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Previously we observed that neural cell adhesion molecule (NCAM) deficiency in beta tumor cells facilitates metastasis into distant organs and local lymph nodes. Here, we show that NCAM-deficient beta cell tumors grew leaky blood vessels with perturbed pericyte-endothelial cell-cell interactions and deficient perivascular deposition of ECM components. Conversely, tumor cell expression of NCAM in a fibrosarcoma model (T241) improved pericyte recruitment and increased perivascular deposition of ECM molecules. Together, these findings suggest that NCAM may limit tumor cell metastasis by stabilizing the microvessel wall. To directly address whether pericyte dysfunction increases the metastatic potential of solid tumors, we studied beta cell tumorigenesis in primary pericyte-deficient Pdgfb(ret/ret) mice. This resulted in beta tumor cell metastases in distant organs and local lymph nodes, demonstrating a role for pericytes in limiting tumor cell metastasis. These data support a new model for how tumor cells trigger metastasis by perturbing pericyte-endothelial cell-cell interactions.

Pancreatic cancer (PC) is a lethal malignancy that lacks specific diagnostic markers. The present study explores the diagnostic potential of the most differentially overexpressed secretory mucin MUC5AC alone and in combination with CA19-9 using multi-center training and validation sets. The expression of MUC5AC in benign pancreatic pathologies, PC precursor lesions, primary PC tissues and metastatic lesions was evaluated by immunohistochemistry. Circulating MUC5AC levels were measured using sandwich ELISA assay developed in-house, and CA19-9 was measured using radioimmunoassay. A combined training set (n=346) was used to evaluate the diagnostic (n=241) and predictive (n=105, total samples 201 from pre- and post-surgical and chemotherapy set) significance of MUC5AC. Results were further validated with a pre-defined cut-off value using independent sets from the Mayo Clinic (n=94) and the University of Pittsburgh Medical Center (n=321). Tissue expression analyses indicated the de novo expression of MUC5AC in pancreatic intraepithelial precursor lesions 1A (PanIN1A); the expression was maintained through all stages of progression to invasive adenocarcinoma. The median circulating MUC5AC levels in patients with resectable early-stage PC (EPC) (stage 1/2; 67.2 ng/ml, IQR: 23.9-382.1) and unresectable late-stage PC (LPC) (stage 3/4; 389.7 ng/ml, IQR: 87.7-948.6) were significantly higher compared with (P-value ≤0.0001) benign controls (BC) (7.2 ng/ml, IQR: 0.4-26.5) and (P-value ≤0.0001) chronic pancreatitis (CP) controls (8.4 ng/ml, IQR: 1.5-19.2). In the diagnostic training set (n=241), MUC5AC efficiently differentiated EPC from healthy controls (HC) (83%/80% sensitive (SN)/specific (SP)), BC (67%/87% SN/SP), and CP (83%/77% SN/SP). Independent validation sets from the Mayo Clinic and UPMC confirmed the diagnostic potential of MUC5AC to differentiate EPC from BC (68%/73%; 65%/83%) and CP (68%/79%; 65%/72%). Furthermore, MUC5AC and CA19-9 combination significantly improved (p-value < 0.001) the diagnostic accuracy for differentiating resectable cases from controls. MUC5AC is a valuable diagnostic biomarker, either alone or in combination with CA19-9, to differentiate PC from CP and benign controls.

The presence of remaining insulin-positive cells in type 1 diabetes (T1D) is well-known. These cells are part of islets or appear as extra-islet insulin-positive cells scattered in the exocrine parenchyma. The latter are poorly described, and the presence of scattered endocrine cells expressing other islet hormones than insulin has not been explored. This study aimed to compare the extra-islet insulin- or glucagon-positive cells concerning their frequency, transcription-factor expression, and mitotic activity in subjects with and without T1D. Multispectral imaging was used to examine extra-islet cells by staining for insulin, glucagon, ARX, PDX1, and Ki67. This was done in well-preserved pancreatic tissue obtained from heart-beating organ donors with or without T1D. In three T1D donors, lobes with insulin-containing islets (ICI) were found. Within these, a higher frequency of extra-islet insulin-positive cells was observed compared to lobes with insulin-deficient islets (IDI). Increased frequency of glucagon-positive extra-islet cells was observed in donors with T1D (median 53 cells/mm 2 ) when compared with non-diabetic donors (11 cells/mm 2 , p  = 0.004). Proliferating endocrine cells were present in donors with, and without T1D, as demonstrated by Ki67-positive staining (0–3% of the cells expressing insulin or glucagon). The reduced frequency of extra-islet insulin-positive cells in lobes with IDI in donors with T1D suggests that the pathological mechanism causing beta cell demise in T1D affects entire lobes. The presence of an increased frequency of glucagon-positive extra-islet cells supports the notion of a preserved capacity to regenerate the endocrine pancreas in donors with T1D.

Islet transplantation is an established clinical procedure for select patients with type 1 diabetes and severe hypoglycemia to stabilize glycemic control. Post-transplant, substantial beta cell mass is lost, necessitating multiple donors to maintain euglycemia. A potential strategy to augment islet engraftment is the co-transplantation of islets with multipotent mesenchymal stem cells to capitalize upon their pro-angiogenic and anti-inflammatory properties. Herein, we examine the in vitro and in vivo effect of co-culturing murine islets with human adipose-derived mesenchymal stem cells (Ad-MSCs). Islets co-cultured with Ad-MSCs for 48 hours had decreased cell death, superior viability as measured by membrane integrity, improved glucose stimulated insulin secretion and reduced apoptosis compared to control islets. These observations were recapitulated with human islets, albeit tested in a limited capacity. Recipients of marginal mouse islet mass grafts, co-transplanted with Ad-MSCs without a co-culture period, did not reverse to normoglycemia as efficiently as islets alone. However, utilizing a 48-hour co-culture period, marginal mouse islets grafts with Ad-MSCs achieved a superior percent euglycemia rate when compared to islets cultured and transplanted alone. A co-culture period of human islets with human Ad-MSCs may have a clinical benefit improving engraftment outcomes.

Aims/hypothesisPancreatic islets produce non-coding microRNAs (miRNAs) that regulate islet cell function and survival. Our earlier investigations revealed that human islets undergo significant damage due to various types of stresses following transplantation and release miRNAs. Here, we sought to identify and validate exosomal miRNAs (exo-miRNAs) produced by human islets under conditions of cellular stress, preceding loss of cell function and death. We also aimed to identify islet stress signalling pathways targeted by exo-miRNAs to elucidate potential regulatory roles in islet cell stress.MethodsHuman islets were subjected to proinflammatory cytokine and hypoxic cell stress and miRNA from exosomes was isolated for RNA sequencing and analysis. Stress-induced exo-miRNAs were evaluated for kinetics of expression and release by intact islets for up to 48 h exposure to cytokines and hypoxia. A subset of stress-induced exo-miRNAs were assessed for recovery and detection as biomarkers of islet cell stress in a diabetic nude mouse xenotransplant model and in patients undergoing total pancreatectomy with islet auto-transplantation (TPIAT). Genes and signalling pathways targeted by stress-induced exo-miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and direct interactions of miRNAs with downstream signalling targets were validated in human islet cells using the miRNA Tests for Read Analysis and Prediction (MirTrap) system.ResultsGlobal exo-miRNA sequencing revealed that 879 miRNA species were released from human islets and 190 islet exo-miRNAs were differentially expressed in response to proinflammatory cytokines, hypoxia or both. Release of exo-miRNAs hsa-miR-29b-3p and hsa-miR-216a-5p was detected within 6 h of exposure to cytokines and hypoxia. The remaining subset of stress-induced exo-miRNAs, including hsa-miR-148a-3p and islet cell damage marker hsa-miR-375, showed delayed release at 24–48 h, correlating with apoptosis and cell death. Stress and damage exo-miRNAs were significantly elevated in the circulation in human-to-mouse xenotransplant models and in human transplant recipients. Elevated blood exo-miRNAs negatively correlated with post-transplant islet function based on comparisons of stress and damage exo-miRNA indices with Secretory Unit of Islet Transplant Objects (SUITO) indices. KEGG analysis and further validation of exo-miRNA targets by MirTrap analysis revealed significant enrichment of islet mRNAs involved in phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signalling pathways.Conclusions/interpretationThe study identifies exo-miRNAs differentially expressed and released by islets in response to damage and stress. These exo-miRNAs could serve as potential biomarkers for assessing islet damage and predicting outcomes in islet transplantation. Notably, exo-miRNAs 29b-3p and 216a-5p could be detected in islets prior to damage-released miRNAs and indicators of cellular apoptosis and death. Thus, these stress-induced exo-miRNAs may have potential diagnostic value for detecting early islet stress prior to progressive loss of islet cell mass and function. Further investigations are warranted to investigate the utility of these exo-miRNAs as early indicators of islet cell stress during prediabetic conditions.

Background There is a paucity of evidence regarding incidence and predictors of survival in pancreatic neuroendocrine tumors (PNETs) ≤2 cm in size. Methods Patients having undergone resection for nonfunctioning PNETs were selected from the SEER database (1988–2009) and an institutional pathology database (1996–2012). PNETs ≤2 cm were compared with PNETs >2 cm. Data were analyzed with χ 2 tests, ANOVA, the Kaplan–Meier method, log rank tests, and Cox proportional hazard, and binary logistic regression. Results The incidence of PNETs ≤2 cm in the United States has increased by 710.4 % over the last 22 years. Rates of extrapancreatic extension, nodal metastasis, and distant metastasis in PNETs ≤2 cm in the SEER database were 17.9, 27.3, and 9.1 %, respectively. The rate of nodal metastasis in our institutional series was 5.7 %. Disease-specific survival at 5, 10, and 15 years for PNETs ≤2 cm was 91.5, 84.0, and 76.8 %. Decreased disease-specific survival was not associated with nodal metastasis, but rather with high grade [moderately differentiated, hazard ratio (HR) 37.2, 95 % confidence interval (CI) 2.7–518.8; poorly differentiated, HR 94.2, 95 % CI 4.9–1,794.4; reference, well differentiated], and minority race (Asian, HR 30.2, 95 % CI 3.1–291.7; Black, HR 60.1, 95 % CI 2.1–1,027.9; reference, White). Conclusions Pancreatic neuroendocrine tumors ≤2 cm are increasingly common, and the most significant predictors of disease-specific survival are grade and race. The SEER database excludes PNETs considered to be benign, and rates of extrapancreatic extension, nodal metastasis, and distant metastasis are overestimated. Small size, however, does not preclude malignant behavior.

Islet transplantation represents a promising therapeutic approach for type 1 diabetes through restoration of endogenous insulin production. However, efficient purification of islets from surrounding exocrine tissue remains a critical challenge, as current methodologies often compromise purity, yield, or islet viability. We exploited the differential expression of CD99 between murine pancreatic exocrine tissue (high expression) and islets (negligible expression) to develop a novel immunomagnetic negative selection protocol. Expression patterns were validated using immunofluorescence, immunohistochemistry, and western blot. Subsequently, streptavidin-conjugated magnetic beads coupled with biotinylated anti-CD99 antibodies were employed to selectively deplete CD99-positive exocrine cells from pancreatic digests, thereby enriching viable islets. This approach achieved a remarkable increase in islet purity from 10.4 ± 3.9% to 93.0 ± 1.4% (P < 0.0001). Purified islets maintained structural integrity and demonstrated robust glucose-stimulated insulin secretion in vitro, comparable to islets isolated via conventional Ficoll density gradient centrifugation (P > 0.05). In a syngeneic transplantation model, 400 islet equivalents effectively reversed streptozotocin-induced diabetes, with therapeutic efficacy equivalent to Ficoll-purified islets (P > 0.05). Our CD99-targeted immunomagnetic negative selection offers a novel, highly specific, and effective alternative for obtaining high-quality islets, as demonstrated by their excellent functional performance both in vitro and in vivo.

Islet transplantation (ITx) is advancing rapidly, with clinical trials of stem cell derived islets demonstrating short-term insulin independence. However, long-term insulin independence may still require multiple infusions. We examined the effects of sequential ITx on portal venous pressure and factors influencing acute pressure changes across 693 intraportal ITx procedures in 298 adults (44% M); who received up to 5 procedures, at the University of Alberta Hospital over 24 years. We assessed acute portal pressure changes per infusion, using linear mixed-effects models to compare sequential pre-infusion pressures to baseline and assess relationships between pressure changes and packed cell volume (PCV), purity, islet dose (IE/kg) and estimated total liver volume (eTLV). Portal pressure transiently increased, similarly, after each infusion by an overall median 2.0 mmHg (IQR 1.0,4.0). PCV exhibited the strongest relationship with change in pressure, with1mmHg increase per 1 mL of PCV, when adjusted for other parameters (Coefficient = 1.0 [95%CI 0.81,1.21];p < 0.0001). Conversely, higher purity and larger eTLV were associated with smaller increases in pressure. A significant interaction term indicated that the effect that PCV has on pressure change may be moderated at higher purities. Our work provides insights from deceased-donor intraportal ITx that can inform the design of future clinical protocols for ITx.

Systemic inflammation is related to hyperglycemia in diabetes mellitus (DM). C-C chemokine motif ligand (CCL) 4 is upregulated in type 1 & type 2 DM patients. This study aimed to investigate if CCL4 could be a potential target to improve blood sugar control in different experimental DM models. Streptozotocin-induced diabetic mice, Lepr db /JNarl diabetic mice, and C57BL/6 mice fed a high fat diet were used as the type 1 DM, type 2 DM, and metabolic syndrome model individually. Mice were randomly assigned to receive an anti-CCL4 neutralizing monoclonal antibody. The pancreatic β-cells were treated with streptozotocin for in vitro experiments. In streptozotocin-induced diabetic mice, inhibition of CCL4 controlled blood sugar, increased serum insulin levels, increased islet cell proliferation and decreased pancreatic interleukin (IL)-6 expression. In the type 2 diabetes and metabolic syndrome models, CCL4 inhibition retarded the progression of hyperglycemia, reduced serum tumor necrosis factor (TNF)-α and IL-6 levels, and improved insulin resistance via reducing the phosphorylation of insulin receptor substrate-1 in skeletal muscle and liver tissues. CCL4 inhibition directly protected pancreatic β-cells from streptozotocin stimulation. Furthermore, CCL4-induced IL-6 and TNF-α expressions could be abolished by siRNA of CCR2/CCR5. In summary, direct inhibition of CCL4 protected pancreatic islet cells, improved insulin resistance and retarded the progression of hyperglycemia in different experimental models, suggesting the critical role of CCL4-related inflammation in the progression of DM. Future experiments may investigate if CCL4 could be a potential target for blood sugar control in clinical DM.

Herein, we characterized the percentage of tacrolimus to the combined sirolimus and tacrolimus trough levels (tacrolimus %) observed during islet transplant-associated immune suppression therapy with post-transplant skin cancer. Although trough levels of tacrolimus and sirolimus were not different (P = 0.79, 0.73, respectively), high tacrolimus % resulted in a 1.32-fold increase in skin cancer odds when adjusting for age, sex, body mass index (BMI), and use of mycopheonlate mofetil (MMF; p = 0.039). Skin cancer patients were likely to have been older but not differ significantly (mean difference 12 years, P = 0.056), but age was significantly associated with a 1.22-fold increase in adjusted skin cancer odds (P = 0.046). BMI was inversely associated with skin cancer, with an adjusted odds ratio (OR) of 0.40 (P = 0.022). High tacrolimus % (>35) resulted in a 4.6-fold increase in skin cancer frequency, whereas sirolimus above 75% of the combined therapy led to a 5.2-fold increase in islet graft dysfunction (IGD) events/year. By calculating the maximum safe exposure (MSE) to tacrolimus % according to patient age and BMI, we found that cumulative months spent above MSE was predictive of skin cancer (1.20-fold increase, P = 0.003). Individuals exceeding the MSE for 1 year were 9.2 times more likely to develop skin cancer (P = 0.008). Results suggest that strategies targeting immunosuppression ratios based on age and BMI may minimize cancer risk while improving graft survival and function. Graphical Abstract