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Patient-derived mini-colons enable long-term modeling of tumor–microenvironment complexity

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Abstract

Existing organoid models fall short of fully capturing the complexity of cancer because they lack sufficient multicellular diversity, tissue-level organization, biological durability and experimental flexibility. Thus, many multifactorial cancer processes, especially those involving the tumor microenvironment, are difficult to study ex vivo. To overcome these limitations, we herein implemented tissue-engineering and microfabrication technologies to develop topobiologically complex, patient-specific cancer avatars. Focusing on colorectal cancer, we generated miniature tissues consisting of long-lived gut-shaped human colon epithelia (‘mini-colons’) that stably integrate cancer cells and their native tumor microenvironment in a format optimized for real-time, high-resolution evaluation of cellular dynamics. We demonstrate the potential of this system through several applications: a comprehensive evaluation of drug effectivity, toxicity and resistance in anticancer therapies; the discovery of a mechanism triggered by cancer-associated fibroblasts that drives cancer invasion; and the identification of immunomodulatory interactions among different components of the tumor microenvironment. Similar approaches should be feasible for diverse tumor types.

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Fig. 1: CRC cells recapitulate patient-specific tumor dynamics in mini-colons.
Fig. 2: CRC mini-colons allow the integrated assessment of drug efficacy and toxicity ex vivo.
Fig. 3: CAFs rewire the CRC transcriptome boosting EMT and invasiveness in mini-colons.
Fig. 4: CAF-induced CRC invasiveness is mediated by an autocrine proinflammatory program that promotes metalloproteinase activity.
Fig. 5: Mini-colons can recreate immunomodulatory interactions among CRC, CAFs and TILs.

Data availability

The RNA-Seq data generated in this work were deposited to the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under accession code GSE226723. Other publicly available data can be found at MSigDB (https://www.gsea-msigdb.org/gsea/msigdb/), cBioPortal (TCGA data, https://www.cbioportal.org) and the CRC–TME atlas (https://crc-tme.com). Source data are provided with this paper.

Code availability

All code used for data analysis is available from GitHub (https://github.com/LorenzoLF/Mini-colon_bioengineering) and Zenodo (https://zenodo.org/records/11103096)41.

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Acknowledgements

We thank O. Mitrofanova, B. Elçi and Y. Tinguely for assistance on microdevice fabrication, D. Dutta and S. Li for inputs on organoid work and J. Prébandier for administrative assistance. We acknowledge support from the following EPFL core facilities: CMi, PTBTG, HCF, BIOP, FCCF, BSF and GECF. This work was funded by the Swiss Cancer Research foundation (KFS-5103-08-2020), the Personalized Health and Related Technologies Initiative from the Swiss Federal Institutes of Technology Board and EPFL.

Author information

Authors and Affiliations

  1. Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences and School of Engineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    L. Francisco Lorenzo-Martín, Nicolas Broguiere, Jakob Langer, Lucie Tillard & Matthias P. Lutolf

  2. Institute of Human Biology (IHB), Roche Pharma Research and Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland

    Mikhail Nikolaev & Matthias P. Lutolf

  3. Department of Oncology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

    George Coukos & Krisztian Homicsko

  4. Ludwig Institute Branch at the University of Lausanne, Lausanne, Switzerland

    George Coukos & Krisztian Homicsko

  5. Swiss Cancer Center Leman, Lausanne, Switzerland

    George Coukos & Krisztian Homicsko

  6. Agora Translational Research Center, Lausanne, Switzerland

    George Coukos & Krisztian Homicsko

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  1. L. Francisco Lorenzo-Martín
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  5. Mikhail Nikolaev
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Contributions

L.F.L.-M. conceptualized the study, designed experiments, carried out the experimental and bioinformatic work, analyzed the data, performed artwork design and wrote the manuscript. N.B. carried out the isolation of CRC and TME cells. J.L. produced the microfluidic devices, optimized hydrogel patterning and generated mini-colon histological sections. L.T. carried out experimental work. M.N. designed and developed the first minigut system. G.C. helped to conceptualize the work. K.H. provided the colorectal tumor biopsies and histological sections. M.P.L. conceptualized the work, designed experiments and carried out the final editing of the manuscript.

Corresponding authors

Correspondence to L. Francisco Lorenzo-Martín or Matthias P. Lutolf.

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Competing interests

The EPFL has filed for patent protection (EP16199677.2, PCT/EP2017/079651 and US20190367872A1) on the scaffold-guided organoid technology used here and M.P.L. and M.N. are named as inventors on those patents. M.P.L. is a shareholder in Doppl SA, which is commercializing those patents. The other authors declare no competing interests.

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Peer review information

Nature Biotechnology thanks Eduard Batlle, Toshiro Sato and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 CRC mini-colons generate topobiologically complex patient-specific tumor avatars.

a, Dimensions (in μm) of the micropatterned mini-colon geometry used in this work. b, Brightfield and fluorescence images of a tumor-bearing CRC mini-colon (patient #NW) showing the presence of nuclei (cyan), cancer cells (green), and MKI67 (Ki-67)+ cells (magenta). A close-up is shown in Fig. 1d. Scale bar, 75 μm. c, Hematoxylin-eosin stained sections of a mini-colon with emergent tumors (arrowheads, patient #NW). The zoomed-in area (bottom) is indicated with a dashed rectangle (top). Scale bars, 100 μm (top) and 35 μm (bottom). d, Heatmap showing the main pathogenic mutations (red cells) found in the indicated patients. e, Size of tumors emerged in mini-colons seeded with CRC cells from the indicated patients. P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 3 for each patient). Data represent mean ± SEM. f, Brightfield images of CRC-only mini-colons and conventional organoids of the indicated patients. Scale bars, 100 μm. g, Hematoxylin-eosin stained sections of CRC tumors in vivo (left) and the corresponding CRC mini-colons generated from cells isolated from their respective biopsies (right). Scale bars, 100 μm.

Source data

Extended Data Fig. 2 CRC cells outcompete the healthy epithelium and expand throughout the mini-colon.

a, Brightfield time-course images of a healthy mini-colon without cancer cells. Scale bar, 100 μm. b, Fluorescence time-course images of a mini-colon bearing patient #NW CRC cells. Red and green fluorescence signal indicate healthy and cancer cells, respectively. Scale bar, 100 μm. c, Brightfield time-course images of conventional organoid cultures generated from the same mix of CRC and healthy colon cells that is used for the establishment of CRC mini-colons (patient #NW). Scale bar, 100 μm.

Extended Data Fig. 3 CAFs promote patient-specific CRC remodeling.

a, Brightfield time-course images showing CAF invasion of the stromal hydrogel and CRC-CAF interaction at the CRC mini-colon (patient #MS). The zoomed-in areas are indicated with dashed squares. Scale bar, 200 μm. b, Brightfield and fluorescence composite images of drug responses in CRC mini-colons with and without CAFs treated with SN-38 (patient #NW). Green fluorescence signal indicates cancer cells. Time after drug treatment initiation is indicated on the left. Scale bar, 100 μm. c, Size of tumors in CRC mini-colons treated with SN-38 under the indicated conditions (patient #NW). *P = 0.0486, **P = 0.0097 (two-way ANOVA and Sidak’s multiple comparisons test; n = 3 mini-colons per condition). d, Brightfield and immunofluorescence images of a CAF-induced invasive front in a CRC mini-colon (patient #MS). Magenta and cyan fluorescence signals indicate laminin-5 (laminin-332) and DAPI stainings, respectively. Scale bar, 40 μm. e, Brightfield image showing a 40-day-old CRC mini-colon (patient #MS) co-cultured with autologous CAFs. Scale bar, 100 μm. f, Brightfield images of conventional organoid cultures (patient #MS) in the absence and presence of autologous CAFs. Scale bar, 150 μm. g-i, Quantitation of invasive front reach (g), mini-colon area (h), and epithelium thickness (i) of CRC mini-colons from patient #MS cultured in the absence and presence of autologous CAFs. *P = 0.0251, ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 24 invasive fronts (g), 3 mini-colons (h), and 18 inter-crypt epithelium sections (i) for each group). In c, and g-i, data represent mean ± SEM.

Source data

Extended Data Fig. 4 CAFs remodel CRC behavior through paracrine factors.

a-c, Quantitation of invasive front area (a), reach (b), and epithelium thickness (c) of CRC mini-colons from patient #NW cultured in the absence and presence of autologous CAFs. ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 3 mini-colons (a), 24 invasive fronts (b), and 18 inter-crypt epithelium sections (c) for each group). d, Brightfield images of CRC mini-colons integrated by the cells of the indicated patients. Scale bar, 100 μm. e, Brightfield images of CRC mini-colons (patient #MS) cultured with control or autologous CAF-conditioned medium. Scale bar, 100 μm. f,g, Quantitation of invasive front area (f) and reach (g) of CRC mini-colons from patient #MS cultured with control or autologous CAF-conditioned medium. **P = 0.0097, ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 3 mini-colons (f) and 24 invasive fronts (g) for each group). In a-c, f and g, data represent mean ± SEM.

Source data

Extended Data Fig. 5 CAFs elicit transcriptomic rearrangements in CRC cells.

a, Volcano plot showing the differentially-expressed genes in patient #MS CRC mini-colons cultured with autologous CAFs. Statistically significant up- and down-regulation events are highlighted in red and blue, respectively. Specific genes mentioned in the main text are indicated. b, Heatmap showing the overlap** differential expression events in patient #MS CRC mini-colons cultured with autologous CAFs and CAF-conditioned medium (CM). The number of differentially expressed genes and the z-score are indicated. c, Venn diagrams showing the overlap in positively (top panel) and negatively (bottom panel) enriched hallmark gene signatures in patient #MS CRC mini-colons cultured with autologous CAFs and CAF-conditioned medium. d, Expression distribution of the indicated genes across mono- and co-cultures of patient #MS CRC cells and CAFs. Cell-type labels can be found in Fig. 3h. e, Single-sample GSEA of the CAF-induced CRC signature in healthy and cancer samples from the TCGA CRC cohort. ***P < 0.0001 (two-tailed Mann–Whitney test; n = 51 and 592 for healthy and CRC samples, respectively). Violins represent data distribution, median and quartiles.

Source data

Extended Data Fig. 6 CAFs promote metalloproteinase activity in CRC cells.

a, Western blot showing the expression of the indicated proteins in CRC organoids in control and CAF-conditioned medium conditions. b, Western blot-based quantitation of MAPK1/3 (ERK1/2) phosphorylation in CRC organoids in control and CAF-conditioned medium conditions. ***P < 0.0001 (two-tailed unpaired t test; n = 6 for each group). c,d, Quantitation of the invasive front area (c) and epithelium thickness (d) in CRC mini-colons co-cultured with autologous CAFs and treated with the indicated compounds. *P = 0.0357; **P = 0.0015 (c), 0.0096 (d); ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 3 mini-colons (c) and 18 inter-crypt epithelium sections (d) for each group). e, Brightfield images of CAF invasion of the stromal hydrogel when CRC mini-colons are treated with the indicated compounds. The maximum reach is indicated with an arrow. Scale bar, 150 μm. f, Quantitation of CAF invasion of the stromal hydrogel 7 days after CRC mini-colons are treated with the indicated compounds. ***P = 0.0002 (two-tailed unpaired t test; n = 6 for each group). g, qRT-PCR based quantitation of MMP7 mRNA in the indicated knockdown cell lines. ***P < 0.0001 (one-way ANOVA and Tukey’s multiple comparisons test; n = 3 for each group). h, Quantitation of the invasive front reach in the indicated CRC mini-colon knockdowns co-cultured with autologous CAFs. **P = 0.0012 (day 7), 0.0093 (day 15); ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 24 invasive fronts for each group). In b-d and f-h, data represent mean ± SEM. Ctrl, control; CM, conditioned medium; KDa, kilodalton. In all panels, data refer to patient #MS.

Source data

Extended Data Fig. 7 CAFs promote an autoinflammatory program in CRC cells.

a, Gene interaction network of the genes induced by CAFs and CAF-conditioned medium in CRC mini-colons. The zoomed-in area is indicated with a dashed rectangle. b, Quantitation of the invasive front reach in CRC mini-colons treated with the indicated molecules. *P = 0.0119, ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 24 invasive fronts for each group). c, Signal intensity profile of MMP7 and CDH1 (E-cadherin) immunofluorescent stainings along IL1B-induced invasive fronts in CRC mini-colons. The tip of the invasive front is located at 100 μm. n = 3 for each staining. d,e, Quantitation of the invasive front reach (d) and epithelium thickness (e) in CRC mini-colons co-cultured with autologous CAFs and treated with the indicated compounds. **P = 0.0025; ***P = 0.0007 (e), < 0.0001 (d) (two-way ANOVA and Sidak’s multiple comparisons test; n = 24 invasive fronts (d) and 18 inter-crypt epithelium sections (e) for each group). f, Brightfield images of CRC mini-colons treated with the indicated compounds. Scale bar, 100 μm. In b-e, data represent mean ± SEM. In all panels, data refer to patient #MS.

Source data

Extended Data Fig. 8 Mini-colons allow the evaluation of TIL functionality.

a, Cell type distribution in the TIL populations from the indicated patients. b, Brightfield image showing TIL infiltration into the stromal hydrogel from the TIL reservoir (patient #MS). The zoomed-in area is indicated with a dashed rectangle. Scale bar, 125 μm. c, Quantitation of TIL infiltration into the stromal hydrogel in the absence and presence of cancer cells (patient #MS). ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 6 for each group). d, Brightfield images of the stromal hydrogel and TIL reservoir boundary of mini-colons with different loads of cancer cells (indicated on the left, patient #MS). The progression of TILs into the stromal hydrogel is indicated with an arrow. Scale bar, 125 μm. e, Brightfield and fluorescent composite image of TILs reaching and interacting with the CRC mini-colon (patient #MS). Green fluorescence signal indicates cancer cells. Scale bar, 20 μm. f, Brightfield images of CRC mini-colons from the indicated patients co-cultured with autologous TILs at the indicated times. Scale bar, 60 μm. g, Luminescence-based quantitation of the change in normalized CRC viability in mini-colons from the indicated patients after 4 days of interaction with autologous TILs. The interaction was measured once TILs had physically reached the mini-colon. Data were normalized to the signal obtained the first day of measurement, which was given a value of 1. **P = 0.0022 (#NS), 0.0059 (#TF) (one-way ANOVA and Tukey’s multiple comparisons test; n = 3 for each group). h, Luminescence-based quantitation of CRC viability of MSS CRC mini-colons from the indicated patients co-cultured with autologous TILs and CAFs, and in the absence or presence of atezolizumab. NS, No significant differences. P = 0.2542 (#NS), 0.5782 (#TF) (two-tailed unpaired t test; n = 3 for each group). In c, g and h, data represent mean ± SEM.

Source data

Extended Data Fig. 9 Mini-colons allow the recreation of complex immune tumor microenvironments.

a, Schematic of the distribution of TILs, DCs, CAFs and CRC cells in mini-colon devices at the moment of seeding. b, Brightfield images capturing the main morphological differences between control monocytes and monocyte-derived dendritic cells. Scale bar, 10 μm. c, Flow cytometry histograms showing the expression of differentiation markers in control and DC-differentiated monocytes. d,e, Quantitation of the indicated cytokines in culture medium recovered from CRC mini-colons co-cultured with autologous TILs and CAFs, and in the absence or presence of dendritic cells. *P = 0.0340 (IL1B), 0.0385 (TNF); **P = 0.0045 (IL6), 0.0095 (IL8); ***P < 0.0001 (two-way ANOVA and Sidak’s multiple comparisons test; n = 3 for each group). f, Brightfield images of CRC mini-colons co-cultured with autologous TILs and CAFs, and in the absence or presence of dendritic cells. Zoomed-in images are shown in Fig. 5k. Scale bar, 100 μm. In d and e, data represent mean ± SEM. Data refer to patient #MS. DC, dendritic cells.

Source data

Extended Data Fig. 10 CRC mini-colons allow the integration of vasculature and the study of angiogenesis.

a, Schematic of the distribution of endothelial cells, CAFs and CRC cells in mini-colon devices at the moment of seeding. b, Brightfield time-course images of vascularized CRC mini-colons (patient #NW) showing angiogenesis events. Bottom panels are zoomed-in areas of top panels. Scale bars, 175 μm.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. Relevant clinical parameters of the patients involved in this study. The patient ID, sex, age, colorectal cancer disease stage (TNM), anatomical region where the sample was taken from, histological type and microsatellite stability status are indicated. Supplementary Table 2. Differentially expressed genes in CRC mini-colons cultured in CAF-conditioned medium (a) or with CAFs (b). Differentially expressed genes were identified using Limma-Voom (two-sided moderated t-test). logFC, log2 fold change; AveExpr, average expression; t, moderated t statistic; P.Value, raw P value; adj.P.Val, Benjamini–Hochberg-adjusted P value for multiple comparisons; B, log odds. Supplementary Table 3. Hallmark functional enrichments in CRC mini-colons cultured in CAF-conditioned medium (a,b) or with CAFs (c,d). The GSEA-derived enrichment parameters are indicated. ES, enrichment score; NES, normalized enrichment score; NOM p-val, nominal P value (two-sided Kolmogorov–Smirnov test); FDR q-val, FDR-adjusted P value for multiple comparisons; FWER p-val, familywise error rate-adjusted P value.

Supplementary Video 1

A 3D visualization of CRC tumors in a mini-colon. A 3D immunofluorescence projection of CRC tumors in a mini-colon. Green and cyan signals indicate cancer cells and nuclei, respectively.

Supplementary Video 2

Early CRC tumor development in a mini-colon. A 60-h time-lapse video of CRC tumor development in a mini-colon. A green signal indicates cancer cells.

Supplementary Video 3

Engagement between T and CRC cells in a mini-colon. A 12-h time-lapse video of TILs attacking a CRC mini-colon tumor.

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Lorenzo-Martín, L.F., Broguiere, N., Langer, J. et al. Patient-derived mini-colons enable long-term modeling of tumor–microenvironment complexity. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02301-4

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