Decellularized extracellular matrix as scaffold for cancer organoid cultures of colorectal peritoneal metastases

Luca Varinelli, Marcello Guaglio, Silvia Brich, Susanna Zanutto, Antonino Belfiore, Federica Zanardi, Fabio Iannelli, Amanda Oldani, Elisa Costa, Matteo Chighizola, Ewelina Lorenc, Simone P Minardi, Stefano Fortuzzi, Martina Filugelli, Giovanna Garzone, Federica Pisati, Manuela Vecchi, Giancarlo Pruneri, Shigeki Kusamura, Dario Baratti, Laura Cattaneo, Dario Parazzoli, Alessandro Podestà, Massimo Milione, Marcello Deraco, Marco A Pierotti, Manuela Gariboldi, Luca Varinelli, Marcello Guaglio, Silvia Brich, Susanna Zanutto, Antonino Belfiore, Federica Zanardi, Fabio Iannelli, Amanda Oldani, Elisa Costa, Matteo Chighizola, Ewelina Lorenc, Simone P Minardi, Stefano Fortuzzi, Martina Filugelli, Giovanna Garzone, Federica Pisati, Manuela Vecchi, Giancarlo Pruneri, Shigeki Kusamura, Dario Baratti, Laura Cattaneo, Dario Parazzoli, Alessandro Podestà, Massimo Milione, Marcello Deraco, Marco A Pierotti, Manuela Gariboldi

Abstract

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.

Keywords: ECM stiffness; colorectal cancer; decellularized extracellular matrix; engineered disease model; extracellular matrix (ECM); organoids; peritoneal metastasis.

© The Author(s) (2022). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.

Figures

Figure 1
Figure 1
Establishment and characterization of human PM-derived organoids. (A) Comparative histochemistry (HC) and IHC analysis of PM-derived organoids and their tissue of origin. Scale bar, 100 µm. Surgical samples and the derived C3 and C4 organoids (passage numbers: P10 and P14, respectively) were developed from patients S16-8598 and S17-3963 who underwent CRS–HIPEC for PM (Supplementary Table S1). (B) Quantitative analysis for the expression of CRC markers and Ki-67 in C3 and C4 organoids versus their corresponding tumor of origin. Positive cells were measured as the percentage of CK AE1/AE3+, CK20+, CK19+, CDX2+, and Ki-67+ cells on the total number of cells present in regions free of dead cells. Three high-magnification (40×) fields per experiment were counted. Data are shown as median ± standard deviation (SD) of CK AE1/AE3+, CK20+, CK19+, CDX2+, and Ki-67+ cells present in each FFPE sample. Cells were counted using Qupath software. The experiment was performed in triplicate. (C) Micrographs showing a glandular-like branched (left) and a spherical-like cohesive (right) organoid. Scale bar, 100 µm. (D) IHC analysis of organoids (top) and their tissue of origin (bottom), using LGR5 immunostaining. Scale bar, 100 µm. (E) Quantitative counts of the percentage of LGR5+ cells in C3 and C4 organoids versus their corresponding tumor of origin. Positive cells were measured as the percentage of LGR5+ cells on the total number of cells present in regions free of dead cells. Three high-magnification (40×) fields per experiment were counted, and data are presented as median ± SD of LGR5+ cells present in each FFPE sample. Cells were counted using Qupath software. The experiment was performed in triplicate. (F) Summary of cancer-related genes with acquired mutations in TDO with respect to their tumor of origin (red boxes). The percentage of similarity was reported. Passage numbers of the organoid lines were: C1, P11; C2, P13; C3, P10; C4, P14; C6, P10.
Figure 2
Figure 2
Establishment of 3D-dECM scaffolds from peritoneal cavity. (A) DNA quantification of normal and neoplastic peritoneal tissue samples before (fresh) and after the decellularization treatment (dECM). Student's t-test (***P < 0.001). (B) IF analysis of normal and neoplastic peritoneal samples before and after decellularization using the WGA (red) staining. The samples were counterstained with DAPI (blue). Scale bar, 100 µm. (C) IHC analysis of fresh peritoneum-derived tissues and the corresponding decellularized samples using H&E staining and vimentin, pan-Cytokeratin, and collagen-IV immunostaining. Scale bar, 200 µm. (D) Van Gienson, Masson's Trichrome, and Alcian–PAS staining on fresh and decellularized peritoneum-derived samples. Scale bar, 200 µm.
Figure 3
Figure 3
Morphological and mechanical properties of PM-derived 3D-dECMs. (A) Confocal and polarized light microscopy analysis of peritoneum-derived 3D-dECM samples. Confocal images (left panel) were obtained by scanning different areas of the samples (area ∼1 mm2), which were then assembled into a single mosaic figure. Scale bar, 200 µm (confocal images) and 50 µm (light microscopy images). (B) Topography analysis of peritoneum-derived 3D-dECMs. Phase, height, and peak force error images of both normal and neoplastic decellularized matrices are shown. Scale bar, 1 µm. (C) Quantification of collagen-IV and sGAG on fresh and decellularized peritoneal tissues. Student's t-test (***P < 0.001 and **P < 0.01). (D) Distribution of the YM values obtained for each patient and condition (normal and neoplastic). Violin-plots: each dot represents the median YM value extracted from a single measurement with Point and Shot made by ∼225 force curves. Pt, patient; KPa, kilo Pascal. (E) Statistical analysis results of the YM value for each patient and condition tested. The bars and error bars represent mean of the median YM values and effective SD of the mean. The percentages represent the relative stiffening of the neoplastic ECM. Student's t-test (**P < 0.01).
Figure 4
Figure 4
Peritoneum-derived 3D-dECM scaffolds support colonization, infiltration, and proliferation of PM-derived organoids, maintaining the stem cell pool. (A) H&E staining of decellularized matrices derived from normal (top) or neoplastic (bottom) peritoneum repopulated with PM-derived organoids (C1). Scale bar, 50 µm. The repopulation experiments were performed in triplicate. (B) Number of cells from PM-derived organoids grown on normal and neoplastic 3D-dECMs after 5, 12, and 21 days. Data are presented as median ± SD of three fields per experiment, counted using Qupath software. One-way ANOVA (***P < 0.001). (C) IF analysis of 3D-dECMs derived from normal (top) and neoplastic (bottom) peritoneum repopulated with organoids (C1) using Ki-67 (green) and collagen-IV (red) antibodies. The samples were counterstained with DAPI (blue). Scale bar, 20 µm. (D) Proliferation rate of organoids measured as the percentage of Ki-67+ cells present in fields devoid of dead cells. Data are presented as median ± SD of five fields per experiment (40× magnification), counted using Qpath software. One-way ANOVA (***P < 0.001). (E) IF analysis of 3D decellularized matrices derived from normal (top) and neoplastic (bottom) peritoneum repopulated with organoids (C1) using LGR5 (green) and collagen-IV (red) antibodies. The samples were counterstained with DAPI (blue). Scale bar, 20 µm. (F) Amount of stem cells in organoids, measured as the percentage of LGR5+ cells present in fields devoid of dead cells. Data are presented as median ± SD of five fields per experiment (40× magnification), counted using Qupath software. One-way ANOVA (**P < 0.01).
Figure 5
Figure 5
Ex vivo engineered PM lesions are comparable to PMs in vivo. (A) Percentage of YAP+ and TAZ+ cells in PM-derived TDO (C3). Data are presented as median ± SD of three fields per experiment (40× magnification), counted using Qupath software. (B) Comparative HC and IHC images of organoids (C1) versus their corresponding tumor of origin and the ex vivo engineered PM lesion. Samples were analyzed for the expression of CRC-specific markers and Ki-67. Scale bar, 100 µm. Images in the first two lanes were previously published (Bozzi et al., 2017). (C) Quantitative counts of the percentage of CRC marker-positive and Ki-67+ cells in C1 organoids versus their corresponding tumor of origin and the ex vivo engineered PM lesion. Data are presented as median ± SD of three fields per experiment, counted using Qupath software. One-way ANOVA did not show differences among the three groups. (D) HC comparison of PM surgical sample and neoplastic peritoneum-derived 3D-dECMs repopulated with PM-derived organoids (C1). Asterisks and arrows indicate the main morphological features. Scale bar, 20 µm.
Figure 6
Figure 6
Gene expression analysis of engineered PM lesions. (A) Percentages of upregulated and downregulated genes belonging to the Matrisome dataset. (B) Unsupervised hierarchical clustering of the organoids according to the expression of the top DEGs included in Naba Secreted Factors and in Naba Collagens categories. (C) FCs of genes belonging to the indicated gene sets among the top 100 deregulated genes. Gene ranks for relative FC are shown on the x-axis and the logFCs on the y-axis. (D) Box plots showing the expression of genes selected for their involvement in the indicated processes of the Naba Matrisome datasets. Median and interquartile range are displayed as horizontal lines. Black squares in the bottom panel indicate which category the genes belong to. (E) Expression of genes selected for their involvement in the indicated processes of GO biological process and KEGG databases. Median and interquartile range are displayed as horizontal lines. Black squares in the bottom panel indicate which category the genes belong to. (F) Expression of genes selected for their involvement in the indicated processes, using a selection of genes related to the following biological processes: cell–cell/cell–matrix interactions, extracellular matrix stiffness, and drug resistance. Median and interquartile range are displayed as horizontal lines. Black squares in the bottom panel indicate which category the genes belong to.
Figure 7
Figure 7
3D-dECM scaffolds decrease the efficacy of HIPEC treatments. (A) Dose–response curve of C1 and C3 organoids cultured in Matrigel and treated with MMC at different concentrations at 42.5°C for 1 h. (B) Immunoblots of C1 and C3 organoids treated with MMC at 3 µM and 10 µM, respectively. Vinculin was used as loading control. (C) IHC analysis of C1 and C3 organoids cultured in Matrigel and on neoplastic peritoneum-derived 3D-dECMs, after in vitro HIPEC treatments, using Ki-67 immunostaining. Scale bar, 50 µm.
Figure 8
Figure 8
3D-dECMs reduce HIPEC-induced apoptosis in TDO. (A) Proliferation rate of PM-derived organoids (C1, left panel; C3, right panel) measured as the percentage of Ki-67+ cells present in fields free of dead cells. Data are presented as median ± SD of five fields per experiment (40× magnification), counted using Qupath software. One-way ANOVA (**P < 0.01). (B) Percentage of apoptotic organoids (C1, left panel; C3, right panel) measured as the percentage of cCASPASE3+ cells present in selected fields. Data are presented as median ± SD of five fields per experiment (40× magnification), counted using Qupath software. One-way ANOVA (**P < 0.01). (C) IF analysis of C1 and C3 organoids cultured in Matrigel and on neoplastic peritoneum-derived 3D-dECMs, after HIPEC treatments, using cCASPASE3 antibody (green). The samples were counterstained with WGA (red) and DAPI (blue). Scale bar, 20 µm.

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