CN114736860B - Monocyte or macrophage loaded with attenuated salmonella and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a monocyte or macrophage loaded with attenuated salmonella, a preparation method and application thereof. Specifically, by loading the engineered attenuated salmonella into monocytes or macrophages, the loaded cells are recruited to the tumor microenvironment, effectively avoiding bacterial off-targeting into normal organs. The camouflage of the cell wrap also effectively avoids premature exposure of the bacteria. It acts as an effective immune effector by directly killing tumor cells or releasing intracellular engineering bacteria to activate/modulate the immune system to indirectly inhibit tumors.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a monocyte or macrophage loaded with attenuated salmonella, and a preparation method and application thereof.
Background
Since the 19 th century, william Coley, an attempt was made to treat patients with tumors using heat-inactivated gram-positive bacteria (Streptococcus) and gram-negative bacteria (Salmonella marc), an increasing number of microorganisms have been developed and engineered for tumor treatment (Coley WB,1991,Clinical Orthopaedics and Related Research,262 (262): 3-11). These microorganisms, often referred to as "oncolytic bacteria" including salmonella, listeria, escherichia coli, are capable of achieving high levels of colonization （Gurbatri CR,2020,Science Translational Medicine,12(530);Zhou S,2018,Nature Reviews Cancer,18(12): 727-743;Suh S,2019,Advanced Science,6(3): 1801309）. at the tumor site after administration by injection due to their own facultative anaerobic nature and characteristic features of the tumor microenvironment, including the internal hypoxic environment of the tumor, immunosuppressive environment and the release of large numbers of necrotic cells, whereas such oncolytic bacteria often also cause varying degrees of liver and spleen damage to the body due to their own toxicity. For example, VNP20009, an attenuated strain of Salmonella typhimurium (hereinafter abbreviated as VNP), has received much attention because of its lower toxicity and good preclinical tumor inhibiting effects (Clairmont C,2000,Journal of Infectious Diseases,181:1996-2002). Although a significant reduction in VNP compared to the original salmonella toxicity was achieved by deletion of both genes (purI and msbB), in preclinical mouse experiments, tumor-inhibiting treatment by intravenous or intraperitoneal injection of VNP20009 still resulted in a degree of toxic side effects due to the presence and accumulation of the strain in normal organs, such as liver injury, splenomegaly, and weight loss in mice. Although the use of intratumoral injection can reduce the occurrence （Chowdhry S,2019,Nature Medicine,25(7): 1057-1063;Toso J F,2002,Journal of Clinical Oncology,20(1): 142-152）, of this injury, this clearly greatly limits the scope of application of microbiological therapies. In clinical phase I experiments, VNP exhibited favorable safety but was not well-behaved in terms of effectiveness (Toso J. F,2002,Journal of Clinical Oncology, 20 (1): 142-152). Therefore, how to further improve the tumor targeting of VNP oncolytic bacteria in vivo and their tumor inhibition effect is a very challenging problem.
In recent years, various types of leukocytes, including macrophages, neutrophils, T cells, have been used as potent tumor drug delivery vehicles （Xie Z,2017,Small,13(10) ;Xue J,2017,Nature Nanotechnology,12(7): 692-700 ;Huang B,2015,Science Translational Medicine,7(291): 291ra94）. for their unique homing effect to the tumor area, and such immune cells can reach the tumor area of a patient after crossing various barriers by sensing the presence of tumor-associated chemokines or cytokines, such as colony stimulating factor 1 (CSF-1), tumor Necrosis Factor (TNF), and the hint of chemokine ligand 5 (CCL 5) (Salmon H,2019,Nature Reviews Cancer,19 (4): 215-227). One of the major challenges of cell drug delivery is limited by the drug loading of individual cells and the high effective therapeutic dose required, which often requires injection of large amounts of cells for treatment (10-7-10-8 levels ）（Xue J,2017,Nature Nanotechnology,12(7): 692-700;Huang B,2015,Science Translational Medicine,7(291): 291ra94）, on mice are required to correspond to complicated procedures and expensive treatment costs, therefore, how to improve the cell drug delivery platform represented by macrophages to achieve cell treatment simplicity, low dose and effectiveness is a constant search of researchers).
Disclosure of Invention
Aiming at the dilemma of the current tumor treatment, the invention provides a technology for carrying out tumor targeted treatment by using macrophage immune cells as attenuated salmonella vectors, so as to realize 1) reducing the toxic and side effects caused by single injection of attenuated salmonella due to off-target; 2) Avoiding premature exposure of attenuated salmonella and rapid elimination. Finally, the titer of the attenuated salmonella in the tumor is improved, and the tumor inhibition effect is improved through the combination of macrophages and oncolytic bacteria.
In order to solve the problems in the prior art, the invention provides the following technical scheme: the preparation method of the monocyte or macrophage loaded with the attenuated salmonella comprises the following steps: the method comprises the steps of loading attenuated salmonella with macrophages, wherein the macrophages are macrophage RAW264.7 or primary macrophages PEMF derived from abdominal cavity and blood, the attenuated salmonella is attenuated salmonella typhimurium VNP20009 and derivative or genetically modified strains thereof, and tumor targeting is not changed, and the strain （ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ）; comprises the strains which do not carry or carry the expression plasmids for expressing the therapeutic genes and the foreign genes of the tracer genes.
Furthermore, the attenuated salmonella is an attenuated salmonella and a derivative or genetically modified strain thereof, wherein the attenuated salmonella contains an expression plasmid capable of expressing a therapeutic gene and a tracer gene exogenous gene, the therapeutic gene is a coding gene of a protein with therapeutic effect which can be expressed in the attenuated salmonella, including but not limited to an apoptosis anti-tumor gene, an angiogenesis inhibitor gene or an immune checkpoint blocker antibody gene （ZL201110360656.0;202111278413.2;202210011915.7;202210070594.8;202210181926.X;202210182870.X;202210182455.4;202210182222.4;Acta Pharmaceutica Sinica B 2021,11(10):31653177;Theranostics 2017, 7(8):2250-2260;Sci Rep 2016, 21;6:34178;American Journal of Cancer Research 2015, 5(2):792-801;Current Gene Therapy 2014, 14(2):75-85;Mol Cell Proteomics. 2011,10(6):M111.009399;Cancer Biology & Therapy 2005,8(4):840-845）; which are disclosed in the previous application, and the tracer gene is a gene capable of expressing fluorescent protein RFP or luciferase tracer protein LuxCDABE in the attenuated salmonella, including but not limited to a gene derived from green fluorescent protein GFP or red fluorescent protein RFP and a fluorescent protein derived therefrom (SEQ ID No. 5), and a luciferase protein gene (SEQ ID No. 4).
Furthermore, the attenuated salmonella carries expression plasmids capable of expressing therapeutic genes and trace genes in the attenuated salmonella, and promoters commonly used in the attenuated salmonella are arranged on the plasmids, including but not limited to J23100 constitutive promoters or NirB promoters, adhE promoters and promoters for amplifying SifB promoters; the plasmid contains an element AT for preventing plasmid loss; the sequence of the amplification plasmid loss prevention element AT is a nucleotide sequence shown in SEQ ID No. 1; the sequence of the amplified SifB promoter PsifB is a nucleotide sequence shown as SEQ ID No. 2; the sequence of the J23100 constitutive promoter is a nucleotide sequence shown as SEQ ID No. 3; the sequence of the PsifB upstream primer is the nucleotide sequence shown in SEQ ID No. 6; the sequence of the PsifB downstream primer is the nucleotide sequence shown as SEQ ID No. 7.
Primary monocytes or macrophages purified by RAW264.7 macrophage cell lines induced with 25-500 ng/mL LPS for 4-48 hours or by 2-4 days combined adherence culture stimulated by 5% starch broth intraperitoneal injection were co-cultured with attenuated Salmonella at a ratio of 1:5-1:100, respectively, for 30-150 minutes, and then treated with 50-100 μg/mL gentamicin for 30-60 minutes to kill extracellular bacteria. The number of viable VNPs after phagocytosis of macrophages was calculated using dilution plating to record the number of intracellular active load strains and cell activity under different treatment conditions and used for the determination of the final co-culture time and calculation of the actual dose of cells following.
The invention relates to a preparation method of monocytes or macrophages loaded with attenuated salmonella, which comprises the following steps: under the condition of macrophages and attenuated salmonella VNP20009 or derivative and mutant strains thereof, VNP20009 capable of stably and constitutively expressing red fluorescence RFP (nucleotide sequence shown as SEQ ID No. 5) is constructed based on electrotransformation of specific plasmids, and is marked as VNP-RFP or VNP20009 bacteria VNP-LuxCDABE for expressing luciferase LuxCDABE (nucleotide sequence shown as SEQ ID No. 4), wherein the constitutive strong promoter J23100 and the plasmid loss prevention original AT (nucleotide sequence shown as SEQ ID No. 1) are used in the plasmids.
Further, untreated RAW264.7 is M0 type macrophage, and after induction with 25-500 ng/mL LPS for 4-48 hours, RAW264.7 is M1 type macrophage, or primary monocytes or macrophages obtained by 2-4 days of combined adherent culture purification by 5% starch broth intraperitoneal injection stimulation. The RAW264.7 or primary macrophage after LPS induction treatment and the attenuated salmonella expressing the tracer protein or therapeutic protein are respectively co-cultured for 30, 60, 90, 120 and 150 minutes in the ratio of 1:5-1:100, and the cells are treated for 30-60 minutes by 50-100 mug/mL gentamicin to kill the attenuated salmonella free or adhered outside the cells. The number of viable VNPs after phagocytosis of macrophages was calculated using dilution plating to record the number of intracellular active load strains and cell activity under different treatment conditions and used for the determination of the final co-culture time and calculation of the actual dose of cells following.
Specifically, the correlation of the quantity of attenuated salmonella of RAW264.7 phagocytosis expression tracer protein or therapeutic protein and the co-culture time is quantitatively detected by fluorescence; obtaining a linear relation between the number of attenuated salmonella expressing the tracer protein or carrying the therapeutic protein and the corresponding fluorescence intensity by a gradient dilution method, and effectively and indirectly calculating the number of the attenuated salmonella expressing the tracer protein or carrying the therapeutic protein in a macrophage cell; the enzyme label instrument detects the fluorescence intensity (550 nm,585 nm) of the tracer protein, and the total bacterial number loaded by each 100 macrophages is calculated by combining the linear relationship of the bacterial number and the fluorescence. Further, a method for evaluating and tracing the loading efficiency and the macrophage activity of the attenuated salmonella in the macrophages loaded with the attenuated salmonella, transferring the attenuated salmonella loaded monocytes or macrophages to 0.3% triton x-100 in PBS configuration for standing for 10-15 min to perforate the macrophages to release the intracellular attenuated salmonella; coating LB plate with the resistance of the kanamycin after gradient dilution, analyzing and detecting the number of live attenuated salmonella loaded by monocytes or macrophages and the correlation of the loading efficiency and the co-culture time; detecting the activity state of the cells after 30, 60, 90, 120 and 150 minutes of co-culture of macrophages and attenuated salmonella by using a phenol blue assay; to further verify that macrophages were efficiently loaded with attenuated salmonella, after obtaining the macrophages loaded with attenuated salmonella, the cells were fixed with 4% paraformaldehyde, at room temperature, protected from light, left to stand for 30 min, washed with pbs 23 times, macrophage scaffold and cell nuclei were stained with phalloidin and DAPI according to manufacturer's instructions, respectively, at room temperature, protected from light, left to stand for 30 min, washed with pbs 23 times, and observed and photographed using a positive fluorescence microscope.
The invention relates to a method for evaluating and tracing the bacterial loading efficiency and tissue distribution and level of mononuclear cells or macrophages loaded with attenuated salmonella, wherein fluorescent protein or luciferase tracing protein capable of being expressed or quantitatively detected is loaded in the mononuclear cells or the macrophages, and the bacterial loading efficiency and tissue distribution and level of the mononuclear cells or the macrophages loaded with the attenuated salmonella are evaluated and traced.
Further, common optical dyes including DiR near infrared dyes label monocytes or macrophages, quantitatively detect the optical dyes, evaluate and track the tissue distribution and level of the attenuated salmonella-loaded monocytes or macrophages.
Further, untreated M0 type macrophages RAW264.7, after 4-48 hours of induction with 25-500 ng/mL LPS, RAW264.7 was converted to M1 type macrophages; or primary monocytes or macrophages obtained by purification by 5% starch broth intraperitoneal injection stimulation for 2-4 days in combination with an adherent culture. Co-culturing RAW264.7 or primary mononuclear cells or macrophages subjected to LPS induction treatment with attenuated salmonella expressing a tracer protein or a therapeutic protein in a ratio of 1:5-1:100 for 30-150 minutes, respectively, and quantitatively detecting the correlation of the amount of the attenuated salmonella phagocytosis of the tracer protein or the therapeutic protein by RAW264.7 and the co-culture time after treating cells with 50-100 mug/mL gentamicin for 30-60 minutes to kill extracellular free or adherent attenuated salmonella; obtaining a linear relation between the number of attenuated salmonella expressing the tracer protein or carrying the therapeutic protein and the corresponding fluorescence intensity by a gradient dilution method, and effectively and indirectly calculating the number of the attenuated salmonella expressing the tracer protein or carrying the therapeutic protein in a macrophage cell; the enzyme label instrument detects the fluorescence intensity (550 nm,585 nm) of the tracer protein, and the total bacterial number loaded by each 100 macrophages is calculated by combining the linear relationship of the bacterial number and the fluorescence.
Transferring the attenuated salmonella-loaded monocytes or macrophages to PBS-configured 0.3% Triton X-100 for 10-15 min to punch the macrophages to release intracellular attenuated salmonella; coating LB plate with the resistance of the kanamycin after gradient dilution, analyzing and detecting the number of live attenuated salmonella loaded by monocytes or macrophages and the correlation of the loading efficiency and the co-culture time; detecting the activity state of the cells after 30, 60, 90, 120 and 150 minutes of co-culture of macrophages and attenuated salmonella by using a phenol blue assay; to further verify that macrophages were efficiently loaded with attenuated salmonella, after obtaining the macrophages loaded with attenuated salmonella, the cells were fixed with 4% paraformaldehyde, at room temperature, protected from light, left to stand for 30 min, washed with pbs 23 times, macrophage scaffold and cell nuclei were stained with phalloidin and DAPI according to manufacturer's instructions, respectively, at room temperature, protected from light, left to stand for 30 min, washed with pbs 23 times, and observed and photographed using a positive fluorescence microscope.
The invention relates to application of mononuclear cells or macrophages loaded with attenuated salmonella in preparing antitumor drugs.
Further, the application comprises that the monocyte or macrophage loaded with the attenuated salmonella is singly used for preparing the anti-tumor medicament, or is combined with the existing anti-tumor medicament to form a composition, or is combined with the existing chemical medicament treatment, traditional Chinese medicine treatment, biological treatment and physical treatment method to be used in the anti-tumor treatment.
The titer of the attenuated salmonella in the tumor is improved, the toxic and side effects of the attenuated salmonella on normal tissues and organs are reduced, and meanwhile, the chemotactic capacity and phagocytic capacity of macrophages are improved by loading the attenuated salmonella, so that the tumor treatment effect is improved; furthermore, the attenuated salmonella expresses the therapeutic protein or the tracer protein, so that the anti-tumor effect of the monocyte or the macrophage loaded with the attenuated salmonella can be further improved or the tissue distribution and the level of the monocyte or the macrophage loaded with the attenuated salmonella can be traced.
The beneficial effects are that: the invention can solve the two problems at the same time, and meets the requirement of the field on a low-toxicity, high-efficiency and economic composition for treating solid tumors. The invention has the advantages that by obtaining the mononuclear cells or macrophages loaded with the attenuated salmonella, after the tumor-bearing mice are administrated by intravenous injection, the mononuclear cells or the macrophages carry the attenuated salmonella to the internal tumor area due to the natural tumor targeting property and the high-efficiency permeability, thereby avoiding the attenuated salmonella from being rapidly cleared due to long-time exposure in the circulatory system of the organism in the early stage, reducing the accumulation of the attenuated salmonella in normal organs, and finally realizing the high-efficiency enrichment in the tumor area.
Compared with the prior art, the invention has the following advantages:
(1) Although the attenuated salmonella is modified based on the characteristics of tumor microenvironment, such as regulation and control of the growth of thallus hypoxia and targeting of tumor specific markers, the targeting of thallus to tumors in mice can be effectively improved, and the increase of the targeting in tumors takes the thallus reaching tumor areas as a precondition.
Taking VNP20009 as an example, phase I clinical results show that after VNP20009 is injected into tumor patients intravenously, no VNP20009 exists in tumor areas of most patients and no trace of bacterial colonization exists. But by using the intratumoral injection mode to treat VNP20009, the high-titer VNP20009 can be obtained in the tumor area of the patient, and the non-attenuated salmonella can also effectively infect the tumor area of the cancer patient and realize effective inhibition of the tumor. This suggests that both salmonella and post-attenuation VNP20009 have the ability to colonize within tumors. However, in tumor patients, VNP20009 administered intravenously cannot reach the tumor, probably because VNP20009 acts as attenuated salmonella, and although strong toxicity of salmonella is avoided, it is therefore also more easily cleared by the immune system of the human body. It can be seen that achieving the accessibility of VNP20009 in human tumors from none to high is a more urgent problem to solve than low to high targeting in mouse/canine tumors. In addition, preclinical studies show that although the effect of anti-tumor therapy based on attenuated salmonella is remarkable, there are still non-negligible toxic and side effects, and how to avoid these toxic and side effects is an urgent problem to be solved.
(2) In most cases, there is a strong innate immune response in the body and invading microorganisms are rapidly cleared by macrophages and neutrophil Antigen Presenting Cells (APCs). However, salmonella can produce a range of self-protective measures, including increasing bacterial antimicrobial peptide resistance gene expression and inhibiting intracellular lysosomal protein expression to survive macrophage-mediated phagocytosis. Then, due to the sustained intracellular stimulation and proliferation of the bacteria, expansion and rupture of macrophages is initiated, eventually leading to delayed release of the bacteria. Therefore, the VNP20009 oncolytic bacteria are hidden in the macrophages in advance to avoid the premature exposure of the thalli in the human body, and are released after being transported to a tumor area by the macrophages, which is probably an effective strategy for realizing the enrichment of the oncolytic bacteria in the tumor of the human body, and also suggests the clinical application potential of the novel platform provided by the invention.
(3) One of the major challenges of cell-based drug delivery is limited by the drug loading per cell and the high effective therapeutic doses required, which often requires injection of large numbers of cells (10 7-108 cells/mouse) for treatment, which in turn corresponds to complicated procedures and expensive treatment costs. The novel therapeutic strategy provided herein greatly reduces the cell injection dose required based on the efficient loading of immune cells into oncolytic bacteria and the self-proliferative capacity of oncolytic bacteria. The method can effectively treat mouse tumor by only 10 5 of loaded cells, and the injection amount required by human body is only 2.4X10 7 cells (=1X 10 5 X (3/37) X (60/0.02)) by means of FDA guidance. The cell amount required by the novel cell therapy is only 1/100-1/1000 of that of the traditional cell therapy, and in fact, effective treatment effect can be obtained by virtue of the continuous proliferation effect of thalli as long as a small amount of loaded cells reach the tumor area and release attenuated salmonella.
(4) The monocyte or macrophage loaded with the attenuated salmonella disclosed by the invention can not only effectively load the attenuated salmonella, but also effectively release the attenuated salmonella. Attenuated salmonella can enhance chemotactic, phagocytic and ROS, NO levels of monocytes or macrophages. Tumor cells are capable of accelerating the release of oncolytic bacteria from monocytes or macrophages. The application effect of the invention does not obviously reduce the weight loss, spleen enlargement and liver inflammation lesion of a tumor model induced by oncolytic bacteria treatment, effectively avoids the toxic and side effects of oncolytic bacteria treatment, reduces the distribution and titer of bacteria in normal tissues and organs, has higher tumor targeting and effectively improves the tumor treatment effect. Achieves the curative effect and the improvement of toxic and side effects which cannot be achieved by the mixed treatment of monocytes or macrophages and oncolytic bacteria.
Drawings
Brief description of the drawings:
VNP: attenuated mice injured salmonella VNP20009; RFP: red fluorescent protein; VNP-RFP: VNP carries RFP gene plasmid; RAW264.7 (VNP) the macrophage cell line RAW264.7 loaded with VNP; PEM Φ (VNP): VNP-loaded mouse primary peritoneal macrophages PEM Φ; mΦ (VNP): two types of cells, RAW264.7 (VNP) and PEM phi (VNP), are collectively referred to as RAW264.7 (VNP); VNP-LuxCDABE: VNP carries LuxCDABE gene plasmids.
FIG. 1 is a diagram showing the loading of the VNP with macrophage RAW264.7 according to the present invention;
Fig. 1A is a fluorescent microscope photograph of the present invention indicating the loading of VNP by macrophages. 1. Non-loaded VNP macrophages were photographed; 2. after 60 min co-incubation, VNP macrophages were loaded for photography.
FIG. 1B shows the total loading of VNP with macrophage RAW264.7 and the time dependence of cell activity on co-culture. 1. (left) average total VNP number change per 100 macrophages; 2. (right) macrophage cell activity change (n=5).
FIG. 1C shows the time-varying relationship between the effective loading and co-cultivation of the macrophage RAW264.7 on the VNP. 1. (left) average total change in active VNP per 100 macrophages; 2. the ratio of total number of active VNP in macrophages to total number of VNP loaded in cells varies (right). (n=5, < P <0.05, < P <0.01, < P < 0.0001).
FIG. 2 is a graph of the loading of VNP with primary peritoneal macrophages PEM phi according to the present invention;
Fig. 2A is a fluorescent microscope photograph of the present invention indicating the loading of VNP by macrophages. 1. Non-loaded VNP macrophages were photographed; 2. after 60 min co-incubation, VNP macrophages were loaded for photography.
FIG. 2B shows the relationship between macrophage PEM phi and total VNP loading and cell activity and co-culture time. 1. (left) average total VNP number change per 100 macrophages; 2. (right) macrophage cell activity change (n=5).
FIG. 2C shows the time dependence of macrophage PEM phi on VNP availability and co-cultivation in accordance with the present invention. 1. (left) average total change in active VNP per 100 macrophages; 2. the ratio of total number of (right) macrophage-active VNP to total number of intracellular-loaded VNP varies (n=5, ns indicates no significant difference, P <0.05, P <0.001, P < 0.0001).
FIG. 3 is a graph of the release of the loaded VNP macrophages RAW264.7 (VNP) and PEM phi (VNP) versus intracellular VNP according to the present invention.
FIG. 3A is a schematic representation of RAW264.7 (VNP-RFP) and PEMP Φ (VNP) versus intracellular VNP release assay design of the present invention, cultured using a 3.0 micron Transwell plate. 1. Co-culturing RAW264.7 or PEM phi with tumor cells (B16F 10) in a lower chamber in direct contact, culturing RAW264.7 (VNP-RFP) or PEM phi (VNP-RFP) in an upper chamber, and recording as a "RAW264.7 (VNP) upper chamber" or "PEM phi (VNP) upper chamber" group; 2. co-culturing RAW264.7 (VNP-RFP) or PEM phi (VNP-RFP) and tumor cells (B16F 10) in direct contact in a lower chamber, culturing RAW264.7 or PEM phi in an upper chamber, and marking as a group of 'RAW 264.7 upper chamber' or 'PEM phi upper chamber'; the amount of VNP-RFP in the culture broth was quantitatively determined by fluorescence after 12 hours.
FIG. 3B is a graph of the invention for detecting and comparing the number of VNP-RFPs in different groups. 1. "RAW264.7 (VNP) upper chamber" group; 2. "RAW264.7 upper chamber" group; 3. a "PEM Φ (VNP) upper chamber" group; 4. a "PEM Φ upper chamber" set.
FIG. 3C shows the bacterial titer of RAW264.7 (VNP) and PEMAI (VNP) cells prepared according to the present invention, respectively, cultured in media of different pH values (7.4 and 6.7) adjusted with dilute hydrochloric acid, releasing intracellular VNP, and collecting the culture supernatant at different times for plating.
FIG. 3D is a graph of the correlation between the number of VNP strains released from RAW264.7 (VNP) and PEM phi (VNP) cells and the incubation time in (C) analyzed according to the present invention. 1. ph=6.7; 2. ph=7.4; a. RAW264.7 (VNP) group; b. PEM Φ (VNP) group. (n=4, ns represents no significant difference, P < 0.0001);
FIG. 4 is a graph showing the detection of VNP-loaded macrophage RAW264.7 (VNP) and PEM phi (VNP) intracellular VNP release process according to the present invention.
Fig. 4A is a schematic diagram showing the experimental design of the release process detection of VNP-loaded macrophages RAW264.7 (VNP) and PEM Φ (VNP) intracellular VNP according to the present invention. RAW264.7 (VNP-psifB-RFP) or PEM phi (VNP-psifB-RFP) was incubated in medium supplemented with 50. Mu.g/mL gentamicin. The amount of VNP released outside the cells was counted by fluorescence microscopy at the indicated time points.
FIG. 4B shows the average number of paraVNP per 100 cell fields in supernatants at various time points counted by fluorescence microscopy according to the invention. 1. RAW264.7 (VNP-psifB-RFP); 2. PEM phi (VNP-psifB-RFP). (n=4, ns indicates no significant difference, P < 0.05)
Fig. 5 is a graph of VNP growth, cell invasion and killing capacity assays released by RAW264.7 (VNP) and PEM Φ (VNP) of the present invention.
FIG. 5A shows the growth curves of VNP after various treatments according to the present invention in LB medium. 1. Untreated VNP; 2. VNP released by RAW264.7 (VNP); 3. VNP released by PEM Φ (VNP) (n=4).
FIG. 5B shows the number of viable intracellular bacteria 4 hours after infection of B16F10 cells with VNP after various treatments by plate counting according to the present invention. 1. Untreated VNP; 2. VNP released by RAW264.7 (VNP); 3. VNP released by PEM Φ (VNP) (n=5).
FIG. 5C shows the apoptosis level of B16F10 after 4 hours of infection of B16F10 cells with VNP after various treatments by flow cytometry detection according to the present invention. 1. Untreated VNP; 2. VNP released by RAW264.7 (VNP); 3. VNP released by PEM Φ (VNP) (n=3, ns indicates no significant difference, < 0.0001).
FIG. 6 is a graph showing the phagocytic capacity of the loaded VNP macrophages RAW264.7 (VNP) and PEMP Φ (VNP) of the present invention;
FIG. 6A shows the phagocytosis of fluorescent microspheres by flow test cells after 4 hours of co-culture of empty or bacterial loaded macrophages with fluorescent microspheres (PE) at a ratio of 1:10, respectively, with the red portal segment being the population of macrophages that significantly phagocytize the microspheres; a. RAW264.7; b. RAW264.7 (VNP); c. PEMΦ; d. PEM Φ (VNP).
Fig. 6B is a graph comparing the percent change in macrophages with strong phagocytic capacity for quantitative analysis in accordance with the present invention. 1. RAW264.7; 2. RAW264.7 (VNP); 3. PEMΦ;4. PEM Φ (VNP). (n=5, ns indicates no significant difference, P <0.05, P < 0.0001).
FIG. 7 is a graph showing the chemotactic potential of the loaded VNP macrophages of the present invention, RAW264.7 (VNP) and PEM phi (VNP).
FIG. 7A shows the chemotaxis of macrophage perforation in the upper and lower layers of the upper and lower chambers of a 8.0 micron Transwell plate of the present invention after 9-12h of tumor cell addition and either no-load or bacteria-loaded macrophages in the upper chamber, and after 16h of stationary culture. 1. The lower chamber is not added with cells, and the upper chamber is added with RAW264.7; 2. B16F10 cells were added to the lower chamber, and RAW264.7 was added to the upper chamber; 3. the lower chamber was cell free and the upper chamber was RAW264.7 (VNP); 4. B16F10 cells were added to the lower chamber, and RAW264.7 (VNP) was added to the upper chamber; 5. the lower chamber had no cells added and the upper chamber had PEM phi added; 6. B16F10 cells were added in the lower chamber and PEM Φ in the upper chamber; 7. the lower chamber had no cells added and the upper chamber had PEM Φ (VNP); 8. the lower chamber was filled with B16F10 cells and the upper chamber was filled with PEM Φ (VNP).
FIG. 7B is a randomly selected microscopic photograph of field counts and statistically comparing cell chemotaxis in accordance with the present invention. 1. RAW264.7 is added into the upper chamber; 2. upper chamber addition RAW264.7 (VNP); 3. adding PEM phi into the upper chamber; 4. upper chamber addition PEM Φ (VNP); a. the lower chamber is free of added cells; b. B16F10 cells were added in the lower chamber (n=5, ns indicates no significant difference, P <0.05, P < 0.0001).
FIG. 8 is a graph showing the indirect killing ability of the loaded VNP macrophages RAW264.7 (VNP) and PEMP phi (VNP) of the present invention against tumor cells;
FIG. 8A is a schematic representation of the experimental design for indirect killing ability of RAW264.7 (VNP) and PEMAPhi (VNP) of the present invention against tumor cells, cultured using a 0.4 μm Transwell plate. 1. No cells were added to the upper chamber and marked as "blank control"; 2. l929 cells were added to the upper chamber and designated as "normal control"; 3. the upper chamber was filled with RAW264.7 or PEM Φ cells, designated as "RAW264.7 group" or "PEM Φ group"; 4. the upper chamber is added with RAW264.7 (VNP) or PEM phi (VNP), which is marked as "RAW264.7 (VNP) group" or "PEM phi (VNP) group"; the cell status of the lower chamber was examined after culturing for 12 hours.
FIG. 8B is a graph showing the comparison of the proliferation level of tumor cells in the lower chamber using the CCK8 assay of the present invention. 1. Blank control group; 2. normal control group; 3. RAW264.7 group; 4. RAW264.7 (VNP) group; 5. PEM Φ set; 6. PEM Φ (VNP) group. (ns represents no significant difference, P <0.05, P <0.01, P < 0.0001).
FIG. 8C is a graph of the detection of intracellular ROS level change using the ROS detection kit of the present invention. 1. RAW264.7;2. RAW264.7 (VNP); 3. PEMΦ;4. PEM Φ (VNP) (n=5). FIG. 8D shows the detection of changes in cellular NO production levels using the NO detection kit of the present invention. 1. RAW264.7;2. RAW264.7 (VNP); 3. PEMΦ;4. PEM Φ (VNP). (n=5, < P <0.05, < P <0.01, < P < 0.0001)
FIG. 9 is a graph showing in vivo attenuation efficacy assessment and tumor targeting detection of VNP-loaded macrophages RAW264.7 (VNP) and PEM phi (VNP) of the present invention; FIG. 9A is a schematic illustration of the experimental procedure of the present invention, wherein prepared VNP or RAW264.7 (VNP) or PEM phi (VNP) was injected into B16F10 tumor bearing mice by tail vein, PBS was used as negative control, and dead mice were examined at 3 hours, 1 day, 3 days, 6 days, and 12 days after injection treatment; 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP); fig. 9B is a comparison of body weight changes in mice 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP). (n=16); fig. 9C is a comparison of liver injury 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP); black arrows indicate the sites where significant damage to the liver occurs; the scale is 5 mm; figure 9D is a statistical comparison of the number of significant lesions of the liver 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP) (n=5); fig. 9E is a comparison of splenomegaly 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP); the scale is 5 mm; fig. 9F is a comparison of spleen weights 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP); p <0.01, P <0.001, P < 0.0001) (n=5).
FIG. 10 shows the extent of liver injury due to side effects in B16F10 tumor-bearing mice treated with VNP-loaded macrophages RAW264.7 (VNP) and PEM phi (VNP) according to the present invention.
Fig. 10A is an H & E stained section of liver 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP); the white arrow points are obvious lesion areas; the scale is 100. Mu.m.
FIG. 10B is a comparison of serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) concentrations 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP). (n=5, ×p < 0.01).
FIG. 11 is a graph showing in vivo organ strain titers of VNP-loaded macrophage RAW264.7 (VNP) and PEM phi (VNP) of the present invention (time nodes include 3 hours, 1 day, 3 days, 6 days, 12 days post-administration);
11A is the VNP biodistribution of the loaded VNP macrophages RAW264.7 (VNP) and PEM phi (VNP) of the present invention during treatment of B16F10 tumor-bearing mice. 1. VNP; 2. RAW264.7 (VNP); 3. PEM Φ (VNP); a. a tumor; b. liver; c. spleen; d. a lung; e. a kidney; f. and (3) a heart. (n=4 or 5)
11B is a comparison of VNP titer ratio in liver versus spleen for tumor at day 6 post-treatment according to the present invention. 1. VNP; 2. RAW264.7 (VNP); 3. PEM Φ (VNP); a. tumor/liver; b. tumor/spleen. (n=5, ns indicates no significant difference, P <0.05, P <0.01, P <0.001, P < 0.0001)
FIG. 12 is a graph of H & E stained sections of organs of a VNP-loaded macrophage RAW264.7 (VNP) and PEM phi (VNP) treated B16F10 tumor-bearing mouse of the present invention after 1 day. 1. PBS; 2. VNP; 3. RAW264.7 (VNP); 4. PEM Φ (VNP); the scale is 40. Mu.m.
FIG. 13 is a graph showing the comparison of the effects of VNP-loaded macrophage RAW264.7 (VNP) and PEM phi (VNP) treatment of B16F10 tumor-bearing mice of the present invention ;1、PBS;2、VNP;3、RAW264.7;4、RAW264.7(VNP);5、PEMΦ;6、PEMΦ(VNP).（n=9,*P<0.05,**P<0.01,****P<0.0001）
FIG. 14 is a graph of a tumor targeted in vivo imaging analysis of DiR-labeled macrophages PEM phi (VNP-LuxCDABE) loaded with VNP-LuxCDABE of the present invention. FIG. 14A is a photograph of in vivo imaging analysis of DiR-labeled macrophages PEM phi (VNP-LuxCDABE) loaded with VNP-LuxCDABE of the present invention at designated time points during treatment of B16F10 tumor bearing mice. 1. Near infrared fluorescence and bright field superposition imaging of VNP-LuxCDABE;2, PEM phi (VNP-LuxCDABE) near infrared fluorescence and bright field superposition imaging; 2, superimposed imaging of PEM phi (VNP-LuxCDABE) biological light and bright field; 3. PEM phi biological light and bright field superposition imaging; white circles indicate tumor sites. FIG. 14B is a graph showing the analysis of macrophage density based on near infrared fluorescence intensity at various time points of tumor sites according to the present invention. 1. PEMΦ; 2. PEM phi (VNP-LuxCDABE). (n=3 or 4); fig. 14C is a graph showing VNP strain density based on bioluminescence intensity analysis of tumor sites at various time points according to the present invention. 1. VNP-LuxCDABE; 2. PEM phi (VNP-LuxCDABE). (n=3 or 4); fig. 14D is near infrared fluorescence and bright field superimposed imaging of tumors and individual organs 36 hours after treatment according to the present invention. 1. PEMΦ; 2. PEM phi (VNP-LuxCDABE); FIG. 14E shows the near infrared fluorescence intensity ratios of tumors and individual organs at 36 hours after treatment according to the present invention. 1. PEMΦ; 2. PEM phi (VNP-LuxCDABE). (n=4, ns indicates no significant difference, P <0.05, P < 0.001).
Fig. 15 is an analysis of VNP-loaded macrophage PEM Φ (VNP) tumor targeting mechanism of the present invention. FIG. 15A is a comparison of serum CCL2 chemokine concentrations in tumor-bearing mice of the invention and tumor-free mice. 1. Tumor-free mice; 2. B16F10 tumor-bearing mice. (n=4); FIG. 15B is a comparison of serum and tumor CCL2 chemokine concentrations of VNP-loaded macrophage PEM phi (VNP) treated B16F10 tumor-bearing mice of the present invention. 1. PBS; 2. PEMΦ; 3. PEM Φ (VNP); a. serum; b tumor lysate. (n=4 or 5, < P <0.05, < P <0.01, < P < 0.001).
FIG. 16 is a graph of an analysis of the attenuation mechanism of the loaded VNP macrophage PEM phi (VNP) of the present invention; fig. 16A is a schematic of experimental procedure of the present invention, whereby a simple mixture of loading VNP macrophages, PEM Φ (VNP) and PEM Φ cells and VNP strains, PEM Φ+vnp, was administered via the tail vein and their toxicity and tumor targeting were evaluated. Control with either volume PBS or single VNP (n=4 or 5); fig. 16B is a comparison of liver injury 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. PEMΦ+VNP; 4. PEM Φ (VNP); black arrows indicate the sites where significant damage to the liver occurs; the scale is 10 mm; figure 16C is a statistical comparison of the number of significant lesions of the liver 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. PEMΦ+VNP; 4. PEM Φ (VNP); fig. 16D is a comparison of splenomegaly 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. PEMΦ+VNP; 4. PEM Φ (VNP); the scale is 10 mm; fig. 16E is a comparison of spleen weights 1 day after treatment according to the present invention. 1. PBS; 2. VNP; 3. PEMΦ+VNP; 4. PEM Φ (VNP); FIG. 16F is a comparison of VNP titers in spleen, liver and tumor at day 6 post-treatment according to the present invention. 1. VNP; 2. PEMΦ+VNP; 3. PEM Φ (VNP); a. spleen; b. liver; c. a tumor; fig. 16G is a comparison of VNP titer ratio in liver versus spleen for tumor at day 6 post-treatment according to the invention. 1. VNP; 2. PEMΦ+VNP; 3. PEM Φ (VNP); a. tumor/spleen; b. tumor/liver. (ns represents no significant difference, P <0.05, P <0.01, P < 0.001).
Detailed Description
The invention will be described in detail below with reference to the drawings and the detailed description. The following examples are illustrative of the invention but are not intended to limit the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.
Example 1
Preparation and optimization of loaded attenuated salmonella macrophages
The attenuated salmonella in the invention is attenuated salmonella typhimurium VNP20009 and derivative or genetically modified strains thereof, and the tumor targeting is not changed, including but not limited to the above-mentioned strains （ZL201410209851.7,ZL201610946268.3,ZL201610945015.4,ZL201610945021.X,202010182038.0;Acta Pharmaceutica Sinica B 2021,11(10):31653177;phoP/phoQ）, which have been applied for the invention, and the attenuated salmonella typhimurium VNP20009 is taken as an example only.
Construction of RFP expression bacterium containing AT element
The plasmid carrying the exogenous genes for expressing the therapeutic genes and the tracer genes is electrically transformed into attenuated salmonella typhimurium VNP20009 to construct the attenuated salmonella capable of expressing the genes for expressing the therapeutic proteins and the tracer proteins, the used promoters are promoters commonly used for attenuated salmonella, including but not limited to J23100 constitutive promoters or NirB promoters, adhE promoters and promoters for amplifying SifB promoters, and the plasmid contains an anti-plasmid loss element AT. For example, a VNP20009 bacterium (VNP-RFP) expressing red fluorescent RFP was constructed, and a constitutive strong promoter J23100 and a plasmid loss preventing original AT were used in the plasmid in order to make the experimental result easier to observe and stabilize.
For example, RAW264.7 and VNP-RFP (VNP strain with RFP fluorescent expression plasmid) induced by 100 ng/mL LPS for 12h were co-cultured at a ratio of 1:10 for different times, and then cells were treated with 50 μg/mL gentamicin for 60 minutes to kill extracellular bacteria. We found that RAW264.7 cells could successfully load VNP-RFP (FIG. 1A). Fluorescence quantitative detection based on a linear relationship between fluorescence intensity and bacterial number showed that total amount of VNP-RFP phagocytosed by RAW264.7 cells was positively correlated with co-incubation time (fig. 1B, left). In addition, the number of viable VNP-RFP after phagocytosis of macrophages was calculated using a dilution plating, and the results showed that the number of viable VNP-RFP loaded in RAW264.7 cells was also positively correlated with the co-cultivation time (fig. 1C, left), during which the loading efficiency (number of viable bacteria/total number of bacteria in RAW 264.7) peaked at 60 minutes (220±13 CFU (mean±sem)/100 cells) (fig. 1C, right), and macrophages showed acceptable cell viability (higher than 90%) (fig. 1B, right). Therefore, the selection of moi=10 was recommended under the treatment culture conditions, and the RAW264.7 (VNP) obtained after 60min co-culture was used for the subsequent study, and the number of intracellular active bacteria was also considered as an effective reference at the time of injection.
(1) Fumbling of macrophage lineage loading VNP conditions: untreated RAW264.7 was used as M0 type macrophages, and RAW264.7 after 4-48 hours of LPS induction was used as M1 type macrophages at 25-500 ng/mL. Primary monocytes or macrophages PEM Φ obtained by LPS-induced treatment of RAW264.7 or purification by 5% starch broth intraperitoneal injection stimulation for 2-4 days in combination with an adherent culture method were co-cultured with VNP-RFP at a ratio of 1:5-1:100 for 30, 60, 90, 120, 150 minutes, respectively, and after 30-60 minutes of treatment of cells with gentamicin at 50-100 μg/mL to kill extracellular free or adherent VNP-RFP, the correlation of the amount of RAW264.7 phagocytizing VNP-RFP with co-culture time was quantitatively examined by fluorescence. The linear relation between the bacterial strain VNP-RFP thallus number and the corresponding fluorescence intensity is obtained by a gradient dilution method, and the linear relation is used for effectively and indirectly calculating the intracellular VNP-RFP number of macrophages. The RFP fluorescence intensity (550 nm,585 nm) was measured by a microplate reader, and the total bacterial count loaded per 100 macrophages was calculated in combination with the linear relationship of strain number and fluorescence. Subsequently, RAW264.7 (VNP-RFP) was transferred to PBS-configured 0.3% Triton X-100 and allowed to stand for 10-15 min to punch macrophages to release intracellular VNP strain. LB plates with the resistance to the kanamycin are coated after the gradient dilution, and the correlation between the loading efficiency and the co-culture time of the number of the live VNP-RFPs loaded by RAW264.7 is analyzed and detected. The active status of the cells was examined after 30, 60, 90, 120, 150 minutes of co-culture of macrophages with VNP using the tebuconazole blue assay. To further verify that macrophages were efficiently loaded with VNP, after obtaining VNP-loaded macrophages, cells were fixed with 4% paraformaldehyde (room temperature, protected from light, resting 30 min), washed 23 times with PBS, macrophage scaffold and cell nuclei were stained with phalloidin and DAPI according to manufacturer's instructions (room temperature, protected from light, resting 30 min), washed 23 times with PBS, and observed and photographed using an overhead fluorescence microscope.
(2) Fumbling of primary macrophage (PEM Φ) loading VNP conditions:
Preparing 5% starch broth (1.8% nutrient broth+5% soluble starch+redistilled water), heating and stirring, sterilizing with high pressure steam (115 deg.C for 30 min) after starch is completely dissolved, packaging in 1.5 ml EP tube, storing at 4 deg.C, and heating for dissolving before use. The 7-10 week old female/male C57 mice were given intraperitoneal injection of 1ml starch broth, sacrificed after 23: 23 d and the peritoneal macrophages were extracted, high purity primary peritoneal macrophages were obtained by the adherent method, and after digestion with lidocaine at 4℃for 5-8 min to obtain cell suspensions, used in the subsequent experiments. The subsequent experimental procedure is as described in I). The M phi is the representative name of RAW264.7 (M1) and PEM phi macrophages.
The invention also determines the loading condition of the mouse blood or abdominal primary macrophage PEMF to the VNP-RFP. The invention firstly prepares high-purity blood or abdominal macrophages through starch broth stimulation, and then purifies the macrophages by using an adherent culture, thereby ensuring that the purity of the macrophages obtained each time is higher than 98 percent. Similarly, co-culture with PEMF and VNP-RFP at a ratio of 1:10 for different times, respectively, followed by treatment of the cells with 50 μg/mL gentamicin for 60 minutes to kill extracellular bacteria is illustrative. PEMFs can also be efficiently loaded with VNP-RFP strains (fig. 2A), but unlike RAW264.7 cells, the effective VNP load of PEMF cells continues to decrease over time (fig. 2C, right). The number of viable VNP strains loaded in PEMF cells was sufficient (510±10 CFU (mean±sem)/100 cells) at 60 minutes (fig. 2C, left), at which time the corresponding PEMF (VNP) cell viability was still higher than 85%, meeting the subsequent experimental requirements (fig. 2B). Therefore, the selection of moi=10 was recommended under the treatment culture conditions, and PEMF (VNP) obtained after co-culture of 60 min was used for the subsequent study, and the number of active bacteria in the cell was also considered as a valid reference at the time of injection.
Example 2
Feature detection of loaded attenuated salmonella macrophages
(1) Intracellular strain release:
In vitro studies confirm release of mΦ (VNP) (including RAW264.7 (VNP) and PEM Φ (VNP)) to the loaded VNP: after obtaining mΦ (VNP) according to the above method, direct/indirect contact of mΦ (VNP) to tumor cells was achieved using a Transwell plate, i.e. tumor cells were spread in the lower chamber of a 3.0 mm Transwell plate (through which large pore size cells could not pass, but bacteria could still pass effectively), mΦ (VNP-RFP) cells were added in the upper chamber of group mΦ (VNP-RFP) UP, and mΦ cells were added in the lower chamber. In contrast, MΦ (VNP-RFP) Down was added to the upper chamber and MΦ (VNP-RFP) cells were added to the lower chamber. After placing the culture medium in an incubator for stationary culture 12 h, the supernatant was collected and subjected to gradient dilution to coat a carbaryl-resistant LB plate for colony count statistics. In vitro studies confirm changes in VNP biological activity released via mΦ (VNP): bioscreen C was used to dynamically examine the growth curves of different VNPs in LB medium. Briefly, 10 μl of VNP suspension (OD 600 = 1.0) was inoculated into 1 mL LB medium and 300 μl of solution per well was inoculated into Bioscreen C multiwell plates. The multiwell plates were incubated at 37 ℃ for 30 hours. OD values were measured every 30 minutes under a brown filter with a wavelength of 600 nm. Finally, the real-time growth curve of the strain is obtained. B16F10 cells (2.0×10 5) were seeded into 12-well plates and incubated for 6-8 hours until the cells adhered to the walls. The harvested normal VNP or released VNP strain was then co-cultured with cells at moi=100 for 4 hours. All cells in the plates were collected, washed and resuspended in binding buffer, stained with 1 μg APC-conjugated Annexin V protein, and placed on ice for 30 minutes in darkness. All samples were analyzed for apoptosis using flow cytometry after 1 μl PI was added.
(2) Chemotaxis detection:
The chemotactic capacity of mΦ was compared with mΦ (VNP) using classical Transwell experiments. Tumor cells B16F10 were added separately in the lower chamber using 8.0 mm Transwell plates. After the cells in the lower chamber had adhered, the supernatant was removed and 700ul of cell culture medium (DMEM, 10% fetal bovine serum, 100 mg/L gentamicin) was added. MΦ and MΦ (VNP) were obtained as described above, the cell concentration was adjusted by using DMEM (containing 100 mg/mL gentamicin), 100 mL cell suspension was added to the upper chamber, and the culture was allowed to stand under 37℃and 5% carbon dioxide. 16 After h, the upper chamber was removed, the upper cells of the chamber were carefully wiped off with a cotton swab, the chamber was washed 23 times with PBS, and the chamber was placed in 4% paraformaldehyde to fix 2030 min. After the fixation, the cells were washed with PBS 23 times to remove residual paraformaldehyde, and then stained in crystal violet at room temperature in the dark for 30 min. And (3) washing with PBS to remove residual staining solution, airing, and photographing by using a positive fluorescent microscope to record the condition of cells at the lower layer of the cell.
(3) Indirect killing ability detection of tumor cells:
Indirect contact of macrophages with tumor cells was achieved using a 0.4 mm Transwell plate to test the indirect killing ability of macrophages on tumor cells after VNP loading. Tumor cells B16F10 were added in the 0.4 mm Transwell cell lower chamber, approximately 60,000 cells/well, respectively. After culturing 12 h cells in the lower chamber, the supernatant was removed and 1,200 ml of cell culture medium (DMEM, 10% fetal bovine serum, 100 mg/L gentamicin) was added. MΦ, MΦ (VNP) and L929 were obtained as described above, the cell concentration was adjusted to 1X 10 6/mL using DMEM (10% fetal bovine serum, 100 mg/L gentamicin), 200 mL cell suspensions were added to the upper chamber, and the upper chamber was incubated under 37℃5% carbon dioxide without adding any cells as a blank. 12 After h, the cell was removed. In order to determine whether the loading group would affect tumor cell proliferation, the proliferation of lower-house tumor cells in the LDH assay was determined using CCK8 assay kit according to the manufacturer's instructions, with no additional upper-house cell group as a blank. By means of the reactive oxygen species detection kit and the cellular NO detection kit, the changes in the levels of Reactive Oxygen Species (ROS) and NO production of the cells after co-culturing macrophages and VNP for 60 minutes at 1:10 were detected according to the instructions provided by the kit to analyze the possible mechanism of enhanced killing of tumor cells after loading the macrophages with VNP.
(4) Phagocytic capacity change:
After 60 minutes of co-culture of MΦ and VNP at 1:10, gentamicin killed extracellular residual strains, and cells were mixed with fluorescent microspheres at 1:10, and were allowed to stand for 4h in a 37℃cell incubator. The supernatant was aspirated and washed 3 times with PBS to remove non-phagocytized fluorescent microspheres. After carefully blowing the collected cells, the phagocytosis of fluorescent microspheres by macrophages was examined in a flow-through manner.
(5) Change in antitumor activity of cell releasing strain:
Growth curves for different VNPs in LB medium (including normal VNP and VNP released by macrophages) were obtained using Bioscreen C. Briefly, 10 μl of VNP suspension (od600=1.0) was inoculated into 1mL LB medium and 300 μl of solution per well was inoculated into Bioscreen C multiwell plates. The multiwell plates were incubated at 37 ℃ for 30 hours. OD values were measured every 30 minutes under a brown filter with a wavelength of 600 nm. The data were summarized to analyze and compare changes in bacterial growth activity. B16F10 cells (2.0×10 5) were seeded into 12-well plates and incubated for 6-8 hours until the cells adhered to the walls. The collected unprocessed VNP or cell-released VNP strain was then co-cultured with cells at moi=100 for 4 hours. All cells in the plates were collected, washed and resuspended in binding buffer, stained with 1 μg APC-conjugated Annexin V protein, and left to stand for 30 minutes in the dark. All samples were added with 1 μl PI and after gentle mixing were analyzed for apoptosis using a flow cytometer.
Example 3
Comparison of tumor inhibition effect
And constructing an in-situ melanoma tumor-bearing mouse model. Specifically, the experimental animal mice are female C57BL/6 mice of 6-8 weeks of age. Mice were randomly assigned to different groups. 100mL cells were inoculated in the right forelimb axilla of mice, 100ml containing cells 2X 10 5 (C57 BL/6, B16F 10) for oncologic therapy. Treatment is performed after the tumor of the mice grows to about 80-160 mm 3. Attenuated salmonella or macrophage-loaded attenuated salmonella was administered to tumor-bearing mice via tail vein injection, and all strains treated by tail vein injection were activated twice prior to injection.
Bacterial titers were adjusted to 5X 10 6/mL with PBS and 100: 100mL was injected intraperitoneally. After obtaining macrophages loaded with the corresponding VNP according to the previous method, PBS was adjusted to cell concentration of 2.5×10 6/mL (RAW 264.7) or 1×10 6/mL (PEM Φ), tail vein injection 100 mL.
The calculation of tumor volume was performed according to the formula v=length×width 2 ×0.52. Tumor sizes were measured at intervals for mice of a particular group and used to plot tumor growth after calculation. Values are expressed as mean±sem.
Example 4
Attenuation effect comparison:
(1) Intratumoral titer detection:
To examine the dynamic distribution of intracellular bacteria in vivo, RAW264.7 (VNP-RFP) and PEM Φ (VNP-RFP) cells were prepared as described above, and 100 μl PBS, 5×10 5 VNP-RFP、2.5 × 105 RAW264.7 (VNP-RFP) or 1×10 5 PEM Φ (VNP) cells were injected into B16F10 tumor-bearing mice by tail vein. After the mice were sacrificed at the designed time point, the organs and tumor tissues of the mice were obtained, and after disruption by means of a tissue mill, 0.3% triton x-100 was added, and 15-20 min was left at room temperature to punch cells, freeing VNP, and shaking was reversed several times during the period. After dilution in appropriate proportions, the samples were applied to a carbaryl resistant LB plate assay to calculate the VNP titres in each organ in mice. The change in tumor/spleen targeting and tumor/liver targeting was calculated and compared. For more visual observation, PEM Φ (VNP-LuxCABDE) was harvested as before and cells were incubated with Near Infrared (NIR) fluorochrome DiR for 45 minutes.
PEM Φ, PEM Φ (VNP-LuxCDABE) cells (1×10 5) or strain VNP-LuxCDABE (5×10 5) in 100 μl PBS were injected into a20 tumor bearing mice via the tail vein. Fluorescence signals of LuxCDABE and DiR were detected using an in vivo imaging system.
(2) Histopathology, blood routine and blood biochemical analysis:
Blood is collected from the eyes for routine examination. After allowing the blood to stand at room temperature for 30 minutes, the supernatant was carefully aspirated at 4℃at 3000 rpm for 15 minutes. Serum obtained from blood was cryopreserved at-80 ℃ until blood biochemical index and ELISA were determined. Conventional blood tests, blood biochemical analysis, H & E staining of tumor, heart, liver, spleen, lung and kidney sections and fluorescent immunostaining of tumor-sectioned macrophages were prepared by the marshman service biosystems.
(3) Enzyme-linked immunosorbent assay (ELISA)
At 24 hours post-dose, mice were bled to obtain serum and sacrificed to collect tumors. The obtained tumor was added to the tissue lysate at 10 mg tumor tissues/50 μl tissue lysate and homogenized using a tissue homogenizer. The supernatant was collected by centrifugation. Serum and tumor tissue lysates were collected by the mouse CCL2 ELISA kit for CCL2 cytokine detection.
Test example 1
Method for detecting release of monocyte or macrophage intracellular strain loaded with attenuated salmonella
With the aid of a Transwell chamber of 3.0 mm, after RAW264.7 (VNP-RFP) or PEMF (VNP-RFP) was obtained according to the method described above, it was placed in DMEM medium supplemented with 50 mg/L gentamicin and 10% serum for continuous culture, 12 h after which antibiotics in the culture broth were removed, and 16 h were co-cultured with tumor cells in direct contact group (MF (VNP) DOWN group) or indirect isolation (MF (VNP) UP group) (fig. 3A). As a result, it was found that direct contact with tumor cells accelerated release of intracellular VNP by macrophages, and that MF (VNP) DOWN intracellular VNP-RFP was still biologically active after release and proliferated rapidly in culture, indicating that tumor cells accelerated strain release (fig. 3B). Since tumor microenvironments are usually acidic (pH 6.5-6.8), to explore the effect of acidity on intracellular bacterial release, the invention simultaneously cultures RAW264.7 (VNP-RFP) and PEMF (VNP-RFP) cells prepared in different pH (7.4 and 6.7) media conditioned with dilute hydrochloric acid (fig. 3C). The results showed that the acidic environment had no significant effect on the release and proliferation of the strain (FIG. 3D). To further verify the effective release behavior of cells against bacteria, the present invention constructs an attenuated salmonella strain that conditionally expresses RFP (designated VNP-psifB-RFP) in macrophages based on amplification of the SifB promoter. Amplification SifB the promoter is a classical salmonella pathogenic island II (SPI-2) promoter, which is activated by intracellular hypoxia, low pH and low phosphate environments. VNP-psifB-RFP expresses RFP only after phagocytosis by macrophages. Next, RAW264.7 (VNP-psifB-RFP) and PEMF (VNP-psifB-RFP) cells were prepared and cultured in a medium to which gentamicin was added (FIG. 4A). After 3 hours, VNP-psifB-RFP with red fluorescence appeared outside the cells was observed and counted by fluorescence microscopy. Since VNP-psifB-RFP activates and expresses RFP only in the intracellular environment, the extracellular activated strain is undoubtedly released from the cells. The sustained slow release of bacteria was then recorded during the remaining test period (3-15 hours) and the results again confirm that bacteria were effectively released from the cells (fig. 4B).
Test example 2
Method for detecting activity of attenuated salmonella released after immune cell loading
RAW264.7 (VNP-RFP) and PEMF (VNP-RFP) -released VNP strains were collected and compared to untreated VNP for growth rate in LB at 37 ℃ (FIG. 5A). The 3 strains were simultaneously co-cultured with B16F10 cells at moi=100. After 1 hour incubation, cells were washed with PBS and then treated with 50. Mu.g/ml gentamicin 1h to remove residual VNP. Cells were lysed with 0.3% Triton X-100 and the number of VNP in tumor cells was determined by plating 10,000-fold dilutions of cell lysates on LB plates (fig. 5B). After co-culturing the 3 strains with B16F10 cells for 4 hours at moi=100, tumor cells were examined for apoptosis using classical apoptosis detection (annexin V and Propidium Iodide (PI) staining) flow cytometry. The results showed that there was no significant difference in the in vitro proliferative activity of the cell-released VNP and normal VNP and the ability to invade and kill tumor cells (FIG. 5C).
The method for detecting the phagocytic capacity of immune cells loaded with the attenuated salmonella is to incubate RAW264.7/RAW264.7 (VNP) and PEMF/PEMF (VNP) cells with fluorescent microspheres for 4 hours in a ratio of 1:10. Flow cytometry detects the percentage of macrophages that phagocytize the microspheres. The results indicate that macrophages co-cultured with VNP are able to phagocytose greater amounts of fluorescent microspheres in the same time than control macrophages, suggesting that they have a greater phagocytic capacity (fig. 6a, b).
Test example 3
Method for detecting chemotactic capacity of immunocyte loaded with attenuated salmonella
Comparing the chemotactic capacities of RAW264.7/PEMF and RAW264.7 (VNP)/PEMF (VNP) by using a classical Transwell experiment, and adding I) RAW264.7 to an upper chamber respectively; II) RAW264.7 (VNP); III) PEMF; IV) PEMF (VNP) with B16F10 tumor cells added in the lower chamber, and no cells added in the lower chamber as negative control, more cells were able to actively cross the small pores towards the lower chamber tumor cells than the RAW264.7 group, and more cells were able to actively cross the small pores than the RAW264.7 (VNP) group, indicating a greater chemotactic capacity than the RAW264.7 (VNP) group (FIGS. 7A, B).
Test example 4
Method for detecting indirect killing capacity of attenuated salmonella-loaded monocytes or macrophages on tumor cells
Tumor cells (B16F 10) were spread in the lower chamber of the 0.4 mm Transwell chamber, and I) L929 was added to the upper chamber; II) RAW264.7 or PEMF cells; III) RAW264.7 (VNP) or PEMF (VNP) cells, with no cells added as a blank, no cells added in the lower chamber, and the above-described types of cells added in the upper chamber as background controls, to eliminate interference of the cell background in the upper chamber (FIG. 8A). Following co-cultivation 12 h, the proliferation capacity of the lower tumor cells was examined separately by means of CCK 8. The results of the assay showed that the proliferation capacity (cell viability) of ventricular tumor cells in the RAW264.7 (VNP) and PEMF (VNP) groups was significantly weaker than that of the RAW264.7 and PEMF groups (fig. 8B). These results indicate that RAW264.7 (VNP) and PEMF (VNP) have significant tumor cell indirect killing ability, and PEMF (VNP) has stronger killing ability. Activated macrophages are able to achieve indirect killing of tumor cells by producing ROS with NO, whereas co-culture of macrophages with VNP can increase the level of ROS with NO production by cells, both ROS with NO of RAW264.7 (VNP) or PEMF (VNP) are significantly elevated compared to the original RAW264.7 or PEMF cells (fig. 8c, d), which may be one of the reasons for enhanced macrophage killing effects after co-culture.
Test example 5
The method for detecting tumor targeting, attenuation, biodistribution and treatment effects in the immune cell body loaded with the attenuated salmonella comprises the following steps of:
The B16F10 tumor-bearing mice were injected with VNP or RAW264.7 (VNP) or PEMF (VNP) via tail vein, respectively, based on the macrophage effective loading dose obtained in the previous experiment and the effective dose and tolerance dose of VNP for treating tumor in mice, wherein VNP was administered via tail vein at a dose of 5×10 5 CFU per mouse, corresponding to RAW264.7 (VNP) injection at 2.5×10 5 cells per mouse, PEMF (VNP) injection at 1×10 5 cells per mouse (fig. 9A). To investigate whether MF (VNP) cells could reduce acute toxicity and inflammatory response caused by VNP in mice, the present invention first assessed changes in body weight and morphological characteristics of liver and spleen 24 hours after different treatments. The results demonstrate that VNP-induced weight loss, splenomegaly, and liver inflammatory lesions can be significantly reduced using this strategy (fig. 9B-F). Liver H & E stained sections showed a reduced liver injury area in RAW264.7 (VNP) group compared to PEMF (VNP) group (fig. 10A). The liver injury index ALT (alanine aminotransferase) and AST (aspartic acid aminotransferase) test results in the hematological test showed that the RAW264.7 (VNP) group and PEMF (VNP) group significantly alleviated acute liver injury of VNP (fig. 10B). All these results indicate that MF (VNP) cell treatment is effective in avoiding side effects caused by single administration of VNP. The lower inflammatory response in vivo after MF (VNP) cell treatment may be due to reduced strain titers in normal organs. To verify this hypothesis, the present invention compares the in vivo colonization of VNP by dilution plating. The results indicated that no matter which macrophages were used to load VNP, less VNP was observed in the liver and spleen (fig. 11A). Day 6 VNP colonization in each tissue was chosen as representative. Tumor targeting refers to the ratio of bacterial titer in tumors to bacterial titer in normal organs, used to assess the differences in tumor-specific delivery effects of VNP between these groups. By comparison, the tumor/liver targeting was improved by 7.8-48.8-12.2-fold compared to the tumor/spleen targeting of the single VNP group in the RAW264.7 (VNP) group, while the tumor/liver targeting was improved by 6.9-51.7-fold and 2.1-12.9-fold compared to the tumor/spleen targeting of the single VNP group in the PEMF (VNP) group (fig. 11B). These results highlight the increase in tumor targeting of macrophage-mediated VNP delivery. Furthermore, VNP titers were also reduced in other organs (including heart, lung and kidney) in the MF (VNP) group compared to the single VNP group (fig. 11A), while H & E staining of these organ sections did not show significant histological defect differences between these groups, indicating negligible toxicity induced by treatment of these organs by the cell preparation (fig. 12). The present invention also evaluates the antitumor effect among different treatment groups and finds that macrophages alone have relatively potent antitumor ability, as is readily understood, because the present invention uses activated M1-like macrophages with tumor killing effect. The VNP group significantly inhibited tumor progression compared to the PBS group, while the MF (VNP) group all showed better antitumor activity (fig. 13). These results indicate that this new strategy not only avoids the side effects caused by a single administration of VNP, but also effectively improves the therapeutic effect on tumors.
The results show that compared with any single treatment mode, the PEM phi (VNP) has obviously improved anti-tumor effect. Tumor fold growth time was significantly prolonged (fig. 13, table 1). Tumor fold growth time was 2.178 ± 0.1503 days in PBS group, 3.511 ± 0.1543 days in VNP20009 group, 2.803± 0.1331 days in RAW264.7 group, 4.483 ± 0.3191 days in RAW264.7 (VNP) group, 3.216 ± 0.2046 days in PEM Φ group, 4.663 ± 0.4282 in PEM Φ (VNP) group (fig. 13, table 1). Compared with the PBS group, the VNP20009 group has a tumor fold growth time prolonged by 61.2%, the RAW264.7 group has a tumor fold growth time prolonged by 28.7%, the RAW264.7 (VNP) group has a tumor fold growth time prolonged by 105.8%, the PEM phi group has a tumor fold growth time prolonged by 47.7%, and the PEM phi (VNP) group has a tumor fold growth time of 114.1%. Compared with the VNP20009 group, the tumor fold growth time of the RAW264.7 group and the PEMEphi is shorter than that of the VNP20009 group, the tumor fold growth time of the RAW264.7 (VNP) group is prolonged by 27.7%, and the tumor fold growth time of the RAW264.7 (VNP) group is prolonged by 32.8%; compared with the RAW264.7 group, the VNP20009 group has a tumor fold growth time prolonged by 25.3%, the RAW264.7 (VNP) group has a tumor fold growth time prolonged by 59.9%, the PEM phi group has a tumor fold growth time prolonged by 14.7%, and the RAW264.7 (VNP) group has a tumor fold growth time prolonged by 66.4%; tumor fold growth time was prolonged by 4.0% in PEM Φ (VNP) group compared to RAW264.7 (VNP); the tumor fold growth time was increased by 39.4% for the RAW264.7 (VNP) group compared to the PEM Φ group, and 45.0% for the PEM Φ (VNP) group. The extension of tumor fold growth time (114.1%) for the PEM Φ (VNP) group was greater than the theoretical value of the sum of the tumor fold growth time extension values for the two groups, PEM Φ and VNP20009 (47.7% +61.2% = 108 9%); the extension of tumor fold growth time (105.8%) in the RAW264.7 (VNP) group was greater than the theoretical value of the sum of the tumor fold growth time extension values of the two groups, RAW264.7 and VNP20009 (28.7% +61.2% = 89.9%).
Compared with any single treatment mode, the PEM phi (VNP) has significantly increased anti-tumor effect and significantly prolonged tumor delay time (FIG. 13 and Table 1). The tumor delay time of PBS group was 2.130 ±0.1590 days, VNP20009 group was 3.261 ± 0.2619 days, the tumor delay time of RAW264.7 group was 2.416± 0.1682 days, the tumor delay time of RAW264.7 (VNP) group was 4.321 ± 0.2676 days, the tumor delay time of PEM Φ group was 2.781± 0.2083 days, and the tumor delay time of PEM Φ (VNP) group was 4.613 ± 0.3337 days (fig. 13, table 1). Compared with the PBS group, the tumor delay time of the VNP20009 group is prolonged by 53.1%, the tumor delay time of the RAW264.7 group is prolonged by 13.4%, the tumor delay time of the RAW264.7 (VNP) group is prolonged by 102.9%, the tumor delay time of the PEM phi group is prolonged by 30.6%, and the tumor delay time of the PEM phi (VNP) group is prolonged by 116.6%. Compared with the VNP20009 group, the tumor delay time of the RAW264.7 group and the PEM phi is shorter than that of the VNP20009 group, the tumor delay time of the RAW264.7 (VNP) group is prolonged by 32.5%, and the tumor delay time of the PEM phi (VNP) group is prolonged by 41.5%; compared with the RAW264.7 group, the tumor delay time of the VNP20009 group is prolonged by 25.3%, the tumor delay time of the RAW264.7 (VNP) group is prolonged by 78.8%, the tumor delay time of the PEM phi group is prolonged by 15.1%, and the tumor delay time of the RAW264.7 (VNP) group is prolonged by 90.9%; the tumor delay time of PEM Φ (VNP) group was prolonged by 4.0% compared to RAW264.7 (VNP), the difference was not much; the tumor delay time of the RAW264.7 (VNP) group was extended by 55.4% and the tumor delay time of the PEM Φ (VNP) group was extended by 65.9% compared to the PEM Φ group. The extension of tumor delay time of PEM Φ (VNP) group (116.6%) was much larger than the theoretical value of the sum of the tumor delay time extension values of the two groups of PEM Φ and VNP20009, respectively (30.6% +53.1% = 83.7%); the extension range of the tumor delay time (102.9%) of the RAW264.7 (VNP) group is far greater than the theoretical value (13.4% +53.1% = 66.5%) of the sum of the respective tumor delay time extension values of the RAW264.7 and VNP20009 groups; the results thus strongly demonstrate that the monocyte or macrophage line loaded with attenuated salmonella engineering bacteria (RAW 264.7) produces a much better synergistic therapeutic effect than the combined use of attenuated salmonella and monocyte or macrophage line (RAW 264.7).
The results thus strongly demonstrate that the monocyte or macrophage line loaded with attenuated salmonella engineering bacteria (RAW 264.7) produces a much better synergistic therapeutic effect than the combined use of attenuated salmonella and monocyte or macrophage line (RAW 264.7).
TABLE 1
| PBS | VNP | RAW264.7 | RAW264.7(VNP) | PEMΦ | PEMΦ(VNP) | |
| Tumor doubling time (Tian) | 2.178±0.1503 | 3.511±0.1543 | 2.803±0.1331 | 4.483±0.3191 | 3.216±0.2046 | 4.663±0.4282 |
| Tumor delay time (Tian) | 2.130±0.1590 | 3.261±0.2619 | 2.416±0.1682 | 4.321±0.2676 | 2.781±0.2083 | 4.613±0.3337 |
Data are shown as mean±sem
Test example 6
The invention relates to an immune cell living body imaging detection method for loading attenuated salmonella, which comprises the following steps:
PEMF (VNP-LuxCDABE) was obtained by co-incubation of luciferase-expressing VNP-LuxCDABE with PEMF labeled with DiR dye, PEMF cells (1X 10 5), PEMF (VNP-LuxCDABE) cells (1X 10 5) or strain VNP-LuxCDABE (5X 10 5 CFU) were injected into A20 tumor-bearing mice via the tail vein and near infrared fluorescence and bioluminescence imaging were performed at the indicated time points using the IVIS ® Lumina III in vivo imaging system. The results showed that near infrared fluorescence signals were first present in spleen, liver region after administration, near infrared fluorescence signals of PEMF and PEMF (VNP-LuxCDABE), biological optical signals of VNP-LuxCDABE and PEMF (VNP-LuxCDABE) were detected in tumor region for 12 hours (FIG. 14A). 24 hours after administration, the infrared fluorescence intensity was higher at the tumor sites of PEMF (VNP-LuxCDABE) group compared to PEMF group, indicating that PEMF (VNP-LuxCDABE) had stronger tumor enrichment capacity than PEMF alone (fig. 14B); VNP-LuxCDABE was similar to the intensity of the tumor site bioluminescence of PEMF (VNP-LuxCDABE) group (FIG. 14C). The tumor and individual organs infrared fluorescence imaging analysis showed a higher proportion of infrared fluorescence signals at the tumor site and a lower proportion of liver and spleen signals than for PEMF alone, PEMF (VNP-LuxCDABE), further demonstrating the tumor targeting ability of immune cells loaded with attenuated salmonella (fig. 14d, e). The verification method of tumor targeting in vivo attributing tumor to tumor site chemokine recruitment of immune cells loaded with attenuated salmonella is to detect serum chemokine CCL2 levels of normal mice and A20 tumor-bearing mice and serum and tumor chemokine CCL2 levels of A20 mice injected with PBS, PEMF and PEMF (VNP) respectively by ELISA. The results show that a20 tumor-bearing mice have significantly higher serum CCL2 levels than normal mice (fig. 15A) and high CCL2 levels facilitate macrophage recruitment to the tumor area. In addition, serum and tumor CCL2 levels were higher in tumor-bearing mice of PEMF (VNP) group compared to PBS and PEMF group (fig. 15B), explaining to some extent the reason that PEMF (VNP) was more potent than PEMF tumor enrichment.
Test example 7
The in vivo attenuation effect of immune cells loaded with attenuated salmonella according to the present invention is attributed to macrophage immune cell targeting carrying effect by injecting B16F10 tumor-bearing mice with pemf+vnp (simple mixed group) and PEMF (VNP) (effective loaded group) via tail vein, respectively, and evaluating their toxicity and tumor targeting (fig. 16A). Comparison of liver and spleen on day 1 found that significant liver injury and splenomegaly occurred in mice in both VNP and pemf+vnp groups, while these toxic side effects were significantly alleviated in PEMF (VNP) groups (fig. 16B-E). Day 6 liver, spleen, intratumoral strain titers and tumor/spleen targeting, tumor/liver targeting indicated that the VNP group and pemf+vnp group exhibited similar intra-visceral titers. Whereas intrahepatic and splenic strain titers of PEMF (VNP) were significantly reduced and tumor/spleen targeting was significantly increased compared to pemf+vnp group (fig. 16f, g). This suggests that injecting a mixture of macrophages and VNP directly into mice, rather than loading, does not reduce the toxicity of VNP nor the accumulation of VNP in the liver and spleen.
The mononuclear cells or macrophages loaded with the attenuated salmonella can be independently used for preparing antitumor drugs, can also be combined with the existing antitumor drugs to form a composition, or can be used in combination with the existing chemical drug treatment, traditional Chinese medicine treatment, biological treatment and physical treatment methods in antitumor treatment.
While the foregoing description illustrates and describes the preferred embodiments of the present invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of numerous other combinations, modifications, and adaptations within the scope of the present invention, as long as the foregoing is apparent to those skilled in the art from the foregoing teachings or is within the generic and descriptive scope of the invention. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
While the foregoing description illustrates and describes the preferred embodiments of the present invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of numerous other combinations, modifications, and adaptations within the scope of the present invention, as long as the foregoing is apparent to those skilled in the art from the foregoing teachings or is within the generic and descriptive scope of the invention. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Sequence listing
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gcggtagagc atgcgccatc ttatgctgat gtgattaaat ttggttctaa aaagagtctt 780
tgcattatcg ataatcctgt tgatttgacg tccgcagcga caggtgcggc tcatgatgtt 840
tgtttttacg atcagcgagc ttgtttttct gcccaaaaca tatattacat gggaaatcat 900
tatgaggaat ttaagttagc gttgatagaa aaacttaatc tatatgcgca tatattaccg 960
aatgccaaaa aagattttga tgaaaaggcg gcctattctt tagttcaaaa agaaagcttg 1020
tttgctggat taaaagtaga ggtggatatt catcaacgtt ggatgattat tgagtcaaat 1080
gcaggtgtgg aatttaatca accacttggc agatgtgtgt accttcatca cgtcgataat 1140
attgagcaaa tattgcctta tgttcaaaaa aataagacgc aaaccatatc tatttttcct 1200
tgggagtcat catttaaata tcgagatgcg ttagcattaa aaggtgcgga aaggattgta 1260
gaagcaggaa tgaataacat atttcgagtt ggtggatctc atgacggaat gagaccgttg 1320
caacgattag tgacatatat ttctcatgaa aggccatcta actatacggc taaggatgtt 1380
gcggttgaaa tagaacagac tcgattcctg gaagaagata agttccttgt atttgtccca 1440
taataggtaa aagtatggaa aatgaatcaa aatataaaac catcgaccac gttatttgtg 1500
ttgaaggaaa taaaaaaatt catgtttggg aaacgctgcc agaagaaaac agcccaaaga 1560
gaaagaatgc cattattatt gcgtctggtt ttgcccgcag gatggatcat tttgctggtc 1620
tggcggaata tttatcgcgg aatggatttc atgtgatccg ctatgattcg cttcaccacg 1680
ttggattgag ttcagggaca attgatgaat ttacaatgtc tataggaaag cagagcttgt 1740
tagcagtggt tgattggtta actacacgaa aaataaataa cttcggtatg ttggcttcaa 1800
gcttatctgc gcggatagct tatgcaagcc tatctgaaat caatgcttcg tttttaatca 1860
ccgcagtcgg tgttgttaac ttaagatatt ctcttgaaag agctttaggg tttgattatc 1920
tcagtctacc cattaatgaa ttgccggata atctagattt tgaaggccat aaattgggtg 1980
ctgaagtctt tgcgagagat tgtcttgatt ttggttggga agatttagct tctacaatta 2040
ataacatgat gtatcttgat ataccgttta ttgcttttac tgcaaataac gataattggg 2100
tcaagcaaga tgaagttatc acattgttat caaatattcg tagtaatcga tgcaagatat 2160
attctttgtt aggaagttcg catgacttga gtgaaaattt agtggtcctg cgcaattttt 2220
atcaatcggt tacgaaagcc gctatcgcga tggataatga tcatctggat attgatgttg 2280
atattactga accgtcattt gaacatttaa ctattgcgac agtcaatgaa cgccgaatga 2340
gaattgagat tgaaaatcaa gcaatttctc tgtcttaaaa tctattgaga tattctatca 2400
ctcaaatagc aatataagga ctctctatga aatttggaaa ctttttgctt acataccaac 2460
ctccccaatt ttctcaaaca gaggtaatga aacgtttggt taaattaggt cgcatctctg 2520
aggagtgtgg ttttgatacc gtatggttac tggagcatca tttcacggag tttggtttgc 2580
ttggtaaccc ttatgtcgct gctgcatatt tacttggcgc gactaaaaaa ttgaatgtag 2640
gaactgccgc tattgttctt cccacagccc atccagtacg ccaacttgaa gatgtgaatt 2700
tattggatca aatgtcaaaa ggacgatttc ggtttggtat ttgccgaggg ctttacaaca 2760
aggactttcg cgtattcggc acagatatga ataacagtcg cgccttagcg gaatgctggt 2820
acgggctgat aaagaatggc atgacagagg gatatatgga agctgataat gaacatatca 2880
agttccataa ggtaaaagta aaccccgcgg cgtatagcag aggtggcgca ccggtttatg 2940
tggtggctga atcagcttcg acgactgagt gggctgctca atttggccta ccgatgatat 3000
taagttggat tataaatact aacgaaaaga aagcacaact tgagctttat aatgaagtgg 3060
ctcaagaata tgggcacgat attcataata tcgaccattg cttatcatat ataacatctg 3120
tagatcatga ctcaattaaa gcgaaagaga tttgccggaa atttctgggg cattggtatg 3180
attcttatgt gaatgctacg actatttttg atgattcaga ccaaacaaga ggttatgatt 3240
tcaataaagg gcagtggcgt gactttgtat taaaaggaca taaagatact aatcgccgta 3300
ttgattacag ttacgaaatc aatcccgtgg gaacgccgca ggaatgtatt gacataattc 3360
aaaaagacat tgatgctaca ggaatatcaa atatttgttg tggatttgaa gctaatggaa 3420
cagtagacga aattattgct tccatgaagc tcttccagtc tgatgtcatg ccatttctta 3480
aagaaaaaca acgttcgcta ttatattagc taaggagaaa gaaatgaaat ttggattgtt 3540
cttccttaac ttcatcaatt caacaactgt tcaagaacaa agtatagttc gcatgcagga 3600
aataacggag tatgttgata agttgaattt tgaacagatt ttagtgtatg aaaatcattt 3660
ttcagataat ggtgttgtcg gcgctcctct gactgtttct ggttttctgc tcggtttaac 3720
agagaaaatt aaaattggtt cattaaatca catcattaca actcatcatc ctgtcgccat 3780
agcggaggaa gcttgcttat tggatcagtt aagtgaaggg agatttattt tagggtttag 3840
tgattgcgaa aaaaaagatg aaatgcattt ttttaatcgc ccggttgaat atcaacagca 3900
actatttgaa gagtgttatg aaatcattaa cgatgcttta acaacaggct attgtaatcc 3960
agataacgat ttttatagct tccctaaaat atctgtaaat ccccatgctt atacgccagg 4020
cggacctcgg aaatatgtaa cagcaaccag tcatcatatt gttgagtggg cggccaaaaa 4080
aggtattcct ctcatcttta agtgggatga ttctaatgat gttagatatg aatatgctga 4140
aagatataaa gccgttgcgg ataaatatga cgttgaccta tcagagatag accatcagtt 4200
aatgatatta gttaactata acgaagatag taataaagct aaacaagaga cgcgtgcatt 4260
tattagtgat tatgttcttg aaatgcaccc taatgaaaat ttcgaaaata aacttgaaga 4320
aataattgca gaaaacgctg tcggaaatta tacggagtgt ataactgcgg ctaagttggc 4380
aattgaaaag tgtggtgcga aaagtgtatt gctgtccttt gaaccaatga atgatttgat 4440
gagccaaaaa aatgtaatca atattgttga tgataatatt aagaagtacc acatggaata 4500
tacctaatag atttcgagtt gcagcgaggc ggcaagtgaa cgaatcccca ggagcataga 4560
taactatgtg actggggtga gtgaaagcag ccaacaaagc agcagcttga aagatgaagg 4620
gtataaaaga gtatgacagc agtgctgcca tactttctaa tattatcttg aggagtaaaa 4680
caggtatgac ttcatatgtt gataaacaag aaattacagc aagctcagaa attgatgatt 4740
tgattttttc gagcgatcca ttagtgtggt cttacgacga gcaggaaaaa atcagaaaga 4800
aacttgtgct tgatgcattt cgtaatcatt ataaacattg tcgagaatat cgtcactact 4860
gtcaggcaca caaagtagat gacaatatta cggaaattga tgacatacct gtattcccaa 4920
catcggtttt taagtttact cgcttattaa cttctcagga aaacgagatt gaaagttggt 4980
ttaccagtag cggcacgaat ggtttaaaaa gtcaggtggc gcgtgacaga ttaagtattg 5040
agagactctt aggctctgtg agttatggca tgaaatatgt tggtagttgg tttgatcatc 5100
aaatagaatt agtcaatttg ggaccagata gatttaatgc tcataatatt tggtttaaat 5160
atgttatgag tttggtggaa ttgttatatc ctacgacatt taccgtaaca gaagaacgaa 5220
tagattttgt taaaacattg aatagtcttg aacgaataaa aaatcaaggg aaagatcttt 5280
gtcttattgg ttcgccatac tttatttatt tactctgcca ttatatgaaa gataaaaaaa 5340
tctcattttc tggagataaa agcctttata tcataaccgg aggcggctgg aaaagttacg 5400
aaaaagaatc tctgaaacgt gatgatttca atcatctttt atttgatact ttcaatctca 5460
gtgatattag tcagatccga gatatattta atcaagttga actcaacact tgtttctttg 5520
aggatgaaat gcagcgtaaa catgttccgc cgtgggtata tgcgcgagcg cttgatcctg 5580
aaacgttgaa acctgtacct gatggaacgc cggggttgat gagttatatg gatgcgtcag 5640
caaccagtta tccagcattt attgttaccg atgatgtcgg gataattagc agagaatatg 5700
gtaagtatcc cggcgtgctc gttgaaattt tacgtcgcgt caatacgagg acgcagaaag 5760
ggtgtgcttt aagcttaacc gaagcgtttg atagttga 5798
<210> 5
<211> 714
<212> DNA
<213> Artificial sequence (sequence of fluorescent protein RFP)
<400> 5
ttagttcagc ttatgaccca gtttgctcgg caggtcgcaa taacgcgcaa ccgccacctc 60
gtgctgttcc acgtaggttt ccttgtccgc ctctttaata cgttccagac gatgatcaac 120
atagtacacg cccggcatct tcaggttttt cgccggcttt ttgctacgat aggtggtttt 180
gaagttgcag atcaggtgac cgccaccaac cagcttcagc gccatgtcgc tacgaccttc 240
caggccacca tccgccgggt acagcatctc ggtgttcgct tcccagccca gggtcttttt 300
ctgcatcacc ggaccgttgc tcggaaagtt aacgccacga attttcacgt tatagatcag 360
gcaaccgtct tgcaggctgg tatcctgggt cgcggtcaga acgccaccgt cctcgtaggt 420
ggtcacacgt tcccaggtga agccctccgg aaagctctgt ttaaagaaat ccggaatacc 480
ctgggtatgg ttgatgaagg tacggctgcc gtacataaag ctggtcgcca gaatatcgaa 540
cgcaaacggc agcgggccac cttcaaccac cttgatacgc atggtctggg tgccctcata 600
cggtttaccc tcgccttcgc tggtgcactt aaaatggtgg ttgttaacgg taccttccat 660
gtacagtttc atgtgcatgt tctccttgat cagctcttcg cctttgctaa ccat 714
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (PsifB sequence of upstream primer)
<400> 6
caaaatccct tataagaatt ctgccctacc gctaaacatc 40
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence (PsifB sequence of downstream primer)
<400> 7
tgtccaacac tcaatggcat ccacaagtga ttatatgata 40
Claims (8)
1. A monocyte or macrophage loaded with attenuated salmonella, wherein: loading attenuated salmonella with macrophages, wherein the macrophages are macrophage line RAW264.7 or primary macrophages from abdominal cavity and blood, the attenuated salmonella is attenuated salmonella typhimurium VNP20009 and derivative or genetically modified strains thereof, and the strains are expression plasmids which do not carry or carry exogenous genes for expressing therapeutic genes and tracer genes; the therapeutic gene is a coding gene of a protein with therapeutic effect, which can be expressed in attenuated salmonella, and comprises an apoptosis antitumor gene, an angiogenesis inhibitor gene or an immune checkpoint blocker antibody gene, the tracer gene is a gene which can express fluorescent protein RFP or luciferase tracer protein LuxCDABE in attenuated salmonella, and comprises a gene derived from green fluorescent protein GFP or red fluorescent protein RFP and fluorescent protein derived from the green fluorescent protein GFP or the red fluorescent protein RFP, and the sequence of the luciferase protein gene LuxCDABE is a nucleotide sequence shown in SEQ ID No. 4; the sequence of the fluorescent protein RFP is a nucleotide sequence shown in SEQ ID No. 5; the expression plasmid is provided with promoters commonly used in attenuated salmonella, including J23100 constitutive promoter, nirB promoter, adhE promoter or promoter for amplifying SifB promoter environmental influence; the plasmid contains an element AT for preventing plasmid loss; the sequence of the amplification plasmid loss prevention element AT is a nucleotide sequence shown in SEQ ID No. 1; the sequence of the amplified SifB promoter PsifB is a nucleotide sequence shown as SEQ ID No. 2; the sequence of the J23100 constitutive promoter is a nucleotide sequence shown as SEQ ID No. 3; the sequence of the PsifB upstream primer is the nucleotide sequence shown in SEQ ID No. 6; the sequence of the PsifB downstream primer is the nucleotide sequence shown as SEQ ID No. 7.
2. A method of preparing an attenuated salmonella-loaded monocyte or macrophage of claim 1, wherein: primary monocytes or macrophages purified by RAW264.7 macrophage cell lines induced with 25-500ng/mL LPS for 4-48 hours or by 2-4 days combined with wall-attached culture stimulated by 5% starch broth intraperitoneal injection are co-cultured with attenuated Salmonella at a ratio of 1:5-1:100, respectively, for 30-150 minutes, and then cells are treated with 50-100 μg/mL gentamicin for 30-60 minutes to kill extracellular bacteria; the number of viable VNPs after phagocytosis of macrophages was calculated using dilution plating to record the number of intracellular active load strains and cell activity under different treatment conditions and used for the determination of the final co-culture time and calculation of the actual dose of cells following.
3. The method for evaluating and tracing the bacterial loading efficiency and tissue distribution level of monocytes or macrophages according to claim 1, characterized by comprising the steps of: loading fluorescent protein or luciferase tracer protein capable of being expressed or quantitatively detected in monocytes or macrophages, wherein the fluorescent protein or luciferase tracer protein comprises VNP20009 bacteria VNP-RFP capable of stably and constitutively expressing red fluorescent RFP or VNP20009 bacteria VNP-LuxCDABE capable of expressing luciferase LuxCDABE, and evaluating and tracing the bacterial loading efficiency and tissue distribution and level of the monocytes or macrophages loaded with attenuated salmonella; the tracer gene is a gene capable of expressing fluorescent protein RFP or luciferase tracer protein LuxCDABE in attenuated salmonella, including but not limited to genes derived from green fluorescent protein GFP or red fluorescent protein RFP and fluorescent proteins derived therefrom.
4. A method of assessing and tracing the bacterial loading efficiency and tissue distribution level of monocytes or macrophages as set forth in claim 3 wherein: the mononuclear cells or macrophages are labeled with a common optical dye including DiR near infrared dye, the optical dye is quantitatively detected, and the tissue distribution and level of the mononuclear cells or macrophages loaded with attenuated salmonella are evaluated and tracked.
5. The method for evaluating and tracing the bacterial loading efficiency and tissue distribution level of monocytes or macrophages as set forth in claim 4, wherein: untreated M0 type macrophages RAW264.7, after 4-48 hours of LPS induction at 25-500ng/mL, RAW264.7 was converted to M1 type macrophages; or purifying the obtained primary monocytes or macrophages by 5% starch broth intraperitoneal injection stimulation for 2-4 days in combination with an adherent culture method; co-culturing RAW264.7 or primary mononuclear cells or macrophages subjected to LPS induction treatment with attenuated salmonella expressing a tracer protein or therapeutic protein in a ratio of 1:5-1:100 for 30-150 minutes, respectively, and quantitatively detecting the correlation of the amount of the attenuated salmonella phagocytosis of the tracer protein or therapeutic protein by RAW264.7 and the co-culture time after treating cells with 50-100 mug/mL gentamicin for 30-60 minutes to kill extracellular free or adherent attenuated salmonella; obtaining a linear relation between the number of attenuated salmonella expressing the tracer protein or carrying the therapeutic protein and the corresponding fluorescence intensity by a gradient dilution method, and effectively and indirectly calculating the number of the attenuated salmonella expressing the tracer protein or carrying the therapeutic protein in a macrophage cell; the enzyme label instrument detects the fluorescence intensity of the tracer protein, and the total bacterial number loaded by each 100 macrophages is calculated by combining the linear relationship of the bacterial number and the fluorescence.
6. The method for evaluating and tracing the bacterial loading efficiency and tissue distribution level of monocytes or macrophages as set forth in claim 5, wherein: transferring the attenuated salmonella-loaded monocytes or macrophages to PBS-formulated 0.3% Triton X-100 for 10-15min to perforate the macrophages to release intracellular attenuated salmonella; coating LB plate with the resistance of the kanamycin after gradient dilution, analyzing and detecting the number of live attenuated salmonella loaded by monocytes or macrophages and the correlation of the loading efficiency and the co-culture time; detecting the activity state of the cells after 30, 60, 90, 120 and 150 minutes of co-culture of macrophages and attenuated salmonella by using a phenol blue assay; to further verify that macrophages were efficiently loaded with attenuated salmonella, after obtaining the macrophages loaded with attenuated salmonella, the cells were fixed with 4% paraformaldehyde, at room temperature, protected from light, left to stand for 30min, washed with pbs 23 times, macrophage scaffold and cell nuclei were stained with phalloidin and DAPI according to manufacturer's instructions, respectively, at room temperature, protected from light, left to stand for 30min, washed with pbs 23 times, and observed and photographed using a positive fluorescence microscope.
7. Use of the attenuated salmonella-loaded monocytes or macrophages of any one of claims 1 to 6 for the preparation of an antitumor medicament.
8. The use according to claim 7, characterized in that: the application comprises that the mononuclear cells or macrophages loaded with the attenuated salmonella are singly used for preparing an anti-tumor medicament, or are combined with the existing anti-tumor medicament to form a composition, or are combined with the existing chemical medicament treatment, traditional Chinese medicine treatment, biological treatment and physical treatment methods for anti-tumor treatment; the titer of the attenuated salmonella in the tumor is improved, and the side effect of oncolytic bacteria treatment is reduced and the tumor inhibition effect is improved through the combination of macrophages and oncolytic bacteria.
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| CN1562375A (en) * | 2004-03-22 | 2005-01-12 | 中国医学科学院血液学研究所 | Combination of medication produced from attenuated salmonella bacteria possessing effect of radiation protection and anti tumour |
| CN106434511A (en) * | 2016-11-02 | 2017-02-22 | 南京大学 | Gene deleted attenuated salmonella typhimurium mutant strain and construction method thereof and application of gene deleted attenuated salmonella typhimurium mutant strain in improvement of melanoma targeting performance |
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| CN1562375A (en) * | 2004-03-22 | 2005-01-12 | 中国医学科学院血液学研究所 | Combination of medication produced from attenuated salmonella bacteria possessing effect of radiation protection and anti tumour |
| CN106434511A (en) * | 2016-11-02 | 2017-02-22 | 南京大学 | Gene deleted attenuated salmonella typhimurium mutant strain and construction method thereof and application of gene deleted attenuated salmonella typhimurium mutant strain in improvement of melanoma targeting performance |
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