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The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hedgehog ligand produced by neoplastic cells reprograms cancer-associated fibroblasts (CAFs) to provide a supportive niche for the acquisition of a chemo-resistant, cancer stem cell (CSC) phenotype via FGF5 expression and production of fibrillar collagen. Stromal treatment of patient-derived xenografts with smoothened inhibitors (SMOi) downregulates CSC markers expression and sensitizes tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. These studies identify Hedgehog signaling to CAFs as a novel mediator of CSC plasticity and an exciting new therapeutic target in TNBC.

While PDL1 expression is frequent in TNBC, it was not independently prognostic. There were differences in outcome depending on the cellular compartment of PDL1 expression. These data provide further impetus for investigating the utility of immune checkpoint therapies in TNBC, given the clinical significance of tumour-infiltrating lymphocytes (TILs) and PDL1 expression in this cohort.

Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basallike phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n ¼ 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial-stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis.

ObjectivesThrombotic and microvascular complications are frequently seen in deceased COVID‐19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation‐induced endothelial activation remains highly contentious.MethodsHere, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial–endothelial cell barrier.ResultsWe show that primary human endothelial cells express very low levels of the SARS‐CoV‐2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS‐CoV‐2. We also show that SARS‐CoV‐2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co‐culture model endothelial cells are not infected with SARS‐CoV‐2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM‐1 expression and releasing pro‐inflammatory cytokines.ConclusionsTaken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS‐CoV‐2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS‐CoV‐2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS‐CoV‐2 pathogenesis by sensing adjacent infection and mounting a pro‐inflammatory response to SARS‐CoV‐2.

These results implicate familial factors in susceptibility to the triggering of narrowly defined postpartum depressive episodes in women with recurrent major depression. They suggest that a postnatal onset definition of within 6-8 weeks of delivery may be optimal in studies of the triggering of depressive illness by childbirth.