Coordinated single-cell tumor microenvironment dynamics reinforce pancreatic cancer subtype.

Bulk analyses of pancreatic ductal adenocarcinoma (PDAC) samples are complicated by the tumor microenvironment (TME), i.e. signals from fibroblasts, endocrine, exocrine, and immune cells. Despite this, we and others have established tumor and stroma subtypes with prognostic significance. However, understanding of underlying signals driving distinct immune and stromal landscapes is still incomplete. Here we integrate 92 single cell RNA-seq samples from seven independent studies to build a reproducible PDAC atlas with a focus on tumor-TME interdependence. Patients with activated stroma are synonymous with higher myofibroblastic and immunogenic fibroblasts, and furthermore show increased M2-like macrophages and regulatory T-cells. Contrastingly, patients with 'normal' stroma show M1-like recruitment, elevated effector and exhausted T-cells. To aid interoperability of future studies, we provide a pretrained cell type classifier and an atlas of subtype-based signaling factors that we also validate in mouse data. Ultimately, this work leverages the heterogeneity among single-cell studies to create a comprehensive view of the orchestra of signaling interactions governing PDAC.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

Whey Protein Supplementation Improves the Glycemic Response and May Reduce Non-Alcoholic Fatty Liver Disease Related Biomarkers in Women with Polycystic Ovary Syndrome (PCOS).

Polycystic ovary syndrome (PCOS) increases type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) with insulin resistance. We hypothesized that a 35 g whey preload would improve insulin sensitivity and glucose handling while reducing biomarkers associated with NAFLD. Twenty-nine age-matched women (CON = 15, PCOS = 14) completed oral glycemic tolerance tests following baseline (Day 0) as well as an acute (Day 1) and short-term whey supplementation (Day 7). Whey had an interaction effect on glucose (p = 0.02) and insulin (p = 0.03), with glucose remaining stable and insulin increasing with whey supplementation. Insulin sensitivity (p < 0.01) improved with whey associated with increased glucagon secretion (p < 0.01). Alanine aminotransferase (ALT), and aspartate aminotransferase (AST) remained unchanged, but "day" had an effect on the AST:ALT ratio (p = 0.04), whereas triglycerides and sex hormone binding globulin overall were greater in the PCOS group (p < 0.05). Total cholesterol decreased in PCOS (by 13%) and CON (by 8%) (NS). HepG2 cells treated with plasma from participants before and after whey decreased lipid accumulation in the PCOS group after whey (p < 0.05). Whey provided an insulinogenic and glycemic homeostatic effect in women with PCOS with the potential to combat NAFLD-consequences.

Whey protein preload enhances the active GLP-1 response and reduces circulating glucose in women with polycystic ovarian syndrome.

Polycystic ovary syndrome (PCOS) increases risk for development of type 2 diabetes. Whey protein ingestion before a carbohydrate load attenuates blood glucose. For our exploratory, case-control study design, we hypothesized that 35 g whey protein isolate (WPI) preloading would increase postprandial incretins and reduce hyperglycemia in women with PCOS. Twenty-nine age-matched women (PCO = 14 and CON = 15) completed oral glycemic tolerance tests (OGTT) following baseline (Day 0) as well as 35 g WPI acute (Day 1) and short-term supplementation (Day 7). Eight venous samples were collected during each test for quantification of glucose, and enteropancreatic hormones and to calculate area under the curve (AUC). Data was analyzed via repeated measures ANCOVA with significance set at P< .05. "Day x time x group" significantly influenced glucose (P = .01) and insulin changes (P = .03). In both groups, AUCglu were significantly lower on Day 7 than Day 0 (P< .05). Postprandial glucose excursions were lower on Days 1 and 7 than Day 0 in PCO and CON. Both, PCO and CON exhibited greater insulin changes on Days 1 and 7 compared to Day 0 (P< .05). AUCglucagon were higher on Days 1 and 7 than on Day 0 (P< .05). Changes in active GLP-1 were higher on Day 1 than Day 0 (P= .03). Overall, we showed that WPI preloading augmented insulin release and consequently lowered circulating glucose in women with and without PCOS. This insulinogenic effect can be attributed to enhanced active GLP-1 levels. We concluded that the incretin-mimetic effect of WPI may aid women with PCOS in achieving glycemic homeostasis.

KRAS drives immune evasion in a genetic model of pancreatic cancer.

Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.

Comparative single-cell RNA sequencing (scRNA-seq) reveals liver metastasis-specific targets in a patient with small intestinal neuroendocrine cancer.

Genomic analysis of a patient's tumor is the cornerstone of precision oncology, but it does not address whether metastases should be treated differently. Here we tested whether comparative single-cell RNA sequencing (scRNA-seq) of a primary small intestinal neuroendocrine tumor to a matched liver metastasis could guide the treatment of a patient's metastatic disease. Following surgery, the patient was put on maintenance treatment with a somatostatin analog. However, the scRNA-seq analysis revealed that the neuroendocrine epithelial cells in the liver metastasis were less differentiated and expressed relatively little SSTR2, the predominant somatostatin receptor. There were also differences in the tumor microenvironments. RNA expression of vascular endothelial growth factors was higher in the primary tumor cells, reflected by an increased number of endothelial cells. Interestingly, vascular expression of the major VEGF receptors was considerably higher in the liver metastasis, indicating that the metastatic vasculature may be primed for expansion and susceptible to treatment with angiogenesis inhibitors. The patient eventually progressed on Sandostatin, and although consideration was given to adding an angiogenesis inhibitor to her regimen, her disease progression involved non-liver metastases that had not been characterized. Although in this specific case comparative scRNA-seq did not alter treatment, its potential to help guide therapy of metastatic disease was clearly demonstrated.

Tandem Duplications May Supply the Missing Genetic Alterations in Many Triple-Negative Breast and Gynecological Cancers.

In this issue of Cancer Cell, Menghi et al. show that tandem duplications (TDs) are a common genetic feature of gynecological and triple-negative breast cancers. They also provide evidence that these TDs generate genetic alterations driving these cancers, which, if substantiated, will fundamentally change our understanding of their oncogenesis.

Development of Patient-Derived Gastric Cancer Organoids from Endoscopic Biopsies and Surgical Tissues.

BACKGROUND: Organoids are three-dimensional in vitro models of human disease developed from benign and malignant gastrointestinal tissues with tremendous potential for personalized medicine applications. We sought to determine whether gastric cancer patient-derived organoids (PDOs) could be safely established from endoscopic biopsies for rapid drug screening. METHODS: Patients underwent esophagogastroduodenoscopy (EGD) for surveillance or staging and had additional forceps biopsies taken for PDO creation. Cancer tissues from operative specimens were also used to create PDOs. To address potential tumor heterogeneity, we performed low-coverage whole-genome sequencing of endoscopic-derived PDOs with paired surgical PDOs and whole-tumor lysates. The stability of genomic alterations in endoscopic organoids was assessed by next-generation sequencing and nested polymerase chain reaction (PCR) assay. The feasibility and potential accuracy of drug sensitivity screening with endoscopic-derived PDOs were also evaluated. RESULTS: Gastric cancer PDOs (n = 15) were successfully established from EGD forceps biopsies (n = 8) and surgical tissues (n = 7) from five patients with gastric adenocarcinoma. Low-coverage whole-genomic profiling of paired EGD and surgical PDOs along with whole-tumor lysates demonstrated absence of tumor heterogeneity. Nested PCR assay identified similar KRAS alterations in primary tumor and paired organoids. Drug sensitivity testing of endoscopic-derived PDOs displayed standard dose-response curves to current gastric cancer cytotoxic therapies. CONCLUSIONS: Our study results demonstrate the feasibility of developing gastric cancer PDOs from EGD biopsies. These results also indicate that endoscopic-derived PDOs are accurate surrogates of the primary tumor and have the potential for drug sensitivity screening and personalized medicine applications.

The change in metabolic heat production is a primary mediator of heat acclimation in adults.

PURPOSE: This study examined whether heat acclimation (HA) results in either predominate improvements in heat dissipation or reduced endogenous heat production via individual components of the human heat balance equation. METHODS: Twelve healthy inactive subjects (5 females, mean ± SD): age 28 ± 6y, 77.9 ± 2kg), completed a 10-day HA (42°C, 28% RH) hyperthermia clamp (90min/day exercise, ∆1.5°C in rectal temperature (Tre)) and control workload matched (CON: 23°C, 42% RH) protocols in a counterbalanced design separated by at least 2 mo. Pre-and post-HA were matched for external work rate (EXWR; day 1 and day 10 first 30min at 118 ± 29W, last 60min at 11 ± 5W); and metabolic heat production (Hprod; day 1 and day 9, first 30min at 296 ± 26Wm-2, last 60min 187 ± 33Wm-2). RESULTS: When Pre- and post- HA was matched for Hprod, there was no difference during the first 30 or last 60min of exercise for metabolic energy expenditure (MEE 363 ± 70/ 195 ± 32Wm-2), Hprod (296 ± 67/ 187 ± 33Wm-2) or Tre (∆2.1 ± 0.5°C). When pre- and post-HA was EXWR equivalent, HA significantly attenuated MEE during the first 30 and last 60min (303 ± 49/ 174 ± 35Wm-2), Hprod (241 ± 44/ 168 ± 33, W·m-2), and ∆Tre (∆1.3 ± 0.4°C) (each P < 0.0001). When ∆Tre, ∆Tsk, ∆Tb were each normalized per 100W Hprod, no differences were found for any pre-to post-HA comparison. Heat loss required (Ereq) to maintain steady state internal temperature (Ereq = 220 ± 32Wm-2), maximal capacity of the climate for evaporative heat loss (Emax = 266 ± 56Wm-2), evaporative heat loss from skin (Esk = 207 ± 38Wm-2) or skin wettedness (Ereq/Emax = 0.88 ± 0.23Wm-2) were not different among each condition during the last 60min. CONCLUSION: The mechanisms that underlie heat acclimation are not wholly attributed to heat dissipation enhancements per se, but are significantly influenced by metabolic heat production alterations under uncompensable heat stress environments.

Three-dimensional macroporous graphene scaffolds for tissue engineering.

The assembly of carbon nanomaterials into three-dimensional (3D) porous scaffolds is critical to harness their unique physiochemical properties for tissue engineering and regenerative medicine applications. In this study, we report the fabrication, characterization, and in vitro cytocompatibility of true 3D (>1 mm in all three dimensions), macroscopic (3-8 mm in height and 4-6 mm in diameter), chemically cross-linked graphene scaffolds prepared via radical initiated thermal cross-linking of single- and multiwalled graphene oxide nanoribbons (SWGONRs and MWGONRs). SWGONR and MWGONR scaffolds possess tunable porosity (∼65-80%) and interconnected macro-, micro-, and nanoscale pores. Human adipose derived stem cells (ADSCs) and murine MC3T3 preosteoblast cells show good cell viability on SWGONR and MWGONR scaffolds after 1, 3, and 5 days comparable to 3D poly(lactic-co-glycolic) acid (PLGA) scaffolds. Confocal live-cell imaging showed that cells were metabolically active and could spread on SWGONR and MWGONR scaffolds. Immunofluorescence imaging showed the presence of focal adhesion protein vinculin and expression of cell proliferation marker Ki-67 suggesting that cells could attach and proliferate on SWGONR and MWGONR scaffolds. These results indicate that cross-linked SWGONR and MWGONR scaffolds are cytocompatible and opens-avenues toward the development of 3D multifunctional graphene scaffolds for tissue engineering applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 73-83, 2017.