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Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic cancers worldwide. EOC cells educate tumor-associated macrophages (TAM) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumor microenvironment (TME). In addition, tumor cells frequently activate Notch1 receptors on endothelial cells (EC) to facilitate metastasis. However, further work is required to establish whether the endothelium also influences the education of recruited monocytes. Here, we report that canonical Notch signaling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature; this was associated with stronger cytotoxic activity of T cells and decreased tumor burden. Mechanistically, CXCL2 was identified as a novel Notch/RBPJ target gene that regulated the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. Together, these findings indicate that EOC cells induce the tumor endothelium to secrete CXCL2 to establish an immunosuppressive microenvironment.Significance:Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.

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Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes into the peritoneum, which contributes to poor prognosis. Metastatic spreading is promoted by cancer cell plasticity, yet its regulation by the microenvironment is incompletely understood. Here we show that the presence of hyaluronan and proteoglycan link protein-1 (HAPLN1) in the extracellular matrix enhances tumor cell plasticity and PDAC metastasis. Bioinformatic analysis showed that HAPLN1 expression is enriched in the basal PDAC subtype and associated with worse overall patient survival. In a mouse model for peritoneal carcinomatosis, HAPLN1-induced immunomodulation favored a more permissive microenvironment, which accelerated the peritoneal spread of tumor cells. Mechanistically, HAPLN1, via hyaluronic acid synthesis and signaling, promoted adoption of a highly plastic cancer cell state, facilitating EMT, stemness, invasion and immunomodulation in a paracrine manner. Extracellular HAPLN1 modifies cancer cells as well as fibroblasts, rendering them immunomodulatory. We identify HAPLN1 as a prognostic marker and a driver for peritoneal metastasis in PDAC.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic cancers worldwide. EOC cells educate tumor-associated macrophages (TAM) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumor microenvironment (TME). In addition, tumor cells frequently activate Notch1 receptors on endothelial cells (EC) to facilitate metastasis. However, further work is required to establish whether the endothelium also influences the education of recruited monocytes. Here, we report that canonical Notch signaling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature; this was associated with stronger cytotoxic activity of T cells and decreased tumor burden. Mechanistically, CXCL2 was identified as a novel Notch/RBPJ target gene that regulated the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. Together, these findings indicate that EOC cells induce the tumor endothelium to secrete CXCL2 to establish an immunosuppressive microenvironment. Significance: Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.

Epithelial ovarian cancer (EOC) is one of the most lethal gynaecological cancers worldwide. EOC cells educate tumour-associated macrophages (TAMs) through CD44-mediated cholesterol depletion to generate an immunosuppressive tumour microenvironment (TME). In addition, tumour cells frequently activate Notch1 receptors on endothelial cells (ECs) to facilitate metastasis. However, little is known whether the endothelium would also influence the education of recruited monocytes. Here, we report that canonical Notch signalling through RBPJ in ECs is an important player in the education of TAMs and EOC progression. Deletion of Rbpj in the endothelium of adult mice reduced infiltration of monocyte-derived macrophages into the TME of EOC and prevented the acquisition of a typical TAM gene signature. This was associated with stronger cytotoxic activity of T cells and decreased tumour burden. Mechanistically, we identified CXCL2 as a novel Notch/RBPJ target gene. This angiocrine factor regulates the expression of CD44 on monocytes and subsequent cholesterol depletion of TAMs. Bioinformatic analysis of ovarian cancer patient data showed that increased CXCL2 expression is accompanied by higher expression of CD44 and TAM education. As such, EOC cells employ the tumour endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment.

ObjectiveFibrotic organ responses have recently been identified as long-term complication in diabetes. Indeed, insulin resistance and aberrant hepatic lipid accumulation represent driving features of progressive non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis and non-alcoholic steatohepatitis (NASH) to fibrosis. Effective pharmacological regimens to stop progressive liver disease are still lacking to-date.MethodsBased on our previous discovery of transforming growth factor beta-like stimulated clone (TSC)22D4 as a key driver of insulin resistance and glucose intolerance in obesity and type 2 diabetes, we generated a TSC22D4-hepatocyte specific knockout line (TSC22D4-HepaKO) and exposed mice to control or NASH diet models. Mechanistic insights were generated by metabolic phenotyping and single cell liver sequencing.ResultsHepatic TSC22D4 expression was significantly correlated with markers of liver disease progression and fibrosis in both murine and human livers. Indeed, hepatic TSC22D4 levels were elevated in human NASH patients as well as in several murine NASH models. Specific genetic deletion of TSC22D4 in hepatocytes led to reduced liver lipid accumulation, improvements in steatosis and inflammation scores and decreased apoptosis in mice. Single cell RNA sequencing revealed a distinct gene signature identifying an upregulation of mitochondrial-related processes. An enrichment of genes involved in the TCA cycle, mitochondrial organization, and triglyceride metabolism underscored the hepatocyte-protective phenotype and overall decreased liver damage as seen in mouse models.ConclusionsTogether, our data uncover a new connection between targeted depletion of TSC22D4 and intrinsic metabolic processes in progressive liver disease. Cell-specific reduction of TSC22D4 improves hepatic steatosis, inflammation and promotes hepatocyte survival thus paving the way for further preclinical therapy developments.

Vascular smooth muscle cells (VSMCs) have been cultured for decades to study the role of these cells in cardiovascular disorders. The most common source of VSMCs is the rat aorta. Here we show the adaptation of this method to isolate and culture mouse aortic VSMCs. The advantage of this method is that there are many more transgenic mouse lines available compared to rats. The protocol consists of the isolation of the aorta, the liberation of vascular cells by the action of collagenase, culturing of VSCMs, and analyzing filamentous actin and alpha smooth muscle actin by fluorescence microscopy. VSCMs can be further used to study mechanisms underlying cardiovascular diseases. Graphic abstract: Figure 1.Working steps.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecological cancers worldwide. Notch1 receptors (N1ICD) are frequently activated in Endothelial cells (ECs) of human tumors to facilitate metastasis. Using VE-Cadherin-CreERT2 transgenic mice to induce gene recombination exclusively in ECs, we explored the role of Notch signaling in ECs in vivo. RbpjiΔEC (VE-Cad-CrERT2xRbpjflox) mice inhibit the canonical Notch signaling upon tamoxifen administration. We modeled metastatic epithelial ovarian cancer (EOC) by injecting the mouse ovarian cancer cell line ID8 intraperitoneally. After four weeks of tumor growth, we observed a significant decreased tumor burden and reduced infiltration of monocyte-derived macrophages into the TME upon EC-specific Rbpj deletion. In vitro, we also confirmed a decreased infiltration of human monocytes through an EC barrier upon Rbpj deletion in human ECs. We isolated tumor-associated macrophages (TAMs) from RbpjΔEC tumor bearing mice and their littermate controls and performed microarray analysis. TCs educate TAMs through CD44-mediated cholesterol depletion. Several bioinformatic analyses showed that TAM from RbpjiΔEC mice were not able to induce the expected signature of TAMs in metastatic EOC because cholesterol depletion cannot take place. This indicates that the TC-induced macrophage education requires the presence of Rbpj in ECs. In vitro experiments showed that CD44 expression was downregulated in monocytes co-cultured with ECs lacking RBPJ. Analysis of the angiocrine factors regulated by Notch signaling revealed CXCL2 as a novel Rbpj-mediated Notch target gene: activation of Notch1 in ECs in vitro induced the production of the chemotactic cytokine CXCL2, whereas Rbpj-deletion (CRISPR-Cas9 technology) in N1ICD transduced ECs abolished CXCL2 expression. In addition, CXCL2 stimulation increased CD44 expression. In keeping with this, the analysis of publicly available ovarian cancer patient’s datasets we found that CXCL2 expression correlates with CD44 expression, suggesting that CXCL2 induces CD44 also in patients. CD44 is a co-receptor of a complex involving CXCR2 and CD74, which without CD44 it is not possible to signal. CD74 expression has been associated with worse prognosis in metastatic ovarian cancer and associated with an immunosuppressive phenotype in macrophages. Bioinformatic analyses of publicly available data showed that Rbpj in ECs is necessary for the immunosuppressive role of CD74. Finally, T cells sorted from RbpjiΔEC tumor bearing mice showed stronger cytotoxic activity (LDH-cytotoxicity assay) than those sorted from their littermate controls. In summary, ovarian cancer cells employ the tumor endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment. Citation Format: Ronja Mülfarth, Elisenda Alsina-Sanchis, Iris Moll, Sarah Böhn, Adrian Stögbauer, Francesca De Angelis Rigotti, Lena Wiedmann, Jacqueline Taylor, Andreas Fischer, Juan Rodriguez Vita. Endothelial Notch activation is essential for the education of tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1564.

Pancreatic Ductal Adenocarcinoma (PDAC) frequently metastasizes into the peritoneum forming peritoneal carcinomatosis, which are so far not treatable effectively. Metastasis-initiating cells need to acquire beneficial traits including cell plasticity, immune evasion, dormancy state control and organ colonization. These characteristics can be summarized in broad terms into two main processes, epithelial-to-mesenchymal transition (EMT) and stemness. Hyaluronic acid (HA), an extracellular matrix component, is a crucial factor in regulating these processes in PDAC, but it is so far not successfully targetable. Analyzing publicly available databases by gene set enrichment analysis (GSEA), a signature related to HA binding was enriched in tumor samples compared to normal tissue. Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1) was the top contributor to the enrichment score, being the 8th most enriched gene overall. We found that higher HAPLN1 expression correlated with shorter overall survival and that HAPLN1high patients had both, basal subtype and EMT signatures enriched. Moreover, these patients had a signature for peritoneal metastasis significantly enriched, suggesting a higher risk for peritoneal carcinomatosis. To study the role of HAPLN1 on PDAC in vitro, we stably overexpressed HAPLN1 in the murine PDAC cell line KPC. KPC-HAPLN1 cells expressed more EMT markers, more stem-related genes and changed the proteoglycan production from Aggrecan to Versican, which is known to be pro-metastatic. We found that spheroid formation, a feature of stemness, was improved in KPC-HAPLN1 vs KPC. Additionally, embedding these spheroids into matrigel led to an increased invasion of KPC-HAPLN1 cells. KPC-HAPLN1 cells improved KPC cell invasion capacities when co-cultured, indicating a paracrine effect. In vivo, intraperitoneal injection of luciferase expressing KPC cells resulted in higher luciferase activity when tumor cells expressed HAPLN1. Analyzing the peritoneal lavage (PL) from these mice, we obtained significantly more tumor cells in KPC-HAPLN1 injected mice. RNAseq data of tumor cells isolated from tumor nodules and PL showed that KPC-HAPLN1 cells acquired an increased metastatic potential and a strong immunomodulatory phenotype. Thus, we evaluated the immune cell composition of the PL by flow cytometry. Neutrophil and monocyte percentages were drastically reduced in KPC-HAPLN1 bearing mice. On the contrary, these mice had a significant increase in macrophages, which showed a reduction in pro-inflammatory gene expression. We conclude that HAPLN1 expression in tumor cells promotes a hyperplastic phenotype that facilitates invasion and colonization of the peritoneum, among others by creation of a pro-tumoral immune microenvironment. Citation Format: Lena Wiedmann, Francesca De Angelis Rigotti, Nuria Vaquero-Siguero, Elisa Donato, Elisa Espinet, Andreas Trumpp, Andreas Fischer, Juan Rodriguez-Vita. HAPLN1 increases peritoneal carcinomatosis by inducing tumor cell hyperplasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 960.

Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke major phenotypic changes in lung fibroblasts to form a metastatic niche that supports malignant growth. Colonization of the lungs by cancer cells confers an inflammatory phenotype in associated fibroblasts, where IL-1α and IL-1β, secreted by breast cancer cells, induceCXCL9andCXCL10production in metastasis-associated fibroblasts via NF-κB signaling. These paracrine interactions fuel the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells with stem/progenitor cell properties and high tumor-initiating ability when co-transplanted with fibroblasts. CXCR3-expressing cancer cells show high JNK signaling that drives IL-1α/β expression. Thus, CXCR3 marks a population of breast cancer cells that induces CXCL9/10 production in fibroblast, but can also respond to and benefit from these chemokines. Importantly, disruption of this intercellular JNK-IL-1-CXCL9/10-CXCR3 axis significantly reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for this molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis.

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ‐specific manner. Insulin flux across the endothelium to muscle cells is a rate‐limiting process influencing insulin‐mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non‐obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC‐specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high‐fat diet‐induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes., Insulin flux from blood plasma to muscle cells across the endothelium is a critical step in insulin‐mediated lowering of blood glucose levels. This study highlights the role of Notch signaling in regulating systemic glucose homeostasis.