Most patients with colorectal cancer die as a result of the disease spreading to other organs. However, no prevalent mutations have been associated with metastatic colorectal cancers. Instead, particular features of the tumour microenvironment, such as lack of T-cell infiltration, low type 1 T-helper cell (T1) activity and reduced immune cytotoxicity or increased TGFβ levels predict adverse outcomes in patients with colorectal cancer. Here we analyse the interplay between genetic alterations and the tumour microenvironment by crossing mice bearing conditional alleles of four main colorectal cancer mutations in intestinal stem cells. Quadruple-mutant mice developed metastatic intestinal tumours that display key hallmarks of human microsatellite-stable colorectal cancers, including low mutational burden, T-cell exclusion and TGFβ-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFβ unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFβ signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFβ in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the T1-effector phenotype. Immunotherapies directed against TGFβ signalling may therefore have broad applications in treating patients with advanced colorectal cancer.

The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient‐derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage‐tracing cassettes knocked in the LGR5 locus. Analysis of LGR5‐EGFP + cells isolated from organoid‐derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage‐tracing experiments showed that LGR5+ CRC cells self‐renew and generate progeny over long time periods that undergo differentiation toward mucosecreting‐ and absorptive‐like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.

Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occur in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA Polymerase 1 subunit A -POLR1A. Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest to CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5-tumor cells. Therefore, a common architecture in CRC is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.

The standard of care for advanced colorectal cancer (CRC) includes treatment with chemotherapeutic drugs that target the cell proliferation machinery 1 . In CRC patients with overt metastases, chemotherapy initially halts tumor growth but, almost inevitably, disease progresses after some cycles of treatment. Adjuvant chemotherapy is also administered to eliminate minimal residual disease, yet it only diminishes the risk of relapse by 10-25% 2 . Previous studies have shown that patient-derived organoids predict responses to chemotherapy 3-6 . Therefore, we used them as models to investigate the mechanisms behind the limited benefit of these treatments. Whereas CRC organoids expand from highly proliferative Lgr5+ tumor cells, we discovered that lack of optimal stem cell growth conditions specifies a latent Lgr5+ cell population. These cells expressed the gene Mex3a, were largely insensitive to chemotherapy and regenerated the organoid culture after treatment. In mouse models of metastatic latency, Mex3a+ cells contributed marginally to metastatic outgrowth. However, after chemotherapy treatment, Mex3a+ cells produced large cell clones that regenerated metastatic disease. Using lineage-tracing analysis combined with single cell profiling, we showed that drug-tolerant persister Mex3a+ cells downregulate the WNT/Lgr5+ stem cell program immediately after chemotherapy and adopt a transient regenerative state

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