Cancer associated fibroblasts promote growth of primarily cultured pancreatic ductal adenocarcinoma cells <i>in vitro</i> and tumor formation in patient-derived tumor xenograft model_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

CHEN Yonghua ¹ , LI Cheng ² , LIANG Yan ³ , ZHOU Li ³ , YANG Zhen ³ ,  LIU Xubao ¹

1. Department of Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu 610041, P. R. China;
2. Rehabilitation Medicine Center, West China Hospital, Sichuan University, Chengdu 610041, P. R. China;
3. Core Facility of West China Hospital, Sichuan University, Chengdu 610041, P. R. China;

Corresponding author：

LIU Xubao, Email: xbliu@medmail.com.cn

Keywords：

cancer associated fibroblasts; pancreatic ductal adenocarcinoma; primary culture; patient-derived tumor xenograft

DOI：

10.7507/1007-9424.201907116

Video：

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Objective To optimize the culture method of human primary pancreatic ductal adenocarcinoma (PDAC) cells and cancer associated fibroblasts (CAFs) and investigate the effect of CAFs on the growth of primary PDAC cells in vitro and tumor formation in patient-derived xenograft (PDX) model.Methods The PDAC specimens were collected and primarily cultured. In order to observe the effect of CAFs on the growth of primary PDAC cells in vitro, the CAFs were co-cultured with primary PDAC cells consistently and the alone cultured primary PDAC cells served as the control. Then, these cells were injected into the shoulder blades of NOG mice in order to develop the PDX model.Results When the primary PDAC cells separated from the CAFs, the proliferation capacity of the primary PDAC decreased rapidly in the passage culture in vitro, and the most cells were terminated within 5 generations. By contrast, when the CAFs co-cultured with the primary PDAC cells, the proliferation capacity of primary PDAC cells were preserved, which could be stably transferred to at least 10 generations. The tumors of NOG mice were detected during 2–3 weeks after injecting the mixed cells (primary PDAC plus CAFs), while had no tumor formation after injecting CAFs alone. The rate of tumor was 92.9% (13 cases) in the primary PDAC plus CAFs group, which was higher than that of the CAFs alone group (64.3%, 9 cases), but there was no statistical difference because of the small sample size. The volume of tumor in the primary PDAC plus CAFs group at 2, 4, 6, and 8 weeks after the tumor cells injection was significantly larger than that in the CAFs alone group at the corresponding time point, the differences were statistically significant (P<0.01).Conclusions The CAFs could promote the growth of primary PDAC cells in vitro. This new method of co-culture CAFs with primary PDAC could improve the success rate of primary PDAC cells culture and improve the success rate of PDX model in NOG mice.

Citation： CHEN Yonghua, LI Cheng, LIANG Yan, ZHOU Li, YANG Zhen, LIU Xubao. Cancer associated fibroblasts promote growth of primarily cultured pancreatic ductal adenocarcinoma cells in vitro and tumor formation in patient-derived tumor xenograft model. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2020, 27(3): 278-283. doi: 10.7507/1007-9424.201907116 Copy

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28.	Hohwieler M, Müller M, Frappart PO, et al. Pancreatic progenitors and organoids as a prerequisite to model pancreatic diseases and cancer. Stem Cells Int, 2019, 2019: 9301382.
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32.	Nicolle R, Blum Y, Marisa L, et al. Pancreatic adenocarcinoma therapeutic targets revealed by tumor-stroma cross-talk analyses in patient-derived xenografts. Cell Rep, 2017, 21(9): 2458-2470.
33.	Huang L, Holtzinger A, Jagan I, et al. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids. Nat Med, 2015, 21(11): 1364-1371.
34.	董丽华, 傅士龙, 韩素萍. 人上皮性卵巢癌组织中肿瘤相关成纤维细胞的培养. 南京医科大学学报(自然科学版), 2011, 31(7): 935-939.
35.	彭巍, 徐小慧, 季江, 等. 大肠癌肿瘤相关成纤维细胞原代培养方法的优化. 中华实验外科杂志, 2013, 30(5): 954-957.

1. Borazanci E, Dang CV, Robey RW, et al. Pancreatic cancer: “A Riddle Wrapped in a Mystery inside an Enigma”. Clin Cancer Res, 2017, 23(7): 1629-1637.
2. Kamisawa T, Wood LD, Itoi T, et al. Pancreatic cancer. Lancet, 2016, 388(10039): 73-85.
3. Raufi AG, Manji GA, Chabot JA, et al. Neoadjuvant treatment for pancreatic cancer. Semin Oncol, 2019, 46(1): 19-27.
4. Singhi AD, Koay EJ, Chari ST, et al. Early detection of pancreatic cancer: opportunities and challenges. Gastroenterology, 2019, 156(7): 2024-2040.
5. Strobel O, Neoptolemos J, Jäger D, et al. Optimizing the outcomes of pancreatic cancer surgery. Nat Rev Clin Oncol, 2019, 16(1): 11-26.
6. Kleger A, Perkhofer L, Seufferlein T. Smarter drugs emerging in pancreatic cancer therapy. Ann Oncol, 2014, 25(7): 1260-1270.
7. Unger FT, Witte I, David KA. Prediction of individual response to anticancer therapy: historical and future perspectives. Cell Mol Life Sci, 2015, 72(4): 729-757.
8. No authors listed. China plans large center for PDX models. Cancer Discov, 2014, 4(2): 136-137.
9. Hwang CI, Boj SF, Clevers H, et al. Preclinical models of pancreatic ductal adenocarcinoma. J Pathol, 2016, 238(2): 197-204.
10. Aparicio S, Hidalgo M, Kung AL. Examining the utility of patient-derived xenograft mouse models. Nat Rev Cancer, 2015, 15(5): 311-316.
11. Ehrenberg KR, Gao J, Oppel F, et al. Systematic generation of patient-derived tumor models in pancreatic cancer. Cells, 2019, 8(2): pii: E142.
12. 宋书铮, 俞继卫. 癌相关成纤维细胞及其在胃肠道肿瘤演进中的作用. 中国普外基础与临床杂志, 2014, 21(10): 1311-1315.
13. Richards KE, Zeleniak AE, Fishel ML, et al. Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells. Oncogene, 2017, 36(13): 1770-1778.
14. Hessmann E, Patzak MS, Klein L, et al. Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer. Gut, 2018, 67(3): 497-507.
15. von Ahrens D, Bhagat TD, Nagrath D, et al. The role of stromal cancer-associated fibroblasts in pancreatic cancer. J Hematol Oncol, 2017, 10(1): 76.
16. Thomas D, Radhakrishnan P. Tumor-stromal crosstalk in pancreatic cancer and tissue fibrosis. Mol Cancer, 2019, 18(1): 14.
17. Lee J, Yakubov B, Ivan C, et al. Tissue transglutaminase activates cancer-associated fibroblasts and contributes to gemcitabine resistance in pancreatic cancer. Neoplasia, 2016, 18(11): 689-698.
18. Murata T, Mekada E, Hoffman RM. Reconstitution of a metastatic-resistant tumor microenvironment with cancer-associated fibroblasts enables metastasis. Cell Cycle, 2017, 16(6): 533-535.
19. Paulson AS, Tran Cao HS, Tempero MA, et al. Therapeutic advances in pancreatic cancer. Gastroenterology, 2013, 144(6): 1316-1326.
20. Henriksen A, Dyhl-Polk A, Chen I, et al. Checkpoint inhibitors in pancreatic cancer. Cancer Treat Rev, 2019, 78: 17-30.
21. Gao D, Chen Y. Organoid development in cancer genome discovery. Curr Opin Genet Dev, 2015, 30: 42-48.
22. Cantrell MA, Kuo CJ. Organoid modeling for cancer precision medicine. Genome Med, 2015, 7(1): 32.
23. Knudsen ES, Balaji U, Mannakee B, et al. Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility. Gut, 2018, 67(3): 508-520.
24. Gao D, Vela I, Sboner A, et al. Organoid cultures derived from patients with advanced prostate cancer. Cel, 2014, 159(1): 176-187.
25. Boj SF, Hwang CI, Baker LA, et al. Organoid models of human and mouse ductal pancreatic cancer. Cell, 2015, 160(1-2): 324-338.
26. Unger C, Kramer N, Walzl A, et al. Modeling human carcinomas: physiologically relevant 3D models to improve anti-cancer drug development. Adv Drug Deliv Rev, 2014, 79-80: 50-67.
27. 李程, 陈拥华, 麦刚, 等. 精准医疗时代胰腺癌个体化类器官研究现状与应用前景. 中国普外基础与临床杂志, 2016, 23(10): 1272-1275.
28. Hohwieler M, Müller M, Frappart PO, et al. Pancreatic progenitors and organoids as a prerequisite to model pancreatic diseases and cancer. Stem Cells Int, 2019, 2019: 9301382.
29. Clevers H, Bender E. Q&A: Hans Clevers. Banking on organoids. Nature, 2015, 521(7551): S15.
30. D’Agosto S, Andreani S, Scarpa A, et al. Preclinical modelling of PDA: Is organoid the new black? Int J Mol Sci, 2019, 20(11): pii: E2766.
31. Vennin C, Murphy KJ, Morton JP, et al. Reshaping the tumor stroma for treatment of pancreatic cancer. Gastroenterology, 2018, 154(4): 820-838.
32. Nicolle R, Blum Y, Marisa L, et al. Pancreatic adenocarcinoma therapeutic targets revealed by tumor-stroma cross-talk analyses in patient-derived xenografts. Cell Rep, 2017, 21(9): 2458-2470.
33. Huang L, Holtzinger A, Jagan I, et al. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids. Nat Med, 2015, 21(11): 1364-1371.
34. 董丽华, 傅士龙, 韩素萍. 人上皮性卵巢癌组织中肿瘤相关成纤维细胞的培养. 南京医科大学学报(自然科学版), 2011, 31(7): 935-939.
35. 彭巍, 徐小慧, 季江, 等. 大肠癌肿瘤相关成纤维细胞原代培养方法的优化. 中华实验外科杂志, 2013, 30(5): 954-957.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Cancer associated fibroblasts promote growth of primarily cultured pancreatic ductal adenocarcinoma cells in vitro and tumor formation in patient-derived tumor xenograft model

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