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Blockade of the TGFβ signaling pathway has emerged from preclinical studies as a potential treatment to enhance the efficacy of immune checkpoint inhibition in advanced colorectal cancer (CRC) and several other types of cancer. However, clinical translation of first-generation inhibitors has shown little success. Here, we report the synthesis and characterization of HYL001, a potent inhibitor of TGFβ receptor 1 (ALK5), that is approximately 9 times more efficacious than the structurally related compound galunisertib, while maintaining a favorable safety profile. HYL001 in combination with immune checkpoint blockade (anti-PD1) eradicates liver metastases generated in mice by microsatellite stable, aggressive colorectal cancer tumors at doses where galunisertib is ineffective., Competing Interests: The authors declare the following competing financial interest(s): DT, DB, JM, AR, and EB hold a patent on the synthesis and use of HYL001. E.B. is author in a patent describing bispecific antibodies to target cancer stem cells. The laboratory of E.B. has received research funding from MERUS and INCYTE. E.B. has received honoraria for consulting from Genentech., (© 2024 The Authors. Published by American Chemical Society.)

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs., (© 2024. Crown.)

A substantial proportion of cancer patients do not benefit from platinum-based chemotherapy (CT) due to the emergence of drug resistance. Here, we apply elemental imaging to the mapping of CT biodistribution after therapy in residual colorectal cancer and achieve a comprehensive analysis of the genetic program induced by oxaliplatin-based CT in the tumor microenvironment. We show that oxaliplatin is largely retained by cancer-associated fibroblasts (CAFs) long time after the treatment ceased. We determine that CT accumulation in CAFs intensifies TGF-beta activity, leading to the production of multiple factors enhancing cancer aggressiveness. We establish periostin as a stromal marker of chemotherapeutic activity intrinsically upregulated in consensus molecular subtype 4 (CMS4) tumors and highly expressed before and/or after treatment in patients unresponsive to therapy. Collectively, our study underscores the ability of CT-retaining CAFs to support cancer progression and resistance to treatment., (© 2023. The Author(s).)

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years 1 . Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5 + stem-like tumour cells 2-4 , and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1 high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5 + tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5 + cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a + cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a + cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a + cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP + fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Colorectal cancer (CRC) is currently the third leading cause for cancer-related mortality. Cancer stem cells have been implicated in colorectal tumor growth, but their specific role in tumor biology, including metastasis, is still uncertain. Methods: Increased expression of L1CAM, CXCR4 and NODAL was identified in tumor section of patients with CRC and in patients-derived-organoids (PDOs). The expression of L1CAM, CXCR4 and NODAL was evaluated using quantitative real-time PCR, western blotting, immunofluorescence, immunohistochemistry and flow cytometry. The effects of the L1CAM, CXCR4 and NODAL on tumor growth, proliferation, migration, invasion, colony-formation ability, metastasis and chemoresistance were investigated both in vitro and in vivo . Results: We found that human colorectal cancer tissue contains cancer stem cells defined by L1CAM high /CXCR4 high expression that is activated by Nodal in hypoxic microenvironment. This L1CAM high /CXCR4 high population is tumorigenic, highly resistant to standard chemotherapy, and determines the metastatic phenotype of the individual tumor. Depletion of the L1CAM high /CXCR4 high population drastically reduces the tumorigenic potential and the metastatic phenotype of colorectal tumors. Conclusion: In conclusion, we demonstrated that a subpopulation of migrating L1CAM high /CXCR4 high is essential for tumor progression. Together, these findings suggest that strategies aimed at modulating the Nodal signaling could have important clinical applications to inhibit colorectal cancer-derived metastasis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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 occurs 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 I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5 + and LGR5 - tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 (T H 1) 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 T H 1-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., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs)., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profiles have defined subtypes displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-β signaling. Likewise, we find that all poor-prognosis CRC subtypes share a gene program induced by TGF-β in tumor stromal cells. Using patient-derived tumor organoids and xenografts, we show that the use of TGF-β signaling inhibitors to block the cross-talk between cancer cells and the microenvironment halts disease progression.

Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with β-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.

A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse., (Copyright © 2011 Elsevier Inc. All rights reserved.)