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Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.

Cancer-associated fibroblasts (CAF) may exert tumor-promoting and tumor-suppressive functions, but the mechanisms underlying these opposing effects remain elusive. Here, we sought to understand these potentially opposing functions by interrogating functional relationships among CAF subtypes, their mediators, desmoplasia, and tumor growth in a wide range of tumor types metastasizing to the liver, the most common organ site for metastasis. Depletion of hepatic stellate cells (HSC), which represented the main source of CAF in mice and patients in our study, or depletion of all CAF decreased tumor growth and mortality in desmoplastic colorectal and pancreatic metastasis but not in nondesmoplastic metastatic tumors. Single-cell RNA-Seq in conjunction with CellPhoneDB ligand-receptor analysis, as well as studies in immune cell-depleted and HSC-selective knockout mice, uncovered direct CAF-tumor interactions as a tumor-promoting mechanism, mediated by myofibroblastic CAF-secreted (myCAF-secreted) hyaluronan and inflammatory CAF-secreted (iCAF-secreted) HGF. These effects were opposed by myCAF-expressed type I collagen, which suppressed tumor growth by mechanically restraining tumor spread, overriding its own stiffness-induced mechanosignals. In summary, mechanical restriction by type I collagen opposes the overall tumor-promoting effects of CAF, thus providing a mechanistic explanation for their dual functions in cancer. Therapeutic targeting of tumor-promoting CAF mediators while preserving type I collagen may convert CAF from tumor promoting to tumor restricting.

Patients with high-risk, nonmuscle-invasive bladder cancer (NMIBC) frequently relapse after standard intravesical bacillus Calmette-Guerin (BCG) therapy and may have a dismal outcome. The mechanisms of resistance to such immunotherapy remain poorly understood. Here, using cancer cell lines, freshly resected human bladder tumors, and samples from cohorts of patients with bladder cancer before and after BCG therapy, we demonstrate 2 distinct patterns of immune subversion upon BCG relapse. In the first pattern, intracellular BCG infection of cancer cells induced a posttranscriptional downregulation of HLA-I membrane expression via inhibition of autophagy flux. Patients with [HLA-I.sup.-]deficient cancer cells following BCG therapy had a myeloid immunosuppressive tumor microenvironment (TME) with epithelial-mesenchymal transition (EMT) characteristics and dismal outcomes. Conversely, patients with [HLA-I.sup.-]proficient cancer cells after BCG therapy presented with [CD8.sup.+] T cell tumor infiltrates, upregulation of inflammatory cytokines, and immune checkpoint-inhibitory molecules. The latter patients had a very favorable outcome. We surmise that HLA-I expression in bladder cancers at relapse following BCG does not result from immunoediting but rather from an immune subversion process directly induced by BCG on cancer cells, which predicts a dismal prognosis. HLA-I scoring of cancer cells by IHC staining can be easily implemented by pathologists in routine practice to stratify future treatment strategies for patients with urothelial cancer., Introduction Bladder cancer is a heterogeneous disease that displays invasive and noninvasive histological features of the urothelium, as well as a wide spectrum of molecular alterations and subtypes (1, 2). [...]

Pathogens utilize features of the host response as cues to regulate virulence gene expression. Salmonella enterica serovar Typhimurium (ST) sense Toll-like receptor (TLR)-dependent signals to induce Salmonella Pathogenicity Island 2 (SPI2), a locus required for intracellular replication. To examine pathogenicity in the absence of such cues, we evaluated ST virulence in mice lacking all TLR function (Tlr2(-/-)xTlr4(-/-)xUnc93b1(3d/3d)). When delivered systemically to TLR-deficient mice, ST do not require SPI2 and maintain virulence by replicating extracellularly. In contrast, SPI2 mutant ST are highly attenuated after oral infection of the same mice, revealing a role for SPI2 in the earliest stages of infection, even when intracellular replication is not required. This early requirement for SPI2 is abolished in MyD88(-/-)xTRIF(-/-) mice lacking both TLR- and other MyD88-dependent signaling pathways, a potential consequence of compromised intestinal permeability. These results demonstrate how pathogens use plasticity in virulence strategies to respond to different host immune environments.

In mice, provision of butyrate--a short-chain fatty acid produced by commensal microorganisms during starch fermentation--facilitates extrathymic generation and differentiation of Foxp3.sup.+ regulatory T cells, demonstrating that metabolic by-products are sensed by cells of the immune system and affect the balance between pro- and anti-inflammatory cells. Bacterial butyrate influences immune balance Several lines of evidence indicate that subsets of commensal microbes shape the gut immune system. For instance, colonization with Clostridia promotes extrathymic generation of regulatory T (T.sub.reg) cells that have a central role in the suppression of inflammatory and allergic responses. However, the molecular basis of such microbe-mediated T.sub.reg induction remains unknown. Two papers in this issue of Nature show that the colonic microbial fermentation product butyrate significantly accelerates the differentiation of colonic T.sub.reg cells and ameliorates colitis in conjunction with an increase in histone H3 acetylation at the Foxp3 promoter. This finding links butyrate to the induction of functional T.sub.reg cells in the colonic mucosa, and also provides molecular insight into the therapeutic application of butyrate. Intestinal microbes provide multicellular hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community. Regulatory T cells (T.sub.reg cells) expressing transcription factor Foxp3 have a key role in limiting inflammatory responses in the intestine.sup.1. Although specific members of the commensal microbial community have been found to potentiate the generation of anti-inflammatory T.sub.reg or pro-inflammatory T helper 17 (T.sub.H17) cells.sup.2,3,4,5,6, the molecular cues driving this process remain elusive. Considering the vital metabolic function afforded by commensal microorganisms, we reasoned that their metabolic by-products are sensed by cells of the immune system and affect the balance between pro- and anti-inflammatory cells. We tested this hypothesis by exploring the effect of microbial metabolites on the generation of anti-inflammatory T.sub.reg cells. We found that in mice a short-chain fatty acid (SCFA), butyrate, produced by commensal microorganisms during starch fermentation, facilitated extrathymic generation of T.sub.reg cells. A boost in T.sub.reg-cell numbers after provision of butyrate was due to potentiation of extrathymic differentiation of T.sub.reg cells, as the observed phenomenon was dependent on intronic enhancer CNS1 (conserved non-coding sequence 1), essential for extrathymic but dispensable for thymic T.sub.reg-cell differentiation.sup.1,7. In addition to butyrate, de novo T.sub.reg-cell generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of histone deacetylase (HDAC) inhibition, but not acetate, which lacks this HDAC-inhibitory activity. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory mechanisms., Author(s): Nicholas Arpaia [sup.1] [sup.2] , Clarissa Campbell [sup.1] [sup.2] , Xiying Fan [sup.1] [sup.2] , Stanislav Dikiy [sup.1] [sup.2] , Joris van der Veeken [sup.1] [sup.2] , Paul deRoos [...]

Various one- and two-dimensional NMR experiments have been found to be extremely useful for assigning the proton and carbon NMR spectra of glycidol. The technique provides extremely valuable information aiding in the complete assignment of the peaks.