Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis.

In combination with cell intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged high-plex single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with specific malignant subtypes and neoadjuvant chemotherapy/radiotherapy. We developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid co-culture system. Overall, this study demonstrates that characterization of the tumor microenvironment using high-plex single-cell spatial transcriptomics allows for identification of molecular interactions that may play a role in the emergence of chemoresistance and establishes a translational spatial biology paradigm that can be broadly applied to other malignancies, diseases, and treatments.

Cause-specific mortality among patients with cirrhosis in a population-based cohort study in Ontario (2000-2017).

BACKGROUND: Although patients with cirrhosis are at increased risk of death, the exact causes of death have not been reported in the contemporary era. This study aimed to describe cause-specific mortality in patients with cirrhosis in the general population. METHODS: Retrospective cohort study using administrative health care data from Ontario, Canada. Adult patients with cirrhosis from 2000-2017 were identified. Cirrhosis etiologies were defined as HCV, HBV, alcohol-associated liver disease (ALD), NAFLD, or autoimmune liver disease/other with validated algorithms. Patients were followed until death, liver transplant, or end of study. Primary outcome was the cause of death as liver-related, cardiovascular disease, non-hepatic malignancy, and external causes (accident/self-harm/suicide/homicide). Nonparametric analyses were used to describe the cumulative incidence of cause-specific death by cirrhosis etiology, sex, and compensation status. RESULTS: Overall, 202,022 patients with cirrhosis were identified (60% male, median age 56 y (IQR 46-67), 52% NAFLD, 26% alcohol-associated liver disease, 11% HCV). After a median follow-up of 5 years (IQR 2-12), 81,428 patients died, and 3024 (2%) received liver transplant . Patients with compensated cirrhosis mostly died from non-hepatic malignancies and cardiovascular disease (30% and 27%, respectively, in NAFLD). The 10-year cumulative incidence of liver-related deaths was the highest among those with viral hepatitis (11%-18%) and alcohol-associated liver disease (25%), those with decompensation (37%) and/or HCC (50%-53%). Liver transplant occurred at low rates (< 5%), and in men more than women. CONCLUSIONS: Cardiovascular disease and cancer-related mortality exceed liver-related mortality in patients with compensated cirrhosis.

Macrophage depletion blocks congenital SARM1-dependent neuropathy.

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified 2 rare NMNAT2 missense variants in 2 brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model to mirror the human syndrome and found that Nmnat2V98M/R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induced inflammatory neuropathy and highlight the potential of immune therapy as a treatment for this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

Features of fibrosis regression abound in "non-cirrhotic" patients with resected hepatocellular carcinoma.

Cirrhosis is a major risk factor for developing hepatocellular carcinoma (HCC). However, many surgically resected HCCs are presumably non-cirrhotic. The dynamic nature of chronic liver disease leads to periods of hepatic repair and fibrosis regression. We hypothesize that most resected HCCs, including those from non-cirrhotic patients, exhibit features of fibrosis regression in their background liver, suggesting previously more advanced liver disease. We reviewed the histology of 37 HCC resections performed between 2005-2020, including 30 from non-cirrhotic patients. The non-neoplastic liver was evaluated for features of liver disease and of the hepatic repair complex (HRC). CD34 immunohistochemistry was performed as a marker of sinusoidal capillarization. CD34 staining was evaluated manually and also by a digital image classifier algorithm. Overall, 28 cases (76%) had a high number of fibrosis regression and hepatic repair features (≥4 out of 8 features). Amongst the 30 non-cirrhotic patients, 21 (70%) showed a high number of repair features. Relative CD34 expression was increased in cases with a high number (≥4) of HRC features versus a low number (≤3) of features (p = 0.019). High HRC cases were more likely to exhibit nodular circumferential CD34 staining (p = 0.019). Our findings suggest that most resected HCC from non-cirrhotic patients display features of fibrosis regression in their background liver. Thus many, if not most, HCC patients who are "non-cirrhotic" may in fact have regressed cirrhosis. This finding reinforces that patients with regressed cirrhosis continue to be at high risk for HCC.

Objective Structured Assessment of technical skill (OSATS) in the Surgical Skills and Technology Elective Program (SSTEP): Comparison of peer and expert raters.

BACKGROUND: A major challenge with Competency Based Medical Education (CBME) is that of increased assessment burden on faculty. To reduce this burden, the accuracy and reliability of peer-assessment for surgical skills requires further exploration. METHODS: Forty-two second year medical students were video recorded while performing a simple interrupted suture and an instrument tie. Four novice raters underwent a short training session on the use of the Objective Structured Assessment of Technical Skills (OSATS) checklists. Videos of the suturing task were then independently assessed by the four novice raters and two expert raters on two occasions. Agreement between novice and expert rater scores was calculated using the intraclass correlation coefficient (ICC). RESULTS: For both simple interrupted suturing (ICC = 0.78, CI = 0.66-0.86, p < 0.001) and instrument ties (ICC = 0.87, CI = 0.80-0.92, p < 0.001), there was good agreement between novice and expert raters. CONCLUSIONS: Novice raters can be taught to use the OSATS checklists to assess peers on simple suturing and instrument tying tasks.

Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

Peripheral nerve resident macrophages share tissue-specific programming and features of activated microglia.

Whereas microglia are recognized as fundamental players in central nervous system (CNS) development and function, much less is known about macrophages of the peripheral nervous system (PNS). Here, by comparing gene expression across neural and conventional tissue-resident macrophages, we identified transcripts that were shared among neural resident macrophages as well as selectively enriched in PNS macrophages. Remarkably, PNS macrophages constitutively expressed genes previously identified to be upregulated by activated microglia during aging, neurodegeneration, or loss of Sall1. Several microglial activation-associated and PNS macrophage-enriched genes were also expressed in spinal cord microglia at steady state. We further show that PNS macrophages rely on IL-34 for maintenance and arise from both embryonic and hematopoietic precursors, while their expression of activation-associated genes did not differ by ontogeny. Collectively, these data uncover shared and unique features between neural resident macrophages and emphasize the role of nerve environment for shaping PNS macrophage identity.

Celecoxib affects estrogen sulfonation catalyzed by several human hepatic sulfotransferases, but does not stimulate 17-sulfonation in rat liver.

Celecoxib is known to alter the preferred position of SULT2A1-catalyzed sulfonation of 17ß-estradiol (17ß-E2) and other estrogens from the 3- to the 17-position. Understanding the effects of celecoxib on estrogen sulfonation is of interest in the context of the investigational use of celecoxib to treat breast cancer. This study examined the effects on celecoxib on cytosolic sulfotransferases in human and rat liver and on SULT enzymes known to be expressed in liver. Celecoxib's effects on the sulfonation of several steroids catalyzed by human liver cytosol were similar but not identical to those observed previously for SULT2A1. Celecoxib was shown to inhibit recombinant SULT1A1-catalyzed sulfonation of 10nM estrone and 4µM p-nitrophenol with IC50 values of 2.6 and 2.1µM, respectively, but did not inhibit SULT1E1-catalyzed estrone sulfonation. In human liver cytosol, the combined effect of celecoxib and known SULT1A1 and 1E1 inhibitors, quercetin and triclosan, resulted in inhibition of 17ß-E2-3-sulfonation such that the 17-sulfate became the major metabolite: this is of interest because the 17-sulfate is not readily hydrolyzed by steroid sulfatase to 17ß-E2. Investigation of hepatic cytosolic steroid sulfonation in rat revealed that celecoxib did not stimulate 17ß-E2 17-sulfonation in male or female rat liver as it does with human SULT2A1 and human liver cytosol, demonstrating that rat is not a useful model of this effect. In silico studies suggested that the presence of the bulky tryptophan residue in the substrate-binding site of the rat SULT2A homolog instead of glycine as in human SULT2A1 may explain this species difference.

Lymphoid Aggregates Remodel Lymphatic Collecting Vessels that Serve Mesenteric Lymph Nodes in Crohn Disease.

Early pathological descriptions of Crohn disease (CD) argued for a potential defect in lymph transport; however, this concept has not been thoroughly investigated. In mice, poor healing in response to infection-induced tissue damage can cause hyperpermeable lymphatic collecting vessels in mesenteric adipose tissue that impair antigen and immune cell access to mesenteric lymph nodes (LNs), which normally sustain appropriate immunity. To investigate whether analogous changes might occur in human intestinal disease, we established a three-dimensional imaging approach to characterize the lymphatic vasculature in mesenteric tissue from controls or patients with CD. In CD specimens, B-cell-rich aggregates resembling tertiary lymphoid organs (TLOs) impinged on lymphatic collecting vessels that enter and exit LNs. In areas of creeping fat, which characterizes inflammation-affected areas of the bowel in CD, we observed B cells and apparent innate lymphoid cells that had invaded the lymphatic vessel wall, suggesting these cells may be mediators of lymphatic remodeling. Although TLOs have been described in many chronic inflammatory states, their anatomical relationship to preestablished LNs has never been revealed. Our data indicate that, at least in the CD-affected mesentery, TLOs are positioned along collecting lymphatic vessels in a manner expected to affect delivery of lymph to LNs.

Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1.

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17ß-estradiol (17ß-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3ß, 17ß-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low µM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17ß-dihydroequilenin (17ß-Eqn), 17ß-dihydroequilin (17ß-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17ß-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17ß-Eq was stimulated at celecoxib concentrations below 40 µM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.

Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival.

The transcription factor Gata6 regulates proliferation and differentiation of epithelial and endocrine cells and cancers. Among hematopoietic cells, Gata6 is expressed selectively in resident peritoneal macrophages. We thus examined whether the loss of Gata6 in the macrophage compartment affected peritoneal macrophages, using Lyz2-Cre x Gata6(flox/flox) mice to tackle this issue. In Lyz2-Cre x Gata6(flox/flox) mice, the resident peritoneal macrophage compartment, but not macrophages in other organs, was contracted, with only a third the normal number of macrophages remaining. Heightened rates of death explained the marked decrease in peritoneal macrophage observed. The metabolism of the remaining macrophages was skewed to favor oxidative phosphorylation and alternative activation markers were spontaneously and selectively induced in Gata6-deficient macrophages. Gene expression profiling revealed perturbed metabolic regulators, including aspartoacylase (Aspa), which facilitates generation of acetyl CoA. Mutant mice lacking functional Aspa phenocopied the higher propensity to death and led to a contraction of resident peritoneal macrophages. Thus, Gata6 regulates differentiation, metabolism, and survival of resident peritoneal macrophages.

ESRRA-C11orf20 is a recurrent gene fusion in serous ovarian carcinoma.

Every year, ovarian cancer kills approximately 14,000 women in the United States and more than 140,000 women worldwide. Most of these deaths are caused by tumors of the serous histological type, which is rarely diagnosed before it has disseminated. By deep paired-end sequencing of mRNA from serous ovarian cancers, followed by deep sequencing of the corresponding genomic region, we identified a recurrent fusion transcript. The fusion transcript joins the 5' exons of ESRRA, encoding a ligand-independent member of the nuclear-hormone receptor superfamily, to the 3' exons of C11orf20, a conserved but uncharacterized gene located immediately upstream of ESRRA in the reference genome. To estimate the prevalence of the fusion, we tested 67 cases of serous ovarian cancer by RT-PCR and sequencing and confirmed its presence in 10 of these. Targeted resequencing of the corresponding genomic region from two fusion-positive tumor samples identified a nearly clonal chromosomal rearrangement positioning ESRRA upstream of C11orf20 in one tumor, and evidence of local copy number variation in the ESRRA locus in the second tumor. We hypothesize that the recurrent novel fusion transcript may play a role in pathogenesis of a substantial fraction of serous ovarian cancers and could provide a molecular marker for detection of the cancer. Gene fusions involving adjacent or nearby genes can readily escape detection but may play important roles in the development and progression of cancer.