将胰腺导管腺癌重编程为多能性
1Centre for Regenerative Medicine, Institute of Regeneration and Repair, Institute of Stem Cell Research,The University of Edinburgh, 2King Abdulaziz City for Science and Technology Health Sector (KACST), 3Cancer Early Detection Advanced Research Center, Knight Cancer Institute,Oregon Health & Science University, 4Brenden-Colson Canter for Pancreatic Care,Oregon Health & Science University, 5Department of Surgery,Oregon Health & Science University, 6Department of Molecular & Medical Genetics,Oregon Health & Science University
Summary
本方案描述了胰腺导管腺癌（PDAC）和正常胰腺导管上皮细胞重编程为诱导多能干细胞（iPSC）。我们提供从制备慢病毒到建立稳定的iPSC细胞系的优化和详细的分步程序。
Abstract
使用转录因子生成诱导多能干细胞（iPSC）已经从几乎任何分化的细胞类型中实现，并被证明对研究和临床应用非常有价值。有趣的是，癌细胞（如胰腺导管腺癌 （PDAC））的 iPSC 重编程已被证明可以恢复侵袭性 PDAC 表型并覆盖癌症表观基因组。PDAC衍生的iPSCs的分化可以概括PDAC从其早期胰腺上皮内瘤变（PanIN）前体的进展，揭示PDAC进展早期发生的分子和细胞变化。因此，PDAC衍生的iPSC可用于对PDAC的早期阶段进行建模，以发现早期检测诊断标志物。这对于PDAC患者尤为重要，由于缺乏早期PanIN阶段的可靠生物标志物，他们通常在晚期转移阶段被诊断出来。然而，将癌细胞系（包括PDAC）重编程为多能性仍然具有挑战性、劳动密集型且不同细胞系之间差异很大。在这里，我们描述了一种更一致的方案，用于使用双反子慢病毒载体从各种人类PDAC细胞系中生成iPSC。所得的 iPSC 细胞系稳定，对重编程因子或诱导药物的外源表达没有依赖性。总体而言，该协议有助于生成广泛的PDAC衍生的iPSC，这对于发现更具特异性和代表性的PDAC病例的早期生物标志物至关重要。
Introduction
胰腺导管腺癌 （PDAC） 是最致命的恶性肿瘤之一，由于该疾病的无症状性质，早期诊断仍然具有挑战性。大多数PDAC患者在晚期转移期被诊断为晚期转移期，此时可用的治疗选择非常有限1,2。这主要是由于早期阶段缺乏可靠的生物标志物，例如那些可以方便地检测为释放到血液中的蛋白质的生物标志物。
PDAC可以在其进展的早期播散，当PDAC位于胰腺时，更好的预后与早期癌症检测有关3。然而，只有不到十分之一的PDAC患者被诊断为预后良好，允许手术切除。尽管如此，少数可切除肿瘤患者也容易在 12 个月内复发4。
在过去的五十年中，手术技术、患者护理和治疗方式取得了显着进步 5,6。然而，手术切除的PDAC患者的5年生存率仅上升至17%。尽管如此，这仍然优于未切除的患者，后者几乎保持不变 （0.9%）4,7。化疗是唯一的替代PDAC治疗。然而，这种选择非常有限，因为绝大多数 PDAC 患者对化疗药物（如吉西他滨 7,8）表现出强烈的耐药性。其他药物，如厄洛替尼，仅适用于一小部分具有特定突变的PDAC患者，其中大多数表现出厄洛替尼耐药性9。在大多数PDAC患者中，与化疗相关的不良副作用是这种治疗的另一个缺点10。最近，有前途的策略表明，免疫检查点抑制剂 （ICI） 和小分子激酶抑制剂 （SMKI） 可有效治疗 PDAC，但对这些靶向治疗的持久反应仍然仅限于少数患者11,12。总体而言，PDAC特异性早期生物标志物的发现可以为早期诊断和治疗铺平新的途径。
PDAC由非浸润性胰管上皮增生引起的胰腺上皮内肿瘤（pancreatic intraepithelial tumors， PanIN）前体病变发展而来13,14。虽然 PanIN 的形成是由癌基因突变（如 KRAS）启动的，但进展为 PDAC 需要额外的遗传和表观遗传改变。据预测，PanIN通过不同阶段发展为侵入性PDAC大约需要10年13,15,16,17。这个时间范围提供了一个很好的机会，可以从早期PDAC诊断中受益。因此，已经进行了广泛的研究，以建立肿瘤异种移植动物模型和类器官培养物来研究PDAC的进展18,19,20,21。这些模型对于研究PDAC的侵袭阶段非常有用，尽管不是从早期PanIN阶段的过渡。因此，开发能够概括PanIN阶段早期进展的实验模型非常重要，以便能够发现早期检测生物标志物。
使用四种转录因子 OCT4、SOX2、KLF4 和 c-MYC （OSKM） 将体细胞重编程为诱导多能干细胞 （iPSC） 说明了细胞可塑性的程度22。癌细胞的可塑性已被充分证明，将人类癌细胞重编程为 iPSC 已成功用于将细胞重置为其原始细胞状态，从而消除癌症进展过程中积累的许多表观遗传损伤 23,24,25,26,27,28,29.因此，使用这种重编程策略来操纵癌细胞身份的可能性在治疗癌症方面具有巨大的希望30,31。事实上，我们之前已经证明，来自PDAC的iPSC的分化可以概括PDAC在早期PanIN阶段的进展32。通过鉴定PDAC早期至中期特异性的基因和通路，确定了可用于临床早期PDAC诊断的候选生物标志物32,33。然而，使用单个 iPSC 细胞系发现的生物标志物在大多数 PDAC 患者中的覆盖率有限32。从其他PDAC患者中生成iPSC细胞系的挑战已经阻止了发现更可靠生物标志物的能力。这是由于许多技术因素造成的，包括OSKM递送的异质性，因为只有一小部分人原代PDAC细胞包含所有四种因子并成功响应重编程。本文介绍了使用更有效、更一致的双慢病毒递送OSKM对原代PDAC细胞进行重编程的详细方案。
Protocol
所有实验方案均已获得OHSU机构审查委员会的批准。所有方法均按照相关准则和规定进行。所有针对 PDX 肿瘤的动物工作均在 OHSU 机构动物使用和护理委员会 （IACUC） 的批准下进行。该方案在来自患者来源的异种移植物 （PDX） 的原代 PDAC 细胞、从 61 岁女性腺癌患者的胰腺组织中分离出的具有上皮形态的 BxPc3 细胞系、源自正常人胰管上皮的 H6C7 永生化上皮细胞系和源自健康个体皮肤活检的原代人?…
Representative Results
图 1 显示了来自 PDAC、BXPc3、H6C7 和 hFib 细胞的 iPSC 集落形态的代表性图像。PDAC-iPSC 菌落在重编程的第 25 天开始形成。在重编程的第 40 天鉴定出具有更成熟的 ESC 样形态的稳健 iPSC 集落（图 1）。同样，BxPc3-iPSCs的形成始于第23天，并在第35天变得更加成熟。H6C7-iPSC的形成与PDAC-iPSC相似，并在第45天开始建立。hFib-iPSC 集落在重编程…
Discussion
为了促进使用 iPSC 重编程来研究癌症进展，已经建立了一个用于重编程胰腺癌细胞的稳健方案。到目前为止，将癌细胞重编程为多能性已被证明非常具有挑战性，因为只有少数研究成功地从癌细胞中生成了iPSC 32、36、37、38、39、40、<sup class=…
Divulgations
The authors have nothing to disclose.
Acknowledgements
A.S 和 J.K 感谢英国癌症研究中心和 OHSU 的资助（CRUK-OHSU 项目奖 C65925/A26986）。A.S 获得 MRC 职业发展奖 （MR/N024028/1） 的支持。A.A.由阿卜杜勒阿齐兹国王科技城的博士奖学金（奖学金参考1078107040）资助。J.K 由 MRF 新研究者资助 （GCNCR1042A） 和 Knight CEDAR 资助 （68182-933-000， 68182-939-000）。我们感谢Keisuke Kaji教授提供重编程载体pSIN4-EF1a-O2S和pSIN4-CMV-K2M。出于开放获取的目的，作者已将知识共享署名 （CC BY） 许可应用于本提交的任何作者接受的手稿版本。
Materials
| 2-Mercaptoethanol (50 mM) | Thermo Fisher | 31350010 |
| Alexa Fluor 488 anti- human TRA-1-60-R | BioLegend | 330613 |
| Bovine Pituitary Extract (BPE) | Thermo Fisher | 13028014 |
| BxPc3 | ATCC | CRL-1687 |
| Cholera Toxin from Vibrio cholerae | Merck | C8052-1MG |
| Collagen, Type I solution from rat tail | Merck | C3867 |
| Completed Defined K-SFM | Thermo Fisher | 10744-019 |
| Corning Costar TC-Treated Multiple Well Plates | Merck | CLS3516 |
| Corning syringe filters | Merck | CLS431231 |
| Corning tissue-culture treated culture dishes | Merck | CLS430599 |
| Day Impex Virkon Disinfectant Virucidal Tablets | Thermo Fisher | 12328667 |
| Dulbecco′s Phosphate Buffered Saline (PBS) | Merck | D8537 |
| Fetal Calf Serum (FCS) | Thermo Fisher | 10270-106 |
| Fugene HD Transfection Reagent | Promega | E2312 |
| Gelatin solution, Type B, 2% in H2O | Merck | G1393-100ML |
| Glasgow Minimum Essential Media (GMEM) | Merck | G5154 |
| Human EGF Recombinant Protein | Thermo Fisher | PHG0311 |
| Human FGF-basic (FGF-2/bFGF) (154 aa) Recombinant Protein, PeproTech | Thermo Fisher | 100-18B |
| Human Pancreatic Duct Epithelial Cell Line (H6c7) | Kerafast | ECA001-FP |
| iMEF feeder cells | iXcells Biotechnologies | 10MU-001-1V |
| Keratinocyte Serum Free Media (KSFM) | Thermo Fisher | 17005-042 |
| KnockOut DMEM | Thermo Fisher | 10829018 |
| KnockOut serum Replacement | Thermo Fisher | 10828028 |
| L-Glutamine (200 mM) | Thermo Fisher | 25030-024 |
| MEM Non-Essential Amino Acids Solution (100x) | Thermo Fisher | 11140050 |
| Millex-HP 0.45 μM syringe Filter Unit (Sterile) | Merck | SLHP033RS |
| Opti-MEM Reduced Serum Medium | Thermo Fisher | 31985062 |
| pMDG | AddGene | 187440 |
| Polybrene (Hexadimethrine bromide) | Merck | H9268-5G |
| pSIN4-CMV-K2M | AddGene | 21164 |
| pSIN4-EF2-O2S | AddGene | 21162 |
| psPAX2 | AddGene | 12260 |
| pWPT-GFP | AddGene | 12255 |
| RPMI 1640 Medium (ATCC modification) | Thermo Fisher | A1049101 |
| Sodym Pyruvate | Thermo Fisher | 11360-039 |
| Sterile Syringes for Single Use (60 mL) | Thermo Fisher | 15899152 |
| TrypLE Express Enzyme (1x), phenol red | Thermo Fisher | 12605036 |
| UltraPure 0.5M EDTA, pH 8.0 | Thermo Fisher | 15575020 |
| Y-27632 (Dihydrochloride) | STEMCELL Technologies | 72304 |
References
- Rahib, L., et al. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Recherche en cancérologie. 74 (11), 2913-2921 (2014).
- Hu, J. X., et al. Pancreatic cancer: A review of epidemiology, trend, and risk factors. World Journal of Gastroenterology. 27 (27), 4298 (2021).
- Howlader, N., et al. SEER cancer statistics review, 1975-2013. National Cancer Institute. 19, (2016).
- Bengtsson, A., Andersson, R., Ansari, D. The actual 5-year survivors of pancreatic ductal adenocarcinoma based on real-world data. Scientific Reports. 10 (1), 16425 (2020).
- He, J., et al. 2564 resected periampullary adenocarcinomas at a single institution: trends over three decades. HPB. 16 (1), 83-90 (2014).
- Dusch, N., et al. Factors predicting long-term survival following pancreatic resection for ductal adenocarcinoma of the pancreas: 40 years of experience. Journal of Gastrointestinal Surgery. 18 (4), 674-681 (2014).
- Principe, D. R., et al. The current treatment paradigm for pancreatic ductal adenocarcinoma and barriers to therapeutic efficacy. Frontiers in Oncology. 11, 688377 (2021).
- Papademetrio, D. L., et al. Interplay between autophagy and apoptosis in pancreatic tumors in response to gemcitabine. Targeted Oncology. 9 (2), 123-134 (2014).
- Ng, S. S., Tsao, M. S., Nicklee, T., Hedley, D. W. Effects of the epidermal growth factor receptor inhibitor OSI-774, Tarceva, on downstream signaling pathways and apoptosis in human pancreatic adenocarcinoma 1 supported by the National Cancer Institute of Canada and the Pat Myhal Fund for Pancreatic Cancer Research. Molecular Cancer Therapeutics. 1 (10), 777-783 (2002).
- Sultana, A., et al. Meta-analyses of chemotherapy for locally advanced and metastatic pancreatic cancer. Journal of Clinical Oncology. 25 (18), 2607-2615 (2007).
- Sun, J., Russell, C. C., Scarlett, C. J., McCluskey, A. Small molecule inhibitors in pancreatic cancer. RSC Medicinal Chemistry. 11 (2), 164-183 (2020).
- Jiang, H., et al. Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nature Medicine. 22 (8), 851-860 (2016).
- Hruban, R. H., et al. Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions. The American Journal of Surgical Pathology. 25 (5), 579-586 (2001).
- Maitra, A., Hruban, R. H. Pancreatic cancer. Annual Review of Pathology: Mechanisms of Disease. 3, 157-188 (2008).
- Vincent, A., Herman, J., Schulick, R., Hruban, R. H., Goggins, M. Pancreatic cancer. The Lancet. 378 (9791), 607-620 (2011).
- Yachida, S., et al. Distant metastasis occurs late during the genetic evolution of pancreatic cancer. Nature. 467 (7319), 1114-1117 (2010).
- Lennon, A. M., et al. The early detection of pancreatic cancer: what will it take to diagnose and treat curable pancreatic neoplasia. Recherche en cancérologie. 74 (13), 3381-3389 (2014).
- Rubio-Viqueira, B., et al. An in vivo platform for translational drug development in pancreatic cancer. Clinical Cancer Research. 12 (15), 4652-4661 (2006).
- Li, C., et al. Identification of pancreatic cancer stem cells. Recherche en cancérologie. 67 (3), 1030-1037 (2007).
- Hermann, P. C., et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 1 (3), 313-323 (2007).
- Boj, S. F., et al. Organoid models of human and mouse ductal pancreatic cancer. Cell. 160 (1-2), 324-338 (2015).
- Takahashi, K., Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126 (4), 663-676 (2006).
- Suvà, M. L., et al. Reconstructing and reprogramming the tumor-propagating potential of glioblastoma stem-like cells. Cell. 157, 580-594 (2014).
- Kotini, A. G., et al. Stage-specific human induced pluripotent stem cells map the progression of myeloid transformation to transplantable leukemia. Cell Stem Cell. 20 (3), 315-328 (2017).
- Stricker, S. H., et al. Widespread resetting of DNA methylation in glioblastoma-initiating cells suppresses malignant cellular behavior in a lineage-dependent manner. Genes Development. 27 (6), 654-669 (2013).
- Chao, M. P., et al. Human AML-iPSCs reacquire leukemic properties after differentiation and model clonal variation of disease. Cell Stem Cell. 20 (3), 329-344 (2017).
- Aparicio, L. A., et al. Clinical implications of epithelial cell plasticity in cancer progression. Cancer letters. 366 (1), 1-10 (2015).
- Grimont, A., Leach, S. D., Chandwani, R. Uncertain beginnings: acinar and ductal cell plasticity in the development of pancreatic cancer. Cellular and Molecular Gastroenterology and Hepatology. 13 (2), 369-382 (2022).
- Greenspan, L. J., Weinstein, B. M. To be or not to be: endothelial cell plasticity in development, repair, and disease. Angiogenesis. 24 (2), 251-269 (2021).
- Papapetrou, E. P. Patient-derived induced pluripotent stem cells in cancer research and precision oncology. Nature Medicine. 22 (12), 1392-1401 (2016).
- Zhang, X., Cruz, F. D., Terry, M., Remotti, F., Matushansky, I. Terminal differentiation and loss of tumorigenicity of human cancers via pluripotency-based reprogramming. Oncogene. 32 (18), 2249-2260 (2013).
- Kim, J., et al. An iPSC line from human pancreatic ductal adenocarcinoma undergoes early to invasive stages of pancreatic cancer progression. Cell Reports. 3 (6), 2088-2099 (2013).
- Kim, J., et al. Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers. Science Translational Medicine. 9 (398), (2017).
- Susac, L., et al. Structure of a fully assembled tumor-specific T cell receptor ligated by pMHC. Cell. 185 (17), 3201-3213 (2022).
- Yu, J., et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 324, 797-801 (2009).
- Carette, J. E., et al. Generation of iPSCs from cultured human malignant cells. Blood, The Journal of the American Society of Hematology. 115 (20), 4039-4042 (2010).
- Choi, S. M., et al. Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells. Blood, The Journal of the American Society of Hematology. 118 (7), 1801-1805 (2011).
- Hochedlinger, K., et al. Reprogramming of a melanoma genome by nuclear transplantation. Genes & Development. 18 (15), 1875-1885 (2004).
- Hu, K., et al. Efficient generation of transgene-free induced pluripotent stem cells from normal and neoplastic bone marrow and cord blood mononuclear cells. Blood, The Journal of the American Society of Hematology. 117 (14), e109-e119 (2011).
- Iskender, B., Izgi, K., Canatan, H. Reprogramming bladder cancer cells for studying cancer initiation and progression. Tumor Biology. 37, 13237-13245 (2016).
- Khoshchehreh, R., et al. Epigenetic reprogramming of primary pancreatic cancer cells counteracts their in vivo tumourigenicity. Oncogene. 38 (34), 6226-6239 (2019).
- Kim, H. J., et al. Establishment of hepatocellular cancer induced pluripotent stem cells using a reprogramming technique. Gut and Liver. 11 (2), 261 (2017).
- Lin, S. L., et al. Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA. 14 (10), 2115-2124 (2008).
- Miyoshi, N., et al. Defined factors induce reprogramming of gastrointestinal cancer cells. Proceedings of the National Academy of Sciences. 107 (1), 40-45 (2010).
- Singovski, G., et al. In vivo epigenetic reprogramming of primary human colon cancer cells enhances metastases. Journal of Molecular Cell Biology. 8 (2), 157-173 (2016).
- Zhao, H., et al. A highly optimized protocol for reprogramming cancer cells to pluripotency using nonviral plasmid vectors. Cellular Reprogramming (Formerly" Cloning and Stem Cells". 17 (1), 7-18 (2015).
- Lo, C. A., et al. Quantification of protein levels in single living cells). Cell Reports. 13 (11), 2634-2644 (2015).
- Yu, J., et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 324 (5928), 797-801 (2009).
- Prösch, S., et al. Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro. Biological Chemistry Hoppe-Seyler. 377 (3), 195-201 (1996).
- Mehta, A. K., Majumdar, S. S., Alam, P., Gulati, N., Brahmachari, V. Epigenetic regulation of cytomegalovirus major immediate-early promoter activity in transgenic mice. Gene. 428 (1-2), 20-24 (2009).
- Chen, X., et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell. 133 (6), 1106-1117 (2008).
- Kim, J., Chu, J., Shen, X., Wang, J., Orkin, S. H. An extended transcriptional network for pluripotency of embryonic stem cells. Cell. 132 (6), 1049-1061 (2008).
- Chronis, C., et al. Cooperative binding of transcription factors orchestrates reprogramming. Cell. 168 (3), 442-459 (2017).
- Li, D., et al. Chromatin accessibility dynamics during iPSC reprogramming. Cell Stem Cell. 21 (6), 819-833 (2017).
- Soufi, A., Donahue, G., Zaret, K. S. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell. 151 (5), 994-1004 (2012).
- Carey, B. W., et al. Reprogramming of murine and human somatic cells using a single polycistronic vector. Proceedings of the National Academy of Sciences of the United States of America. 106, 157-162 (2009).
Citer Cet Article
Alshaikh, A., Grygoryev, D., Keith, D., Sheppard, B., Sears, R. C., Kim, J., Soufi, A. Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency. J. Vis. Exp. (204), e65811, doi:10.3791/65811 (2024).