Metastatic colorectal cancer (mCRC) is one of the deadliest cancers with very poor response to immune checkpoint blockade (ICB). Standard therapies employ chemotherapy combined with epidermal growth factor receptor (EGFR) blocking antibodies, which are only effective in a fraction of patients with RAS/RAF wild-type tumours. We have previously shown that EGFR deletion in myeloid cells of CRC, rather than in the cancer cells themselves, reduces tumour growth. Here, we investigate to which extent EGFR blockade in myeloid cells increases anti-tumour immunity, thus sensitising CRC to ICB. Using a syngeneic preclinical CRC liver metastasis model based on the transplantation of murine RAS mutant CRC organoids into mice lacking EGFR in myeloid cells, we observe a reduction in metastasis development accompanied by increased intratumoural T-cell infiltration. Importantly, we demonstrate that EGFR deletion reduces the capacity of granulocytic myeloid-derived suppressor cells (G-MDSCs) to suppress CD4+ T-cell proliferation. RNA-seq analysis of sorted MDSCs and T-cells uncovered an EGFR-dependent signature involved in immunosuppression, which in proficient-mismatch-repair (pMMR) CRC patients is associated with worse overall survival. Therapeutically, lifting immunosuppression by EGFR deletion in myeloid cells sensitised tumours to anti-PD-L1 treatment, thus preventing liver metastasis development. These results imply that anti-EGFR therapies combined with ICB might be successful in preventing metastasis of RAS mutated CRC with high infiltration of suppressive EGFR+ myeloid cells.

Adenovirus type 5 (Ad5) is a well-established platform for gene transfer but relies on the Coxsackie and Adenovirus Receptor (CAR), limiting efficacy in CAR-deficient tissues and influencing biodistribution through interactions with blood cells. Human adenovirus 52 (Ad52) encodes two fibers: a long CAR-binding fiber (F52L) and a short fiber (F52s) that engages sialylated glycoconjugates. Here, we engineered a chimeric Ad5/52s vector by replacing the Ad5 fiber with F52s. Ad5/52s production was initially inefficient due to delayed replication, but optimization of harvest timing markedly improved infectious titers and particle quality. In vitro, Ad5/52s efficiently transduced different Schwann cell lines and other Ad5-refractory lines. A CAR-independent tropism was demonstrated for Ad5/52s, along with the ability to transduce all tested CAR-positive cell lines. In vivo, systemic delivery revealed strikingly reduced liver tropism and rapid loss of infectivity in circulation, despite comparable genome persistence. Fractionation experiments implicated plasma factors as mediators of Ad5/52s inactivation. Bioinformatic analysis identified a putative thrombin-sensitive cleavage motif within F52s, and mutation (R67N) conferred enhanced viral stability in blood. The Ad5/52s platform highlights both opportunities for targeted local applications and the potential to rationally modulate systemic safety profiles.

Accumulating evidence indicates that peritoneal macrophages, comprising resident peritoneal macrophages (resMØ) and monocyte-derived nonresident macrophages (moMØ), contribute to peritoneal tumor progression by promoting tumor cell proliferation and migration and driving immunosuppression. However, the mechanisms governing the expansion of resMØs and moMØs, as well as their differential contributions to the peritoneal macrophage pool in tumor-bearing mice and to tumor growth, remain to be elucidated. Using a mouse model of colorectal cancer peritoneal metastasis, induced by intraperitoneal injection of tumor organoids derived from primary tumors in genetically engineered mice carrying Apc, Kras, Tgfbr2, and Trp53 mutations, and recapitulating human-like metastatic colorectal cancer, we investigated the origin, expansion, and function of peritoneal macrophages during metastatic tumor growth. Our data support that the low inflammatory status of the peritoneal cavity during colorectal cancer peritoneal tumor growth restrains monocyte recruitment and the differentiation of Tim4- resMØs and moMØs while enabling a marked, proliferation-driven expansion of Tim4+ resMØs. Tumor-induced Tim4+ resMØs displayed a migratory and protumor transcriptomic signature characterized by the activation of genes encoding key protumorigenic molecules and potential immunotherapeutic targets, including Adora2a/Adora2b, Arg1, Ido2, Acod1, Mmp12, Cd274, Pdcd1lg2, Spp1, Trem1, and Vegfa. Correspondingly, during peritoneal colorectal cancer tumor growth, Tim4+TREM1+ resMØs migrated to the omentum, the principal peritoneal target organ for metastasis, and promoted colorectal cancer peritoneal tumor progression. These findings may help lead to the development of immunotherapies for colorectal cancer peritoneal metastasis that target tumor-associated peritoneal macrophages.

Abstract Mismatch repair-deficient (MMRd) tumors respond well to immune checkpoint blockade (ICB) while MMR-proficient (MMRp) tumors from the same cell type do not. We hypothesized that disrupting MMR would make immunologically cold MMRp tumors more immunogenic and responsive to ICB. Initial attempts to knockout individual MMR genes had a significant but weak impact on growth of implanted tumors because the development of microsatellite instability (MSI) and chromosomal instability (CIN) was slow relative to tumor growth and progression. However, simultaneous knockout of two MMR genes, Mlh1 and Msh2, in multiple mouse cancer cell lines and organoids (colorectal (CRC), breast (TNBC), and melanoma) led to rapid CD8⁺ T cell-mediated tumor growth inhibition or even rejection of subcutaneous and orthotopic tumor implants. Tumors that were not rejected could be ablated by treatment with anti-PD1. Mlh1-/-Msh2-/- tumors proliferated like wild-type tumors in vitro. In vivo tumor control was immune-mediated and depended on CD8 T cells since it was abrogated in immunodeficient NSG mice or by depletion of CD8 T cells, but not CD4 T cells or NK cells. Delayed or concurrent challenge of mice that reject Mlh1-/-Msh2-/- tumors with MMRp tumors also led to their rejection, indicating that exposure to MMRd tumors induces immune reactivity and memory to the MMRp tumor. Surprisingly, mice that rejected Mlh1-/-Msh2-/- CRC or TNBC tumors were also protected against heterologous CRC or TNBC cancers and even against B16F10 implants, suggesting that exposure to MMRd tumors led to CD8 T cell immunity that was independent of tumor antigens. In fact, knockout of B2m in Mlh1-/-Msh2-/- CRC tumors, which eliminated MHC class I expression and antigen presentation to CD8 T cells, did not affect tumor rejection, suggesting that immune protection was not T cell receptor-mediated. scRNA-seq, qRT-PCR and flow cytometry revealed profound remodeling of the tumor immune microenvironment in Mlh1-/-Msh2-/- tumors, characterized by up-regulation of interferon response genes by the tumor, an expansion of cytotoxic CD8⁺ T cells that express NK activating receptors, reduction in infiltrating neutrophils and a shift in myeloid populations toward less immunosuppressive, but more phagocytic, myeloid cells. Antitumor immunity generated by exposure to Mlh1-/-Msh2-/- tumors was driven by NK activating receptor recognition by CD8⁺ T cells of NK ligands on tumor cells, since protection was abrogated by treating tumor-bearing mice with an antibody to an NK activating receptor. Although no small molecule inhibitors of MMR have been identified, in vivo tumor-targeted knockdown of Mlh1 and Msh2 using EpCAM-targeted aptamer-siRNAs suppressed tumor growth. Thus, disrupting MMR in MMRp cancer cells induces a novel, potent CD8 T cell protective immune response that is mediated by an NK activating receptor that recognizes the genotoxic stress of unrepaired DNA damage in the tumor, rather than by T cell receptor recognition of tumor antigens, that strongly protects mice from MMRp and even unrelated tumors. These results in mice suggest that therapeutic strategies that make MMRp tumors MMRd could be developed to treat immunologically cold tumors or sensitize them to ICB. Citation Format: Haiwei Zhang, Ayijiang Yisimayi, Bowen Gu, Yuting Wang, Wayne M. Yokoyama, Sytse J. Piersma, Eduard Batlle, Daniele V. Tauriello, Judy Lieberman. Disrupting mismatch repair makes mismatch repair-proficient tumors immunogenic and induces CD8 T cell-dependent, but T cell receptor-independent, immune control of heterologous tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB082.

BACKGROUND:Colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, with clinical progress limited by intratumoural cellular diversity and the absence of robust, informative markers. OBJECTIVE:Leveraging the heterogeneity of cancer-associated fibroblasts (CAFs) within the tumour microenvironment, this study aims to identify and evaluate candidate biomarkers to support patient stratification and improve prediction of therapeutic responses. DESIGN:We applied a multiomic approach integrating single-cell RNA sequencing, computational cell deconvolution and protein-level assessment from patient tumours, complemented by in vitro and in vivo preclinical models, to characterise stromal populations linked to CRC progression and treatment resistance. RESULTS:Retrospective analysis of over 3000 patient samples across multiple cohorts identified a distinct subset of CAFs expressing collagen triple helix repeat containing 1 (CTHRC1). CTHRC1(+) CAFs were associated with increased transforming growth factor-beta (TGF-beta) signalling and poor clinical outcomes in early and advanced disease stages. CTHRC1(+) CAFs enabled stratification of both mismatch repair-deficient/microsatellite instability (dMMR/MSI) and mismatch repair-proficient/microsatellite stability (pMMR/MSS) tumours into immune-inflamed and poorly immunogenic subtypes. Retrospective analysis of several clinical trials revealed that CTHRC1(+) CAFs are linked to resistance to immune checkpoint inhibitors in MSI and MSS tumours, suggesting therapeutic potential for combining TGF-beta blockade with immunotherapy. CONCLUSION:CTHRC1-expressing CAFs represent clinically relevant biomarkers that link molecular profiling with diagnostic pathology. Our findings support the potential incorporation of CTHRC1(+) CAF assessment into routine histopathological workflows, pending prospective validation, and suggest a framework for stroma-informed CRC stratification, particularly in patients with stroma-rich, treatment-resistant tumours and pMMR/MSS with limited therapeutic options.

Inhibitors of the oncogene KRAS hold promise for treating metastatic colorectal cancer (mCRC). In this study, we show that a selective, covalent small-molecule inhibitor of the active (ON) conformation of RAS-G12D, RMC-9945, exerts durable disease control in preclinical colorectal cancer models of early liver metastasis, but its therapeutic activity was diminished in the advanced metastatic disease. RMC-9945-treated metastases underwent a transition from a poor prognosis-associated Emp1+ transcriptional state to a WNT-driven Lgr5+ stem cell-like state that withstands the absence of RAS-G12D activity. This cell state change occurred within hours of RAS(ON) inhibitor treatment through a shift in transcription factor usage that involved limited chromatin remodeling. Forced conversion of metastatic cells to the Lgr5+ state through RAS-G12D inhibition, followed by genetic ablation of this population, reduced metastatic burden and prolonged survival in a mouse mCRC model. Overall, these preclinical findings demonstrate a central role for oncogenic KRAS in governing cellular plasticity in mCRC. SIGNIFICANCE:We show that inhibition of oncogenic KRAS in preclinical models of advanced mCRC exerts a limited benefit, primarily due to the reversion of tumor cells to a stem cell-like state. Our findings highlight the context-dependent effects of oncogenic KRAS mutations and underscore cell plasticity as a therapeutic opportunity. See related commentary by Eng and Yilmaz et al., p. 201.

Transforming growth factor β (TGF-β) signaling in the tumor microenvironment predicts resistance to immune checkpoint blockade (ICB). While TGF-β inhibition enhances ICB efficacy in murine cancer models, clinical trials have yet to demonstrate benefit, underscoring the need to better understand its immunoregulatory roles across disease contexts. Using mouse models of advanced colorectal cancer and patient-derived data, we demonstrate that TGF-β impairs antitumor immunity at multiple levels in liver metastases. It acts directly on T cells to block recruitment of peripheral memory CD8+ T cells, thereby limiting the effectiveness of ICB. Concurrently, TGF-β instructs tumor-associated macrophages to suppress clonal expansion of newly arrived T cells by inducing SPP1 expression. This extracellular matrix protein promotes collagen deposition and accumulation of tumor-associated macrophages and fibroblasts, ultimately driving ICB resistance. Our findings reveal how TGF-β coordinates immunosuppressive mechanisms across innate and adaptive immune compartments to promote metastasis, offering new avenues to improve immunotherapy in colorectal cancer.

Olfactory sensory neurons (OSNs) use olfactory receptor (OR)-specific patterns of endoplasmic reticulum (ER) stress to transform OR sequence identity into axon guidance precision. However, during neuronal differentiation, OSNs transiently co-express random combinations of OR genes, which could generate unpredictable ER stress signatures that could lead to axon miswiring. Here, we show that post-transcriptional OR silencing by the transiently expressed and cytoplasmic RING and KH domain protein Mex3a, decouples OR transcription from OR protein-induced ER stress, until the onset of singular OR transcription. Consequently, conditional Mex3a deletion results in premature ER stress during the polygenic stage of OR transcription, which biases OR choice toward the OR alleles that are transcribed first and perturbs the specificity of OR-regulated axon targeting, disrupting the glomerular map of odor representation in the olfactory bulb. Our experiments reveal the critical role of post-transcriptional gene regulation in a fundamental cellular pathway that influences the assembly of neuronal circuits.

Intestinal epithelial cells are segregated into proliferative crypts and differentiated regions. This organization relies on specific signals, including Wnt3a, which regulates cell proliferation within crypts, and Eph/Ephrin, which dictates cell positioning along the crypt-villus axis. However, studying how the spatial distributions of these signals influences crypt-villus organization is challenging both in vitro and in vivo. Here we show that micropatterns of Wnt3a can govern the size, shape and long-range organization of crypts in vitro. By adjusting the spacing between Wnt3a ligand patterns at the microscale over large surfaces, we override endogenous Wnt3a to precisely control the distribution and long-range order of crypt-like regions in primary epithelial monolayers. Additionally, an agent-based model integrating Wnt3a/BMP feedback and Eph/Ephrin repulsion effectively replicates experimental tissue compartmentalization, crypt size, shape, and organization. This combined experimental and computational approach offers a framework to study how signaling pathways help organize intestinal epithelial tissue.

Colorectal cancers (CRCs) present across a range of differentiation grades, which impact patient outcome and management; however, the molecular features and drivers of differentiation status are not fully understood. To address this, 84 commonly used human CRC cell lines were grown as xenografts in mice, revealing models of low-grade (LG) and high-grade (HG) CRC. Transcriptional profiling revealed coordinate downregulation of multiple transcription factors involved in intestinal development and differentiation, markers of colonic lineage-specific differentiation, and effectors of normal functions of the colonic epithelium in HG tumours. Mechanistically, multiple genes suppressed in HG tumours harboured promoter methylation, indicative of stable epigenetic silencing. Furthermore, markers of LGR5+ colon stem cells were suppressed in HG tumours, while markers of cell proliferation, fetal-like intestinal stem cells, and non-canonical cell types including mesenchymal cells were increased. These changes manifested in HG cell line displaying increased proliferation, migration and metastatic capacity. Importantly, CRC cell line-derived transcriptional profiles of differentiation grade were reflected in LG and HG patient-derived tumour organoids and primary CRCs, revealing cell lines accurately model differentiation grade. The models and tumour differentiation-related properties identified herein may inform new approaches for tailored CRC treatments based on tumour grade.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Only a subset of patients treated with immune checkpoint inhibitors (CPIs) respond to the treatment, and distinguishing responders from non-responders is a major challenge. Many proposed biomarkers of CPI response and survival probably represent alternative measurements of the same aspects of the tumor, its microenvironment or the host. Thus, we currently ignore how many truly independent biomarkers there are. With an unbiased analysis of genomics, transcriptomics and clinical data of a cohort of patients with metastatic tumors (n = 479), we discovered five orthogonal latent factors: tumor mutation burden, T cell effective infiltration, transforming growth factor-beta activity in the microenvironment, prior treatment and tumor proliferative potential. Their association with CPI response and survival was observed across all tumor types and validated across six independent cohorts (n = 1,491). These five latent factors constitute a frame of reference to organize current and future knowledge on biomarkers of CPI response and survival.

Despite extensive research and improvements in understanding colorectal cancer (CRC), its metastatic form continues to pose a substantial challenge, primarily owing to limited therapeutic options and a poor prognosis. This Review addresses the emerging focus on metastatic CRC (mCRC), which has historically been under-studied compared with primary CRC despite its lethality. We delve into two crucial aspects: the molecular and cellular determinants facilitating CRC metastasis and the principles guiding the evolution of metastatic disease. Initially, we examine the genetic alterations integral to CRC metastasis, connecting them to clinically marked characteristics of advanced CRC. Subsequently, we scrutinize the role of cellular heterogeneity and plasticity in metastatic spread and therapy resistance. Finally, we explore how the tumour microenvironment influences metastatic disease, emphasizing the effect of stromal gene programmes and the immune context. The ongoing research in these fields holds immense importance, as its future implications are projected to revolutionize the treatment of patients with mCRC, hopefully offering a promising outlook for their survival.

The emergence of drug resistance is the most substantial challenge to the effectiveness of anticancer therapies. Orthogonal approaches have revealed that a subset of cells, known as drug-tolerant 'persister' (DTP) cells, have a prominent role in drug resistance. Although long recognized in bacterial populations which have acquired resistance to antibiotics, the presence of DTPs in various cancer types has come to light only in the past two decades, yet several aspects of their biology remain enigmatic. Here, we delve into the biological characteristics of DTPs and explore potential strategies for tracking and targeting them. Recent findings suggest that DTPs exhibit remarkable plasticity, being capable of transitioning between different cellular states, resulting in distinct DTP phenotypes within a single tumour. However, defining the biological features of DTPs has been challenging, partly due to the complex interplay between clonal dynamics and tissue-specific factors influencing their phenotype. Moreover, the interactions between DTPs and the tumour microenvironment, including their potential to evade immune surveillance, remain to be discovered. Finally, the mechanisms underlying DTP-derived drug resistance and their correlation with clinical outcomes remain poorly understood. This Roadmap aims to provide a comprehensive overview of the field of DTPs, encompassing past achievements and current endeavours in elucidating their biology. We also discuss the prospect of future advancements in technologies in helping to unveil the features of DTPs and propose novel therapeutic strategies that could lead to their eradication.

Blockade of the TGFβ signalling 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 known 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 favourable safety profile. HYL001 in combination with immune checkpoint blockade (anti-PD1) eradicates liver metastases generated in mice by microsatellite stable, aggressive colorectal cancer tumours at doses where galunisertib is ineffective. ![Figure][1] ### Competing Interest Statement 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. [1]: pending:yes

Colorectal cancer tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is unknown. Using CRC patient derived organoids (PDOs), we found that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing scRNA-seq expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

Supplementary Materials and Methods