CN113424799A

CN113424799A - Construction method and application of PDX model based on osteogenic niche microenvironment modification

Info

Publication number: CN113424799A
Application number: CN202110720889.0A
Authority: CN
Inventors: 曾东风; 贾乙; 付文莹
Original assignee: Chinese Peoples Liberation Army Army Specialized Medical Center
Current assignee: Chinese Peoples Liberation Army Army Specialized Medical Center
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2021-09-24
Anticipated expiration: 2041-06-28
Also published as: CN113424799B

Abstract

The invention belongs to the technical field, and particularly relates to a construction method and application of a PDX model based on ossification niche microenvironment modification. The construction method comprises the steps of placing human umbilical cord mesenchymal cells on the biologically derived bones to induce and differentiate into osteoblasts, obtaining the biologically derived bones loaded with the osteoblasts, planting the biologically derived bones under the skin of a mouse, and then inoculating primary leukemia cells or tumor cells on the planting parts of the biologically derived bones under the skin of the mouse. The invention constructs a novel microenvironment modified PDX model, and provides a disease research model which has high transplantation success rate and stable passage and can effectively retain the biological characteristics of primary cells for researching drug resistance formation mechanism and drug screening of leukemia or tumor cells.

Description

Construction method and application of PDX model based on osteogenic niche microenvironment modification

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a construction method and application of a PDX model based on osteogenic niche microenvironment modification.

Background

A Patient-Derived tumor Xenograft (PDX) model is a model which can effectively research the biological characteristics and drug resistance of malignant tumors and can screen potential sensitive drugs, the PDX model refers to that fresh tumor tissues of patients are transplanted to immunodeficient mice after being treated and grow by depending on the microenvironment provided by the mice, the model can keep the basic characteristics of primary tumor cells, and a good in vivo model is provided for the research of tumors. Compared with the traditional tumor cell line transplantation model, the PDX tumor model can be more approximate to the cytogenetics and molecular biology characteristics of a human body, and an effective in-vivo animal model is provided for the research of tumor pathogenesis and the drug screening. Therefore, the PDX model has important transformational medical implications for tumor biological and genetic evaluation and treatment selection in tumor patients.

The PDX model is widely applied to clinical drug screening and clinical basic research of leukemia, but for leukemia, the obtained tissue is usually cell suspension, and an effective microenvironment is lacked as a co-transplantation component in the PDX transplantation process, so the transplantation and tumorigenicity effect and the genetic property of the PDX model are not maintained as good as possible. In order to improve the successful efficiency of the transplantation of the PDX model and simulate the effect of the real microenvironment in a patient body on the tumor, related researches propose different improved schemes aiming at the PDX model. Her Z et al have reported that the construction of leukemia PDX models is carried out by using genetically modified NNPG mice (NOD-NPG-IL2Rgc) to replace traditional NPG mice, and in addition, NSG mice expressing human SCF, GM-CSF and IL-3 transgenes (NSG-S) are applied to PDX models, and the models simulate and secrete the expression of factors related to human microenvironment, so as to promote the implantation of primary leukemia cells.

Although these PDX models mimic the modified microenvironment of human secreted cytokines, successful engraftment of leukemic cells can be promoted to some extent compared to conventional PDX. However, because the artificial mouse is not a direct solid microenvironment result simulating the leukemia hematopoietic niche, the final implantation effect and the maintenance of the leukemia characteristics after transplantation can not achieve the solid tumor transplantation effect, and the experimental cost is high, and the acquisition time and the number of passages of experimental mice are limited, so that the wide application of the artificial mouse is limited. In a blood tumor PDX transplantation model simulating a solid tumor, in the research of a PDX model of lymphoma accompanied with bone marrow invasion, a Michael Wang team tries to apply a fetal cartilage component as a microenvironment modification component of the tumor transplantation model and inoculate a cell suspension-liquefied lymphoma cell into a pre-transplanted fetal cartilage PDX mouse, and as a result, the tumorigenic time of the lymphoma PDX model is obviously shortened, and the biological characteristics of the tumor of a simulated patient are obviously improved. However, due to ethical factors related to the acquisition of fetal cartilage, the popularization of the method is limited. The existing leukemia PDX model graft is a cell suspension, and is lack of the support of a matrix component of a solid tumor graft or a hematopoietic niche in bone marrow tissue, so that the tumor formation rate is low, the stable passability is poor, and even a partially improved PDX model is limited in application due to overhigh cost or ethical factors.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a method for constructing a PDX model modified by an osteogenic niche microenvironment; the invention also aims to provide application of the PDX model in screening anti-leukemia drugs and anti-tumor drugs.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a method for constructing a PDX model modified by an osteogenic niche microenvironment comprises the following steps:

A. placing human umbilical cord mesenchymal cells on a biological derived bone for co-culture, adding an osteogenic induction culture medium to differentiate the cells into bone marrow cells, and obtaining the biological derived bone loaded with the bone marrow cells;

B. bone marrow cell-loaded, biologically-derived bone was implanted subcutaneously in mice, and after 1 week, primary tumor cells were inoculated into the implanted sites of the subcutaneously biologically-derived bone in mice.

As one of the preferable technical schemes, the preparation method of the biological derived bone comprises the following steps: processing the cancellous bone of the medullary end of the long shaft of the pig into bone blocks, then putting the bone blocks into 30% hydrogen peroxide, soaking for 72h at 38 ℃, changing the solution once every 24h, then putting the bone blocks into double distilled water for soaking for 30min, soaking for 24h in ethanol, soaking for 24h in acetone, soaking for 30min in double distilled water, finally drying for 8h at room temperature, carrying out vacuum freeze drying, and sterilizing with ethylene oxide.

As one of the preferred technical solutions, the primary tumor cell is a leukemia cell or a solid tumor cell.

As one of the preferred technical solutions, the primary tumor cell is derived from a human or an animal.

As one of the preferred technical schemes, the method for co-culturing the umbilical cord mesenchymal cells and the biological derived bones comprises the steps of placing the biological derived bones in a buffer solution for soaking for 2 days, soaking in a serum-free culture medium for 3 days, soaking with fetal calf serum, incubating for 2 hours at 37 ℃, adding umbilical cord mesenchymal cell suspension, standing for 4-6 hours, and adding a complete culture medium.

2. The constructed PDX model is applied to screening anti-leukemia and anti-tumor drugs.

The invention has the beneficial effects that:

because the existing leukemia PDX model graft is a cell suspension and lacks the support of a matrix component of a solid tumor graft or a hematopoietic niche in bone marrow tissue, the tumor formation rate is low, the stable passability is poor, and even a partially improved PDX model is limited in application due to overhigh cost or ethical factors. The invention designs an osteogenic niche which takes a biological derived bone as a basic structure as a carrier, constructs a novel microenvironment-modified PDX model by combined transplantation of human body microenvironment-related cell components such as stromal cells, osteoblasts and leukemia cells or tumor cells, and provides a disease research model which has high transplantation success rate and stable passage and can effectively retain the biological characteristics of primary cells for researching a drug resistance formation mechanism and drug screening of the leukemia or tumor cells.

Drawings

FIG. 1 is a graph showing the morphological results of a bio-derived bone, wherein 1A is an appearance graph of the bio-derived bone, and 1B is a 400X enlarged view;

FIG. 2 is a graph of a leukemia PDX model mouse inoculated with bio-derived bone;

FIG. 3 is a peripheral blood and bone marrow smear of P1 generation leukemia PDX model mouse, wherein A is peripheral blood smear, in which immature granulocyte can be seen, and B is bone marrow smear;

FIG. 4 is a graph of mouse leukemia PDX model mouse tumorigenesis of P1 generation graft derived bone;

FIG. 5 is a flow chart of the immunophenotyping results of the PDX model-passaged mice, wherein A is the immunophenotype of primary leukemia bone marrow cells, B is the immunophenotype of leukemia bone marrow cells at P1 generation, and C is the immunophenotype of leukemia bone marrow cells at P2 generation.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Example 1

Preparation of bio-derived bone

Processing spongy bone at medullary end of long shaft of pig into bone pieces of 1cm × 0.8cm × 0.8cm, soaking the processed bone pieces in 30% hydrogen peroxide solution at 38 deg.C for 72 hr, and changing the solution every 24 hr. Soaking in double distilled water for 30min, soaking in ethanol for 24h, soaking in acetone for 24h, soaking in double distilled water for 30min, drying at room temperature for 8h, vacuum freeze drying, sterilizing by fumigation with ethylene oxide gas, packaging in sterile plastic bags, and sealing, wherein the form of the bone niche derived from organism is shown as A, B in FIG. 1.

Example 2

Construction method of 'osteogenic niche' microenvironment modified PDX model

(1) Isolation and culture of human umbilical cord mesenchymal cells (MSCs)

Taking sterile healthy fetal umbilical cord about 4-5cm, washing with PBS, separating and removing umbilical cord adventitia tissue and blood vessel tissue, cutting the obtained umbilical cord tissue into small tissue blocks, transferring into 0.1% collagenase IV, digesting at 37 ℃, sieving with a 100-mesh sieve, collecting filtrate containing cells, centrifuging at room temperature at 1200r/min for 10min, removing supernatant, and retaining precipitate; after washing 2 times with PBS, 1X 10⁶Cell density of/mL was inoculated in 10% FBS-containing DMEM/F12 medium at 37 ℃ with 5% CO₂Culturing under saturated humidity environment. After 6-7 days, the cell sap is replaced, the cells which are not attached to the wall are discarded, and then the cell sap is replaced 1 time every 3-4 days. After observing that the cells reached 80% confluence, they were digested with 0.25% pancreatin, at a rate of 1: 3, proportional passage and expansionAnd (5) performing enrichment culture.

(2) Simulation of human bone marrow microenvironment with NPG mice

Placing the biological derived bone niche in a 24-hole plate, inoculating one biological derived bone niche in each hole, soaking for 2 days by PBS, replacing PBS according to the amount of bone residues, removing the bone residues as much as possible, soaking for 3 days by a serum-free L-DMEM culture solution, removing the culture solution, adding a small amount of FBS to infiltrate the bone blocks, and incubating for 2 hours at 37 ℃. Culturing in vitro MSC_SAdjusted to 1-8 x 10⁶Per mL of cell suspension, 20. mu.L of cell suspension per well containing the bioderived bone niche, 5% CO at 37 ℃₂Standing for 4-6h under saturated humidity condition, adding L-DMEM complete culture medium after the cells are fully attached to the biologically derived bones, culturing for 3 days, then changing the bone blocks into a new 24-pore plate to continue culturing, after 3 days, changing the osteogenic induction culture medium, changing the liquid for 1 time in half every 3 days, culturing for 1 week, then taking out, and under aseptic condition, planting the biologically derived bones under the skin of NPG mice of 5-8 weeks old.

(4) Leukemia PDX model construction

Collecting 5-20ml bone marrow cell suspension of leukemia patients, separating mononuclear cells by human lymphocyte separating medium according to the operation flow of Ficoll lymphocyte, washing with PBS, resuspending with normal saline, and counting. Inoculating leukemia single cell suspension to the implantation position of NPG mouse subcutaneous biological derived bone via trocar, transplanting for 8-12 weeks, and observing physiological state index (weight, mental state, appetite, hair, skin ulcer) and subcutaneous graft volume of mouse; taking bone marrow cells, and detecting the leukemia immunophenotype of P1 generation by a flow cytometer. As shown in fig. 2, P1 generation leukemic PDX model mice successfully became tumors, and fig. 4 is a graph of tumors of P1 generation seeded bone-derived murine leukemic PDX model mice. In the PDX mouse model modified by the 'osteogenic niche' microenvironment, the tumor formation rate can reach 50-75 percent, while the tumor formation rate of the unmodified PDX mouse is only 25 percent.

The result of immunostaining peripheral blood cells and bone marrow cells of a leukemia PDX model mouse is shown in fig. 3, and primitive granulocytes with obvious nucleoli can be seen in a peripheral blood smear of a mouse with generation P1 (a in fig. 3), and primitive granulocytes and immature granulocytes with a large number can be seen in a myeloid cytology smear with generation P2, and Auer corpuscles can be easily seen (B in fig. 3).

(6) Continuous passage of PDX model

Collecting primary leukemia cells of P1 generation PDX model mice, counting by resuspension, inoculating to NPG mice subcutaneously planted with biological derived bone according to the method, transplanting for 8-12 weeks, taking bone marrow cells, and detecting leukemia immunophenotype of P2 generation by flow cytometry. The immunophenotyping flow test results of primary and P1 generation and P2 generation PDX model mice are shown in FIG. 5, and the immunophenotyping of primary leukemia bone marrow cells (A in FIG. 5) is CD34+ CD13+ CD7+ and is consistent with the myelocytoimmunophenotyping of P1 generation (B in FIG. 5) and P2 generation (C in FIG. 5).

The P3 generation PDX model mice are cultured according to the same method, and the leukemia cells of the PDX model mice modified by the P2 and P3 generation 'osteogenic niche' microenvironment are found to have peripheral blood leukemia cell proliferation in 30 +/-15 days of modeling. And (3) performing flow immunophenotyping analysis on leukemia cells of PDX mice modified by the microenvironment of the osteogenic niche of the P3 generation, and comparing with a primary cell flow result to find that the phenotype is stably passaged without obvious loss.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Claims

1. A method for constructing a PDX model modified by an osteogenic niche microenvironment is characterized by comprising the following steps:

A. placing human umbilical cord mesenchymal cells on the biological derived bone for co-culture, adding an osteogenic induction culture medium to differentiate the cells into osteoblasts, and obtaining the biological derived bone loaded with the osteoblasts;

B. the biologically derived bone loaded with osteoblasts was implanted subcutaneously in mice, and after 1 week, primary tumor cells were inoculated into the implanted sites of the biologically derived bone subcutaneously in mice.

2. The method of claim 1, wherein the bio-derived bone is prepared by: processing the cancellous bone of the medullary end of the long shaft of the pig into bone blocks, then putting the bone blocks into 30% hydrogen peroxide, soaking for 72h at 38 ℃, changing the solution once every 24h, then putting the bone blocks into double distilled water for soaking for 30min, soaking for 24h in ethanol, soaking for 24h in acetone, soaking for 30min in double distilled water, finally drying for 8h at room temperature, carrying out vacuum freeze drying, and sterilizing with ethylene oxide.

3. The method of claim 1, wherein the primary tumor cell is a leukemia cell or a solid tumor cell.

4. The method of constructing according to claim 1, wherein the primary tumor cell is derived from a human or animal.

5. The method of claim 1, wherein the umbilical cord mesenchymal cells are co-cultured with the bioderived bone by soaking the bioderived bone in a buffer for 2 days, in a serum-free medium for 3 days, then infiltrating with fetal bovine serum, incubating at 37 ℃ for 2h, adding an umbilical cord mesenchymal cell suspension, standing for 4-6h, and then adding complete medium.

6. The PDX model constructed by the construction method of any one of claims 1-5 is applied to screening of anti-leukemia drugs or anti-tumor drugs.