Abstract
Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia. We identified 38 candidate invasion-driver genes, 17 of which, including TCF7L2, TWIST2, MSH2, DCC, EPHB1 and EPHB2 have been previously implicated in colorectal cancer progression. Six invasion-driver genes that have not, to our knowledge, been previously described were validated in vitro using cell proliferation, migration and invasion assays and ex vivo using recellularized human colon. These results demonstrate the utility of our organoid model for studying cancer biology.
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Acknowledgements
We thank the members of M.L.S.'s laboratory and N.A.J. and N.G.C.'s laboratory. We also thank H.E. Varmus for providing laboratory resources and helpful discussions. This work was supported by a US National Cancer Institute Physical Sciences-Oncology Center (NCI-PSOC) Young Investigator trans-network grant to H.J.C. and Z.W., US National Institutes of Health (NIH) grant UH2TR000516 to M.L.S., US National Science Foundation (NSF) grants NSF-1106153 to M.L.S. and NSF GRFP-2011131053 to H.J.C., NIH grant R01GM095990 to X.S., an Arnold O. Beckman Postdoctoral fellowship to H.J.C., a Welch Foundation grant C-0627 at Rice University to Y.M. and the Cancer Prevention Research Institute of Texas (CPRIT) (N.G.C. and N.A.J.).
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H.J.C., Z.W., R.S., N.G.C., N.A.J. and M.L.S. conceived the concept, designed the experiments and wrote the manuscript; H.J.C., Z.W., A.B., D.J.S., P.B., L.W., Y. M., S.A.C., S.C., E.H. and L.C.-G. performed the experiments and data analyses; J.S., Z.W., S.M.L., X.S., N.G.C. and N.A.J. contributed to bioinformatics analyses.
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Supplementary Figure 1 Generation and characterization of primary human colonic epithelial cells (hCECs).
(A) Representative images of hCECs grown in 2D cultures or matrigel 3D cultures. In matrigel cultures, individual hCECs progressively formed organiod-like structures (left to right). Scale bars, 100µ (left panel) and 50µ (right panel) in 2D culture; 100µ in 3D culture. White arrows denote micro-crypt domains formed with the multicellular cyst-like structure, which is the typical behavior of physiologically active intestinal stem cells grown in 3D (B) Cells expressing the stem cell marker Lgr5, epithelial markers cytokeratin 18 and 20, tight junction protein zonula occludens-1 (ZO-1) in condition of co-culture with colonic fibroblasts, colon epithelial cell-specific marker A33, and differentiation markers villin (absorptive cells), mucin 2 (goblet cells) and chromogranin A (enteroendocrine cells), were detected by immunostaining of hCECs in 2D culture. Scale bars, 50µ; blue nuclei, DAPI. (C) Quantification of Lgr5+ cells and functionally differentiated cells in hCECs. Error bars indicate S.E.M. n= 3 independent cultures.
Supplementary Figure 2 The procedure of recellularization.
The acellular colon matrix was physically separated into (A) mucosal (20x and 40x in big and small windows, respectively) and (B) submucosal layers. White arrow highlights intact epithelial crypt niches in the mucosal layer. Scale bars, 1mm. (C) Endothelial cells are shown being loaded into an injection needle and (D) being injected into the mucosa layer. Scale bars, 200 µ. The acellular colon matrix (E) was populated with colonic epithelial cells, endothelial cells and myofibroblasts. (F) A colon after recellularization for 6 weeks. Scale bars, 2 mm. (See Supplementary Video 1, 2 and 3 for detailed procedures.)
Supplementary Figure 3 Characterization of human colonic myofibroblasts and microvascular endothelial cells.
(A) Primary cultured colonic myofibroblasts in light microscope (left) or immunostained with anti-human α smooth muscle actin (right). Scale bars, 100µ (left) and 20µ (right). Purple nuclei, DAPI; More than 90% cells were positively stained with α smooth muscle actin; (B) colonic microvascular endothelial cells in light microscope (left) or immunostained with anti- human CD31 (right). Scale bars, 100 µ (left) and 20 µ (right). Blue nuclei, DAPI. More than 90% cells were positively stained with CD31. n= 3 independent cultures.
Supplementary Figure 4 Electron microscopic characterization of ex vivo colon mucosa.
(A) Scanning electron micrographs of mucosa surface, lamina propria and muscularis mucosa show that crypt niches were retained and intestinal fibers were well preserved in composition and ultrastructure during the de-/re-cellularization process. White arrows denote crypt niches; characteristic weaves (W), coils (C) and struts (S) of colonic fibers of laminin and collagen are present in both decellularized and recellularized matrices. Scale bars, 20 µM. (B) Transmission electron micrographs reveal the continuous intactness of muscularis mucosa (MM) layers in both decellularized matrix and recellularized tissue. |<->| designates the location and thickness of MM layers that were repopulated with fibroblasts (<-) in recellularized colon. Features: collagen fibers (\/); lamina filaments (<-). M, mucosa area; SM, submucosal area; scale bars, 500 nM (300 nM in small windows). All of the recellularized tissues have been cultured for 6-7 weeks when they are processed for electron microscopy. (C) No statistical differences in MM thickness were observed among native, recellularized and recellularized colon tissues. Error bars indicate S.E.M. (n= 6 of independent matrix; difference analysis by one-way ANOVA).
Supplementary Figure 5 Molecular characterization of physiologically active crypts recellularized with hCECs.
Immunostaining of the recellularized crypts showed that they contain cells (yellow denoted by white arrows) double positive for the intestinal stem cell marker Lgr5 (red) and proliferative marker Ki67 (green) (top panel) in addition to differentiated colon epithelial cells (Muc1 positive cells, transit amplifying cells), goblet cells (Muc2 positive cells), and enteroendocrine cells (chromogranin A-positive cells) (bottom panels). All the recellularized tissues had been cultured for 4 weeks before they were terminated from culture and prepared for immunostaining. Blue nuclei, DAPI; Scale bars, 20 µM. n= 3 independent cultures.
Supplementary Figure 6 Molecular characterization of recellularized mucosa.
(A) WNT/β-catenin signaling was restored in recellularized mucosa, detected by immunostaining of β-catenin (green). (B) Immunostaining of proliferative marker Ki67 (yellow) revealed the growth dynamic patterns of recellularized crypts. Blue nuclei, DAPI; scale bars, 20 µM. (C) ELISA experiments were performed to measure MUC2 protein levels. Substantially higher MUC2 levels were observed in the culture medium of recellularized colon compared with those in the medium of hCEC cells or decellularized colon matrices alone (student-t test), indicating the recellularized colon’s physiological function of mucous secretion. All of the recellularized tissues or hCEC cells had been cultured for 4 weeks before they were terminated from culture and prepared for immunostaining or MUC2 quantification. n = 5 independent matrices.
Supplementary Figure 7 Quantification of APC and KRASG12D expression in hCECs.
The psi-LVRH1GP (CMV-H1-APC shRNA-SV40-KRASG12D - neomycin) lentivirus, containing both an APC shRNA and a human KRASG12D expression construct, was transduced into hCEC cells. Protein expression levels of APC and a Myc-DDK tag for KRASG12D in hCEC cells were quantified by Western blotting (+). hCEC cells transduced with the backbone vector served used the control (-). β-actin is loading control.
Supplementary Figure 8 Tumor formation in mice injected with APC-KRASG12D hCECs.
2x106 matrigel-embedded parental or APC-KRASG12D hCECs were subcutaneously injected into each flank of 6-8 week old NSG mice (NOD/Shi-scid/IL-2Rγnull mice). The TGF-β signaling pathway was then blocked through the intraperitoneal injection of a TGF-β receptor inhibitor (TGF-β RI Inhibitor III), beginning at two weeks after injection (100mg/kg) with continuing injections three times per week. Six weeks after injection of APC-KRASG12D hCECs, tumor nodules (>250mm3) were visible at 60-80% of the injection sites, while injection of parental hCECs did not induce any tumors. (A) A representative tumor is shown following H+E staining. Scale bars, 100µ. (B) Quantification of tumor formation following injection of APC-KRASG12D hCECs and parental hCECs. ** P=0.0002, comparison in tumor formation by Fisher’s exact test.
Supplementary Figure 9 Creation of a modified SB transposition system for use in human tissues.
(A) Transposition activity was quantified using a fixed dose of transposon-donor plasmid (500 ng T2/Onc1) and varying doses of SB100X transposase plasmid; error bars indicate S.E.M. The activity of transposase reached its peak at a dose of 100ng. n = 5 independent experiments. (B) Average transposon copy numbers in individual hCEC colonies.
Supplementary Figure 10 Functional validation of ten candidate invasion-driver genes using the ex vivo human colon model.
shRNAs against 10 candidate driver genes that were functionally validated in in vitro cell proliferation, invasion and migration assays were used to downregulated expression of these genes in APC shRNA-expressing hCECs. These cells where then used to recellularize acellular colon matrices and the effects of this downregulated expression on neoplastic cell invasion measured (n= 4). Seven candidate genes, including LATS2, significantly promoted submucosa invasion in the ex vivo human colon model (A) Representative H+E-stained image (left panel) and dual immunostained image of cytokeratin and fibronectin (right panel) from an ex vivo colon recellularized with hCECs expressing shRNA against APC and CAMTA1. Scale bars, 100µ in left panel and 20µ in right panel. (B). Quantification of invasive neoplasia formation in ex vivo CRC models. *P<0.05 compared to APC shRNA-expressing hCECs transfected with control vector by 2-sided student t test. All of the recellularized tissues had been cultured for 7 weeks when they were terminated for quantification of tumorigenesis. n = 3 independent matrices.
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Chen, H., Wei, Z., Sun, J. et al. A recellularized human colon model identifies cancer driver genes. Nat Biotechnol 34, 845–851 (2016). https://doi.org/10.1038/nbt.3586
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DOI: https://doi.org/10.1038/nbt.3586
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