cDC2 plasticity and acquisition of a DC3-like phenotype mediated by IL-6 and PGE2 in a patient-derived colorectal cancer organoids model.

Metastatic colorectal cancer (CRC) is highly resistant to therapy and prone to recur. The tumor-induced local and systemic immunosuppression allows cancer cells to evade immunosurveillance, facilitating their proliferation and dissemination. Dendritic cells (DCs) are required for the detection, processing, and presentation of tumor antigens, and subsequently for the activation of antigen-specific T cells to orchestrate an effective antitumor response. Notably, successful tumors have evolved mechanisms to disrupt and impair DC functions, underlining the key role of tumor-induced DC dysfunction in promoting tumor growth, metastasis initiation, and treatment resistance. Conventional DC type 2 (cDC2) are highly prevalent in tumors and have been shown to present high phenotypic and functional plasticity in response to tumor-released environmental cues. This plasticity reverberates on both the development of antitumor responses and on the efficacy of immunotherapies in cancer patients. Uncovering the processes, mechanisms, and mediators by which CRC shapes and disrupts cDC2 functions is crucial to restoring their full antitumor potential. In this study, we use our recently developed 3D DC-tumor co-culture system to investigate how patient-derived primary and metastatic CRC organoids modulate cDC2 phenotype and function. We first demonstrate that our collagen-based system displays extensive interaction between cDC2 and tumor organoids. Interestingly, we show that tumor-corrupted cDC2 shift toward a CD14+ population with defective expression of maturation markers, an intermediate phenotype positioned between cDC2 and monocytes, and impaired T-cell activating abilities. This phenotype aligns with the newly defined DC3 (CD14+ CD1c+ CD163+) subset. Remarkably, a comparable population was found to be present in tumor lesions and enriched in the peripheral blood of metastatic CRC patients. Moreover, using EP2 and EP4 receptor antagonists and an anti-IL-6 neutralizing antibody, we determined that the observed phenotype shift is partially mediated by PGE2 and IL-6. Importantly, our system holds promise as a platform for testing therapies aimed at preventing or mitigating tumor-induced DC dysfunction. Overall, our study offers novel and relevant insights into cDC2 (dys)function in CRC that hold relevance for the design of therapeutic approaches.

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.

Targeting CAFs to Improve Anti-PD-1 Checkpoint Immunotherapy.

The tumor microenvironment is a complex ecosystem that drives cancer progression and restrains immunity. Although immune checkpoint inhibitors have shown strong potential in a subset of patients, a better understanding of suppressive mechanisms may inspire ways to improve immunotherapeutic efficacy. A new study in this issue of Cancer Research focuses on targeting cancer-associated fibroblasts in preclinical models of gastric tumors. Aiming to rebalance anticancer immunity and enhance treatment responses to checkpoint-blocking antibodies, this work also addresses the potential for multitarget tyrosine kinase inhibitors in treating gastrointestinal cancer. See related article by Akiyama et al., p. 753.

Dendritic cell phenotype and function in a 3D co-culture model of patient-derived metastatic colorectal cancer organoids.

Colorectal cancer (CRC) remains one of the most aggressive and lethal cancers, with metastasis accounting for most deaths. As such, there is an unmet need for improved therapies for metastatic CRC (mCRC). Currently, the research focus is shifting towards the reciprocal interactions within the tumor microenvironment (TME), which prevent tumor clearance by the immune system. Dendritic cells (DCs) play a key role in the initiation and amplification of anti-tumor immune responses and in driving the clinical success of immunotherapies. Dissecting the interactions between DCs and CRC cells may open doors to identifying key mediators in tumor progression, and possible therapeutic targets. This requires representative, robust and versatile models and tools. Currently, there is a shortage of such in vitro systems to model the CRC TME and its tumor-immune cell interactions. Here we develop and establish a dynamic organotypic 3D co-culture system to recapitulate and untangle the interactions between DCs and patient-derived mCRC tumor organoids. To our knowledge, this is the first study investigating human DCs in co-culture with tumor organoids in a 3D, organotypic setting. This system reveals how mCRC organoids modulate and shape monocyte-derived DCs (MoDCs) behavior, phenotype, and function, within a collagen matrix, using techniques such as brightfield and fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting. Our 3D co-culture model shows high viability and extensive interaction between DCs and tumor organoids, and its structure resembles patient tissue sections. Furthermore, it is possible to retrieve DCs from the co-cultures and characterize their phenotypic and functional profile. In our study, the expression of activation markers in both mature and immature DCs and their ability to activate T cells were impacted by co-culture with tumor organoids. In the future, this direct co-culture platform can be adapted and exploited to study the CRC-DC interplay in more detail, enabling novel and broader insights into CRC-driven DC (dys)function.

Multiomics of Colorectal Cancer Organoids Reveals Putative Mediators of Cancer Progression Resulting from SMAD4 Inactivation.

The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Although loss of SMAD4 is a key event in CRC progression, the resulting changes in biological processes in advanced disease and metastasis are not fully understood. Here, we applied a multiomics approach to a CRC organoid model that faithfully reflects the metastasis-supporting effects of SMAD4 inactivation. We show that loss of SMAD4 results in decreased differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFß, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by SMAD4-mutant organoids─DKK3─reduces the antitumor effects of natural killer cells (NK cells). Altogether, our data provide new insights into the role of SMAD4 perturbation in advanced CRC.

High-Dose Intermittent Treatment with the Multikinase Inhibitor Sunitinib Leads to High Intra-Tumor Drug Exposure in Patients with Advanced Solid Tumors.

Patients with advanced cancer refractory to standard treatment were treated with sunitinib at a dose of 300 mg once every week (Q1W) or 700 mg once every two weeks (Q2W). Tumor, skin and plasma concentrations were measured and immunohistochemical staining for tumor cell proliferation (TCP), microvessel density (MVD) and T-cell infiltration was performed on tumor biopsies before and after 17 days of treatment. Oral administration of 300 mg sunitinib Q1W or 700 mg Q2W resulted in 19-fold (range 5-35×) and 37-fold higher (range 10-88×) tumor drug concentrations compared to parallel maximum plasma drug concentrations, respectively. Patients with higher tumor sunitinib concentrations had favorable progression-free and overall survival than those with lower concentrations (p = 0.046 and 0.024, respectively). In addition, immunohistochemistry of tumor biopsies revealed an induction of T-cell infiltration upon treatment. These findings provide pharmacological and biological insights in the clinical benefit from high-dose intermittent sunitinib treatment. It emphasizes the potential benefit from reaching higher tumor drug concentrations and the value of measuring TKI tumor- over plasma-concentrations. The finding that reaching higher tumor drug concentrations provides most clinical benefit in patients with treatment refractory malignancies indicates that the inhibitory potency of sunitinib may be enforced by a high-dose intermittent treatment schedule. These results provide proof of concept for testing other clinically available multitargeted tyrosine kinase inhibitors in a high-dose intermittent treatment schedule.

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.

Lost in translation: Revisiting the use of tyrosine kinase inhibitors in colorectal cancer.

Patients with advanced or metastatic colorectal cancer ((m)CRC) have limited effective treatment options resulting in high mortality rates. A better understanding of the molecular basis of this disease has led to growing interest in small molecule tyrosine kinase inhibitors (TKIs) for its treatment. However, of around 42 TKIs demonstrating preclinical anti-tumour activity, and despite numerous clinical trials, only 1 has been approved for clinical use in mCRC. Clearly, there is a huge gap in the translation of these targeted therapies to the clinic. This underlines the limitations of preclinical models to predict clinical drug efficacy and to fully characterize the mechanism of action. Moreover, several relevant topics remain poorly resolved. Do we know the actual intracellular concentrations that are required for anticancer efficacy, and what range of intra-tumoral drug concentrations is reached in clinical setting? Are the intended targeted kinases responsible for the anti-cancer activity or are other unexpected cellular targets involved? Do we have any idea of the effect of these drugs on the tumour microenvironment and does this help explain therapy success, failure or heterogeneity? In this review, we address these questions and discuss concepts that jointly complicate the clinical translation of TKIs for CRC. Finally, we will argue that an integrated approach with more sophisticated preclinical models and techniques may improve the prediction of clinical treatment efficacy.

Mutant RAS and the tumor microenvironment as dual therapeutic targets for advanced colorectal cancer.

RAS genes are the most frequently mutated oncogenes in cancer. These mutations occur in roughly half of the patients with colorectal cancer (CRC). RAS mutant tumors are resistant to therapy with anti-EGFR monoclonal antibodies. Therefore, patients with RAS mutant CRC currently have few effective therapy options. RAS mutations lead to constitutively active RAS GTPases, involved in multiple downstream signaling pathways. These alterations are associated with a tumor microenvironment (TME) that drives immune evasion and disease progression by mechanisms that remain incompletely understood. In this review, we focus on the available evidence in the literature explaining the potential effects of RAS mutations on the CRC microenvironment. Ongoing efforts to influence the TME by targeting mutant RAS and thereby sensitizing these tumors to immunotherapy will be discussed as well.

Mex3a marks drug-tolerant persister colorectal cancer cells that mediate relapse after chemotherapy.

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.

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.

Overcoming TGFß-mediated immune evasion in cancer.

Transforming growth factor-ß (TGFß) signalling controls multiple cell fate decisions during development and tissue homeostasis; hence, dysregulation of this pathway can drive several diseases, including cancer. Here we discuss the influence that TGFß exerts on the composition and behaviour of different cell populations present in the tumour immune microenvironment, and the context-dependent functions of this cytokine in suppressing or promoting cancer. During homeostasis, TGFß controls inflammatory responses triggered by exposure to the outside milieu in barrier tissues. Lack of TGFß exacerbates inflammation, leading to tissue damage and cellular transformation. In contrast, as tumours progress, they leverage TGFß to drive an unrestrained wound-healing programme in cancer-associated fibroblasts, as well as to suppress the adaptive immune system and the innate immune system. In consonance with this key role in reprogramming the tumour microenvironment, emerging data demonstrate that TGFß-inhibitory therapies can restore cancer immunity. Indeed, this approach can synergize with other immunotherapies - including immune checkpoint blockade - to unleash robust antitumour immune responses in preclinical cancer models. Despite initial challenges in clinical translation, these findings have sparked the development of multiple therapeutic strategies that inhibit the TGFß pathway, many of which are currently in clinical evaluation.

The Therapeutic Potential of Tackling Tumor-Induced Dendritic Cell Dysfunction in Colorectal Cancer.

Colorectal cancer (CRC) is the third most diagnosed malignancy and the second leading cause of cancer-related deaths worldwide. Locally advanced and metastatic disease exhibit resistance to therapy and are prone to recurrence. Despite significant advances in standard of care and targeted (immuno)therapies, the treatment effects in metastatic CRC patients have been modest. Untreatable cancer metastasis accounts for poor prognosis and most CRC deaths. The generation of a strong immunosuppressive tumor microenvironment (TME) by CRC constitutes a major hurdle for tumor clearance by the immune system. Dendritic cells (DCs), often impaired in the TME, play a critical role in the initiation and amplification of anti-tumor immune responses. Evidence suggests that tumor-mediated DC dysfunction is decisive for tumor growth and metastasis initiation, as well as for the success of immunotherapies. Unravelling and understanding the complex crosstalk between CRC and DCs holds promise for identifying key mechanisms involved in tumor progression and spread that can be exploited for therapy. The main goal of this review is to provide an overview of the current knowledge on the impact of CRC-driven immunosuppression on DCs phenotype and functionality, and its significance for disease progression, patient prognosis, and treatment response. Moreover, present knowledge gaps will be highlighted as promising opportunities to further understand and therapeutically target DC dysfunction in CRC. Given the complexity and heterogeneity of CRC, future research will benefit from the use of patient-derived material and the development of in vitro organoid-based co-culture systems to model and study DCs within the CRC TME.

Stromal SOX2 Upregulation Promotes Tumorigenesis through the Generation of a SFRP1/2-Expressing Cancer-Associated Fibroblast Population.

Cancer-associated fibroblasts (CAFs) promote tumor malignancy, but the precise transcriptional mechanisms regulating the acquisition of the CAF phenotype are not well understood. We show that the upregulation of SOX2 is central to this process, which is repressed by protein kinase Cζ (PKCζ). PKCζ deficiency activates the reprogramming of colonic fibroblasts to generate a predominant SOX2-dependent CAF population expressing the WNT regulator Sfrp2 as its top biomarker. SOX2 directly binds the Sfrp1/2 promoters, and the inactivation of Sox2 or Sfrp1/2 in CAFs impaired the induction of migration and invasion of colon cancer cells, as well as their tumorigenicity in vivo. Importantly, recurrence-free and overall survival of colorectal cancer (CRC) patients negatively correlates with stromal PKCζ levels. Also, SOX2 expression in the stroma is associated with CRC T invasion and worse prognosis of recurrence-free survival. Therefore, the PKCζ-SOX2 axis emerges as a critical step in the control of CAF pro-tumorigenic potential.

Combinatorial Immunotherapies for Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is one of the most frequent and deadly forms of cancer. About half of patients are affected by metastasis, with the cancer spreading to e.g., liver, lungs or the peritoneum. The majority of these patients cannot be cured despite steady advances in treatment options. Immunotherapies are currently not widely applicable for this disease, yet show potential in preclinical models and clinical translation. The tumour microenvironment (TME) has emerged as a key factor in CRC metastasis, including by means of immune evasion-forming a major barrier to effective immuno-oncology. Several approaches are in development that aim to overcome the immunosuppressive environment and boost anti-tumour immunity. Among them are vaccination strategies, cellular transplantation therapies, and targeted treatments. Given the complexity of the system, we argue for rational design of combinatorial therapies and consider the implications of precision medicine in this context.

Simultaneous Loss of Both Atypical Protein Kinase C Genes in the Intestinal Epithelium Drives Serrated Intestinal Cancer by Impairing Immunosurveillance.

Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.

TMEM59 potentiates Wnt signaling by promoting signalosome formation.

Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.

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.

Determinants of metastatic competency in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancer types and represents a major therapeutic challenge. Although initial events in colorectal carcinogenesis are relatively well characterized and treatment for early-stage disease has significantly improved over the last decades, the mechanisms underlying metastasis - the main cause of death - remain poorly understood. Correspondingly, no effective therapy is currently available for advanced or metastatic disease. There is increasing evidence that colorectal cancer is hierarchically organized and sustained by cancer stem cells, in concert with various stromal cell types. Here, we review the interplay between cancer stem cells and their microenvironment in promoting metastasis and discuss recent insights relating to both patient prognosis and novel targeted treatment strategies. A better understanding of these topics may aid the prevention or reduction of metastatic burden.