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Breakthrough Moments: Organoid Models of Cancer

2019; Elsevier BV; Volume: 24; Issue: 6 Linguagem: Inglês

10.1016/j.stem.2019.05.006

1934-5909

April Chan, Helen H.N. Yan, Suet Yi Leung,

Cancer Genomics and Diagnostics

Five years ago, Li et al., 2014Li X. Nadauld L. Ootani A. Corney D.C. Pai R.K. Gevaert O. Cantrell M.A. Rack P.G. Neal J.T. Chan C.W. et al.Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.Nat. Med. 2014; 20: 769-777Crossref PubMed Scopus (282) Google Scholar and Gao et al., 2014Gao D. Vela I. Sboner A. Iaquinta P.J. Karthaus W.R. Gopalan A. Dowling C. Wanjala J.N. Undvall E.A. Arora V.K. et al.Organoid cultures derived from patients with advanced prostate cancer.Cell. 2014; 159: 176-187Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar reported the power and unique advantages of cancer organoids. We shine a spotlight on the platform's enormous potential for studying cancer biology and as a preclinical human tumor model that can facilitate drug discovery and personalized therapy. Five years ago, Li et al., 2014Li X. Nadauld L. Ootani A. Corney D.C. Pai R.K. Gevaert O. Cantrell M.A. Rack P.G. Neal J.T. Chan C.W. et al.Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.Nat. Med. 2014; 20: 769-777Crossref PubMed Scopus (282) Google Scholar and Gao et al., 2014Gao D. Vela I. Sboner A. Iaquinta P.J. Karthaus W.R. Gopalan A. Dowling C. Wanjala J.N. Undvall E.A. Arora V.K. et al.Organoid cultures derived from patients with advanced prostate cancer.Cell. 2014; 159: 176-187Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar reported the power and unique advantages of cancer organoids. We shine a spotlight on the platform's enormous potential for studying cancer biology and as a preclinical human tumor model that can facilitate drug discovery and personalized therapy. Until recently, traditional two-dimensional (2D) neoplastic cell lines have been a mainstay of cancer research. However, the culture medium for monolayer cell lines lacks the supporting niche factors for stem cell growth, making it difficult to establish cancer cell lines and severely restricting the molecular repertoire that can be captured. Moreover, normal cell lines or cell lines representing early neoplastic stages were also lacking. Then, in 2009, Sato et al. described the ability to indefinitely culture three-dimensional (3D) organoids in Matrigel from single Lgr5-positive intestinal stem cells under defined culture conditions artificially providing niche factors (R-spondin, EGF, and noggin for small intestines, subsequently adding Wnt for other organs) (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (4148) Google Scholar). In parallel, Ootani et al., 2009Ootani A. Li X. Sangiorgi E. Ho Q.T. Ueno H. Toda S. Sugihara H. Fujimoto K. Weissman I.L. Capecchi M.R. Kuo C.J. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.Nat. Med. 2009; 15: 701-706Crossref PubMed Scopus (617) Google Scholar developed an air-liquid interface (ALI) model with a type I collagen gel as the scaffold to support the growth of intestinal cells from minced tissues, but instead of exogenous growth factors serving as a supplement, residual stromal components from tissue fragments provided the essential niche factors. The next 5 years saw the establishment of numerous long-term organoid culture protocols of normal epithelial cells from an array of different organs, incorporating organ-specific niche factor combinations. Subsequently, in 2014, Gao et al. and Li et al. described major breakthroughs for the use of organoid models in cancer research. Gao et al. cultured organoids from human metastatic prostate tumors from diverse disease sites or circulating tumor cells (Gao et al., 2014Gao D. Vela I. Sboner A. Iaquinta P.J. Karthaus W.R. Gopalan A. Dowling C. Wanjala J.N. Undvall E.A. Arora V.K. et al.Organoid cultures derived from patients with advanced prostate cancer.Cell. 2014; 159: 176-187Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar) by optimizing their own novel protocol for culturing normal prostate epithelial cells (Karthaus et al., 2014Karthaus W.R. Iaquinta P.J. Drost J. Gracanin A. van Boxtel R. Wongvipat J. Dowling C.M. Gao D. Begthel H. Sachs N. et al.Identification of multipotent luminal progenitor cells in human prostate organoid cultures.Cell. 2014; 159: 163-175Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). They successfully generated seven novel organoid lines that harbored prostate-cancer-specific driver alterations, including ETS-translocations, CHD1 loss, and SPOP and FOXA1 mutations, of which the latter three had not been previously represented in 2D prostate cancer cell line models. This landmark study reported the first tumor organoid biobank capturing the molecular diversity of a solid tumor type. They performed comprehensive genomic analysis and documented that apart from driver mutations, the organoids highly recapitulated in vivo prostate cancers in copy number alterations, gene expression subtypes, and histological patterns. Moreover, they were able to engraft in immunodeficient mice to recapitulate the original histological patterns of the patients. Finally, the authors demonstrated that these lines were amenable to drug testing both in vitro and in vivo. Although their initial success rate was only 15%–20%, this study set the scene for numerous subsequent studies describing the generation of large biobank collections of patient-derived organoids from various organs, including primary colon, stomach, gastresophageal, pancreas, liver, breast, etc. (Drost and Clevers, 2018Drost J. Clevers H. Organoids in cancer research.Nat. Rev. Cancer. 2018; 18: 407-418Crossref PubMed Scopus (772) Google Scholar, Yan et al., 2018Yan H.H.N. Siu H.C. Law S. Ho S.L. Yue S.S.K. Tsui W.Y. Chan D. Chan A.S. Ma S. Lam K.O. et al.A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening.Cell Stem Cell. 2018; 23: 882-897.e11Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), with improved success rates ranging from 50%–100% in some organs through the incorporation of specific selection methods. All of these studies consistently illustrated that tumor organoids highly resemble the original tumor tissue and that they are genetically stable over long-term in vitro culture, making them a superior model for drug screening. Indeed, the feasibility of large-scale drug sensitivity screening directly using patient-derived tumor organoids in vitro or mouse xenografts in vivo has already been demonstrated across various tumor types. The initial ALI organoid cultures from the normal intestine, gastric, or pancreatic tissue of neonatal mice could be maintained for >30 days, but it could not be passaged. Li et al. then engineered ALI pancreatic organoids with combination driver mutations of known cancer genes (Li et al., 2014Li X. Nadauld L. Ootani A. Corney D.C. Pai R.K. Gevaert O. Cantrell M.A. Rack P.G. Neal J.T. Chan C.W. et al.Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.Nat. Med. 2014; 20: 769-777Crossref PubMed Scopus (282) Google Scholar). The authors collected organoids from mice bearing KrasG12D LSL and/or p53flox/flox alleles and infected them with adenovirus-expressing Cre-GFP. These organoids were able to proliferate, could be serially passaged, and formed tumors when transplanted subcutaneously. The authors also established a colorectal cancer progression model whereby tamoxifen-treated organoids derived from villin-CreER;Apcflox/flox mice were subsequently transduced with retroviruses expressing mutant Kras, p53 shRNA, and Smad4 shRNA. They observed progressive dysplasia with increasing number of mutations and transformation into cribriform growth reminiscent of in vivo cancers with those four mutations. Subcutaneous xenotranplantation of the quadruple mutant organoids resulted in tumors with poorly differentiated adenocarcinoma morphology, whereas organoids with only Apc loss could not form tumors. Although the strategy of RNA interference and mutant cDNA overexpression by Li et al. does not produce permanent genetic alterations, this study nonetheless established the framework and potential for using normal organoid cultures to model stepwise oncogenic transformation. Shortly thereafter, a study using Crispr-Cas9 to engineer the same four mutations in pure human epithelial intestinal organoids in 3D Matrigel also generated adenocarcinoma in vitro and in vivo (Drost et al., 2015Drost J. van Jaarsveld R.H. Ponsioen B. Zimberlin C. van Boxtel R. Buijs A. Sachs N. Overmeer R.M. Offerhaus G.J. Begthel H. et al.Sequential cancer mutations in cultured human intestinal stem cells.Nature. 2015; 521: 43-47Crossref PubMed Scopus (665) Google Scholar). This enabled follow-up orthotopic transplantation studies that made it possible to examine metastatic potential in a physiological environment. The potential utility of both models continues to evolve to encompass the study of the microenvironment and immune regulation. The group of Li et al. has recently enhanced their ALI organoid model by including niche factor supplementation, which preserves the stromal fibroblasts and a diversity of immune cell types, including the original tumor T cell spectrum, macrophages, and natural killer cells (Neal et al., 2018Neal J.T. Li X. Zhu J. Giangarra V. Grzeskowiak C.L. Ju J. Liu I.H. Chiou S.-H. Salahudeen A.A. Smith A.R. et al.Organoid Modeling of the Tumor Immune Microenvironment.Cell. 2018; 175: 1972-1988.e1916Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). Although the culture cannot go beyond 60 days, it allows a window of opportunity in which to study the effects of immune-modulatory drugs such as anti-PD1 and anti-PDL1. As an alternative approach, peripheral blood lymphocytes (PBMCs) can be artificially co-cultured with autologous pure epithelial tumor organoids to generate tumor-reactive T cells, thus establishing a platform to assess the efficiency of T cell-mediated tumor killing (Dijkstra et al., 2018Dijkstra K.K. Cattaneo C.M. Weeber F. Chalabi M. van de Haar J. Fanchi L.F. Slagter M. van der Velden D.L. Kaing S. Kelderman S. et al.Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids.Cell. 2018; 174: 1586-1598.e1512Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). The continuous development of these platforms will have huge potential applications in cancer research. The ALI model, which conveniently uses one culture medium for all tumor types, requires a shorter time to establish and has a higher success rate, but it is short-term in nature. This ALI model may be further exploited for individual patient drug selection in a precision oncology setting. The Matrigel method with pure epithelial organoids, which has the capability for long-term culture and can be scaled up for large-scale drug screening, offers other unique opportunities for cancer cell biology studies and drug testing. The pure tumor epithelial culture allows a clear delineation of the cancer cell transcriptome, DNA copy number changes, and biallelic inactivation of tumor suppressors, which is often difficult to define in the tumor tissue that is often confounded by normal contamination. Furthermore, the organoids retain their reliance on niche factors, thus offering a clean functional assay of the integrity of key oncogenic signaling pathways (e.g., Wnt, BMP, and EGF), potentially facilitating the discovery of new mechanisms causing perturbation to these pathways. Additionally, the consistent retention of the EBV viral episomes in gastric cancer organoids (Yan et al., 2018Yan H.H.N. Siu H.C. Law S. Ho S.L. Yue S.S.K. Tsui W.Y. Chan D. Chan A.S. Ma S. Lam K.O. et al.A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening.Cell Stem Cell. 2018; 23: 882-897.e11Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) illustrates the possibility for the study of tumor-viral interaction. Recently, pure tumor epithelial cultures have also been demonstrated to capture intra-tumoral heterogeneity of oncogenic amplifications common in chromosome-unstable cancers (Yan et al., 2018Yan H.H.N. Siu H.C. Law S. Ho S.L. Yue S.S.K. Tsui W.Y. Chan D. Chan A.S. Ma S. Lam K.O. et al.A Comprehensive Human Gastric Cancer Organoid Biobank Captures Tumor Subtype Heterogeneity and Enables Therapeutic Screening.Cell Stem Cell. 2018; 23: 882-897.e11Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), potentially enabling the study of paracrine interactions between tumor subpopulations. Yet, many challenges still remain, but continuous improvement of the platform is in progress, such as the availability of better synthetic 3D matrices and highly potent Wnt surrogates, along with optimized methods to favor growth of cancerous instead of normal organoids. Overall, organoid technology holds great promise to completely change the way we study and treat cancer, and future efforts will surely bring this novel technique closer to clinical practice. Organoid Cultures Derived from Patients with Advanced Prostate CancerGao et al.CellSeptember 4, 2014In BriefOrganoids derived from patients with prostate cancer recapitulate the phenotypic and molecular diversity of the disease, providing a valuable resource for mechanistic and therapeutic explorations. Full-Text PDF Open Archive