WO2021227435A1 - Tumor pericytes, isolation method therefor and use thereof - Google Patents

Tumor pericytes, isolation method therefor and use thereof Download PDF

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WO2021227435A1
WO2021227435A1 PCT/CN2020/130723 CN2020130723W WO2021227435A1 WO 2021227435 A1 WO2021227435 A1 WO 2021227435A1 CN 2020130723 W CN2020130723 W CN 2020130723W WO 2021227435 A1 WO2021227435 A1 WO 2021227435A1
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pericytes
tumor
matrigel
colorectal cancer
immortalized
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Dongmei Zhang
Minfeng Chen
Wencai Ye
Jinghua PAN
Yong Li
Tongzheng LIU
Qi QI
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Jinan University
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Definitions

  • the present invention belongs to the field of biotechnology and particularly relates to tumor pericytes, an isolation method therefor and use thereof.
  • TPCs Tumor pericytes
  • TPCs are parietal cells that are located on the periphery of tumor blood vessels and embedded in the basement membrane, which plays a vital role in tumor angiogenesis, maturation and stability, tumor invasion and metastasis, and tolerance of tumor anti-angiogenesis therapy (1-3) .
  • the regulatory effect of TPCs on tumor progression has become a research focus in recent years.
  • one of the biggest obstacles in the field of TPCs research is the lack of TPCs-specific molecular markers, which not only affects the identification of pericytes but also limits the isolation and sorting of TPCs.
  • TPCs unlike other types of cells in tumor tissue, TPCs still lack of specific molecular markers.
  • NG2, CD146, PDGFR ⁇ , CD13 and ⁇ SMA markers such as NG2, CD146, PDGFR ⁇ , CD13 and ⁇ SMA.
  • a current method of isolating pericytes from normal tissues or tumor tissues mainly labels the pericytes using one or more of molecular markers (e.g., NG2, CD146, PDGFR ⁇ , CD13, and ⁇ SMA) , which are highly expressed in pericytes, and then isolates pericytes by magnetic beads sorting or flow sorting (8-10) .
  • the pericytes obtained by magnetic beads sorting or flow sorting based on these molecular markers may contain miscellaneous cells such as fibroblasts and mesenchymal stem cells at the same time, which have the disadvantage of insufficient purity.
  • the expression of molecular markers of TPCs in different regions may be different. Therefore, by the sorting method using one or several of the molecular markers, only a single type of TPCs can be obtained, and the heterogeneity of TPCs cannot be fully reflected.
  • a primary purpose of the present invention is to overcome the current limitations of the separation methods of TPCs by using magnetic bead sorting or flow sorting, and to provide a new method for isolating tumor pericytes. This method provides sufficient numbers, reliable sources, and stable quality of TPCs for basic medicine, biological research and drug development research.
  • Another object of the present invention is to provide TPCs.
  • the present invention for example uses human colorectal cancer blood vessels to isolate and identify TPCs, and other types of solid malignant TPCs obtained by the method described in the present invention are also within the protection scope of the present invention.
  • the present invention also provides a use of the abovementioned TPCs.
  • the present invention mainly achieves the foregoing objects through the following technical solutions:
  • the method for isolating TPCs comprises stripping tumor vessels from fresh solid tumor tissue samples, cutting the tumor vessels into annular fragments, placing the annular fragments of the tumor vessels in a pericyte culture medium pre-coated with matrigel for conditioned culture, and recycling tumor pericytes from the matrigel.
  • the solid tumor may be liver cancer, colorectal cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer or gastric cancer; and preferably, the solid tumor is colorectal cancer.
  • the solid tumor tissue samples are stored in DMEM cell culture medium containing 1% (v/v) penicillin-streptomycin (PS) .
  • PS penicillin-streptomycin
  • the abovementioned tumor specimens containing cancerous and adjacent tissues without large area necrosis are obtained from patients who underwent surgery for solid malignant tumor.
  • the abovementioned tumor samples are washed by DMEM medium containing 1% (v/v) PS to remove contaminants such as residual feces and blood stains.
  • the abovementioned tumor samples are preferably rinsed with phosphate buffer solution containing 1% (v/v) PS pre-cooled at 4°C until there is no blood color.
  • the abovementioned tumor blood vessels stripped from the tumor samples are cut into circular segments under the guidance of a stereo microscope.
  • a diameter of the blood vessel is preferably 30-100 ⁇ m.
  • a length of the circular segment of blood vessel is preferably 4-6 mm.
  • a preparation method of the matrigel comprises: diluting the matrigel by a DMEM medium pre-cooled at 4°C at a volume ratio of 2: 1 to obtain a diluted matrigel solution, and adding PDGF-BB to the diluted matrigel solution at a final concentration of 0.5% ⁇ 2% (w/v) ; More preferably, PDGF-BB at a final concentration of 1% (w/v) was added to the diluted matrigel solution.
  • 100 ⁇ L of the abovementioned matrigel is added to each well of a 24-well plate.
  • the matrigel is incubated at 37 °C for 30 ⁇ 40 min in an incubator before being used.
  • the pericyte culture medium is a basal medium comprising 2%v/v fetal bovine serum, 1%v/v pericyte growth supplement (PGS) , 100U/mL penicillin and 100 ⁇ g/mL streptomycin.
  • PPS pericyte growth supplement
  • the conditioned culture is conducted at a condition of 37 °C, 5%CO 2 and 95%humidity for 13-15 days in an incubator.
  • the recycling comprises: rinsing the matrigel by a phosphate buffer solution, centrifuging to obtain a precipitate and a supernatant, removing the supernatant, and adding a dispase solution to the precipitate for digestion, and stopping the digestion.
  • the matrigel is centrifuged at 1000-2000g for 10-20 min, and more preferably at 1500g for 15 min.
  • a concentration of the dispase solution is 1U/mL.
  • the digestion is conducted at 37 °C for 2-3 h in the incubator.
  • the digestion is stopped by an EDTA solution, which preferably has a concentration of 10 mM.
  • a volume ratio of the dispase solution and the EDTA solution is 1: 4.
  • TPCs were obtained by the above method.
  • the TPCs have the following biological characteristics: high expression of four positive molecular markers including FAP ⁇ , NG2, PDGFR ⁇ and CD146, and no expression of two negative molecular markers including CD31 and MYH11; a subcellular structure with large nuclear proportion and few organelles; and chemotaxis and adhesion to endothelial cells.
  • the TPCs may be used in tumor-related biological research and/or preparation of anti-tumor drugs.
  • the TPCs can be used in the construction of immortalized tumor pericytes.
  • a construction method for immortalized tumor pericytes comprises preparing tumor pericytes by the above method for isolating tumor pericytes, and obtaining the immortalized tumor pericytes by lentiviral transfection of the tumor pericytes with SV40T antigen.
  • Immortalized tumor pericytes are obtained by the construction method.
  • a method for constructing immortalized tumor pericytes monoclonal strains comprises cloning and culturing positive clones of the immortalized tumor pericytes by a limiting dilution method, so as to obtain the immortalized tumor pericytes monoclonal strains.
  • the immortalized tumor pericytes monoclonal strains are obtained by the construction method.
  • the present invention has the following beneficial effects as compared with those in the prior art:
  • the present invention is the first to obtain tumor pericytes by a method comprising stripping tumor vessels and conditioned culture.
  • a method comprising stripping tumor vessels and conditioned culture.
  • the isolation method of TPCs described in the present invention is easier to operate and has lower cost; the types of TPCs obtained by the isolation method of the present invention are more comprehensive, which truly reflect the heterogeneity of TPCs.
  • the TPCs obtained by the present invention are derived from surgical specimens of human colorectal cancer, which truly reflects the biological characteristics of tumor blood vessels. Therefore, the human colorectal cancer pericytes obtained by the present invention are superior to the simulated TPCs obtained previously by co-incubation of normal human cerebral pericytes or bovine retinal pericytes with tumor cells in vitro.
  • the present invention is the first to observe the subcellular structure characteristics of the obtained primary colorectal cancer pericytes and immortalized cell monoclonal strain thereof by transmission electron microscopy, which provides a new reference standard for the identification of human colorectal cancer pericytes.
  • the present invention is the first to infect primary colorectal cancer pericytes with SV40T lentivirus, which is a zero breakthrough in the construction of immortalized human colorectal cancer pericytes model.
  • the present invention is the first to pick out an immortalized colorectal cancer pericyte monoclonal strain, which may serve as a reliable raw material with sufficient quantity and stable quality for studying the biological and medical of human colorectal cancer pericytes.
  • Figure 1 shows a process of isolating human colorectal cancer pericytes.
  • Figure 2 is shows the identification of human colorectal cancer pericytes by flow cytometry.
  • Figure 3 shows the identification of human colorectal cancer pericytes by immunofluorescence.
  • Figure 4 shows the identification of human colorectal cancer pericytes by transmission electron microscopy.
  • Figure 5 is the result of a 3D lumen adhesion assay of HUVEC and human colorectal cancer pericytes, wherein the PKH26 labeled HUVEC is in red fluorescence and the PKH67 labeled human colorectal cancer pericytes is in green fluorescence.
  • Figure 6 shows the structure of pGMLV-SV40T lentiviral plasmid.
  • Figure 7 is the detection result of the expression of SV40T gene by PCR, wherein, lane 1 is Marker, lane 2 is human colorectal cancer pericytes, lane 3 is immortalized human colorectal cancer pericytes (ihTPC) , and lane 4 is immortalized human colorectal cancer pericytes monoclonal strains (ihTPC-1) .
  • Figure 8 shows the proliferation activity of different generations of immortalized human colorectal cancer pericytes (ihTPC) by CCK8 assay.
  • Figure 9 shows the identification of immortalized human colorectal cancer pericytes monoclonal strains by flow cytometry.
  • Figure 10 shows the identification of immortalized human colorectal cancer pericytes monoclonal strains by immunofluorescence.
  • Figure 11 shows the identification of immortalized human colorectal cancer pericytes monoclonal strains by transmission electron microscopy.
  • Figure 12 is the result of a 3D lumen adhesion assay of HUVEC and immortalized human colorectal cancer pericyte monoclonal strains (ihTPC-1) , wherein, the PKH26 labeled HUVEC is in red fluorescence and the PKH67 labeled immortalized human colorectal cancer pericyte monoclonal strain0 is in green fluorescence.
  • ihTPC-1 immortalized human colorectal cancer pericyte monoclonal strains
  • Example 1 Dissection of blood vessels from human colorectal cancer samples, isolation and culture of colorectal cancer pericytes
  • 354248 was dilutedby an ice-cold DMEM blank medium at a 2: 1 ratio to prepare a diluted matrigel solution and PDGF-BB (w/v) at a final concentration of 1 w/v%was added to the diluted matrigel solution. Then, the obtained smear solution was added to a 24-well plate at an amount of 100 ⁇ L/well, and the plate was placed in an incubator at 37°C for 30 min. The blood vessels were cut into a vascular ring with a length of about 5 mm and were added to 1 mL of complete medium of pericytes (PM, Cat. No.
  • PM complete medium of pericytes
  • a basal medium containing 2 v/v%fetal bovine serum, 1 v/v%PGS, 100U/mL of penicillin, and 100 ⁇ g/mL of streptomycin followed by adding the incubate vascular rings to the 24-well plate pre-coated with matrigel.
  • the cells were incubated at 37°C with 5%CO 2 for 14days, after which colorectal cancer vascular cells grew outward and close to the matrigel.
  • the matrigel-coated cells were collected by centrifugation at 1500 ⁇ g for 5 min after washing with PBS, and the matrigel was then digested with 200 ⁇ L of 1 U/mL dispase solution ( Cat. No.
  • tumor vessels with a diameter of 30-100 ⁇ m were obtained; about 3 ⁇ 10 6 of human colorectal cancer vascular cells were collected, which can be used for subsequent culture and identification.
  • Results the results of the flow cytometry analysis are shown in Figure 2.
  • the primary colorectal cancer pericytes highly expressed positive pericytes molecular markers including FAP ⁇ , NG2, PDGFR ⁇ and CD146, but not expressed negative pericytes molecular markers including CD31 and MYH11.
  • the primary antibodies includes: anti-FAP ⁇ -PE (R&D Cat. No.
  • Results the results of the confocal laser scanning microscope are shown in Figure 3.
  • the primary colorectal cancer pericytes highly expressed positive pericytes molecular markers including FAP ⁇ , NG2, PDGFR ⁇ and CD146, but not expressed negative pericytes molecular markers including CD31 and MYH11.
  • the cells were successively dehydrated for 2 times, each time for 10 min, by ethanol solution with a concentration of 30%, 50%, 70%, 90%, 100%after washing with PBS for 3 times. After sucking out the ethanol, the obtained cell mass was transferred onto a capsule with an embedding agent at the bottom and then the capsule was placed in an oven for polymerization at 60 °C for 2 h after filling the capsule with the embedding agent; Finally, the embedded cells were processed for preparation of ultrathin (70 ⁇ 90 nm) sections by LEICA EM UC7 ultra-thin microtome, and after double staining with 2%uranyl acetate (cat. No. SPI-02624) and 1%lead citrate ( Cat. No. HD17800) , the ultrathin sections were observed under a Hitachi h-7650 transmission electron microscope.
  • Example 5 Detection of tendency effects of human colorectal cancer pericytes to endothelial cell lumen by 3D lumen adhesion assay.
  • HUVECs were further labeled with PKH 26 (1: 1000, Sigma Aldrich, Cat. No. PKH26GL) for 3 min at room temperature, and the staining was blocked with sterile 5%BSA. After washing with PBS, the HUVECs were re-suspended with ECM, counted, and added to a 96-well plate pre-coated with matrigel at 3 ⁇ 10 4 cells/well, where the HUVECs began to form tube structure 2 h later. Subsequently, the obtained colorectal cancer pericytes were labeled with PKH67 (Sigma Aldrich, Cat. No.
  • PKH67GL PKH67GL
  • Results the results of the 3D lumen adhesion assay are shown in Figure 5.
  • the primary human colorectal cancer pericytes tended to attach to the lumen of HUVECs.
  • Example 6 Construction of SV40T lentivirus and infection of primary colorectal cancer pericytes and colorectal cancer pericytes monoclonal line
  • lentiviral vectors pGMLV-SV40T, Genomeditech, Cat. No. GM-0220SV01
  • Figure 6 which encoded simian virus 40 Large T Antigen
  • Lipofectamine 2000 lentiviral packaging plasmid VSVG/PMDL/REV at a ratio of 5: 3: 2.
  • the mixture was co-transfected into HEK-293T cells, and virus supernatant was collected, centrifuged to remove impurities 24 h later.
  • primary colorectal cancer pericytes were transferred to a 24-well plate at 1 ⁇ 10 5 cells/well, infected with virus at a MOI of 50 (a total of 5 ⁇ 10 6 viral particles) 6 h later, incubated at 37°C with 5%CO 2 for 24 h in an incubator, and sub cultured after another 48 h with a replaced fresh medium.
  • the human colorectal cancer pericytes transfected with SV40T were diluted to 1 cell per 2 wells by limiting dilution. After 7-10 days of incubation, the positive cells with single-cell clone growth were selected to clone again, which was performed triple times, and finally immortalized human colorectal cancer pericyte monoclonal strain was obtained.
  • RNA of the human colorectal cancer pericytes, the human colorectal cancer pericytes transfected with SV40T and the human colorectal cancer pericytes monoclonal strains transfected with SV40T were extracted using a TRIzol method.
  • PCR primers were designed according to the sequences of SV40T genes, the sequences being list below: forward strand: 5'-CCACAAAGGA-AAAAGCTGCACTGCTA-3'; reverse strand: 5'-TCAACAGCCTGTTGGCATATGGTTTT-3'.
  • the reaction conditions were: 95°C for 5 min, then 35 cycles of 95 °C for 30 s, 60°C for 30 s and 72 °C for 30 s, followed by a terminal 10-min extension phase at 72 °C and permanent at 4 °C.
  • the expression level of SV40T was evaluated by gel electrophoresis.
  • results are shown in Figure 7.
  • the expression level of SV40T in the human colorectal cancer pericytes (lane 1) , the human colorectal cancer pericytes transfected with SV40T (lane 2) and the human colorectal cancer pericytes monoclonal strains transfected with SV40T (lane 3) detected by RT-PCR and the SV40-LT-target band of about 550 bp observed in both lane 2 and 3 indicate that the immortalized colorectal cancer vascular cells and its monoclonal strains were constructed successfully.
  • Results the results are shown in Figure 8.
  • the proliferation activity of non-immortalized human colorectal cancer pericytes was decreased significantly from the 1 st generation to the 10 th generation, and the proliferation activity of the 1 st , 10 th and 25 th generation of the immobilized human colorectal cancer pericytes was similar to that of the 1 st generation colorectal cancer pericytes.
  • mice the immortalized human colorectal cancer pericytes monoclonal strain at a logarithmic growth phase was collected and re-suspended with ice-cold matrigel.
  • Cell suspensions at a density of 1 ⁇ 10 7 cells per mL were subcutaneously injected into the back of ten BALB/C nu/nu nude mice (half male and half female) at 0.2 mL/mouse. After a four-week feeding of the nude mice, the tumor formation situation of the immortalized human colorectal cancer pericytes monoclonal strain in these mice was observed and recorded.
  • Example 10 Identification of the expression of molecular markers in immortalized cell monoclonal strain by flow cytometry.
  • results the results of the flow cytometry experiment are shown in Figure 9.
  • the results showed that, like the primary cultured cells of human colorectal cancer pericytes, the immortalized cell monoclonal strain highly expressed four pericyte positive molecular markers including FAP ⁇ , NG2, PDGFR ⁇ and CD146, but not expressed negative molecular markers including CD31 and MYH11. It can be seen that the immortalized cell strain constructed by the present invention still expressed the same molecular markers as the primary pericytes.
  • Example 11 Identification of the expression of molecular markers in immortalized cell monoclonal strain by immunofluorescence.
  • results the results of the immunofluorescence experiment are shown in Figure 10.
  • the results showed that, like the primary cultured cells of human colorectal cancer vascular pericytes, the immortalized cell monoclonal strain highly expressed four pericyte positive molecular markers including FAP ⁇ , NG2, PDGFR ⁇ and CD146, but not expressed negative molecular markers including CD31 and MYH11. It can be seen that the immortalized cell strain constructed by the present invention still expressed the same molecular markers as the primary pericytes.
  • Results the results of the transmission electron microscope are shown in Figure 11. Like the primary cultured cells of the human colorectal cancer pericytes, the immortalized cell monoclonal strain has subcellular structure characteristics of large nuclear proportion and few organelles.
  • Example 13 Detection of tendency effects of immortalized cell monoclonal strain to endothelial cell lumen by three-dimensional lumen adhesion assay.
  • results the results of the three-dimensional lumen adhesion assay are shown in Figure 12.
  • the immortalized cell monoclonal strain has a tendency to attach to the lumen of HUVEC. It can be seen that the immortalized cell strain constructed by the present invention retains the same biological characteristics of chemotaxis to the lumen of endothelial cells as the primary pericytes.

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Abstract

Provided are a new kind of tumor pericytes, an isolation method thereof and use thereof, which belongs to the field of biotechnology. The method comprises stripping tumor vessels from fresh solid tumor tissue samples, cutting the tumor vessels into annular fragments, placing the annular fragments of the tumor vessels in a pericyte culture medium pre-coated with matrigel for conditioned culture, and recycling tumor pericytes from the matrigel. The TPCs highly expressed positive pericytes molecular markers including FAPα, NG2, PDGFRβ and CD146, but not expressed negative pericytes molecular markers including CD31 and MYH11.

Description

Tumor Pericytes, isolation method therefor and use thereof Technical Field
The present invention belongs to the field of biotechnology and particularly relates to tumor pericytes, an isolation method therefor and use thereof.
Background
Tumor pericytes (TPCs) are parietal cells that are located on the periphery of tumor blood vessels and embedded in the basement membrane, which plays a vital role in tumor angiogenesis, maturation and stability, tumor invasion and metastasis, and tolerance of tumor anti-angiogenesis therapy (1-3) . The regulatory effect of TPCs on tumor progression has become a research focus in recent years. At present, one of the biggest obstacles in the field of TPCs research is the lack of TPCs-specific molecular markers, which not only affects the identification of pericytes but also limits the isolation and sorting of TPCs. Firstly, unlike other types of cells in tumor tissue, TPCs still lack of specific molecular markers. Most studies generally use markers such as NG2, CD146, PDGFRβ, CD13 and αSMA to indicate TPCs (4-7) . Based on this, a current method of isolating pericytes from normal tissues or tumor tissues mainly labels the pericytes using one or more of molecular markers (e.g., NG2, CD146, PDGFRβ, CD13, and αSMA) , which are highly expressed in pericytes, and then isolates pericytes by magnetic beads sorting or flow sorting (8-10) . After searching the patent database, it was found that Chinese patent application CN201611078356.2 (Application Publication Number: CN106589124A; Title of Invention: use of CD146 Monoclonal Antibody in Detection and Isolation of Glioma pericytes) and Chinese patent application CN201810550545.8 (Application Publication Number: CN108715836A; Title of Invention: Isolation and bionic culture of pericytes in tumor tissue) respectively disclosed the use of CD146 single antibodies or CD13, CD140b, CD146, NG2, and αSMA multiple antibodies to label cells and isolate them using flow cytometry assay for TPCs However, molecular markers such as NG2, CD146, PDGFRβ, CD13, and αSMA are not only expressed in TPCs, but also in stromal cells such as tumor-associated fibroblasts, smooth muscle cells, and mesenchymal stem cells (11-13) . Therefore, the pericytes obtained by magnetic beads sorting or flow sorting based on these  molecular markers may contain miscellaneous cells such as fibroblasts and mesenchymal stem cells at the same time, which have the disadvantage of insufficient purity. Moreover, due to the heterogeneity of tumor tissue, the expression of molecular markers of TPCs in different regions may be different. Therefore, by the sorting method using one or several of the molecular markers, only a single type of TPCs can be obtained, and the heterogeneity of TPCs cannot be fully reflected. In addition, due to the lack of effective isolation and culture methods of TPCs, in vitro studies of pericytes usually use methods such as co-incubation of human brain vascular pericytes (HBVPs) with tumor cells or HBVPs transfected with FAPα plasmid to simulate TPCs (14, 15) . However, because of the great difference between cerebral blood vessels and tumor blood vessels in physiological functions and characteristics, the use of tumor cells to educate HBVPs cannot reflect the biological functions of real TPCs. In view of the above situation, it is urgent to provide a new, simple and efficient method for the isolation and culture of TPCs from a new perspective, for enriching TPCs with high purity, high activity and high heterogeneity.
References
1. Ferland-McCollough D, Slater S, Richard J, Reni C, and Mangialardi G. Pericytes, an overlooked player in vascular pathobiology. Pharmacol Ther. 2017; 171: 30-42.
2. Paiva AE, Lousado L, Guerra DAP, Azevedo PO, Sena IFG, Andreotti JP, et al. Pericytes in the Premetastatic Niche. Cancer Res. 2018; 78 (11) : 2779-86.
3. Raza A, Franklin MJ, and Dudek AZ. Pericytes and vessel maturation during tumor angiogenesis and metastasis. Am J Hematol. 2010; 85 (8) : 593-8.
4. Proebstl D, Voisin MB, Woodfin A, Whiteford J, D'Acquisto F, Jones GE, et al. Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. J Exp Med. 2012; 209 (6) : 1219-34.
5. Cooke VG, LeBleu VS, Keskin D, Khan Z, O'Connell JT, Teng Y, et al. Pericyte depletion results in hypoxia-associated epithelial-to-mesenchymal transition and metastasis mediated by met signaling pathway. Cancer Cell. 2012; 21 (1) : 66-81.
6. Hong J, Tobin NP, Rundqvist H, Li T, Lavergne M, Garcia-Ibanez Y, et al. Role of Tumor Pericytes in the Recruitment of Myeloid-Derived Suppressor Cells. J Natl Cancer Inst. 2015; 107 (10) .
7. Murgai M, Ju W, Eason M, Kline J, Beury DW, Kaczanowska S, et al. KLF4-dependent pericytes cell plasticity mediates pre-metastatic niche formation and metastasis. Nat Med.  2017; 23 (10) : 1176-90.
8. EE C, and F D. FACS isolation of endothelial cells and pericytes from mouse brain microregions. Nature protocols. 2018; 13 (4) : 738-51.
9. K Y, EA S, ME O, A N, S R, H H, et al. Loss of Endothelium-Derived Wnt5a Is Associated With Reduced Pericyte Recruitment and Small Vessel Loss in Pulmonary Arterial Hypertension. Circulation. 2019; 139 (14) : 1710-24.
10. Yuan SM, Guo Y, Zhou XJ, Shen WM, Chen HN. PDGFR-β (+) pericytes from infantile hemangioma display the features of mesenchymal stem cells and show stronger adipogenic potential in vitro and in vivo. Int J Clin Exp Pathol. 2014; 7 (6) : 2861-70.
11. Primac I, Maquoi E, Blacher S, Heljasvaara R, Van Deun J, Smeland HY, et al. Stromal integrin alpha11 regulates PDGFR-beta signaling and promotes breast cancer progression. J Clin Invest. 2019; 130.
12. A K, SS DS, OV M, H T, B W, J Z, et al. Specification and Diversification of Pericytes and Smooth Muscle Cells from Mesenchymoangioblasts. Cell reports. 2017; 19 (9) : 1902-16.
13. LE dS, TM M, S KH, and DT C. Mesenchymal Stem Cells and Pericytes: To What Extent Are They Related? Stem cells and development. 2016; 25 (24) : 1843-52.
14. Franco M, Roswall P, Cortez E, Hanahan D, and Pietras K. Pericytes promote endothelial cell survival through induction of autocrine VEGF-A signaling and Bcl-w expression. Blood. 2011; 118 (10) : 2906-17.
15. Chen M, Lei X, Shi C, Huang M, Li X, Wu B, et al. Pericyte-targeting prodrug overcomes tumor resistance to vascular disrupting agents. J Clin Invest. 2017; 127 (10) : 3689-701.
Summary of invention
A primary purpose of the present invention is to overcome the current limitations of the separation methods of TPCs by using magnetic bead sorting or flow sorting, and to provide a new method for isolating tumor pericytes. This method provides sufficient numbers, reliable sources, and stable quality of TPCs for basic medicine, biological research and drug development research.
Another object of the present invention is to provide TPCs. The present invention for example uses human colorectal cancer blood vessels to isolate and identify TPCs, and other types of solid malignant TPCs obtained by the method described in the present invention are also within the protection scope of the present invention.
The present invention also provides a use of the abovementioned TPCs.
The present invention mainly achieves the foregoing objects through the following technical solutions:
The method for isolating TPCs comprises stripping tumor vessels from fresh solid tumor tissue samples, cutting the tumor vessels into annular fragments, placing the annular fragments of the tumor vessels in a pericyte culture medium pre-coated with matrigel for conditioned culture, and recycling tumor pericytes from the matrigel.
The solid tumor may be liver cancer, colorectal cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer or gastric cancer; and preferably, the solid tumor is colorectal cancer.
The solid tumor tissue samples are stored in DMEM cell culture medium containing 1% (v/v) penicillin-streptomycin (PS) .
With informed consent and medical ethics approval, the abovementioned tumor specimens containing cancerous and adjacent tissues without large area necrosis are obtained from patients who underwent surgery for solid malignant tumor.
Before preservation, the abovementioned tumor samples are washed by DMEM medium containing 1% (v/v) PS to remove contaminants such as residual feces and blood stains.
Before being used, the abovementioned tumor samples are preferably rinsed with phosphate buffer solution containing 1% (v/v) PS pre-cooled at 4℃ until there is no blood color.
As a preferred embodiment, the abovementioned tumor blood vessels stripped from the tumor samples are cut into circular segments under the guidance of a stereo microscope.
Before stripping the abovementioned tumor blood vessels, it is preferable to trim and remove the fat tissue attached to the outer diameter of the blood vessel. This process is preferably performed under the guidance of a stereo microscope.
A diameter of the blood vessel is preferably 30-100μm.
A length of the circular segment of blood vessel is preferably 4-6 mm.
As a preferred embodiment, a preparation method of the matrigel comprises: diluting the matrigel by a DMEM medium pre-cooled at 4℃ at a volume ratio of 2: 1 to obtain a diluted matrigel solution, and adding PDGF-BB to the diluted matrigel solution at a final concentration of 0.5%～2% (w/v) ; More preferably, PDGF-BB at a final concentration of 1% (w/v) was added to the diluted matrigel solution.
As a preferred embodiment, 100μL of the abovementioned matrigel is added to each well of  a 24-well plate.
As a preferred embodiment, the matrigel is incubated at 37 ℃ for 30～40 min in an incubator before being used.
As a preferred embodiment, the pericyte culture medium is a basal medium comprising 2%v/v fetal bovine serum, 1%v/v pericyte growth supplement (PGS) , 100U/mL penicillin and 100μg/mL streptomycin.
As a preferred embodiment, the conditioned culture is conducted at a condition of 37 ℃, 5%CO 2 and 95%humidity for 13-15 days in an incubator.
The recycling comprises: rinsing the matrigel by a phosphate buffer solution, centrifuging to obtain a precipitate and a supernatant, removing the supernatant, and adding a dispase solution to the precipitate for digestion, and stopping the digestion.
The matrigel is centrifuged at 1000-2000g for 10-20 min, and more preferably at 1500g for 15 min.
A concentration of the dispase solution is 1U/mL.
The digestion is conducted at 37 ℃ for 2-3 h in the incubator.
The digestion is stopped by an EDTA solution, which preferably has a concentration of 10 mM.
A volume ratio of the dispase solution and the EDTA solution is 1: 4.
All of the isolation process is operated under aseptic conditions in a clean bench.
TPCs were obtained by the above method.
The TPCs have the following biological characteristics: high expression of four positive molecular markers including FAPα, NG2, PDGFRβ and CD146, and no expression of two negative molecular markers including CD31 and MYH11; a subcellular structure with large nuclear proportion and few organelles; and chemotaxis and adhesion to endothelial cells.
The TPCs may be used in tumor-related biological research and/or preparation of anti-tumor drugs.
The TPCs can be used in the construction of immortalized tumor pericytes.
A construction method for immortalized tumor pericytes comprises preparing tumor pericytes by the above method for isolating tumor pericytes, and obtaining the immortalized tumor pericytes by lentiviral transfection of the tumor pericytes with SV40T antigen.
Immortalized tumor pericytes are obtained by the construction method.
A method for constructing immortalized tumor pericytes monoclonal strains comprises  cloning and culturing positive clones of the immortalized tumor pericytes by a limiting dilution method, so as to obtain the immortalized tumor pericytes monoclonal strains.
The immortalized tumor pericytes monoclonal strains are obtained by the construction method.
The present invention has the following beneficial effects as compared with those in the prior art:
(1) The present invention is the first to obtain tumor pericytes by a method comprising stripping tumor vessels and conditioned culture. Compared with a single type of tumor pericytes obtained by magnetic beads sorting or flow sorting based on one or more of molecular markers such as CD13, αSMA, CD146, NG2, PDGFRβ, the isolation method of TPCs described in the present invention is easier to operate and has lower cost; the types of TPCs obtained by the isolation method of the present invention are more comprehensive, which truly reflect the heterogeneity of TPCs.
(2) The TPCs obtained by the present invention are derived from surgical specimens of human colorectal cancer, which truly reflects the biological characteristics of tumor blood vessels. Therefore, the human colorectal cancer pericytes obtained by the present invention are superior to the simulated TPCs obtained previously by co-incubation of normal human cerebral pericytes or bovine retinal pericytes with tumor cells in vitro.
(3) The present invention is the first to observe the subcellular structure characteristics of the obtained primary colorectal cancer pericytes and immortalized cell monoclonal strain thereof by transmission electron microscopy, which provides a new reference standard for the identification of human colorectal cancer pericytes.
(4) The present invention is the first to infect primary colorectal cancer pericytes with SV40T lentivirus, which is a zero breakthrough in the construction of immortalized human colorectal cancer pericytes model.
(5) The present invention is the first to pick out an immortalized colorectal cancer pericyte monoclonal strain, which may serve as a reliable raw material with sufficient quantity and stable quality for studying the biological and medical of human colorectal cancer pericytes.
Brief Description of the Drawings
Figure 1 shows a process of isolating human colorectal cancer pericytes.
Figure 2 is shows the identification of human colorectal cancer pericytes by flow cytometry.
Figure 3 shows the identification of human colorectal cancer pericytes by immunofluorescence.
Figure 4 shows the identification of human colorectal cancer pericytes by transmission electron microscopy.
Figure 5 is the result of a 3D lumen adhesion assay of HUVEC and human colorectal cancer pericytes, wherein the PKH26 labeled HUVEC is in red fluorescence and the PKH67 labeled human colorectal cancer pericytes is in green fluorescence.
Figure 6 shows the structure of pGMLV-SV40T lentiviral plasmid.
Figure 7 is the detection result of the expression of SV40T gene by PCR, wherein, lane 1 is Marker, lane 2 is human colorectal cancer pericytes, lane 3 is immortalized human colorectal cancer pericytes (ihTPC) , and lane 4 is immortalized human colorectal cancer pericytes monoclonal strains (ihTPC-1) .
Figure 8 shows the proliferation activity of different generations of immortalized human colorectal cancer pericytes (ihTPC) by CCK8 assay.
Figure 9 shows the identification of immortalized human colorectal cancer pericytes monoclonal strains by flow cytometry.
Figure 10 shows the identification of immortalized human colorectal cancer pericytes monoclonal strains by immunofluorescence.
Figure 11 shows the identification of immortalized human colorectal cancer pericytes monoclonal strains by transmission electron microscopy.
Figure 12 is the result of a 3D lumen adhesion assay of HUVEC and immortalized human colorectal cancer pericyte monoclonal strains (ihTPC-1) , wherein, the PKH26 labeled HUVEC is in red fluorescence and the PKH67 labeled immortalized human colorectal cancer pericyte monoclonal strain0 is in green fluorescence.
Detailed Embodiments
The present invention will be further described in detail below in combination with the examples, but the embodiments of the present invention are not limited thereto.
Example 1 Dissection of blood vessels from human colorectal cancer samples, isolation and culture of colorectal cancer pericytes
Experimental method: with informed consent and medical ethics approval, human colorectal cancer specimens containing cancerous and adjacent tissues collected from Department of Gastrointestinal Surgery, Guangzhou Overseas Chinese Hospital were obtained from patients  who have been diagnosed with malignant colorectal cancer by clinical imaging, serum carcinoembryonic protein test, biopsy and other technical means. Referring to the process shown in Figure 1, colorectal cancer pericytes were obtained. To be more specific, the obtained specimens were washed with DMEM medium containing 1%v/v PS to remove contaminants such as residual feces and blood stains and then preserved in the DMEM medium. In a clean bench, the human colorectal cancer specimens were placed on a sterile dissection dish containing ice-cold PBS after being washed with 1%v/v PS-containing PBS (1×, PH=7.4) until no blood color was seen, and then under a stereo microscope colorectal mucosa tissues were located to remove the surrounding tissues of submucosal blood vessels by micro-anatomical spring scissor. Subsequently, the submucosal blood vessels with a diameter of fewer than 100μm near the mucosal were cut and placed on an ice-cold conditioned medium. To obtain a smear solution, matrigel (matrigel, 
Figure PCTCN2020130723-appb-000001
Cat. No. 354248) was dilutedby an ice-cold DMEM blank medium at a 2: 1 ratio to prepare a diluted matrigel solution and PDGF-BB (w/v) at a final concentration of 1 w/v%was added to the diluted matrigel solution. Then, the obtained smear solution was added to a 24-well plate at an amount of 100μL/well, and the plate was placed in an incubator at 37℃ for 30 min. The blood vessels were cut into a vascular ring with a length of about 5 mm and were added to 1 mL of complete medium of pericytes (PM, 
Figure PCTCN2020130723-appb-000002
Cat. No. 1201, a basal medium containing 2 v/v%fetal bovine serum, 1 v/v%PGS, 100U/mL of penicillin, and 100μg/mL of streptomycin) followed by adding the incubate vascular rings to the 24-well plate pre-coated with matrigel. The cells were incubated at 37℃ with 5%CO 2 for 14days, after which colorectal cancer vascular cells grew outward and close to the matrigel. Next, the matrigel-coated cells were collected by centrifugation at 1500×g for 5 min after washing with PBS, and the matrigel was then digested with 200μL of 1 U/mL dispase solution (
Figure PCTCN2020130723-appb-000003
Cat. No. 07923) at 37 ℃ for 2 h in an incubator, followed by stopping the digestion with 800μL of EDTA (10 mM) . Finally, the obtained cells were transferred to a 6-well plate after washing with PBS for three times and were continued to culture routinely.
Results: tumor vessels with a diameter of 30-100μm were obtained; about 3×10 6 of human colorectal cancer vascular cells were collected, which can be used for subsequent culture and identification.
Example 2 Identification of the expression of molecular markers of colorectal cancer pericytes by flow cytometry
Experimental method: the human colorectal cancer vascular cells obtained from Example 1 were re-suspended with 100μL of staining buffer. 100μL of cell suspension was then transferred into a 1.5 mL EP tube and 1 μL of Anti-CD32-PE blocking (Miltenyi
Figure PCTCN2020130723-appb-000004
Cat. No. 130-097-521) prototype control solution was added to the tube and incubated on ice for 10 min. Subsequently, the cells were incubated on ice for 30-60 min after addition of antibodies as follows: anti-FAPα-PE (R&D
Figure PCTCN2020130723-appb-000005
Cat. No. FAB3715P) , anti-NG2-PE (Miltenyi
Figure PCTCN2020130723-appb-000006
Cat. No. 130-097-458) , anti-PDGFRβ-PE (Miltenyi
Figure PCTCN2020130723-appb-000007
Cat. No. 130-105-323) , anti-CD146-PE (Miltenyi
Figure PCTCN2020130723-appb-000008
Cat. No. 130-097-939) , anti-CD31-PE (Miltenyi
Figure PCTCN2020130723-appb-000009
Cat. No. 130-110-807) and MYH11 (
Figure PCTCN2020130723-appb-000010
Cat. No. PA5-82526) . Following with washing the cells twice with PBS, the cells were further stained with 1μg/mL DAPI for cell nuclear staining on ice for 10 min. The cells were then washed twice with PBS and re-suspended in 400μL of staining buffer, which were used for flow cytometry analysis on a BD FACS Canto II flow cytometer and the expression level of antibodies was processed using FlowJo VX.
Results: the results of the flow cytometry analysis are shown in Figure 2. The primary colorectal cancer pericytes highly expressed positive pericytes molecular markers including FAPα, NG2, PDGFRβ and CD146, but not expressed negative pericytes molecular markers including CD31 and MYH11.
Example 3 Identification of the expression of molecular markers of human colorectal cancer pericytes by immunofluorescence assay
Experimental method: the human colorectal cancer vascular cells obtained from Example 1 were re-suspended, and the obtained cell suspension was seeded on a laser confocal dish at a density of 1×10 5 cells per dish. 24 h later, the cells were washed with PBS after removing the culture supernatant, fixed with 4%paraformaldehyde in 0.1 M PBS (pH=7.4) for 30 min, permeabilized with 0.1%Triton-X100 for 3 min and then incubated with 5%BSA for 1 hour at room temperature, followed by incubation with primary antibodies overnight at 4℃. The primary antibodies includes: anti-FAPα-PE (R&D
Figure PCTCN2020130723-appb-000011
Cat. No. FAB3715P) , anti-NG2-PE (Miltenyi
Figure PCTCN2020130723-appb-000012
Cat. No. 130-097-458) , anti-PDGFRβ-PE (Miltenyi
Figure PCTCN2020130723-appb-000013
Cat. No. 130-105-323) , anti-CD146-PE (Miltenyi
Figure PCTCN2020130723-appb-000014
Cat. No. 130-097-939) , anti-CD31-PE (Miltenyi
Figure PCTCN2020130723-appb-000015
Cat. No. 130-110-807) and MYH11 (
Figure PCTCN2020130723-appb-000016
Cat. No. PA5-82526) . After one day, the cells were washed with PBS for 3 times, each time for 10 min, followed by staining with Alexa Fluor dye-conjugated secondary antibodies at room temperature for 60 min and  incubation with DAPI for 3 min. Finally, the cells were subsequently observed and imaged under a Zeiss LSM 800 confocal laser scanning microscope.
Results: the results of the confocal laser scanning microscope are shown in Figure 3. The primary colorectal cancer pericytes highly expressed positive pericytes molecular markers including FAPα, NG2, PDGFRβand CD146, but not expressed negative pericytes molecular markers including CD31 and MYH11.
Example 4 Identification of subcellular characteristic of colorectal cancer pericytes by transmission electron microscopy
Experimental method: the human colorectal cancer pericytes obtained from example 1 at a logarithmic growth phase were trypsinized with 0.25%Tyrisin, centrifuged at 800×rpm for 5 min, transferred into an EP tube and washed twice with PBS. In order to fix the cells, 3%glutaraldehyde (ALFAAESAR, USA) was added to the tube overnight at 4℃ and 1%osmium tetroxide (
Figure PCTCN2020130723-appb-000017
Cat. No. 18459) was added to the tube for 30 min at 4℃ after washing with PBS for 3 times, each time for 10 min. Then, the cells were successively dehydrated for 2 times, each time for 10 min, by ethanol solution with a concentration of 30%, 50%, 70%, 90%, 100%after washing with PBS for 3 times. After sucking out the ethanol, the obtained cell mass was transferred onto a capsule with an embedding agent at the bottom and then the capsule was placed in an oven for polymerization at 60 ℃ for 2 h after filling the capsule with the embedding agent; Finally, the embedded cells were processed for preparation of ultrathin (70～90 nm) sections by LEICA EM UC7 ultra-thin microtome, and after double staining with 2%uranyl acetate (
Figure PCTCN2020130723-appb-000018
Cat. No. SPI-02624) and 1%lead citrate (
Figure PCTCN2020130723-appb-000019
Cat. No. HD17800) , the ultrathin sections were observed under a Hitachi h-7650 transmission electron microscope.
Results: the transmission electron microscopy scope images are shown in Figure 4. The primary human colorectal cancer pericytes were characterized by large nuclei and few organelles.
Example 5 Detection of tendency effects of human colorectal cancer pericytes to endothelial cell lumen by 3D lumen adhesion assay.
Experimental method: 3D co-culture assay of HUVECs (
Figure PCTCN2020130723-appb-000020
Cat. No. 8000) and the human colorectal cancer pericytes obtained from Example 1 were performed to examine the biological characteristics of pericytes. Briefly, 20μL of matrigel was added to each well of a 96-well plate, which was then placed in an incubator at 37 ℃ for 30 min. Subsequently, HUVECs  digested with trypsin were collected, washed with PBS for 3 times and suspended with a low serum endothelial cell culture medium containing ECM (
Figure PCTCN2020130723-appb-000021
Cat. No. 1001) , 2%fetal bovine serum, 1%ECGS (Endothelial cell growth factor, 
Figure PCTCN2020130723-appb-000022
Cat. No. 1052) , 100 U/mL of penicillin and 100μg/mL of streptomycin. The obtained HUVECs were further labeled with PKH 26 (1: 1000, Sigma Aldrich, Cat. No. PKH26GL) for 3 min at room temperature, and the staining was blocked with sterile 5%BSA. After washing with PBS, the HUVECs were re-suspended with ECM, counted, and added to a 96-well plate pre-coated with matrigel at 3×10 4 cells/well, where the HUVECs began to form tube structure 2 h later. Subsequently, the obtained colorectal cancer pericytes were labeled with PKH67 (Sigma Aldrich, Cat. No. PKH67GL) according to the method described above, and then added to the matrigel-coated HUVECs at 2×10 4 cells per well. The tendency effects of human colorectal cancer pericytes to endothelial cell lumen was observed under a fluorescence microscopy at 0, 2 and4 h.
Results: the results of the 3D lumen adhesion assay are shown in Figure 5. The primary human colorectal cancer pericytes tended to attach to the lumen of HUVECs.
Example 6 Construction of SV40T lentivirus and infection of primary colorectal cancer pericytes and colorectal cancer pericytes monoclonal line
Experimental method: lentiviral vectors (pGMLV-SV40T, Genomeditech, Cat. No. GM-0220SV01) shown in Figure 6, which encoded simian virus 40 Large T Antigen, was mixed with Lipofectamine 2000 and lentiviral packaging plasmid VSVG/PMDL/REV at a ratio of 5: 3: 2. The mixture was co-transfected into HEK-293T cells, and virus supernatant was collected, centrifuged to remove impurities 24 h later. Subsequently, primary colorectal cancer pericytes were transferred to a 24-well plate at 1×10 5 cells/well, infected with virus at a MOI of 50 (a total of 5×10 6 viral particles) 6 h later, incubated at 37℃ with 5%CO 2 for 24 h in an incubator, and sub cultured after another 48 h with a replaced fresh medium. In a 96-well plate, the human colorectal cancer pericytes transfected with SV40T were diluted to 1 cell per 2 wells by limiting dilution. After 7-10 days of incubation, the positive cells with single-cell clone growth were selected to clone again, which was performed triple times, and finally immortalized human colorectal cancer pericyte monoclonal strain was obtained.
Example 7 Detection of expression level of SV40T in human colorectal cancer pericytes by RT-PCR
Experimental method: the total RNA of the human colorectal cancer pericytes, the human colorectal cancer pericytes transfected with SV40T and the human colorectal cancer pericytes monoclonal strains transfected with SV40T were extracted using a TRIzol method. PCR primers were designed according to the sequences of SV40T genes, the sequences being list below: forward strand: 5'-CCACAAAGGA-AAAAGCTGCACTGCTA-3'; reverse strand: 5'-TCAACAGCCTGTTGGCATATGGTTTT-3'. The reaction conditions were: 95℃ for 5 min, then 35 cycles of 95 ℃ for 30 s, 60℃ for 30 s and 72 ℃ for 30 s, followed by a terminal 10-min extension phase at 72 ℃ and permanent at 4 ℃. The expression level of SV40T was evaluated by gel electrophoresis.
Results: the results are shown in Figure 7. The expression level of SV40T in the human colorectal cancer pericytes (lane 1) , the human colorectal cancer pericytes transfected with SV40T (lane 2) and the human colorectal cancer pericytes monoclonal strains transfected with SV40T (lane 3) detected by RT-PCR and the SV40-LT-target band of about 550 bp observed in both  lane  2 and 3 indicate that the immortalized colorectal cancer vascular cells and its monoclonal strains were constructed successfully.
Example 8 Detection of proliferative responses of human colorectal cancer pericytes by proliferation assay
Experimental method: The 1 st and 10 th generation of the human colorectal cancer pericytes and the 1 st, 10 th and 25 th generation of immobilized human colorectal cancer pericytes were seeded in a 96-well plate at a cell density of 1×10 3 per well. After seeding for 1-7 days, the cell proliferation activity was examined by Cell Counting kit-8 (CCK-8) with the following step: the cell supernatant was replaced by 90μL of fresh conditioned medium and 10μL of CCK8 solution (Beyotime, C0038) , and the cells were placed in an incubator for 60 min. The absorbance was measured at 450 nm by a microplate reader (TECAN F500) , and the cells proliferation rate was calculated according to the absorbance.
Results: the results are shown in Figure 8. The proliferation activity of non-immortalized human colorectal cancer pericytes was decreased significantly from the 1 st generation to the 10 th generation, and the proliferation activity of the 1 st, 10 th and 25 th generation of the immobilized human colorectal cancer pericytes was similar to that of the 1 st generation colorectal cancer pericytes.
Example 9 Detection of tumorigenesis of immortalized human colorectal cancer pericytes monoclonal strain by tumorigenicity assay
Experimental method: the immortalized human colorectal cancer pericytes monoclonal strain at a logarithmic growth phase was collected and re-suspended with ice-cold matrigel. Cell suspensions at a density of 1×10 7 cells per mL were subcutaneously injected into the back of ten BALB/C nu/nu nude mice (half male and half female) at 0.2 mL/mouse. After a four-week feeding of the nude mice, the tumor formation situation of the immortalized human colorectal cancer pericytes monoclonal strain in these mice was observed and recorded.
Results: there was no tumor formed within 4 weeks of inoculation with immortalized human colorectal cancer pericytes monoclonal strain. The results suggested that the immortalized human colorectal cancer pericytes monoclonal strain constructed by the present invention was non-tumorigenic.
Example 10 Identification of the expression of molecular markers in immortalized cell monoclonal strain by flow cytometry.
Experimental method was performed as described in Example 2.
Results: the results of the flow cytometry experiment are shown in Figure 9. The results showed that, like the primary cultured cells of human colorectal cancer pericytes, the immortalized cell monoclonal strain highly expressed four pericyte positive molecular markers including FAPα, NG2, PDGFRβand CD146, but not expressed negative molecular markers including CD31 and MYH11. It can be seen that the immortalized cell strain constructed by the present invention still expressed the same molecular markers as the primary pericytes.
Example 11 Identification of the expression of molecular markers in immortalized cell monoclonal strain by immunofluorescence.
Experimental method was performed as described in Example 3.
Results: the results of the immunofluorescence experiment are shown in Figure 10. The results showed that, like the primary cultured cells of human colorectal cancer vascular pericytes, the immortalized cell monoclonal strain highly expressed four pericyte positive molecular markers including FAPα, NG2, PDGFRβand CD146, but not expressed negative molecular markers including CD31 and MYH11. It can be seen that the immortalized cell strain constructed by the present invention still expressed the same molecular markers as the primary pericytes.
Example 12 Identification of subcellular characteristic of immortalized cell monoclonal strain by transmission electron microscopy
Experimental method was performed as described in Example 4.
Results: the results of the transmission electron microscope are shown in Figure 11. Like the primary cultured cells of the human colorectal cancer pericytes, the immortalized cell monoclonal strain has subcellular structure characteristics of large nuclear proportion and few organelles.
Example 13 Detection of tendency effects of immortalized cell monoclonal strain to endothelial cell lumen by three-dimensional lumen adhesion assay.
Experimental method was performed as described in Example 5
Results: the results of the three-dimensional lumen adhesion assay are shown in Figure 12. Like the primary cultured cells of the human colorectal cancer pericytes, the immortalized cell monoclonal strain has a tendency to attach to the lumen of HUVEC. It can be seen that the immortalized cell strain constructed by the present invention retains the same biological characteristics of chemotaxis to the lumen of endothelial cells as the primary pericytes.
The abovementioned embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited thereto, any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention, which shall be the equivalent replacements, are all included within the scope of the present invention.
Figure PCTCN2020130723-appb-000023
Figure PCTCN2020130723-appb-000024

Claims (10)

  1. A method for isolating tumor pericytes, characterized in comprising stripping tumor vessels from fresh solid tumor tissue samples, cutting the tumor vessels into annular fragments, placing the annular fragments of the tumor vessels in a pericyte culture medium pre-coated with matrigel for conditioned culture, and recycling tumor pericytes from the matrigel.
  2. The method for isolating tumor pericytes according to claim 1, characterized in that,
    a diameter of the tumor vessel is 30-100μm; and
    a length of the annular fragments of the tumor vessels is 4-6mm.
  3. The method for isolating tumor pericytes according to claim 1, characterized in that,
    a preparation method of the matrigel comprises: diluting the matrigel by a DMEM medium pre-cooled at 4℃ at a volume ratio of 2: 1 to obtain a diluted matrigel solution, and adding PDGF-BB to the diluted matrigel solution at a final concentration of 0.5%～2% (w/v) ; and
    the pericyte culture medium is a basal medium comprising 2%v/v fetal bovine serum, 1%v/v pericyte growth supplement (PGS) , 100U/mL penicillin and 100μg/mL streptomycin.
  4. The method for isolating tumor pericytes according to claim 1, characterized in that,
    the conditioned culture is conducted at a condition of 37 ℃, 5%CO2 and 95%humidity for 13-15 days in an incubator;
    the recycling comprises: rinsing the matrigel by a phosphate buffer solution, centrifuging to obtain a precipitate and a supernatant, removing the supernatant, adding a dispase solution to the precipitate for digestion, and stopping the digestion;
    the matrigel is centrifuged at 1000-2000g for 10-20 min;
    a concentration of the dispase solution is 1U/mL;
    the digestion is conducted at 37 ℃ for 2-3 h in the incubator;
    the digestion is stopped by a EDTA solution with a concentration of 10 mM;
    a volume ratio of the dispase solution and the EDTA solution is 1: 4.
  5. The method for isolating tumor pericytes according to claim 1, characterized in that,
    the solid tumor is one of liver, colorectal, breast, cervical, prostate, glioma, melanoma, pancreatic, nasopharyngeal, lung or gastric cancers.
  6. Tumor pericytes, characterized in being prepared by the method for isolating tumor pericytes according to any one of claims 1-5.
  7. The tumor pericytes according to claim 6, characterized in that,
    the tumor pericytes have the following biological characteristics: high expression of four positive molecular markers including FAPα, NG2, PDGFRβ and CD146, and no expression of two  negative molecular markers including CD31 and MYH11; a subcellular structure with large nuclear proportion and few organelles; and chemotaxis and adhesion to endothelial cells.
  8. Use of the tumor pericytes according to claims 6 or 7 in tumor-related biological research and/or preparation of anti-tumor drugs.
  9. A construction method for immortalized tumor pericytes, characterized in preparing tumor pericytes by the method for isolating tumor pericytes according to any one of claims 1 to 5, and obtaining the immortalized tumor pericytes by lentiviral transfection of the tumor pericytes with SV40T antigen.
  10. Immortalized tumor pericytes, characterized in being prepared by the construction method for immortalized tumor pericytes according to claim 9.
PCT/CN2020/130723 2020-05-15 2020-11-23 Tumor pericytes, isolation method therefor and use thereof WO2021227435A1 (en)

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