Single-Cell Analysis Identifies NOTCH3-Mediated Interactions between Stromal Cells That Promote Microenvironment Remodeling and Invasion in Lung Adenocarcinoma.

Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant proportion of patients do not respond. Recent transcriptomic studies to understand determinants of immunotherapy response have pinpointed stromal-mediated resistance mechanisms. To gain a better understanding of stromal biology at the cellular and molecular level in LUAD, we performed single-cell RNA sequencing of 256,379 cells, including 13,857 mesenchymal cells, from 9 treatment-naïve patients. Among the mesenchymal cell subsets, FAP+PDPN+ cancer-associated fibroblasts (CAF) and ACTA2+MCAM+ pericytes were enriched in tumors and differentiated from lung-resident fibroblasts. Imaging mass cytometry revealed that both subsets were topographically adjacent to the perivascular niche and had close spatial interactions with endothelial cells (EC). Modeling of ligand and receptor interactomes between mesenchymal and ECs identified that NOTCH signaling drives these cell-to-cell interactions in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or genetically depleting NOTCH3 levels in mesenchymal cells significantly reduced collagen production and suppressed cell invasion. Bulk RNA sequencing data demonstrated that NOTCH3 expression correlated with poor survival in stroma-rich patients and that a T cell-inflamed gene signature only predicted survival in patients with low NOTCH3. Collectively, this study provides valuable insights into the role of NOTCH3 in regulating tumor stroma biology, warranting further studies to elucidate the clinical implications of targeting NOTCH3 signaling. SIGNIFICANCE: NOTCH3 signaling activates tumor-associated mesenchymal cells, increases collagen production, and augments cell invasion in lung adenocarcinoma, suggesting its critical role in remodeling tumor stroma.

SPATA2 and CYLD inhibit T cell infiltration into colorectal cancer via regulation of IFN-γ/STAT1 axis.

Introduction: Colorectal cancer (CRC) is largely refractory to currently available immunotherapies such as blockade of programmed cell death protein-1 (PD-1). Results: In this study, we identified SPATA2 and its protein partner CYLD as novel regulators of CXC-ligand 10 (CXCL10), a T-cell-attractant chemokine, in CRC. By specifically deleting SPATA2 and CYLD in human and mouse CRC cell lines, we showed that these two proteins inhibit STAT1 accumulation and activation and subsequently CXCL10 expression in tumor cells. At steady-state, STAT1 is highly ubiquitinated in a SPATA2/CYLD-dependent manner. Finally, we demonstrated that tumor-specific deletion of SPATA2 and CYLD enhances anti-PD-1 response in vivo. Discussion: Our data suggest that SPATA2 and CYLD represent two potential novel targets for treatment of immune-excluded, PD-1-resistant tumors.

Fungal Symbionts Produce Prostaglandin E2 to Promote Their Intestinal Colonization.

Candida albicans is a ubiquitous fungal symbiont that resides on diverse human barrier surfaces. Both mammalian and fungal cells can convert arachidonic acid into the lipid mediator, prostaglandin E2 (PGE2), but the physiological significance of fungus-derived PGE2 remains elusive. Here we report that a C. albicans mutant deficient in PGE2 production suffered a loss of competitive fitness in the murine gastrointestinal (GI) tract and that PGE2 supplementation mitigated this fitness defect. Impaired fungal PGE2 production affected neither the in vitro fitness of C. albicans nor hyphal morphogenesis and virulence in either systemic or mucosal infection models. Instead, fungal production of PGE2 was associated with enhanced fungal survival within phagocytes. Consequently, ablation of colonic phagocytes abrogated the intra-GI fitness boost conferred by fungal PGE2. These observations suggest that C. albicans has evolved the capacity to produce PGE2 from arachidonic acid, a host-derived precursor, to promote its own colonization of the host gut. Analogous mechanisms might undergird host-microbe interactions of other symbiont fungi.

Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring.

Maternal immune activation (MIA) contributes to behavioural abnormalities associated with neurodevelopmental disorders in both primate and rodent offspring. In humans, epidemiological studies suggest that exposure of fetuses to maternal inflammation increases the likelihood of developing autism spectrum disorder. In pregnant mice, interleukin-17a (IL-17a) produced by T helper 17 (TH17) cells (CD4+ T helper effector cells involved in multiple inflammatory conditions) induces behavioural and cortical abnormalities in the offspring exposed to MIA. However, it is unclear whether other maternal factors are required to promote MIA-associated phenotypes. Moreover, the underlying mechanisms by which MIA leads to T cell activation with increased IL-17a in the maternal circulation are not well understood. Here we show that MIA phenotypes in offspring require maternal intestinal bacteria that promote TH17 cell differentiation. Pregnant mice that had been colonized with mouse commensal segmented filamentous bacteria or human commensal bacteria that induce intestinal TH17 cells were more likely to produce offspring with MIA-associated abnormalities. We also show that small intestine dendritic cells from pregnant, but not from non-pregnant, females secrete IL-1ß, IL-23 and IL-6 and stimulate T cells to produce IL-17a upon exposure to MIA. Overall, our data suggest that defined gut commensal bacteria with a propensity to induce TH17 cells may increase the risk of neurodevelopmental disorders in the offspring of pregnant mothers undergoing immune system activation owing to infections or autoinflammatory syndromes.

Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas.

Fibroblast growth factor 21 (FGF21) is an important endocrine metabolic regulator expressed in multiple tissues including liver and adipose tissue. Although highest levels of expression are in pancreas, little is known about the function of FGF21 in this tissue. In order to understand the physiology of FGF21 in the pancreas, we analyzed its expression and regulation in both acinar and islet tissues. We found that acinar tissue express 20-fold higher levels than that observed in islets. We also observed that pancreatic FGF21 is nutritionally regulated; a marked reduction in FGF21 expression was noted with fasting while obesity is associated with 3-4 fold higher expression. Acinar and islet cells are targets of FGF21, which when systemically administered, leads to phosphorylation of the downstream target ERK 1/2 in about half of acinar cells and a small subset of islet cells. Chronic, systemic FGF21 infusion down-regulates its own expression in the pancreas. Mice lacking FGF21 develop significant islet hyperplasia and periductal lymphocytic inflammation when fed with a high fat obesogenic diet. Inflammatory infiltrates consist of TCRb+ Thy1+ T lymphocytes with increased levels of Foxp3+ regulatory T cells. Increased levels of inflammatory cells were coupled with elevated expression of cytokines such as TNFα, IFNγ and IL1ß. We conclude that FGF21 acts to limit islet hyperplasia and may also prevent pancreatic inflammation.

A special population of regulatory T cells potentiates muscle repair.

Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.

Tumor cells disseminate early, but immunosurveillance limits metastatic outgrowth, in a mouse model of melanoma.

Although metastasis is the leading cause of cancer-related death, it is not clear why some patients with localized cancer develop metastatic disease after complete resection of their primary tumor. Such relapses have been attributed to tumor cells that disseminate early and remain dormant for prolonged periods of time; however, little is known about the control of these disseminated tumor cells. Here, we have used a spontaneous mouse model of melanoma to investigate tumor cell dissemination and immune control of metastatic outgrowth. Tumor cells were found to disseminate throughout the body early in development of the primary tumor, even before it became clinically detectable. The disseminated tumor cells remained dormant for varying periods of time depending on the tissue, resulting in staggered metastatic outgrowth. Dormancy in the lung was associated with reduced proliferation of the disseminated tumor cells relative to the primary tumor. This was mediated, at least in part, by cytostatic CD8+ T cells, since depletion of these cells resulted in faster outgrowth of visceral metastases. Our findings predict that immune responses favoring dormancy of disseminated tumor cells, which we propose to be the seed of subsequent macroscopic metastases, are essential for prolonging the survival of early stage cancer patients and suggest that therapeutic strategies designed to reinforce such immune responses may produce marked benefits in these patients.