Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

Transforming Growth Factor-ß Signaling in Immunity and Cancer.

Transforming growth factor (TGF)-ß is a crucial enforcer of immune homeostasis and tolerance, inhibiting the expansion and function of many components of the immune system. Perturbations in TGF-ß signaling underlie inflammatory diseases and promote tumor emergence. TGF-ß is also central to immune suppression within the tumor microenvironment, and recent studies have revealed roles in tumor immune evasion and poor responses to cancer immunotherapy. Here, we present an overview of the complex biology of the TGF-ß family and its context-dependent nature. Then, focusing on cancer, we discuss the roles of TGF-ß signaling in distinct immune cell types and how this knowledge is being leveraged to unleash the immune system against the tumor.

Metastatic recurrence in colorectal cancer arises from residual EMP1+ cells.

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. 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 cells2-4, 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 EMP1high 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.

Antitumor T-cell function requires CPEB4-mediated adaptation to chronic endoplasmic reticulum stress.

Tumor growth is influenced by a complex network of interactions between multiple cell types in the tumor microenvironment (TME). These constrained conditions trigger the endoplasmic reticulum (ER) stress response, which extensively reprograms mRNA translation. When uncontrolled over time, chronic ER stress impairs the antitumor effector function of CD8 T lymphocytes. How cells promote adaptation to chronic stress in the TME without the detrimental effects of the terminal unfolded protein response (UPR) is unknown. Here, we find that, in effector CD8 T lymphocytes, RNA-binding protein CPEB4 constitutes a new branch of the UPR that allows cells to adapt to sustained ER stress, yet remains decoupled from the terminal UPR. ER stress, induced during CD8 T-cell activation and effector function, triggers CPEB4 expression. CPEB4 then mediates chronic stress adaptation to maintain cellular fitness, allowing effector molecule production and cytotoxic activity. Accordingly, this branch of the UPR is required for the antitumor effector function of T lymphocytes, and its disruption in these cells exacerbates tumor growth.

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction.

BACKGROUND & AIMS: The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo. METHODS: To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. RESULTS: We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. CONCLUSIONS: Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. IMPACT AND IMPLICATIONS: Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

A single-cell tumor immune atlas for precision oncology.

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients, and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing published data sets of >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying colocalization patterns of immune, stromal, and cancer cells in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immunotherapy.

TGFß drives immune evasion in genetically reconstituted colon cancer metastasis.

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 (TH1) activity and reduced immune cytotoxicity or increased TGFß 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 TGFß-activated stroma. Inhibition of the PD-1-PD-L1 immune checkpoint provoked a limited response in this model system. By contrast, inhibition of TGFß unleashed a potent and enduring cytotoxic T-cell response against tumour cells that prevented metastasis. In mice with progressive liver metastatic disease, blockade of TGFß signalling rendered tumours susceptible to anti-PD-1-PD-L1 therapy. Our data show that increased TGFß in the tumour microenvironment represents a primary mechanism of immune evasion that promotes T-cell exclusion and blocks acquisition of the TH1-effector phenotype. Immunotherapies directed against TGFß signalling may therefore have broad applications in treating patients with advanced colorectal cancer.

MmCMS: mouse models' consensus molecular subtypes of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODS: Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTS: We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONS: When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.

The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA.

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3'UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3'UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A-eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5'-3' end interaction, mediated by both viral and cellular proteins.

Long-lived force patterns and deformation waves at repulsive epithelial boundaries.

For an organism to develop and maintain homeostasis, cell types with distinct functions must often be separated by physical boundaries. The formation and maintenance of such boundaries are commonly attributed to mechanisms restricted to the cells lining the boundary. Here we show that, besides these local subcellular mechanisms, the formation and maintenance of tissue boundaries involves long-lived, long-ranged mechanical events. Following contact between two epithelial monolayers expressing, respectively, EphB2 and its ligand ephrinB1, both monolayers exhibit oscillatory patterns of traction forces and intercellular stresses that tend to pull cell-matrix adhesions away from the boundary. With time, monolayers jam, accompanied by the emergence of deformation waves that propagate away from the boundary. This phenomenon is not specific to EphB2/ephrinB1 repulsion but is also present during the formation of boundaries with an inert interface and during fusion of homotypic epithelial layers. Our findings thus unveil a global physical mechanism that sustains tissue separation independently of the biochemical and mechanical features of the local tissue boundary.

Alterations in the epithelial stem cell compartment could contribute to permanent changes in the mucosa of patients with ulcerative colitis.

OBJECTIVE: UC is a chronic inflammatory disease of the colonic mucosa. Growing evidence supports a role for epithelial cell defects in driving pathology. Moreover, long-lasting changes in the epithelial barrier have been reported in quiescent UC. Our aim was to investigate whether epithelial cell defects could originate from changes in the epithelial compartment imprinted by the disease. DESIGN: Epithelial organoid cultures (EpOCs) were expanded ex vivo from the intestinal crypts of non-IBD controls and patients with UC. EpOCs were induced to differentiate (d-EpOCs), and the total RNA was extracted for microarray and quantitative real-time PCR (qPCR) analyses. Whole intestinal samples were used to determine mRNA expression by qPCR, or protein localisation by immunostaining. RESULTS: EpOCs from patients with UC maintained self-renewal potential and the capability to give rise to differentiated epithelial cell lineages comparable with control EpOCs. Nonetheless, a group of genes was differentially regulated in the EpOCs and d-EpOCs of patients with UC, including genes associated with antimicrobial defence (ie, LYZ, PLA2G2A), with secretory (ie, ZG16, CLCA1) and absorptive (ie, AQP8, MUC12) functions, and with a gastric phenotype (ie, ANXA10, CLDN18 and LYZ). A high rate of concordance was found in the expression profiles of the organoid cultures and whole colonic tissues from patients with UC. CONCLUSIONS: Permanent changes in the colonic epithelium of patients with UC could be promoted by alterations imprinted in the stem cell compartment. These changes may contribute to perpetuation of the disease.

The circadian molecular clock creates epidermal stem cell heterogeneity.

Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.

Inosine modifications in human tRNAs are incorporated at the precursor tRNA level.

Transfer RNAs (tRNAs) are key adaptor molecules of the genetic code that are heavily modified post-transcriptionally. Inosine at the first residue of the anticodon (position 34; I34) is an essential widespread tRNA modification that has been poorly studied thus far. The modification in eukaryotes results from a deamination reaction of adenine that is catalyzed by the heterodimeric enzyme adenosine deaminase acting on tRNA (hetADAT), composed of two subunits: ADAT2 and ADAT3. Using high-throughput small RNA sequencing (RNAseq), we show that this modification is incorporated to human tRNAs at the precursor tRNA level and during maturation. We also functionally validated the human genes encoding for hetADAT and show that the subunits of this enzyme co-localize in nucleus in an ADAT2-dependent manner. Finally, by knocking down HsADAT2, we demonstrate that variations in the cellular levels of hetADAT will result in changes in the levels of I34 modification in all its potential substrates. Altogether, we present RNAseq as a powerful tool to study post-transcriptional tRNA modifications at the precursor tRNA level and give the first insights on the biology of I34 tRNA modification in metazoans.

Iro/IRX transcription factors negatively regulate Dpp/TGF-ß pathway activity during intestinal tumorigenesis.

Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces "tumor-like" overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-ß acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-ß. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-ß. In addition, Irx5 expression confers a growth advantage in the presence of TGF-ß, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-ß activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-ß, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-ß pathway components.

Eph-ephrin signalling in adult tissues and cancer.

Eph receptor tyrosine kinases and their ligands, the ephrins, play key roles in the regulation of migration and cell adhesion during development, thereby influencing cell fate, morphogenesis and organogenesis. Recent findings suggest that Eph signalling also controls the architecture and physiology of different tissues in the adult body under normal and pathological conditions such as cancer. A prime example is the intestinal epithelium where EphB-ephrinB interactions regulate both cell positioning and tumor progression. Here, we will review recent advances on the role of Eph-ephrin signalling in the intestine and other organs.

Mechanisms of metastatic colorectal cancer.

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.

Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer.

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.

Five latent factors underlie response to immunotherapy.

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.

Mutant TP53 switches therapeutic vulnerability during gastric cancer progression within interleukin-6 family cytokines.

Although aberrant activation of the KRAS and PI3K pathway alongside TP53 mutations account for frequent aberrations in human gastric cancers, neither the sequence nor the individual contributions of these mutations have been clarified. Here, we establish an allelic series of mice to afford conditional expression in the glandular epithelium of KrasG12D;Pik3caH1047R or Trp53R172H and/or ablation of Pten or Trp53. We find that KrasG12D;Pik3caH1047R is sufficient to induce adenomas and that lesions progress to carcinoma when also harboring Pten deletions. An additional challenge with either Trp53 loss- or gain-of-function alleles further accelerated tumor progression and triggered metastatic disease. While tumor-intrinsic STAT3 signaling in response to gp130 family cytokines remained as a gatekeeper for all stages of tumor development, metastatic progression required a mutant Trp53-induced interleukin (IL)-11 to IL-6 dependency switch. Consistent with the poorer survival of patients with high IL-6 expression, we identify IL-6/STAT3 signaling as a therapeutic vulnerability for TP53-mutant gastric cancer.