and/or macrophage of the present application, and/or the composition of the present application. For example, the kit may be a packaged composition.

The kit of the present disclosure may comprise the agent, the engineered cell, and/or the composition according to the present disclosure. The agent, the engineered cell, and/or the composition may be comprised in suitable packaging, and written material that can include instructions for use, discussion of experimental studies (such as clinical studies) , listing of side effects, and the like. Such kits may also include information, such as scientific literature references, package insert materials, experimental results (such as clinical trial results) , and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the agent, the engineered cell and/or the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the users (such as health care provider or consumers) . Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may further contain an additional agent. In some embodiments, the agent, engineered cell and/or the composition of the present invention and the additional agent may be provided as separate compositions in separate containers within the kit. In some embodiments, the agent, the engineered cell and/or the composition of the present disclosure and the additional agent are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to users (such as health providers) , including scientists, physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

In one aspect, the present disclosure provides a method for regulating the recruitment of a second immune cell by a first immune cell, comprising regulating the formation and/or function of a migrasome generated by the first immune cell. In another aspect, the present disclosure provides a method for regulating the migration of an immune cell towards a location, comprising regulating the amount and/or function of a migrasome present at or near the location. In another aspect, the present disclosure provides a method for regulating an immune response and/or an immune response mediated biological process, comprising regulating the formation and/or function of a migrasome generated by an immune cell mediating the immune response. In another aspect, the present disclosure provides a method for regulating an immune response and/or an immune response mediated biological process in a subject in need thereof, comprising administering to the subject an effective amount of an immune cell derived migrasome.

In another aspect, the present disclosure provides a method for monitoring an immune response and/or an immune response mediated biological process in a subject, comprising analyzing the presence, amount and/or function of a migrasome obtained from a biological sample of the subject. In another aspect, the present disclosure provides a method for regulating an immune response and/or an immune response mediated biological process in a subject, comprising: i) monitoring the immune response and/or the immune response mediated biological process in the subject according to the present disclosure; and ii) administering a regulating agent according to the result of step i) .

In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome generated by a first immune cell, for use in recruiting a second immune cell to the first immune cell. In another aspect, the present disclosure provides an agent capable of regulating the formation and/or function of a migrasome present at or near a location, for use in regulating the migration of an immune cell towards the location.

In another aspect, the present disclosure provides an isolated migrasome derived from an immune cell. In another aspect, the present disclosure provides an engineered immune cell with altered ability for recruiting a second immune cell comparing to a corresponding unmodified immune cell, the engineered immune cell has been modified to alter its migrasome generation ability. In another aspect, the present disclosure provides an engineered immune cell with altered ability for regulating an immune response and/or an immune response mediated biological process comparing to a corresponding unmodified immune cell, the engineered immune cell has been modified to alter its migrasome generation ability. In another aspect, the present disclosure provides use of the agent according to the present disclosure, the isolated migrasome according to the present disclosure and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the recruitment of a second immune cell to the first immune cell. In another aspect, the present disclosure provides use of the agent according to the present disclosure, the isolated migrasome according to the present disclosure and/or the engineered immune cell according to the present disclosure in the preparation of a regulator for the migration of an immune cell towards the location.

In another aspect, the present disclosure provides a composition, comprising the agent, the isolated migrasome, and/or the engineered cell according to the present disclosure. In another aspect, the present disclosure provides a kit, comprising the agent, the isolated migrasome, the engineered cell and/or the composition according to the present disclosure. In another aspect, the present disclosure provides a method for isolating and/or regulating a migrasome generated by a monocyte, comprising: i) characterizing the migrasome according to a method of the present disclosure; and ii) isolating the characterized migrasome, and/or administering a regulating agent to said characterized migrasome.

In the present disclosure, an immune-cell mediated response may encompass any biological process involving the function or participation of an immune cell. The immune-cell mediated response may comprise an immune response or a non-immune response (such as a response mainly involving other types of cells, but it could be triggered or promoted by an immune cell) .

For example, in the present disclosure, the immune cell mediated process may comprise the recruitment of a second immune cell by a first immune cell. The recruitment of the second immune cell may be increased by promoting the formation and/or function of the migrasome generated by the first immune cell. In some cases, the recruitment of the second immune cell is increased by administering an effective amount of migrasomes derived from the first immune cell. In some cases, the recruitment of the second immune cell is decreased by inhibiting the formation and/or function of the migrasome generated by the first immune cell.

The formation and/or function of a migrasome may be regulated (i.e., promoted or inhibited, as appropriate) by any approach applicable. For example, the formation and/or function of a migrasome may be regulated by regulating migration of the cell generating the migrasome. For example, the formation and/or function of a migrasome may be regulated by regulating the formation of a retraction fiber of the cell generating the migrasome. For example, the formation and/or function of a migrasome may be regulated by regulating the amount and/or function of a tetraspanin protein (including its function fragment, and/or its functional variant) . For example, the formation and/or function of a migrasome may be regulated by regulating the amount of cholesterol in a cell generating the migrasome or in the migrasome. In some cases, promoting the formation and/or function of the migrasomes comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in the immune cell generating the migrasome and/or in the migrasome. For example, this may be achieved by overexpressing the tetraspanin protein, the functional fragment thereof, and/or the functional variant thereof in the immune cell. For example, the tetraspanin may comprise TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.

In some cases, promoting the formation and/or function of the migrasome comprises increasing the amount and/or function of a chemokine in the immune cell generating the migrasomes and/or in the migrasome. For example, the chemokine may comprise CCL2 and/or CXCL12. For example, the chemokine may comprise CXCL12.

In some cases, inhibiting the formation and/or function of the migrasome comprises inhibiting the expression and/or function of a tetraspanin in the immune cell generating the migrasome and/or in the migrasome. Inhibiting the expression and/or function of the tetraspanin may comprise knocking out or knocking down the expression of a gene encoding for the tetraspanin in the immune cell generating the migrasome. The tetraspanin may comprises tetraspanin 4 and/or tetraspanin 9.

According to any aspect of the present disclosure, the immune cell (e.g., the first immune cell, the second immune cell, the local immune cell; including the engineered immune cell of the present disclousre) may comprise a monocyte and/or a macrophage, including their progenitors and/or progenies (i.e., cells derived from the monocytes and/or the macrophage) . For example. the immune cell generating the migrasome (e.g., the first immune cell, or the local immune cell of the present disclosure) may consists essentially of a monocyte and/or macrophage. In some cases, the affected or targeted immune cell (e.g., the second immune cell of the present disclosure, or the immune cell to migrate) comprises a monocyte and/or a macrophage (including their progenitors and/or progenies) . In some cases, the affected or targeted immune cell (e.g., the second immune cell of the present disclosure, or the immune cell to be migrated) consists essentially of a monocyte and/or a macrophage. In some cases, the immune cell generating the migrasome is of the same type as the affected or targeted immune cell. For example, the immune cell generating the migrasome may comprise a monocyte and/or a macrophage, and the affected or targeted immune cell may also comprise a monocyte and/or a macrophage. In some cases, the migrasome generated by the immune cell comprises and/or expresses a chemokine (e.g., CXCL12) .

The present disclosure also provides an engineered immune cell. In some cases, the engineered immune cell has increased ability for recruiting a target immune cell (e.g., the second immune cell of the present disclosure) comparing to a corresponding unmodified immune cell. In some cases, the engineered immune cell has decreased ability for recruiting a target immune cell (e.g., the second immune cell of the present disclosure) comparing to a corresponding unmodified immune cell. In some cases, the engineered immune cell has increased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell. In some cases, the engineered immune cell has decreased ability for regulating the immune response and/or the immune response mediated biological process comparing to a corresponding unmodified immune cell.

In some cases, the engineered cell has increased secretion ability comparing to a corresponding unmodified immune cell. In some cases, the engineered cell has decreased secretion ability comparing to a corresponding unmodified immune cell.

The spatial distribution of pro-angiogenic factors may determine where blood vessels can grow. The mechanisms that determine the spatial distribution of pro-angiogenic factors are currently unclear. This application reveals that highly migratory cells patrol the area of capillary formation in chick embryo chorioallantoic membrane (CAM) . These cells may deposit migrasomes on their migration tracks, creating migrasome-enriched areas. Single-cell sequencing identified that these cells may be monocytes. Depletion of monocytes may impair capillary formation. Quantitative mass spectrometry analysis reveals that monocyte migrasomes may be enriched with pro-angiogenic factors. Purified migrasomes may promote capillary formation and monocyte recruitment in vivo, and endothelium cell tube formation and monocyte chemotaxis in vitro. Knockdown or knockout of TSPAN4 may reduce migrasome formation and impair capillary formation and monocyte recruitment. For example, monocytes may promote angiogenesis via VEGFA and CXCL12 in migrasomes. For example, monocytes may deposit migrasomes enriched in pro-angiogenic factors to promote angiogenesis.

For example, migrasome formation by monocytes may play an essential role in angiogenesis in chicken embryos. For example, extensive formation of migrasomes by highly migratory cells may be on the chorioallantoic membrane (CAM) of chicken embryos from day 9. By single-cell sequencing and antibody staining, these highly migratory cells may be identified as monocytes. Depletion of monocytes may cause impairment of capillary formation, which shows that these cells may play important roles in angiogenesis. Moreover, migrasomes may be capable of inducing angiogenesis in vivo and endothelium cell tube formation in vitro. Knockdown or knockout of TSPAN4, a key gene for migrasome formation in zebrafish and mammalian cells, may block migrasome formation and impairs angiogenesis, which can be rescued by adding back purified migrasomes from wild-type CAM. Furthermore, migrasomes can recruit monocytes in vitro and in vivo. Knockdown or knockout of TSPAN4 significantly may reduce the number of monocytes in the area where capillaries are forming. This defect in monocyte recruitment may be rescued by adding back migrasomes. For example, VEGFA and CXCL12 may be enriched in migrasomes. Addition of migrasomes can rescue the phenotypes cause by knockdown of VEGFA or CXCL12. Thus, migrasomes may carry out their function in recruiting monocytes and promoting angiogenesis through release of CXCL12 and VEGFA, respectively. Based on these data, there may be a “vanguard” model of angiogenesis, in which monocytes prepare a pro-angiogenic microenvironment in advance of angiogenesis by depositing migrasomes enriched in angiogenic factors.

For example, it shows that migrasomes may be generated by monocytes/macrophages in the CAM during chicken embryonic development. For example, it shows that migrasomes may play an important role in angiogenesis by delivering angiogenic factors such as VEGFA and CXCL12 to the area of capillary formation, thus creating a favorable microenvironment for angiogenesis. For example, it shows that monocytes can recruit more monocytes via migrasomes through CXCL12-mediated chemotaxis. This may create a positive feedback loop which can sustain the rapid capillary formation in CAM.

For example, VEGF can be sequestered by the ECM near VEGF-secreting cells, which restricts the diffusion of VEGF and creates a signaling pattern. For example, it shows that, beside this mechanism, migrasomes may represent an additional mechanistic layer which can generate signal patterns and gradients. Using migrasomes as the signal source to guide angiogenesis may have multiple advantages. First, for many angiogenic factors which do not bind to ECM, migrasomes may provide a mechanism for creating spatial and temporal signal patterns. Secondly, multiple different kinds of angiogenic factors can be packed into the same migrasome. These factors, which may contribute to angiogenesis through different yet complementary mechanisms, can undergo synchronized release, thus ensuring the spatial and temporal coordination of diverse regulatory mechanisms for angiogenesis. Finally, the release of angiogenic factors from migrasomes may require the rupture or leakage of migrasomes. This may create a latency effect and provide more opportunity to fine-tune the formation of signal patterns.

For example, it shows the importance of monocytes in angiogenesis during embryonic development. For example, it shows a “vanguard” model for angiogenesis, in which migratory monocytes may serve as forerunners to prepare a favorable microenvironment for angiogenesis in advance of capillary formation (Fig. 7f) .

Beside angiogenesis during embryonic development, there may be the involvement of monocytes in physiological homeostasis of adult tissue and in pathological conditions. For example, in both injury-induced angiogenesis and tumor angiogenesis, monocyte-derived macrophages and tumor-associated macrophages may have been shown to promote angiogenesis by secretion of VEGFA. Therefore, it is possible that migrasome formation may be also involved in these processes.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; r.t., room temperature; and the like.

Materials and Methods

Cultivation of SPF chick eggs

Fertilized SPF eggs (variety: White Leghorn; cleanliness: SPF) are bought from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd. The eggs are incubated in a hatching incubator at 37.5 ℃ with 60-70%humidity. Eggs are turned every 5 minutes.

Reagents

The antibody against VEGFA is generated by ABclonal Technology (Co. WG-04988, China) . The antibody against CXCL12 is from LSBio (Co. LS-B943-100, Seattle, USA) . The anti-Integrinα5 is from Cell Signaling Technology (4705S, Massachusetts, USA) . The antibody against GAPDH is from Proteintech (60060004-1-IG, Rosemont, USA) . The antibody against NDST is from Santa Cruz Biotechnology (sc-374529, Dallas, USA) . The antibody against CPQ is generated by Sigma (HPA023235, Shanghai, China) . The KUL01 antibody is from SouthernBiotech (8420-09, Birmingham, USA) . The antibodies against CD115 (CSF1R) , CD115-Alex488 and CD115-Alex647 are from Bio RAD (MCA5956GA, MCA5956GA488, MCA5956GA647, Hercules, USA) . The antibody against TSG101 is from Abcam (ab125011, Cambridge, USA) . The antibody against Calnexin is from Abcam (ab22595, Cambridge, USA) .

WGA (wheat-germ agglutinin) is from Life Technologies (W11261, Carlsbad, USA) . CellTracker ^TM Red CMTPX is from Invitrogen Life Technologies (C34552, Carlsbad, USA) . GM-CSF (granulocyte-macrophage colony stimulating factor) is from PeproTech (315-03, Cranbury, USA) . Lipo-fectamine ^TM3000 transfection reagent and P3000 reagent are from Invitrogen Life Technologies (L3000015, Carlsbad, USA) . Matrigel Basement Membrane Matrix is from Corning (356234, New York, USA) . Dextran is from Sigma-Aldrich (46945-100MG-F, USA) . PBS liposomes and Clodronate liposomes are from LIPOSOMA research (C-005, P-005, Amsterdam, The Netherlands) . Low melting agarose II is from AMRESCO (0815-25G, USA) .

Phosphate buffered saline is from Cytiva HyClone (SH30256.01, Marlborough, USA) . Endothelial Cell Medium is from ScienCell Research Laboratories (1001, Carlsbad, USA) . 0.25%Trypsin+0.02%EDTA solution is from Cienry (CR-25200, Hangzhou, China) . Penicillin&Streptomycin solution is from GENOM (GNM15140, Hangzhou, China) . GlutaMAX ^TM I (100×) is from Gibco (35050-061, Carlsbad, USA) ; 4%Paraformaldehyde is from DINGGUO CHANGSHENG Biotechnology (ar-0211, Beijing, China) .

Collagenase Type II powder is from Gibco (17101-015, Carlsbad, USA) . Lysosome isolation kit is from Sigma-Aldrich (LYSISO1-1KT, Shanghai, China) .

2xRealStar green power mixture is from Gibco (A311-01, Carlsbad, USA) . TaKaRa MiniBEST Universal RNA Extraction kit is from TaKaRa (9767, Kusatsu, Japan) . Endofree plasmid Midi kit is from CWBIO (CW2105S, Taizhou, China) . TIANgel Midi Purification kit is from Tiangen (DP209-02, Beijing, China) .

Cell culture and treatment

HUVECs are grown in endothelial cell medium (ECM, ScienCell) and used between

passages

3 and 5. HEK293A cells are cultured in DMEM (Gibco Life Technology) supplemented with 10%FBS. Cells are grown at 37 ℃ in a humidified incubator with 5%CO ₂. WGA ^high cells are cultured in RPMI 1640 medium (Gibco) supplemented with 10%v/v fetal calf serum (Sigma-Aldrich, UK) , 2 mM L-glutamine, and 1%v/v (500 U/mL) penicillin/streptomycin. Cells are grown at 37℃ in a humidified incubator with 5%CO ₂.

Migrasome purification

Migrasome purification is performed by iodixanol sucrose density-gradient centrifugation using an Opti-prep kit (LYSISO1, Sigma-Aldrich) . Chorioallantoic membranes are isolated from E9d chicken embryos (approximately 8 embryos for the rescue experiments and 30 embryos for quantitative mass spectrometry analysis) , then subjected to mechanical mincing. The chopped-up CAMs are then treated with collagenase II and trypsin. The samples are centrifuged at 1,000g for 5 min at 4 ℃ to remove the cell bodies, followed by 4,000g for 20 min at 4 ℃ to remove the cell fragments, and finally at 20,000g for 20 min at 4 ℃. The pellet containing the crude migrasome fraction is resuspended and lysed in extraction buffer (Sigma-Aldrich) and then fractionated at 150,000g for 4 h at 4 ℃ in a multistep Optiprep dilution gradient. The gradient is: 3, 5, 8, 12, 16, 19 (sample) , 22.5 and 27%. Fractions are collected and added to 500 μl PBS. Centrifugation is then performed at 20,000g for 30 min at 4 ℃. The pellet is collected, washed once with PBS and centrifuged at 4 ℃, 2,000g for 10 min. The supernatant is collected and centrifuged at 4 ℃, 20,000g for 30 min to obtain migrasomes for TEM observation and injection into embryos.

Tube formation assay

HUVECs are cultured for 24 h then used for the tube formation assay. The cells are first synchronized by incubating them in ECM containing 0.1%FBS for 12 h. The assay is then performed according to the description in the Manual (BD Biosciences) . Briefly, 6-well plates are coated with Matrigel Basement Membrane Matrix (BD Biosciences; diluted in basal ECM at a ratio of 1: 1; 500 μl mixture/well) and incubated at 37 ℃ for 30 min to allow gelation. HUVECs are plated at a density of 2×10 ⁶ cells/well. Cells are incubated at 37 ℃ with 5%CO ₂ within 24 h and pictures are captured with a light microscope (Olympus) .

Observation of migrasome formation in CAM in vivo

First, a square hole (about 1 cm) is cut in the eggshell. Second, WGA is diluted in 1×PBS (1: 500, 200 μl 1×PBS) and then this mixture is added to the top of the CAM. After staining for 20 min at 37.5 ℃ in a humidified incubator with 70%humidity, the egg is placed on a holder with the hole in direct contact with a cover glass, so that the weight of the egg held the CAM in tight contact with the cover glass. Last, the CAM is visualized under a Nikon A1 FV3000 confocal microscope and a Dragonfly Andor spinning disc confocal microscope.

Migrasome delivery by Matrigel or agarose

Fertilized chick eggs are incubated at 37.5 ℃ for 9 days. Then the E8d eggs are windowed. 5 μl (20 μg/μl, 100 μg) of migrasome sediment are embedded in 3 μl low-melting-point agarose or 3 μl Matrigel. After the mixture solidified, it is placed onto the CAM. 48 h later, images are captured by a Leica EZ4W stereomicroscope.

RNAi treatment of CAM and rescue by migrasome delivery

Fertilized chick eggs are incubated at 37.5 ℃ for 8 days. Then the E8d eggs are windowed and the CAM tissue is transfected with 2.5 μg siRNA for target genes (TSPAN4, VEGFA, CXCL12, GenePharma, Shanghai, China) with Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, 100022052) and P3000 (Thermo Fisher Scientific, Waltham, MA, 100022058) according to the manufacturer’s instructions. A pair of platinum electrodes (Nepagene) is used for electroporation. Electroporation (five pulses of 50 ms duration at 20 mV) is used to improve the transfection efficiency. SiNS (GenePharma, Shanghai, China) is used for all control siRNA experiments. The knockdown efficiency of the target gene TSPAN4 is confirmed by quantitative real-time PCR (rtPCR) analysis. After the target gene is knocked down successfully, 5 μl (20 μg/μl, 100 μg) of migrasome sediment are embedded in 3 μl of low-melting-point agarose. After the mixture solidified, it is placed onto the CAM.

Flow cytometry

The mAbs used for flow cytometry are listed in STAR METHODS. Isolation of monocytes is performed by flow cytometry using anti-CD115 antibody. For isolation of WGA ^high cells, briefly, CAMs are stained by WGA in vivo, then treated with collagenase II and trypsin. After removing the red blood cells, the residual cells (about 2×10 ⁷) are sorted by FACS (fluorescence activated cell sorting) . For isolation of CD115 ⁺ cells, cells are isolated from CAMs as described above, then incubated with anti-CD115 antibody (5×10 ⁶ cells in 600 μl 1×PBS, 1: 10, 37 ℃, 20 min) and sorted by FACS. To estimate the efficiency of TSPAN4 knockout in CAM9d, mCherry-positive cells are counted by flow cytometry.

TEM

CAMs isolated from 9d chick embryos are fixed with 2.5%glutaraldehyde + 2.0%paraformaldehyde diluted in 0.1 M Phosphate Buffer (0.1 M Na ₂HPO ₄.12H ₂O, 0.1 M NaH ₂PO ₄.2H ₂O, pH 7.2) . The CAMs are kept at room temperature for 2 h and then at 4 ℃ overnight. After three 10-min washes with 0.1M PB, the CAMs are treated with 1.5%K ₃Fe (CN) ₆ + 1%OsO ₄ (mixed before use) and kept at 4 ℃ for 1.5 h. The CAMs are washed three times with ddH ₂O (10 min each wash) , and then treated with 1%uranyl acetate in water and kept at 4 ℃ overnight. After three 15 min washes in ddH ₂O, the samples are dehydrated in ethanol (50, 70, 80, 90, 100, 100 and 100%; 15 min each) , then treated with 100%ethanol: 100%acetone at a 1: 1 ratio for 8 min, and finally with 100%acetone for 8 min. The CAMs are infiltrated with PON812 resin as follows: 1: 1 resin: acetone, 2 h at room temperature; 2: 1 resin : acetone, 2 h at room temperature; 3: 1 resin : acetone, 2 h at room temperature; resin alone, overnight; and resin alone, 2 h. Each CAM is then placed in the correct orientation on a 3.5-mm culture dish and a capsule filled with resin is placed over the CAM. The resin is polymerized at 37 ℃ for 8 h, 45 ℃ for 24 h and 60 ℃ for 12 h. Sections (70 nm) are cut with a Leica EM UC7 microtome and then stained with uranyl acetate and lead citrate. Images are obtained with a H-7650B TEM at 50-70 KV.

Imaging

To acquire two-dimensional images of vessel sprouting in CAMs in vivo, migrasomes or siRNA are added at the desired embryonic stage, then the CAMs are imaged by a Leica EZ4W stereomicroscope. Time-lapse multiple-view z-stack images (4D) of WGA ^high cells are acquired for statistical analysis of migration and migrasome production. TSPAN4-KD, TSPAN4-KO or Cl-clodronate treatments are applied after windowing at the desired embryonic stage. Then the egg is placed on a holder with the window directly touching a cover glass, so that the weight of the egg kept the CAM in contact with the cover glass. The CAM is imaged by Olympus FV000 confocal microscopy, Nikon A1 confocal microscopy or spinning disk microscopy (Andor Dragonfly) .

Image processing

All of the time-lapse multiple-view z-stack embryo images (4D images) are processed using Imaris software 8.1.4 (Bitplane AG) . Images are processed by Image J to quantify the fluorescence intensity to assess the number of WGA ^high cells, the number of sprouting capillaries or the density of sprouting capillaries. Anima software is used to reconstruct the 3D migrasome structure from FIB-SEM images. To determine the cell migration speed, time-lapse images are acquired by Nikon A1 and analyzed using Imaris software 8.1.4.

Tandem-Mass-Tag (TMT) quantitative mass spectrometry analysis

First, proteins either from migrasomes (case) or from cell bodies (control) are prepared using 8M urea in phosphate buffered saline (PBS) (Wisent, Nanjing, China) containing protease inhibitor cocktail. Second, all samples are sonicated for 2 min and centrifuged, and the supernatant is carefully separated. Protein concentrations are analyzed using the BCA protein assay kit. Third, in-solution digestion is performed. A total of 100 μg of protein extracted from each sample is reduced with 5 mM dithiothreitol (DTT) at room temperature and alkylated with 12.5 mM iodoacetamide (IAM) in the dark at room temperature. Then, the mixture is diluted to 1.5 M urea with PBS and the proteins are digested with trypsin (Promega, Madison, WI, USA) at 37 ℃ overnight. Fourth, the tryptic peptides are desalted using Sep-Pak desalting columns (Waters, Milford, MA, USA) and then the desalted peptides are labeled with 15 μl tandem mass tags (TMT) 10-PLEX reagents (Thermo Fisher Scientific, Waltham, MA, USA) . Three repeats in the control group are marked as 126, 126N and 127C. Three repeats in the case group are marked as 128C, 129N and 129C. These combined TMT-labeled peptides are desalted by Sep-Pak columns and separated with a UPLC 3000 system (Thermo Fisher Scientific, Waltham, MA, USA) with an XBridgeTM BEH300 C18 column (Waters, Milford, MA, USA) at a flow rate of 1 ml/min. The mobile phase A is H ₂O (pH=10) and the mobile phase B is 98%acetonitrile (pH=10) . Peptides are separated with a gradient elution consisting of an increase from 8%to 18%phase B for 30 min, followed by an increase from 18%to 32%phase B for 22 min. Forty-eight fractions are dried by speedvac and recombined to 12 fractions. The fractions are dissolved in 20 μl of 0.1% (v/v) formic acid (FA) and analyzed by LC–MS/MS. Spectra from the mass spectrometer are searched against the UniProt Gallus gallus database using the SEQUEST search engine of Proteome Discoverer software (version 2.3) . The identified proteins are quality monitored, and each protein with more than 5 points and with a specific peptide segment number greater than 2 is judged to be credible and is carried forward for the subsequent quantitative analysis. For the results of the peptide segment search, X _corr needed to be higher than 2.5 for the peptide segment to be judged as credible.

Quantitative real-time PCR (qPCR)

To avoid contamination, all RNA-associated experiments are conducted in a molecular biology laboratory that is specifically designed for clinical diagnosis using molecular techniques, and which includes separate laboratories dedicated to performing each step of the procedure. Total RNA is isolated from CAM tissues with a TaKaRa MiniBEST Universal RNA Extraction Kit (Clontech TaKaRa, Cat#9796, USA) . cDNA is synthesized from 2 μg total RNA using a reverse transcription kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Total RNA is isolated from cells with Trizol reagent (Tiangen, Beijing, China) . qPCR is performed with the Roche LightCycler 480 II System (Roche, Basel, Switzerland) using SYBR green reaction mixture (GenStar, Beijing, China, Cat#A311-101) according to the manufacturer’s instructions. GAPDH and ACTB are used as internal controls for mRNA quantification. TSPAN4 primers are acquired from Primer-Blast and are listed in STAR METHODS.

Western blotting

Whole cell extracts or migrasome extracts are isolated from E9.5 chick embryos using RIPA buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1%Triton X-100, 1%sodium deoxycholate, 0.1%SDS, 5 mM EDTA) or 2%SDS in 1×TBS complemented with protease inhibitors (complete protease inhibitor cocktail tablets, Roche) . After determining the protein concentration of each sample using the BCA kit (Biorad) , 40 μg of lysate is resolved on 10%SDS PAGE gels (Invitrogen) and transferred onto PVDF membranes (Amersham) . Blots are then blocked with 5%milk in 1×PBS for 1 h at room temperature, followed by incubation with antibodies against Integrin α5/NDST1/CPQ/Calnexin/Tsg101/GAPDH/VEGFA/CXCL12/Alix at 4℃ overnight. Binding of HRP-conjugated secondary antibodies is subsequently visualized on the ChemiDoc MP Imaging System (BIO-RAD) .

Immunofluorescence

For immunofluorescent detection of VEGFA or CXCL12 in migrasomes, monocytes are isolated from CAM, and then cultured the monocytes on galectin-coated chambers for about 12 h. Then, the monocytes are stained by WGA488 (1: 2000) at 37 ℃ for 10 min. After that, monocytes are washed with PBS, fixed in 4%paraformaldehyde and permeabilized for 10 min with 0.3%Triton X-100 in 1×PBS. The monocytes are then blocked with 5%milk in 1×PBS for 1 h at room temperature and incubated with anti-VEGFA or CXCL12 antibody at 4℃ overnight. After that, the cells are washed three times with 1×TBST, then incubated with Alexa 561-conjugated IgG antibody (Sigma, USA) for 1 h at room temperature. The cells are finally washed with 1×TBST, and visualized under a Nikon A1 confocal microscope. For immunofluorescence of CSF1R and KULO1 in CAM, the CAM is isolated from E9d. Approximately 1 cm ² of CAM is cut and incubated in diluted WGA buffer (1 μl WGA in 500 μl 1×PBS) at 37℃ for 20 min. Then the CAM is washed with 1×PBS, fixed in 4%paraformaldehyde for 2 h and permeabilized for 2 h with 0.3%Triton X-100 in 1×PBS. After permeabilization, the CAM is blocked with 5%BSA in 1×PBS for 6 h at room temperature and incubated with anti-CSF1R or KUL01 antibody at 4℃ overnight. After that, the CAM is washed three times with 1×TBST and incubated with Alexa 561-conjugated IgG antibody (Sigma, USA) for 1 h at room temperature. The CAM is finally washed with 1×TBST and coated on SuperFrost Plus microscope slides by ProLong TM Diamond Antifade Mountant (P36970, Thermo Fisher Scientific, USA) . Colocalization of WGA ^high signal and CSF1R or KUL0l is visualized with a Nikon A1 confocal microscope.

Yeast phagocytosis by monocytes after GM-CSF stimulation

Monocytes from CAM9d are sorted by FACS after incubation with anti-CD115 antibody (5×10 ⁶ cells in 600 μl 1×PBS, 1: 10, 37℃, 20 min) . Then monocytes (1×10 ⁶) are cultured in chambers coated with 10%w/v gelatin solution. After that, monocytes are stimulated by GM-CSF (final concentration about 20 ng/μl, #315-03, PeproTech, USA) according to the manufacturer’s instructions and stained by WGA (1: 2000, 10 min, 37 ℃) . Yeast cells (strain BY4741, 1×10 ⁷) are stained by CellTracker ^TM Red CMTPX (C34552, Thermo Fisher Scientific, USA, 1: 50000, 95 ℃, 30 min) and added to the chamber. 12 h later, images are acquired by spinning disk microscopy (Andor Dragonfly) .

Monocyte depletion by clodronate liposomes

When chick eggs had developed to embryonic day 6, they are windowed and 2 μl clodronate-liposomes or PBS-liposomes (Liposoma BV, The Netherlands) are microinjected into a vein in the CAM. After 48 h, 7.5 μl clodronate-liposomes or PBS-liposomes are microinjected into a vein of CAM. Then the windows are sealed by Parafilm and incubated at 37.5 ℃ with 60%humidity. The CAMs are imaged by a Leica EZ4W stereomicroscope.

Dextran staining of the blood vessel system

Chick eggs (E9d) are windowed. 2 μl dextran (46945-100MG-F, 25 mg/ml) are diluted by 8 μl 1×PBS and the mixture is microinjected into the vein system. Images are immediately captured by an FV3000 Olympus confocal microscope.

Monocyte tracing

Chick eggs (E9d) are windowed and 20 μl CD115 (MCA5956GA488) are microinjected into the vein system. 20 min, 75 min and 120 min later, images are captured by FV3000 Olympus confocal microscopy.

Transwell chemotaxis assay

Transwell chemotaxis assays are performed with 12-well transwell plates. WGA ^high or WGA ^low cells are isolated by FACS after WGA staining. WGA ^high or WGA ^low cells are seeded in the upper chambers of the 12-well plate at 0.5×10 ⁶ cells/ml in RPMI 1640 medium (Gibco) supplemented with 10%v/v fetal calf serum (Sigma-Aldrich, UK) , 2 mM L-glutamine and 1%v/v (500 U/mL) penicillin/streptomycin. Generally, 12-well plates employed 1.5 ml of this RPMI 1640 medium in the lower chamber and 500 μl in the upper chamber. To identify the chemotaxis response of WGA ^high or WGA ^low cells to migrasomes, 15 μg migrasomes (1.5 μl of 10 μg/μl migrasomes in 1×PBS) are placed in the lower chambers as the case group and 1.5 μl 1×PBS are added in the lower chambers as the negative control group. The plates are then incubated for 6 h. The upper chambers are removed and the cells coating the top side of the polycarbonate membranes (Corning) are thoroughly removed with swabs. Then the polycarbonate membranes are cut off, washed with 1×PBS, fixed in 4%paraformaldehyde for 30 min, and stained by DAPI (1 μg/μl, 1: 1000) for 15 min. Lastly, the polycarbonate membranes are coated on SuperFrost Plus microscope slides with ProLong TM Diamond Antifade Mountant (P36970, Thermo Fisher Scientific, USA) . The number of migrated cells adhered to the underside of the polycarbonate membrane is visualized and counted by Nikon A1 confocal microscopy.

Generation of TSPAN4-KO-mCherry-KI chick embryos by the CRISPR-Cas9 system

A CRISPR/Cas9-based gene editing strategy is used to achieve TSPAN4 gene knockout in chick embryos. A guide RNA (gRNA) coding sequence is cloned into pUC57 vector (Addgene 55132) as the gRNA plasmid backbone, and the empty pUC57 vector is constructed as negative control (Scramble) which did not contain a sgRNA sequence. A sgRNA (Gallus-TSPAN4-gRNA1-Bsa1-F, 5’-TAGGGAAGGTTGAAGACAAACATT-3’ (SEQ ID NO: 1) ; Gallus-TSPAN4-gRNA1-Bsa1-R, 5’-AAACAATGTTTGTCTTCAACCTTC -3’ (SEQ ID NO: 2) ) is then designed to target exon 5 of chick TSPAN4. The sgRNA is inserted into the sgRNA expression cassettes of the pUC57 vector under control of the T7 promoter. Then the vector is introduced by chemical transformation into competent E. coli Top10 for cloning purposes using a kanamycin selectable marker.

To evaluate the TSPAN4 knockout efficiency, a knock-in plasmid is constructed to insert mCherry into the chick TSPAN4 gene under control of the original promoter. mCherry is inserted into TSPAN4 at the position targeted by the sgRNA. Thus, an mCherry-positive signal indicates that native TSPAN4 gene expression is silenced simultaneously. Follow this targeting strategy (Fig. 9a) , the T4-Chick-KO-mcherry-KI plasmid (PM19040-A) is constructed by Biocytogen. The integration detection primers are as follows: PM19040-A-WT-F, 5’-GGTCCAGCACTGATGAGTCCACCTA-3’ (SEQ ID NO: 3) ; PM19040-A-Mut-R, 5’-GGGGAAGGACAGCTTCAAGTAGTCG-3’ (SEQ ID NO: 4) ; PM19040-A-WT-F, 5’-GGTCCAGCACTGATGAGTCCACCTA-3’ (SEQ ID NO: 3) ; PM19040-A-WT-R, 5’-ACCATCTTGCCCAACTTTCGAGTTCA-3’ (SEQ ID NO: 5) .

To generate the TSPAN4-KO chick embryos, a square hole (0.6 cm × 0.6 cm) is cut in the eggshell of gastrulating chick embryos. 1.2 μg of the guide RNAs (target sequences are listed in STAR METHODS) are co-injected with 1 μg Cas9 and 1.6 μg mCherry-KI plasmid into chick embryos at Hamburger Hamilton stage 4 (HH4, embryonic 18 h) using a glass capillary with a tip diameter of 0.1 mm. Then chick embryos are then electroporated using previously described techniques (Sauka‐Spengler and Barembaum, 2008) . The hole is covered by Parafilm and the chick embryos are incubated at 37.5 ℃ in the air under 70%humidity. Knockouts are confirmed by direct FACS for mCherry-positive signal sorting and by in vivo imaging for mCherry-positive signal detection.

Chick CAM angiogenesis assay

Fertilized chicken eggs are incubated at 37.5 ℃ for 9 days and then opened so that the CAM could be observed. For preparation of Matrigel-or agarose-embedded migrasomes, first, 30 μg of purified migrasomes (from CAM of chicken embryos at E9d) are resuspended in 3 μl 1×PBS. Second, the resuspended migrasomes are mixed with either 5 μl Matrigel (356234, BD, USA) or 2%low-melting-point agarose. As the control, 3 μl 1×PBS is mixed with 5 μl Matrigel or 2%low-melting-point agarose. Last, the congealed mixture is placed on the designated side of the CAM in vivo. In the following days, numerous allantoic vessels developed as a “spoked-wheel” pattern. The newly formed vessels are examined and visualized with a photo microscope (Leica EZ4W) . The density or the number of newly formed capillaries in the CAM is quantified with Image J or Image-Pro Plus software (Media Cybernetics) .

Single-cell RNA-seq and data analysis

WGA ^high CAM cells are sorted by FACS into PCR tubes. Single-cell RNA-seq experiments are performed according to the Smart-seq2 protocol with 20 cDNA pre-amplification cycles. Samples are sequenced by Illumina Hiseq 4000 with 150-bp paired-end reads. The transcriptome is quantified by Salmon with the chicken genome reference GRCg6a. Data from cells with more than 1500 genes detected are considered as high quality and are used for subsequent analysis. Further data analysis and visualization used Seurat.

Identification of monocyte-like cells

Annotated homologs of many classical cell-type marker genes may not be found in the chicken reference genome. Therefore, all reported classical and non-classical marker genes for the cell types are collected based on morphology data. The collection of classical and non-classical marker genes contained experimental results and single-cell RNA sequencing results from biocc. hrbmu. edu. cn/CellMarker/. 317 high-expression genes are found in the monocyte-like cell group (Fig. 2g) .

Statistical analysis

Data are expressed as the mean ± standard error of the mean from at least three separate experiments performed in triplicate. Statistical analysis is performed using one-way/two-way ANOVA or two-tailed Student’s t-test. A value of P<0.05 is considered statistically significant. All experiments are performed at least three independent times with similar results.

Example 1 Migrasomes are related to angiogenesis related biological process

Migrasomes can be detected by staining cells with wheat-germ agglutinin (WGA) . To check whether migrasomes are formed in CAM during embryonic development, CAM from 9-day chicken embryos (CAM9d) is stained with WGA ex vivo. WGA staining reveals two types of cells: the majority of cells are large and flat with low WGA signal (WGA ^low) ; the other cells are smaller but with a bright WGA signal (WGA ^high) . It shows the WGA ^high cells form retraction fibers and migrasomes (Fig. 1a) . To further verify this observation, transmission electron microscopy (TEM) analysis is carried out on CAM9d, which reveals many large vesicles, with diameters up to 2 μm, in the extracellular space (Fig. 1b, 1c) . Consistent with the characteristic morphological features of migrasomes, these vesicles contain numerous intralumenal vesicles, and many of them are adjacent to fibers with diameters of about 50-100 nm (Fig. 1b) . To determine the 3D structure of this network of fibers and vesicles, FIB-SEM analysis is carried out. FIB-SEM shows that these vesicles are connected to fibers and, in many cases, the vesicles are localized on the tips of the fibers (Fig. 1d, 1e) , which is another morphological feature of migrasomes.

To directly observe the formation of migrasomes in vivo, an imaging protocol is designed. First, a hole is cut in the eggshell and stained the CAM with WGA. After staining, the egg is placed on a holder with the hole in direct contact with a cover glass, so that the weight of the egg white and yolk keeps the CAM in tight contact with the cover glass. Using time-lapse microscopy, it shows that retraction fibers and migrasomes are indeed formed in CAM of living chicken embryos. Put together, it shows that migrasomes are formed in CAM by WGA ^high cells (Fig. 1f) .

It shows that WGA ^high cells are evenly distributed in the CAM, and most of them are outside the blood vessels (Fig. 1g) . WGA ^high cells are extraordinarily mobile cells: in some cases, they can move as fast as 2 μm/min (Fig. 1h) , and they leave a dense patch of migrasomes in these areas (Fig. 1i) .

Example 2 Migrasomes are generated by monocytes

To investigate the identity of WGA ^high cells, WGA-stained CAM9d is first isolated from chicken embryos then subjected to mechanical mincing. The chopped-up CAMs are then treated with collagenase II and trypsin, and the released cells are collected and subjected to fluorescence activated cell sorting (FACS) . The WGA ^high and WGA ^low cells are collected and cultured in vitro (Fig. 2a, 2b) . WGA ^high and WGA ^low cells have different morphologies (Fig. 2c, 2d) . TEM analysis shows that the migrasomes generated by WGA ^high cells have a similar morphology to the migrasomes observed in vivo. Importantly, WGA ^high cells generate much higher numbers of migrasomes than WGA ^low cells (Fig. 2d) . Similar to in vivo observations, cultured WGA ^high cells move much faster and are smaller than WGA ^low cells (Fig. 2f, Fig. 9a-9c) . These data shows that the WGA ^high cells isolated may be the migrasome-generating WGA ^high cells observed in vivo.

Next, the WGA ^high cells are subjected to single-cell RNA sequencing, which identifies two subsets of cells in the WGA ^high population (Fig. 2g) . The first group is enriched with markers for monocytes, while the second group is enriched with markers for endothelial cells (Fig. 2g, 2h) .

The RNA-seq results, and the fact that the migrasome-forming WGA ^high cells are highly migratory, indicate that these cells could be monocytes. To verify the identity of WGA ^high cells, immunostaining is carried out with KUL01, an anti-macrophage/monocyte monoclonal antibody, and an antibody against CD115, which is expressed by monocytes/macrophages. It shows that indeed the WGA ^high cells stained positive for both antibodies, which indicates that WGA ^high cells are monocytes (Fig. 2k) . To further verify this, CAM is labeled with anti-CD115 antibody, and the CD115-positive cells are sorted out (Fig. 2i) . It shows that these cells are identical to WGA ^high cells in terms of morphology and their ability to form migrasomes (Fig. 2j) . Finally, it shows that WGA ^high cells are highly phagocytic, and the phagocytosis can be further enhanced by treating WGA ^high cells with GM-CSF (Fig. 2l, 2m) . Put together, these results indicate that WGA ^high cells are monocytes.

Example 3 Depletion of monocytes reduces migrasome number in CAM and impairs angiogenesis

A large pool of monocytes may be present inside blood vessels. To determine the origin of migrasome-forming monocytes outside blood vessels, the anti-CD115 antibody is injected into blood vessels. Ten minutes after antibody injection, it shows that there are indeed large numbers of CD115 positive cells lined up on the wall of blood vessels, and there are no CD115 cells outside blood vessels at this time point (Fig. 3a) . Then, 2 hours after injection, many of the CD115-positive cells are found outside the blood vessels, which suggests that the migrasome-forming monocytes in CAM probably come from blood vessels (Fig. 3a) . To test the role of monocytes in migrasome formation, liposomes containing the drug clodronate are used to deplete the monocytes. Clodronate-liposomes are microinjected into the CAM blood vessels (Fig. 3b) . Five days after the injection, the WGA ^high cells are almost completely depleted (Fig. 3c, 3d) ; moreover, the production of migrasomes in CAM is significantly reduced (Fig. 3e, 3f) . When angiogenesis is checked, it shows that the formation of large blood vessels may be not markedly affected by monocyte depletion, but the formation of capillaries may be significantly reduced (Fig. 3g, 3h) . It shows that monocytes contribute to angiogenesis.

Example 4 Monocyte migrasomes contain angiogenesis factors

To study the role of migrasomes in angiogenesis, migrasomes from CAM9.5d are isolated (Fig. 4a) . The purity of the isolated migrasome is analyzed by TEM and by western blot for various migrasome markers. It shows that the isolated migrasomes have the characteristic morphological features of migrasomes (Fig. 4b) ; moreover, migrasome markers are highly enriched in the preparation (Fig. 4c) . Next, tandem-mass-tag (TMT) labelling is carried out followed by quantitative mass spectrometry (Fig. 4d) . The resulting volcano plot shows that the protein composition of migrasomes is markedly different from cell bodies (Fig. 4e) . Known migrasome-enriched proteins such as tetraspanins and integrin β are enriched in CAM migrasomes, while nuclear proteins are depleted (Fig. 4f) , which shows that the Q-MS analysis may be reliable. Next, whether known angiogenesis factors and chemokines are enriched in migrasomes is checked. Indeed, it shows that a host of these factors, including TGF-β3, VEGFA and CXCL12, are enriched in migrasomes (Fig. 4f) . It is worth noting that compared to epithelial cells (the majority of WGA ^low cells) , CXCL12, TGF-β3, and VEGFA are highly expressed in monocytes (Fig. 4g and Fig. 10) .

Next, the enrichment of these angiogenesis factors and chemokines by western blotting and immunostaining is verified. CXCL12 and VEGFA are test. It shows that both proteins are enriched on migrasomes by both western blotting and immunostaining (Fig. 4h, 4i) . Next, cryo-sections of CAM are stained with anti-VEGFA and anti-CXCL12 antibodies. It shows that monocytes are the main VEGFA-and CXCL12-expressing cells (Fig. 4j and Fig. 11) . There may be a discrepancy in the degree of enrichment of VEGFA and CXCL12 signal in western blots and immunostaining. This likely results from an optical illusion created by the method for imaging CAM. The CAM is visualized by z-stack imaging and presented as the maximum intensity projection, which takes the brightest pixel in each layer and displays it in the final 2D image. Thus, thicker objects will look brighter as they have more layers. In this case, the cells are much thicker than migrasomes, and thus appear much brighter when displayed as maximum intensity projection images.

Example 5 Migrasomes induce capillary formation

Next, the role of migrasomes in angiogenesis is tested by adding isolated migrasomes on top of the CAM. To keep the migrasomes in place, migrasomes are mixed with Matrigel and mixture is added to the CAM of 9-day embryos. Since Matrigel may induce angiogenesis by itself, the migrasomes are also delivered by mixing with low-melting-point agarose. In both cases, adding migrasomes significantly induces capillary formation (Fig. 5a-5e) , which indicates that migrasomes are pro-angiogenic. Furthermore, it shows that adding migrasomes significantly enhanced the recruitment of monocyte cells (Fig. 5f, 5g) , which suggests that migrasomes act as a chemoattractant for monocytes.

Example 6 Migrasomes enhance endothelial cell tube formation in vitro

To further verify the capacity of migrasomes to promote capillary formation, the endothelial cell tube formation assay is carried out, which is widely used to assess the differentiation and proliferation of endothelial cells during angiogenesis. Similar to the in vivo result, it shows that adding migrasomes significantly enhances tube formation (Fig. 5h, 5i) . Similarly, the role of migrasomes in recruitment of monocytes is also tested in vitro by transwell assay (Fig. 5j) . It shows that migrasomes markedly enhance the chemotaxis of WGA ^high monocytes; in contrast, migrasomes only slightly enhance the chemotaxis of WGA ^low cells (Fig. 5k, 5l) . Put together, it shows a role for migrasomes in promoting capillary formation and monocyte recruitment.

Example 7 Blocking migrasome formation impairs capillary formation

To further test the role of migrasomes in capillary formation, RNAi knockdown of TSPAN4, an essential gene for migrasome formation is performed. It shows that knockdown of TSPAN4 significantly blocks migrasome formation and reduces capillary formation (Fig. 6a-6e) . This suggests that migrasomes do play important roles in capillary formation. Similarly, knocking down TSPAN4 causes reduced recruitment of monocytes (Fig. 6f, 6g) . To distinguish the roles of TSPAN4 from the role of migrasomes in angiogenesis, a rescue experiment is carried out, in which migrasomes isolated from wild-type CAM are added to the place where the TSPAN siRNAs are delivered. Addition of migrasomes successfully rescues monocyte recruitment and capillary formation, which suggests that TSPAN4 affects capillary formation by affecting migrasome formation (Fig. 6f-6i) .

To further support this observation, a protocol is established to knock out TSPAN4 in the early stage of chicken embryonic development. Knockout of TSPAN4 is achieved by using the Cas9-CRISPR TSPAN4 knock-out system and the mCherry knock-in system simultaneously (va) . In brief, plasmids encoding guide RNAs and Cas9 are injected into chick embryos at the gastrulating stage along with an mCherry knock-in plasmid. The knockout efficiency is indicated by mCherry expression, which may occur in cells where TSPAN4 has been successfully knocked out (Fig. 6j, 6k) . This protocol works well: knockout in more than 70%of cells is routinely achieved (Fig. 6l) . It shows that migrasome formation in TSPAN4-knockout CAM is significantly reduced (Fig. 6m, 6n) . In addition, migrasome formation in vitro is reduced in monocytes isolated from TSPAN4-knockout CAM (Fig. 9b) . Moreover, in agreement with the RNAi knockdown experiment, both recruitment of monocytes and formation of capillaries are significantly reduced (Fig. 6o, 6p, Fig. 9c, 9d) . For example, the defects in TSPAN4-knockout CAM may be rescued. The capillary formation defect is visible in CAM8d. Therefore, in the rescue experiment, migrasomes at 8 days are added and the effects are assessed after 48 hours. For example, knockout of TSPAN4 may cause embryo lethality at 9 days.

Example 8 Migrasomes rescue capillary formation defects in CAM with knockdown of angiogenesis factors

To investigate whether migrasomes affect angiogenesis through enrichment of VEGFA and/or CXCL12, VEGFA and CXCL12 are knocked down by RNAi. It shows that knockdown of VEGFA or CXCL12 significantly reduced capillary formation 48 hours after adding RNAi. For example, isolated migrasomes from wild-type CAM are added and the capillary formation is re-assessed 48 hours after rescue. It shows that adding migrasomes largely rescues capillary formation (Fig. 7a-7c) .

Using a similar protocol, the role of migrasomal CXCL12 is tested in recruitment of monocytes. It shows that CXCL12 is required for recruitment of monocytes and adding migrasomes can rescue the impaired monocyte recruitment in CXCL12 knockdown CAM (Fig. 7d, 7e) .

While exemplary embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A method for regulating angiogenesis and/or an angiogenesis related biological process, comprising regulating the formation and/or function of a migrasome generated by a monocyte and/or macrophage.
The method of claim 1, wherein said angiogenesis related biological process comprises tumor and/or wound healing.
The method of any one of claims 1-2, which increases angiogenesis, and comprises promoting the formation and/or function of said migrasome.
The method of claim 3, wherein said promoting the formation and/or function of said migrasome comprises increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said monocyte, said macrophage and/or in said migrasome.
The method of any one of claims 3-4, wherein said promoting the formation and/or function of said migrasome comprises overexpressing the tetraspanin, the functional fragment thereof, and/or the functional variant thereof in said monocyte, said macrophage and/or in said migrasome.
The method of any one of claims 4-5, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.
The method of any one of claims 3-6, wherein said promoting the function of said migrasome comprises increasing the amount and/or function of an angiogenesis factor in said migrasome.
The method of claim 7, wherein said angiogenesis factor comprises vascular endothelial growth factor (VEGF) and/or transforming growth factor (TGF) .
The method of claim 8, wherein said VEGF comprises VEGFA.
The method of any one of claims 8-9, wherein said TGF comprises TGF-β3.
The method of any one of claims 1-10, which decreases angiogenesis, and comprises inhibiting the formation and/or function of said migrasome.
The method of claim 11, wherein said inhibiting the formation and/or function of said migrasome comprises inhibiting the expression and/or function of a tetraspanin in said monocyte, said macrophage and/or in said migrasome.
The method of claim 12, wherein said inhibiting the expression and/or function of a tetraspanin comprises knocking out or knocking down the expression of a gene encoding for said tetraspanin in said monocyte and/or macrophage.
The method of any one of claims 12-13, wherein said tetraspanin comprises tetraspanin 4.
The method of any one of claims 11-14, wherein said inhibiting the function of said migrasome comprises decreasing the amount and/or function of an angiogenesis factor in said migrasome.
The method of claim 15, wherein said decreasing the amount and/or function of said angiogenesis factor comprises knocking out or knocking down the expression of a gene encoding for said angiogenesis factor in said monocyte and/or said macrophage.
The method of any one of claims 15-16, wherein said decreasing the amount and/or function of said angiogenesis factor comprises treating said migrasome with an agent capable of inhibiting the function of said angiogenesis factor.
The method of claim 17, wherein said agent capable of inhibiting the function of said angiogenesis factor comprises a protease, a small molecule, and/or an antibody capable of inhibiting the activity of said angiogenesis factor.
The method of any one of claims 17-18, wherein said angiogenesis factor comprises vascular endothelial growth factor (VEGF) and/or transforming growth factor (TGF) .
The method of claim 19, wherein said VEGF comprises VEGFA.
The method of any one of claims 19-20, wherein said TGF comprises TGF-β3.
A method for regulating angiogenesis and/or an angiogenesis related biological process in a subject in need thereof, comprising administering to said subject an effective amount of migrasomes generated by a monocyte and/or a macrophage.
The method of claim 22, wherein said angiogenesis related biological process comprises tumor and/or wound healing.
The method of any one of claims 22-23, which increases angiogenesis.
A method for monitoring angiogenesis and/or an angiogenesis related biological process in a subject, comprising analyzing the presence, amount and/or function of a migrasome generated by a monocyte and/or a macrophage in a biological sample of said subject.
The method of claim 25, wherein said angiogenesis related biological process comprises tumor and/or wound healing.
The method of any one of claims 25-26, wherein said biological sample comprises a body fluid sample of said subject.
The method of any one of claims 25-27, wherein said biological sample comprises a blood sample of said subject.
The method of any one of claims 25-28, wherein an increase of the amount of said migrasome indicates an increase of said angiogenesis.
The method of any one of claims 25-29, wherein analyzing the presence, amount and/or function of said migrasome comprises analyzing the presence and/or amount of a marker molecule of said migrasome.
The method of any one of claims 25-30, wherein analyzing the presence, amount and/or function of said migrasome comprises determining the presence and/or amount of Tspan4 ⁺, Integrin ⁺, Pleckstrin Homology (PH) domain ⁺, NDST1 ⁺, PIGK ⁺, CPQ ⁺, EOGT ⁺, KUL01 ⁺ and/or CD115 ⁺ vesicles in said biological sample.
The method of any one of claims 25-31, wherein analyzing the presence, amount and/or function of said migrasome comprises staining said biological sample with wheatgerm agglutinin (WGA) .
The method of any one of claims 25-32, wherein said migrasome is KUL01 ⁺ and/or CD115 ⁺.
A method for regulating angiogenesis and/or an angiogenesis related biological process in a subject, comprising:

i) monitoring the angiogenesis and/or the angiogenesis related biological process in said subject according to any one of claims 25-33; and

ii) administering a regulating agent according to the result of step i) .
The method of any one of claims 1-34, which is an in vitro or ex vivo method.
The method of any one of claims 1-34, which is an in vivo method.
An agent capable of regulating the formation and/or function of a migrasome generated by a monocyte and/or a macrophage, for use in regulating angiogenesis and/or an angiogenesis related biological process.
The agent of claim 37, wherein said angiogenesis related biological process comprises tumor and/or wound healing.
The agent of any one of claims 37-38, which is capable of increasing the formation and/or function of said migrasome, and for use in increasing angiogenesis.
The agent of any one of claims 37-39, which is capable of increasing the amount and/or function of a tetraspanin protein, a functional fragment thereof, and/or a functional variant thereof in said monocyte, said macrophage and/or in said migrasome.
The agent of any one of claims 37-40, which comprises a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
The agent of any one of claims 40-41, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.
The agent of any one of claims 37-42, which is capable of inhibiting the formation and/or function of said migrasome, and for use in decreasing angiogenesis.
The agent of any one of claims 37-43, which is capable of inhibiting the expression and/or function of a tetraspanin in said monocyte, said macrophage and/or in said migrasome.
The agent of any one of claims 37-44, which is capable of knocking out or knocking down the expression of a gene encoding for said tetraspanin in said monocyte and/or macrophage.
The agent of any one of claims 40-45, wherein said tetraspanin comprises tetraspanin 4.
An isolated migrasome generated by a monocyte and/or a macrophage, for use in regulating angiogenesis and/or an angiogenesis related biological process.
An engineered monocyte and/or a macrophage with altered ability for regulating angiogenesis and/or an angiogenesis related biological process comparing to a corresponding unmodified monocyte and/or macrophage, said engineered monocyte and/or macrophage has been modified to alter its migrasome generation ability.
The engineered monocyte and/or macrophage of claim 48, which has increased ability for promoting angiogenesis comparing to a corresponding unmodified monocyte and/or macrophage.
The engineered monocyte and/or macrophage of any one of claims 48-49, which has been modified to have increased ability for generating migrasomes.
The engineered monocyte and/or macrophage of any one of claims 48-50, which has been modified to increase the amount and/or function of a tetraspanin therein.
The engineered monocyte and/or macrophage of any one of claims 48-51, which has been modified to overexpress a tetraspanin protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
The engineered monocyte and/or macrophage of any one of claims 51-52, wherein said tetraspanin comprises TSPAN1, TSPAN2, TSPAN4, TSPAN6, TSPAN7, TSPAN9, TSPAN18, CD82, CD81, TSPAN13, CD53, TSPAN3, TSPAN5 and/or CD37.
The engineered monocyte and/or macrophage of claim 48, which has decreased ability for promoting angiogenesis comparing to a corresponding unmodified monocyte and/or macrophage.
The engineered monocyte and/or macrophage of claim 48 and 54, which has been modified to have decreased ability for generating migrasomes.
The engineered monocyte and/or macrophage of claim 48 and 54-55, which has been modified to decrease the amount and/or function of a tetraspanin therein.
The engineered monocyte and/or macrophage of claim 48 and 54-56, wherein the expression of a gene encoding for a tetraspanin has been knocked out or knocked down.
The engineered monocyte and/or macrophage of any one of claims 56-57, wherein said tetraspanin comprises tetraspanin 4.
Use of the agent according to any one of claims 37-46, the isolated migrasome according to claim 47, and/or the engineered monocyte and/or a macrophage according to any one of claims 48-58 in the preparation of a regulator for angiogenesis and/or the angiogenesis related biological process.
The use of claim 59, wherein said angiogenesis related biological process comprises tumor and/or wound healing.
An agent capable of detecting the presence, amount and/or function of a migrasome generated by a monocyte and/or a macrophage in a biological sample of a subject, for use in monitoring angiogenesis and/or an angiogenesis related biological process in said subject.
The agent of claim 61, wherein said migrasome is KUL01 ⁺ and/or CD115 ⁺.
The agent of any one of claims 61-62, wherein said angiogenesis related biological process comprises tumor and/or wound healing.
Use of the agent according to any one of claims 61-63 in the preparation of an indicator for angiogenesis and/or the angiogenesis related biological process in said subject.
A composition, comprising the agent according to any one of claims 37-46 and 61-63, the isolated migrasome according to claim 47, and/or the engineered monocyte and/or macrophage according to any one of claims 48-58.
The composition of claim 65, which is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.
A kit, comprising the agent according to any one of claims 37-46 and 61-63, the isolated migrasome according to claim 47, the engineered monocyte and/or macrophage according to any one of claims 48-58, and/or the composition according to any one of claims 65-66.