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L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

An Author Correction to this article was published on 30 September 2020

This article has been updated

Abstract

Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAMhigh cells partially overlap with LGR5high stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin–REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAMlow to an L1CAMhigh state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.

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Fig. 1: L1CAM marks chemoresistant organoid-generating cells in human CRC.
Fig. 2: L1CAM is a mediator of organoid and tumor regeneration.
Fig. 3: Disruption of the epithelial niche induces L1CAM expression.
Fig. 4: L1CAM is required for epithelial regeneration after colitis.
Fig. 5: L1CAM is dispensable for adenoma formation but is required for orthotopic tumor engraftment, local expansion, metastasis and chemoresistance.
Fig. 6: Plasticity of the L1CAMhigh phenotype.
Fig. 7: Loss of E-cadherin-dependent cell contact downregulates REST and enables L1CAM expression.
Fig. 8: Epistasis analysis of E-cadherin, REST and L1CAM.

Data availability

ChIP–seq and scRNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA) under accession codes GSE112555 and SRP136919, respectively. Ranked differentially expressed genes in each LGR5L1CAM cluster are listed in Supplementary Table 1. The human genes corresponding to the revival stem cell signature were derived from GSE117783 and are listed in Supplementary Table 2. Source data for Figs. 18 and Extended Data Figs. 16 are provided with this paper. All other data files supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

All single-cell analyses and visualizations were performed in Python with the following open-source algorithms as described above: SEQC (https://github.com/ambrosejcarr/seqc), t-SNE (https://lvdmaaten.github.io/software/), MAGIC (https://github.com/dpeerlab/magic) and the Scikit-learn implementation of a Gaussian mixture model. Computer code is available upon reasonable request. ChIP–seq data analysis was performed in R and data were visualized with IGV.

Change history

  • 30 September 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank E. Batlle, F. Barriga and C. Morral for feedback and technical assistance, M. Schachner (Shantou University Medical College and Rutgers University) for the L1camfl/y mice, members of the Memorial Sloan Kettering Cancer Center core facilities, Colorectal, Hepatobiliary and Gastrointestinal Oncology Services and the Department of Pathology. This work was supported by NIH grants P01-CA129243 (J.M.), P01-CA094060 (J.M.), P30-CA008748 (MSKCC), K08-CA230213 (K.G.), T32-CA009207 (K.G.), T32-GM007739 (K.P.O. and Y.-H.H.) and F30-CA203238 (Y.-H.H.), Department of Defense Innovator Award W81XWH-12-0074 (J.M.), a Damon Runyon Clinical Investigator Award (K.G.), an American Cancer Society Postdoctoral Fellowship, an AACR Basic Cancer Research Fellowship, a Conquer Cancer Foundation of ASCO Young Investigator Award (K.G.), Shulamit Katzman Endowed Postdoctoral Research Fellowships (K.G. and E.E.E.), a Damon Runyon Postdoctoral Fellowship (H.B.), a Burroughs Wellcome Career Award at the Scientific Interface (A.M.L.) and the Alan and Sandra Gerry Metastasis and Tumour Ecosystems Center. K.G. recieves support as an investigator of the Stand Up To Cancer Colorectal Cancer Dream Team (grant number: SU2C AACR-DR22-17).

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Authors and Affiliations

Authors

Contributions

K.G. and J.M. conceived and oversaw the project, performed data interpretation and wrote the manuscript. H.B. designed and performed ChIP experiments. A.M.L., R.S. and D.P. performed scRNA-seq data analysis. Y.K. designed and performed laminin adhesion assays. K.G. and L.H. performed other experiments. K.P.O. and S.W.L. provided reagents, animals and technical assistance with mouse experiments. Y.-H.H., V.P.R. and R.K. performed ChIP–seq data analysis. I.M. prepared scRNA-seq libraries. M.T. and K.M.-T. performed LGR5 and L1CAM immunostaining. E.E.E. assisted with data interpretation. K.G., M.R.W., L.B.S., J.G.-A. and J.S. enabled the procurement of human samples. J.S. oversaw tissue procurement, clinical specimen processing and histopathological data interpretation.

Corresponding author

Correspondence to Joan Massagué.

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J.M. is a science advisor for and owns company stock in Scholar Rock.

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Extended data

Extended Data Fig. 1 Association of L1CAM expression with invasion, post-therapy residual disease and stemness.

a, Immunohistochemistry of serial sections of the primary tumour invasion front in a patient sample (refer to Fig. 1a) showing a cluster of L1CAM-expressing cells (left) invading a CD31 positive blood vessel (right). b, Representative brightfield image of organoids grown from L1CAMhigh (left) or L1CAMlow (right) cells flow-sorted from freshly resected patient CRC liver metastases. Representative of 7 patient tumours. c, Gating strategy for flow cytometry/sorting of epithelial cells from fresh CRC liver metastasis surgical specimens. Bottom right: histogram of mode normalized cell counts based on L1CAM expression. Gates identify L1CAMhigh and L1CAMlow cells in the EpCAM + population. Representative of 7 patient tumours d, Representative image showing subcutaneous tumour growth in mice transplanted with 50,000 organoid-derived flow-sorted L1CAMhigh (left) or L1CAMlow (right) cells. Representative of 5 mice per group. e, In vivo limiting dilution assay. NSG mice were transplanted with the indicated numbers of FACS sorted L1CAMhigh or L1CAMlow cells derived from MSK107Li organoids (n = 8 injections per dilution per group, 2 injections per mouse). Tumour formation was assayed 90 days following injection. Tumour-initiating cell frequency by limiting dilution analysis was 1 in 31,027 for L1CAMhigh cells, 1 in 491,441 for L1CAMlow cells (p = 0.03, χ2 test). f, Day 90 bioluminescent images (n = 8 injections per dilution per group, 2 injections per mouse). g, CRC metastasis organoid-derived xenografts retain patient tumour morphology. Hematoxylin & eosin staining of matched MSK107Li patient CRC liver metastasis (top) and organoid-derived subcutaneous xenograft (bottom) showing similar glandular tumour histology surrounding central necrosis. Representative of 4 tumour/organoid pairs (h, i) L1CAMhigh cells in organoid–derived xenografts retain selective organoid generation capacity. h, Brightfield images and i, viability (luminescence) of organoids grown from flow-sorted L1CAMlow (left) or L1CAMhigh (right) cells plated at 2000 cells/40μL matrigel in organoid media, 14 days following sorting. Boxplots, boxes show 25th-75th percentile with median, whiskers show min-max, n = 9 independently plated wells each, representative of 3 experiments from independent xenografts, two-sided Mann-Whitney U test.

Source data

Extended Data Fig. 2 Relationship between L1CAM expression and stemness.

a, Summary of clinical, genetic and treatment features of the patient-derived organoids assayed by scRNA-seq. Organoids were profiled using MSK-IMPACT next-generation sequencing to determine the presence of known oncogenic mutations based on OncoKB annotations. FOLFOX = 5-fluorouracil, leucovorin, oxaliplatin. (b-g) scRNAseq analysis of 9,974 cells from 4 patient-derived CRC organoids. (b, c) tSNE projection of all cells analysed, b, colored by patient, c, indicating expression levels of LGR5 and L1CAM (red = high, blue = low), d, Population distribution of L1CAM/LGR5 subpopulations identified in each primary tumour or metastasis organoid assayed. e, tSNE projection indicating expression of the revival stem cell signature (red = high, blue = low) (f, g) Violin plots (left) indicating expression of an EMT signature (f) or the KEGG fatty acid metabolism signature (g) comprising the genes shown on the heatmaps (right) in each L1CAM/LGR5 subpopulation. Bars indicated min to max. Heatmaps indicate relative expression of the indicated genes in each cluster. h, Flow cytometric analysis of stem cell marker expression in L1CAMhigh and L1CAMlow epithelial cells from freshly resected human CRC liver metastases, gated as in Figure S1C. Representative (of 12 independent patient tumours) flow cytometry contour plots showing stem cell marker expression in EpCAM+cells. i, Median EphB2, CD133 and CD44 expression in L1CAMhigh and L1CAMlow cells sorted from freshly resected and dissociated patient CRC liver metastases. Boxplots, boxes show 25th-75th percentile with median, whiskers show min-max, n = 12 tumours (one tumour per patient), two-sided Mann-Whitney U test.

Source data

Extended Data Fig. 3 L1CAM is required for laminin binding, survival and organoid regeneration by single cells.

a, L1CAM protein expression by flow cytometry (median ± s.e.m.) of MSK107Li cells 14 days following transduction with plasmids expressing Cas9 alone or together with sgRNA targeting L1CAM, shown as a percentage of the population transduced with Cas9 alone. n = 3 replicates per group, two-sided Student’s t-test. b, FACS sorted cells were seeded at a concentration of 2000 cells/40 µL and permitted to grow for 14 days. Viability assay showing luminescence (mean ± s.e.m.) of each population relative to luminescence on day 0 (dashed line); n = 3 organoid cultures per group, two-sided Mann-Whitney U test. c, Relative caspase-glo luminescence (mean ± s.e.m.) at the indicated timepoints during MSK107Li organoid growth relative to the time of single cell seeding (day 0). Data were normalized to cell viability measured at the same time points. (n = 3 organoid cultures per timepoint, p values compare shL1CAM.1-Dox vs. shL1CAM.1 + Dox and shL1CAM.2-Dox vs. shL1CAM.2 + Dox, two-sided Student’s t tests). d, MSK107Li organoid-derived single cells expressing a doxycycline-inducible L1CAM shRNA or control were seeded in the presence or absence of doxycycline as indicated. After 14 days, culture media was aspirated and replaced with doxycycline-free media, and permitted to grow for a further 14 days prior to measuring cell viability (luminescence, mean ± s.e.m.). n = 3 organoid cultures per timepoint, two-sided Mann-Whitney U test. e, Relative L1CAM mRNA expression (mean ± s.e.m.) in steady-state day 14 organoids, or residual organoid cells following 14 days of treatment with doxycycline and/or irinotecan as indicated. Data were normalized to GAPDH mRNA expression levels. n = 4 organoid cultures, two-sided Student’s t tests. f, Solid phase assay showing binding of 120 nM recombinant human L1CAM-Fc to plates coated with 30 nM of each of the indicated ligands. L1CAM-Fc was detected using HRP-conjugated anti-human IgG, chromogenic substrate was added and OD(450 nM) measured. Mean ± s.e.m., n = 4 wells per condition, representative of 2 independent experiments, two-sided Mann-Whitney U test. g, Dose-response curves showing binding of increasing concentrations of recombinant human L1CAM-Fc to plates coated with 30 nM of each of the indicated ligands. L1CAM-Fc was detected using HRP-conjugated anti-human IgG, chromogenic substrate was added and OD(450 nM) measured. Mean ± s.e.m., n = 5 wells/dose/condition, two-sided Mann-Whitney U test.

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Extended Data Fig. 4 L1CAM is dispensable for adenoma formation but required for orthotopic tumour engraftment, local expansion, metastasis and chemoresistance.

Representative sections of colons from APCΔIEC and L1CAM/APCΔIEC mice stained with hematoxylin & eosin, and antibodies against Ki67, L1CAM or E-cadherin, showing no histopathological differences between the two groups. Representative of 5 mice in each group. (b-g) L1CAM inhibition impairs local tumour expansion and metastasis from murine orthotopic caecal transplants. b, Relative L1cam mRNA expression (mean ± s.e.m) in murine AKP organoids stably transduced with lentivirus directing the expression of doxycycline-inducible shRNA targeting L1cam or control, and treated with or without doxycycline for 48 h. Data were normalized to Gapdh mRNA expression levels. n = 4 organoid cultures, two-sided Student’s t test. c, Dissociated cells derived from AKP organoids transduced with lentivirus directing the expression of tdTomato-luciferase and shRNA against L1cam or control were injected into the caecal submucosa. Mice were monitored until caecal tumours were evident by ex vivo BLI imaging 3 weeks following injection, randomized based on BLI signal, and maintained on or off doxycycline diet for 5 weeks prior to euthanasia. Representative of 7 (-Dox), 13 mice (+Dox). (d-g) Quantification of whole animal or ex vivo BLI signal in each indicated organ per group, normalized to BLI at the time of randomization, is shown. Boxplot, boxes show 25th-75th percentile with median, whiskers show min to max, n = 12, 7, 7, 13 mice per group (left to right), two-sided Mann-Whitney U test. h, L1CAM immunohistochemistry in sections of orthotopic rectal xenografts (representative of 3 animals analysed per group) or i, liver metastases (representative of 3 animals analysed per group) from mice injected with cells derived from MSK107Li organoids expressing doxycycline-inducible shRNA targeting L1CAM. Mice were maintained on or off doxycycline (dox) diet as indicated. j, Representative (n = 8 (-Dox), n = 9 (+Dox)) ex vivo liver bioluminescence images related to i. k, Representative (n = 12 (-Dox, n = 11 (+Dox)) bioluminescent images of orthotopic caecal xenografts, liver and lung metastases derived from MSK121Li organoids expressing doxycycline-inducible shRNA targeting L1CAM, and randomized to treatment with or without doxycycline. (l, m) L1CAM immunohistochemistry in sections of subcutaneous xenograft tumours derived from MSK107Li organoids expressing doxycycline-inducible shRNA targeting L1CAM. Mice were treated with doxycycline diet and/or irinotecan chemotherapy as indicated. Mean ± s.e.m, n = 22, 15, 21, 19, 18, 23, 25, 21 fields from 3 mice per group (left to right), two-sided Mann-Whitney U test.

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Extended Data Fig. 5 Apoptosis and plasticity of L1CAMlow cells.

a, Proportion of L1CAM expressing cells decreases as organoids grow. Flow cytometry mode-normalized histograms (top) and contour plots (bottom) showing L1CAM expression in the L1CAMhigh population in freshly resected, dissociated and flow-sorted CRC primary tumours and liver metastases (grey), and in organoids grown from these L1CAMhigh cells 21 days following initial sorting (red), showing a left shift in the population over time. n = 6 independent patient tumours. b, Organoid generation selects for L1CAM expression in L1CAMlow cells. Flow cytometry mode-normalized histograms (top) and contour plots (bottom) showing L1CAM expression in the L1CAMlow population in freshly resected, dissociated and flow-sorted CRC primary tumours and liver metastases (grey), and in organoids grown from these L1CAMlow cells 21 days following initial sorting (purple), showing a right shift in the population over time. n = 5 independent patient tumours. c, Median L1CAM fluorescence intensity of the unselected population of MSK107Li (top) or MSK121Li (bottom) organoid derived cells pre-sort (grey), and viable cells regenerated from flow-sorted L1CAMhigh (red) or L1CAMlow (purple) populations at the indicated time points after sorting. Histograms indicating the distribution of the populations are shown in Fig. 5c. Representative of 3 independent experiments. d, Flow cytometry density plots showing staining with Annexin V-FITC and propidium iodide (PI) in populations derived from L1CAMhigh or L1CAMlow cells flow-sorted from MSK107Li (top) or MSK121Li (bottom) organoids and analysed 48 h after reseeding. Representative of 3 independent experiments. n = 2807 cells (MSK107Li), 2182 cells (MSK121Li), two-sided χ2 tests. e, Median L1CAM-APC staining intensity of Annexin V-FITC + (apoptotic) or Annexin V-FITC- (non-apoptotic) populations derived from L1CAMhigh or L1CAMlow cells flow-sorted from MSK107Li/MSK121Li organoids and analysed 48 h after reseeding, gated and distributed as in d. Mean ± s.e.m, n = 3 independent organoid cultures, two-sided Student’s t test. f, Gating strategy for isolating mutually exclusive tdTomato+;GFP-;L1CAMhigh and tdTomato-;GFP+;L1CAMlow cells from organoids stably expressing each fluorescent protein.

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Extended Data Fig. 6 Loss of membrane E-cadherin contacts drives L1CAM expression.

a, Epithelial disruption is a potent inducer of L1CAM expression. Relative L1CAM mRNA expression (mean ± s.e.m.) in intact normal colon organoids or organoid-derived single cells 24 h following dissociation. Gene expression was normalized to GAPDH mRNA expression. Organoids were cultured in media containing the indicated cytokines, or in conditioned media derived from DSS-colitis affected mouse colon. n = 4 organoid cultures per condition, two-sided Student’s t test. b, ChIP-PCR using antibodies against REST, or isotype control immunoglobulin in intact MSK107Li organoids or organoid-derived single cells 24 h following dissociation. Fold enrichment (mean ± s.e.m.) compared to the corresponding 2% input is shown. PCR primers (Extended Data Table 3) were selected to amplify immunoprecipitated DNA at the indicated number of residues from the L1CAM transcription site. p values indicate intact α-REST vs. dissociated α-REST, n = 3 independent experiments, two-sided Student’s t tests. c, Top: Venn diagram showing the number of genes in the vicinity of REST binding peaks observed in both MSK107Li and MSK121Li organoids, either in one integrity state or in both. Bottom: Table showing Biocarta pathway terms significantly enriched in the list of genes that lose REST ChIP-seq peaks upon dissociation of both MSK107Li and MSK121Li organoids. n = 2 independent experiments from 2 patient-derived organoids per integrity condition. Poisson-enrichment p value over background tag count. d, qRT-PCR showing relative REST and L1CAM mRNA expression (mean ± s.e.m.) in intact MSK121Li organoids (day 0), 24 h after dissociation and plating as single cells (day 1) and at the indicated time points during organoid regeneration. Organoids were transduced with lentivirus constitutively expressing shRNA targeting REST or control shRNA. Gene expression was normalized to the mRNA expression of GAPDH. Day 1 shCTRL vs. shREST.1: p = 0.01 (REST), p < 0.0001 (L1CAM); shCTRL vs. shREST.2: p = 0.02 (REST), p < 0.0001 (L1CAM); n = 4 organoid cultures per sample per time point, two-sided Student’s t tests. e, Relative mRNA expression (mean ± s.e.m.) of CDH1, REST and L1CAM in intact MSK121Li organoids transduced with lentivirus constitutively expressing shRNA targeting CDH1 or control shRNA. n = 4 organoid cultures per group, two-sided Student’s t tests.

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Supplementary information

Reporting Summary

Supplementary Table 1

Ranked differentially expressed genes in LGR5L1CAM clusters. Human genes corresponding to revival stem cell signature, L1CAM shRNA and sgRNA sequences and ChIP–PCR primers.

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Ganesh, K., Basnet, H., Kaygusuz, Y. et al. L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer. Nat Cancer 1, 28–45 (2020). https://doi.org/10.1038/s43018-019-0006-x

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