Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions.

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

Cancer-on-chip models for metastasis: importance of the tumor microenvironment.

Cancer-on-chip (CoC) models, based on microfluidic chips harboring chambers for 3D tumor-cell culture, enable us to create a controlled tumor microenvironment (TME). CoC models are therefore increasingly used to systematically study effects of the TME on the various steps in cancer metastasis. Moreover, CoC models have great potential for developing novel cancer therapies and for predicting patient-specific response to cancer treatments. We review recent developments in CoC models, focusing on three main TME components: (i) the anisotropic extracellular matrix (ECM) architectures, (ii) the vasculature, and (iii) the immune system. We aim to provide guidance to biologists to choose the best CoC approach for addressing questions about the role of the TME in metastasis, and to inspire engineers to develop novel CoC technologies.

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC is characterized by a desmoplastic, hypoxic and highly immunosuppressive tumor microenvironment (TME), where T-cell infiltration is often lacking (immune desert), or where T cells are located distant from the tumor islands (immune excluded). Converting the TME to an immune-inflamed state, allowing T-cell infiltration, could increase the success of immunotherapy in PDAC. METHOD: In this study, we use the KPC3 subcutaneous PDAC mouse model to investigate the role of tumor-derived sialic acids in shaping the tumor immune landscape. A sialic acid deficient KPC3 line was generated by genetic knock-out of the CMAS (cytidine monophosphate N-acetylneuraminic acid synthetase) enzyme, a critical enzyme in the synthesis of sialic acid-containing glycans. The effect of sialic acid-deficiency on immunotherapy efficacy was assessed by treatment with anti-programmed cell death protein 1 (PD-1) and agonistic CD40. RESULT: The absence of sialic acids in KPC3 tumors resulted in increased numbers of CD4+ and CD8+ T cells in the TME, and reduced frequencies of CD4+ regulatory T cells (Tregs) within the T-cell population. Importantly, CD8+ T cells were able to infiltrate the tumor islands in sialic acid-deficient tumors. These favorable alterations in the immune landscape sensitized sialic acid-deficient tumors to immunotherapy, which was ineffective in sialic acid-expressing KPC3 tumors. In addition, high expression of sialylation-related genes in human pancreatic cancer correlated with decreased CD8+ T-cell infiltration, increased presence of Tregs, and poorer survival probability. CONCLUSION: Our results demonstrate that tumor-derived sialic acids mediate T-cell exclusion within the PDAC TME, thereby impairing immunotherapy efficacy. Targeting sialic acids represents a potential strategy to enhance T-cell infiltration and improve immunotherapy outcomes in PDAC.

Immune function and dysfunction are determined by lymphoid tissue efficacy.

Lymphoid tissue returns to a steady state once each immune response is resolved, and although this occurs multiple times throughout life, its structural integrity and functionality remain unaffected. Stromal cells orchestrate cellular interactions within lymphoid tissue, and any changes to the microenvironment can have detrimental outcomes and drive disease. A breakdown in lymphoid tissue homeostasis can lead to a loss of tissue structure and function that can cause aberrant immune responses. This Review highlights recent advances in our understanding of lymphoid tissue function and remodelling in adaptive immunity and in disease states. We discuss the functional role of lymphoid tissue in disease progression and explore the changes to lymphoid tissue structure and function driven by infection, chronic inflammatory conditions and cancer. Understanding the role of lymphoid tissues in immune responses to a wide range of pathologies allows us to take a fuller systemic view of disease progression.

IRF8 is a transcriptional activator of CD37 expression in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Podoplanin drives dedifferentiation and amoeboid invasion of melanoma.

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilize amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by Rho GTPase signalling. Migrastatic drugs targeting actin polymerization and contractility are therefore a promising treatment option for metastatic melanoma. To predict amoeboid invasion and metastatic potential, biomarkers functionally linked to contractility pathways are needed. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts and is overexpressed in many cancers. We show that podoplanin enhances amoeboid invasion in melanoma. Podoplanin expression in murine melanoma drives rounded cell morphology, increasing motility, and invasion in vivo. Podoplanin expression is increased in a subset of dedifferentiated human melanoma, and in vitro is sufficient to upregulate melanoma-associated marker Pou3f2/Brn2. Together, our data define podoplanin as a functional biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

Fibroblastic reticular cell response to dendritic cells requires coordinated activity of podoplanin, CD44 and CD9.

In adaptive immunity, CLEC-2+ dendritic cells (DCs) contact fibroblastic reticular cells (FRCs) inhibiting podoplanin-dependent actomyosin contractility, permitting FRC spreading and lymph node expansion. The molecular mechanisms controlling lymph node remodelling are incompletely understood. We asked how podoplanin is regulated on FRCs in the early phase of lymph node expansion, and which other proteins are required for the FRC response to DCs. We find that podoplanin and its partner proteins CD44 and CD9 are differentially expressed by specific lymph node stromal populations in vivo, and their expression in FRCs is coregulated by CLEC-2 (encoded by CLEC1B). Both CD44 and CD9 suppress podoplanin-dependent contractility. We find that beyond contractility, podoplanin is required for FRC polarity and alignment. Independently of podoplanin, CD44 and CD9 affect FRC-FRC interactions. Furthermore, our data show that remodelling of the FRC cytoskeleton in response to DCs is a two-step process requiring podoplanin partner proteins CD44 and CD9. Firstly, CLEC-2 and podoplanin binding inhibits FRC contractility, and, secondly, FRCs form protrusions and spread, which requires both CD44 and CD9. Together, we show a multi-faceted FRC response to DCs, which requires CD44 and CD9 in addition to podoplanin.

Molecular mechanisms of dendritic cell migration in immunity and cancer.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells that act to bridge innate and adaptive immunity. DCs are critical in mounting effective immune responses to tissue damage, pathogens and cancer. Immature DCs continuously sample tissues and engulf antigens via endocytic pathways such as phagocytosis or macropinocytosis, which result in DC activation. Activated DCs undergo a maturation process by downregulating endocytosis and upregulating surface proteins controlling migration to lymphoid tissues where DC-mediated antigen presentation initiates adaptive immune responses. To traffic to lymphoid tissues, DCs must adapt their motility mechanisms to migrate within a wide variety of tissue types and cross barriers to enter lymphatics. All steps of DC migration involve cell-cell or cell-substrate interactions. This review discusses DC migration mechanisms in immunity and cancer with a focus on the role of cytoskeletal processes and cell surface proteins, including integrins, lectins and tetraspanins. Understanding the adapting molecular mechanisms controlling DC migration in immunity provides the basis for therapeutic interventions to dampen immune activation in autoimmunity, or to improve anti-tumour immune responses.

High frequency of inactivating tetraspanin C D37 mutations in diffuse large B-cell lymphoma at immune-privileged sites.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

C-type lectin-like receptor 2 (CLEC-2)-dependent dendritic cell migration is controlled by tetraspanin CD37.

Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 (Cd37-/-) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37-/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37-/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper.

Novel Insights into Membrane Targeting of B Cell Lymphoma.

Standard therapy of patients with B cell non-Hodgkin lymphoma (B-NHL) predominantly consists of chemotherapy combined with anti-CD20 (e.g., rituximab) immunotherapy. However, relapse of aggressive B-NHL occurs frequently, and this may coincide with therapy resistance. This demonstrates the urgent need for exploring new lymphoma-targeted therapies. We review here recent insights in the pathophysiology of B-NHL and discuss CD20 and three alternative membrane targets (B cell receptor, immune checkpoints PD-1/PD-L1, tetraspanin CD37) that are currently in the spotlight for B-NHL treatment. Furthermore, we present a novel concept in which the plasma membrane organization of the lymphoma B cell determines the efficacy of membrane-targeted therapies, and this has consequences for treatment application and clinical outcome in patients with B cell lymphoma.

Tetraspanin CD37 protects against the development of B cell lymphoma.

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.

Multispectral imaging reveals the tissue distribution of tetraspanins in human lymphoid organs.

Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20(+) cells in B cell follicles in human spleen and appendix. CD3(+) T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response.

Loading of acute myeloid leukemia cells with poly(I:C) by electroporation.

In this chapter, we describe the technique of electroporation as an efficient method to load primary leukemic cells with the double-stranded RNA (dsRNA) analogue, polyriboinosinic polyribocytidylic acid (poly(I:C)), and detail on the delicate freezing and thawing procedure of primary leukemic cells.Electroporation is a non-viral gene transfer method by which short-term pores in the membrane of cells are generated by an electrical pulse, allowing molecules to enter the cell. RNA electroporation, a technique developed in our laboratory, is a widely used and versatile transfection method for efficient introduction of both coding RNA (messenger RNA) and non-coding RNA, e.g., dsRNA and small interfering (siRNA), into mammalian cells. Accurate cell processing and storage of patient material is essential for optimal recovery and quality of the cell product for downstream applications.

Microdomains in the membrane landscape shape antigen-presenting cell function.

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for coupling to components of intracellular signaling cascades. In the last two decades, specialized membrane microdomains, including lipid rafts and TEMs, have been identified. These domains are preformed structures ("physical entities") that compartmentalize proteins, lipids, and signaling molecules into multimolecular assemblies. In APCs, different microdomains containing immunoreceptors (MHC proteins, PRRs, integrins, among others) have been reported that are imperative for efficient pathogen recognition, the formation of the immunological synapse, and subsequent T cell activation. In addition, recent work has demonstrated that tetraspanin microdomains and lipid rafts are involved in BCR signaling and B cell activation. Research into the molecular mechanisms underlying membrane domain formation is fundamental to a comprehensive understanding of membrane-proximal signaling and APC function. This review will also discuss the advances in the microscopy field for the visualization of the plasma membrane, as well as the recent progress in targeting microdomains as novel, therapeutic approach for infectious and malignant diseases.