Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment.

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ T cell phenotypes. In vivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ T cells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

Activation of innate-adaptive immune machinery by poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer.

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.

Stromal Cells Promote Matrix Deposition, Remodelling and an Immunosuppressive Tumour Microenvironment in a 3D Model of Colon Cancer.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC develops in a complex tumour microenvironment (TME) with both mesenchymal stromal cells (MSCs) and immune infiltrate, shown to alter disease progression and treatment response. We hypothesised that an accessible, affordable model of CRC that combines multiple cell types will improve research translation to the clinic and enable the identification of novel therapeutic targets. A viable gelatine-methacrloyl-based hydrogel culture system that incorporates CRC cells with MSCs and a monocyte cell line was developed. Gels were analysed on day 10 by PCR, cytokine array, microscopy and flow cytometry. The addition of stromal cells increased transcription of matrix remodelling proteins FN1 and MMP9, induced release of tumour-promoting immune molecules MIF, Serpin E1, CXCL1, IL-8 and CXCL12 and altered cancer cell expression of immunotherapeutic targets EGFR, CD47 and PD-L1. Treatment with PD153035, an EGFR inhibitor, revealed altered CRC expression of PD-L1 but only in gels lacking MSCs. We established a viable 3D model of CRC that combined cancer cells, MSCs and monocytic cells that can be used to research the role the stroma plays in the TME, identify novel therapeutic targets and improve the transitional efficacy of therapies.

Cyclophosphamide alters the tumor cell secretome to potentiate the anti-myeloma activity of daratumumab through augmentation of macrophage-mediated antibody dependent cellular phagocytosis.

Multiple Myeloma (MM) is a malignant disorder of plasma cells which, despite significant advances in treatment, remains incurable. Daratumumab, the first CD38 directed monoclonal antibody, has shown promising activity alone and in combination with other agents for MM treatment. Daratumumab is thought to have pleiotropic mechanisms of activity including natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). With the knowledge that CD38-expressing NK cells are depleted by daratumumab, we sought to investigate a potential mechanism of enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) by combining daratumumab with cyclophosphamide (CTX). Cyclophosphamide's immunomodulatory function was investigated by conditioning macrophages with tumor cell secretome collected from cyclophosphamide treated MM cell lines (CTX-TCS). Flow cytometry analysis revealed that CTX-TCS conditioning augmented the migratory capacity of macrophages and increased CD32 and CD64 Fcγ receptor expression on their cell surface. Daratumumab-specific tumor clearance was increased by conditioning macrophages with CTX-TCS in a dose-dependent manner. This effect was impeded by pre-incubating macrophages with Cytochalasin D (CytoD), an inhibitor of actin polymerization, indicating macrophage-mediated ADCP as the mechanism of clearance. CD64 expression on macrophages directly correlated with MM cell clearance and was essential to the observed synergy between cyclophosphamide and daratumumab, as tumor clearance was attenuated in the presence of a FcγRI/CD64 blocking agent. Cyclophosphamide independently enhances daratumumab-mediated killing of MM cells by altering the tumor microenvironment to promote macrophage recruitment, polarization to a pro-inflammatory phenotype, and directing ADCP. These findings support the addition of cyclophosphamide to existing or novel monoclonal antibody-containing MM regimens.

A 3D View of Colorectal Cancer Models in Predicting Therapeutic Responses and Resistance.

Although there have been many advances in recent years for the treatment of colorectal cancer (CRC), it still remains the third most common cause of cancer-related deaths worldwide. Many patients with late stage CRC display resistance to multiple different therapeutics. An important aspect in developing effective therapeutics for CRC patients is understanding the interactions that take place in the tumor microenvironment (TME), as it has been shown to contribute to drug resistance in vivo. Much research over the past 100 years has focused on 2D monolayer cultures or in vivo studies, however, the efficacy in translating these to the clinic is very low. More recent studies are turning towards developing an effective 3D model of CRC that is clinically relevant, that can recapitulate the TME in vitro and bridge the gap between 2D cultures and in vivo studies, with the aim of reducing the use of animal models in the future. This review summarises the advantages and limitations of different 3D CRC models. It emphasizes how different 3D models may be optimised to study cellular and extracellular interactions that take place in the TME of CRC in an effort to allow the development of more translatable effective treatment options for patients.

Stromal Cell PD-L1 Inhibits CD8+ T-cell Antitumor Immune Responses and Promotes Colon Cancer.

Stromal cells of mesenchymal origin reside below the epithelial compartment and provide structural support in the intestine. These intestinal stromal cells interact with both the epithelial cell compartments, as well as infiltrating hematopoietic immune cells. The importance of these cells in regulating immune homeostasis during inflammation is well recognized. However, little is known about their function and phenotype in the inflammatory tumor microenvironment. Using a syngeneic, immunogenic model of colorectal cancer, we showed that TNFα-initiated inflammatory signaling in CT26 colorectal cancer cells selectively induced PD-L1 expression in stromal cells. Using CD274 shRNA and antibody-mediated approaches, we showed that stromal cell PD-L1 potentiated enhanced immunosuppression, characterized by inhibition of activated CD8+ granzyme B-secreting T cells in vitro, and the inhibition of CD8+ effector cells was associated with enhanced tumor progression. Stromal cell immunosuppressive and tumor-promoting effects could be reversed with administration of anti-PD-1 in vivo We validated our findings of stromal cell CD274 expression in two cohorts of clinical samples and also observed PD-L1 induction on human stromal cells in response to exposure to the inflammatory secretome from human colon cancer cells, irrespective of microsatellite instability. Collectively, our data showed that tumor-associated stromal cells support T-cell suppression by PD-L1 induction, which is dependent on colon cancer inflammatory signaling. Our findings reveal a key role of mesenchymal stromal cells PD-L1 in suppression of CD8+ antitumor immune responses and potentiation of colorectal cancer progression. Cancer Immunol Res; 6(11); 1426-41. ©2018 AACR.