Donor Pancreas Research Articles

Abstract B113: Characterization of the epigenomic landscape of the human pancreas and early pancreatic neoplastic lesions

Abstract Study of early pancreas neoplasia in humans had previously been limited by lack of available tissue. Recent work by our group on deceased donor pancreata identified pancreatic intraepithelial neoplasia (PanIN) lesions in non-diseased organs and defined a transcriptomic signature for the microenvironment of these pre-cancerous lesions. Prevalence of PanINs in healthy human tissue was higher than expected and established a novel model to expand understanding of the complex biology of these precursor lesions. Epigenetic changes in chromatin structure and the subsequent effects on gene expression are essential processes in neoplasia that have yet to be described at the single cell level in the human pancreas. We hypothesized that characterization of the epigenomic landscape in donor pancreata would identify differences in chromatin structure and gene expression between healthy human tissue and PanINs, thereby expanding prior knowledge and identifying novel targets of study in the biology of early neoplasia in the pancreas. Single nuclei from five donor pancreata (six total samples including one replicate sample from a single donor pancreas) were isolated for transposase-accessible chromatin preparation followed by high-throughput sequencing (ATAC-seq) using the 10x Genomics platform. Data analysis was conducted using CellRanger, Seurat, Signac, and AUCell to identify and label chromatin peaks in individual nuclei corresponding to acinar, ductal, and PanIN origin according to gene signatures identified by scRNA on matched pancreata (Carpenter et al 2023, Cancer Discovery). Using this method, cells corresponding to these gene signatures, including PanIN-like cells, were indeed identified in preliminary analysis. Differentially accessible regions of the genome were then analyzed for motif enrichment between these cell types. Motifs enriched in cells identified as PanINs compared to either ductal or acinar cells included several transcription factors implicated in tumorigenesis, including members of the AP-1/Fos/Jun family and multiple zinc-finger protein family members, such as the KLF. These results are similar to previously demonstrated essential factors in pancreatic neoplasia. Differentially expressed motifs between ductal and acinar cells included factors associated with differentiation, including Myod1, Ptf1a, and the TCF family of transcription factors. Together, this study demonstrates that PanINs can be identified in human pancreas tissue by ATAC-seq and that differentially expressed motifs can be identified this model, providing opportunity for further integration with transcriptomic information to elucidate detailed understanding of early neoplasia in the pancreas. Citation Format: Jamie N. Mills, Joyce Thompson, Jacqueline Morales, Padma Kadiyala, Ahmed M. Elhossiny, Howard Crawford, Eileen Carpenter, Marina Pasca di Magliano, Filip Bednar, Simone Benitz. Characterization of the epigenomic landscape of the human pancreas and early pancreatic neoplastic lesions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B113.

Lack of association between deceased pancreas donor LFTs and graft failure or patient mortality

Lack of association between deceased pancreas donor LFTs and graft failure or patient mortality

Lack of association between deceased pancreas donor LFTs and graft failure or patient mortality

Lack of association between deceased pancreas donor LFTs and graft failure or patient mortality

Islet transplantation outcomes in type 1 diabetes and transplantation of HLA-DQ8/DR4: results of a single-centre retrospective cohort in Canada

In solid organ transplantation, HLA matching between donor and recipient is associated with superior outcomes. In islet transplantation, an intervention for Type 1 diabetes, HLA matching between donor and recipient is not performed as part of allocation. Susceptibility to Type 1 diabetes is associated with the presence of certain HLA types. This study was conducted to determine the impact of these susceptibility antigens on isletallograft survival. This is a single-centre retrospective cohort study. This cohort of transplant recipients (n=268) received islets from 661 donor pancreases between March 11th, 1999 and August 29th, 2018at the University of Alberta Hospital (Edmonton, AB, Canada). The frequency of the Type 1 diabetes susceptibility HLA antigens (HLA-A24, -B39, -DQ8, -DQ2 and-DQ2-DQA1∗05) in recipients and donors were determined. Recipient and donor HLA antigens were examined in relation to time to first C-peptide negative status/graft failure or last observation point. Taking into account multiple transplants per patient, we fitted a Gaussian frailty survival analysis model with baseline hazard function stratified by transplant number, adjusted for cumulative islet dose and other confounders. Across all transplants recipients of donors positive for HLA-DQ8 had significantly better graft survival (adjusted HRs 0.33 95% CI 0.17-0.66; p=0.002). At first transplant only, donors positive for HLA-DQ2-DQA1∗05 had inferior graft survival (adjusted HR 1.96 95% CI 1.10-3.46); p=0.02), although this was not significant in the frailty analysis taking multiple transplants into account (adjusted HR 1.46 95% CI 0.77-2.78; p=0.25). Other HLA antigens were not associated with graft survival after adjustment for confounders. Our findings suggest islet transplantation from HLA-DQ8 donors is associated with superior graft outcomes. A donor positive for HLA-DQ2-DQA1∗05at first transplant was associated with inferior graft survival but not when taking into account multiple transplants per recipient. The relevance of HLA-antigens on organ allocation needs further evaluation and inclusion in islet transplant registries and additional observational and interventional studies to evaluate the role of HLA-DQ8 in islet graft survival are required. None.

THU377 Arterio-enteric Fistula, A Rare Complication After Pancreatic Transplant

Abstract Disclosure: A.M. Herrera Chancay: None. A. Calderon: None. A. Manavalan: None. Introduction: Areterio-enteric (AE) fistula is a rare but life-threatening complication of a failed pancreas transplant and most often presents with a gastrointestinal (GI) herald bleed. This case illustrates the importance of the prompt recognition and appropriate evaluation of GI bleeding in patients with a failed pancreas transplant and highlights the challenges associated with the management of an AE fistula. Case Description: A 53-year-old man with history of T1D complicated by retinopathy, neuropathy, nephropathy, end-stage renal disease (ESRD) and failed combined pancreas-renal transplant, presented to our hospital with diabetic ketoacidosis secondary to insulin pump malfunction. His course was complicated by a GI bleed. He was initiated on proton pump inhibitors but endoscopy was deferred due to resolution of bleeding. The patient was discharged from the hospital with plans for outpatient endoscopy however, he returned to the emergency room with hematemesis, malena and hemorrhagic shock complicated by cardiac arrest. Emergent endoscopy was performed but the source of bleeding could not be visualized due to large clots and fresh blood. A CT angiogram revealed brisk bleeding from the right common iliac pancreatic transplant graft. Coil embolization was attempted but unsuccessful and the patient was ultimately treated with two stents to the right iliac artery with subsequent resolution of bleeding. The patient was ultimately discharged home in stable condition. Conclusion: During pancreas transplant, the donor pancreas and duodenum are connected by a Roux en Y anastomosis to the small bowel of the recipient. The donor pancreas is placed in the right iliac fossa followed by anastomosis of the recipient right common iliac artery with the donor Y graft (Consisting of the iliac artery, its bifurcation, and the splenic and superior mesenteric arteries). AE fistula is an extremely rare complication of pancreatic transplant with only about 30 reported cases. This occurs in the setting of transplant failure, possibly due to the increased local inflammation. Endoscopy is often unsuccessful in locating the bleeding source. Angiography and stenting of the bleeding vessel seem to offer the most success and should be considered in patients with failed pancreas transplant presenting with GI bleeding. Presentation: Thursday, June 15, 2023

FRI604 Stem-cell Derived Islets For Treating Diabetes

Abstract Disclosure: W.L. Rust: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. P.M. Dalal: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. A.P. Koval: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. J.J. Ratiu: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. S.M. Southard: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. P.S. Strumph: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. C.A. Welsch: Employee; Self; Seraxis Inc. Stock Owner; Self; Seraxis Inc. Background and Aims: We report the development of a novel human multipotent immortal stem cell line, SR1423 that efficiently differentiates to clusters of functional endocrine pancreatic cells. These Synthetic Replacement Endocrine (SRE) clusters contain cell types that recapitulate native islet function in vitro and potently control blood glucose in animal models of type 1 diabetes. Detailed characterization reveals significant distinctions between SRE, native islets and SC-beta cells. SRE clusters are manufactured following GMP requirements in closed vessels with large-scale formats and are being developed for near-term clinical studies. Materials and Methods: SRE was generated by transient expression of the Yamanaka factors in human islet cells harvested from a consented donor pancreas. Rather than screen for pluripotency, this cell line was selected for its ability to differentiate to the definitive endoderm in a first screen, and Pdx1+ pancreatic progenitors in a second screen. SRE does not meet criteria for pluripotency as it fails to express the master control gene for mesoderm specification, brachyury, in response to mesoderm-inducing agents. Comparison against a pluripotent stem cell database demonstrates poor overall differentiation capability for the mesoderm lineage. In contrast, SRE responds to basic differentiation protocols that drive endocrine pancreatic fate choice and generates highly pure populations of cells with endocrine cell characteristics. We therefore characterize SR1423 as a multipotent, and not pluripotent stem cell. In a prospectively designed islet replacement study male NSG mice with streptozotocin-induced diabetes were implanted with SRE clusters to the kidney capsule or gonadal fat pad (as a surrogate for the omentum) and disease progression was monitored. Results: SRE resemble human islets in morphology and distribution of hormone expressing cells. In this mouse model of STZ-induced type 1 diabetes (blood glucose > 180 mg/dL), SRE transplantation resulted in euglycemia 3-4 weeks post-implant; 72 mg/dL +/− 12 SEM when implanted to the kidney capsule (n=13), and 79 mg/dL +/− 15 SEM when implanted to the gonadal fat pad (n=10). Control mice (without SRE implant) in this model of type 1 diabetes (n=2) remained hyperglycemic; 570 mg/dL +/− 20 SEM. Conclusion: These data are consistent with demonstration of proof of principle in this model with resolution of hyperglycemia after transplant of SRE. This is the first description of a novel cell type (non-native, non-SC-beta) with potential clinical therapeutic benefit. SRE clusters are currently in preparation for clinical studies. Presentation: Friday, June 16, 2023

310.4: Developing an algorithm for islet transplant dosing that considers both pancreas donor and islet recipient factors

310.4: Developing an algorithm for islet transplant dosing that considers both pancreas donor and islet recipient factors

A Prospective Study of the Effect of Gastroduodenal Artery Reconstruction on Duodenal Oxygenation and Enzyme Content After Pancreas Transplantation

BackgroundWhole pancreas transplantation provides durable glycemic control and can improve survival rate; however, it can carry an increased risk of surgical complications. One devastating complication is a duodenal leak at the site of enteroenteric anastomosis. The gastroduodenal artery (GDA) supplies blood to the donor duodenum and pancreas but is commonly ligated during procurement. Since we have not had expressive changes in pancreatic back table surgical techniques in the recent decades, we hypothesized whether back table GDA reconstruction, improving perfusion of the donor duodenum and head of the pancreas, could lead to fewer surgical complications in simultaneous pancreas-kidney (SPK) transplants.Material and MethodsBetween 2017 and 2021, we evaluated demographic information, postoperative complications, intraoperative donor duodenum, recipient bowel O2 tissue saturation, and patient morbidity through the Comprehensive Complication Index (CCI®).ResultsA total of 26 patients were included: 13 underwent GDA reconstruction (GDA-R), and 13 had GDA ligation (GDA-L). There were no pancreatic leaks in the GR group compared to 38% (5/13) in the GDA-L group (p = 0.03913). Intraoperative tissue oxygen saturation was higher in the GDA-R group than in the GDA-L (95.18 vs.76.88%, p < 0,001). We observed an increase in transfusion rate in GDA-R (p < 0.05), which did not result in a higher rate of exploration (p = 0.38). CCI® patient morbidity was also significantly lower in the GDA-R group (s < 0.05).ConclusionsThis study identified improved intraoperative duodenal tissue oxygen saturation in the GDA-R group with an associated reduction in pancreatic leaks and CCI® morbidity risk. A larger prospective multicenter study comparing the two methods is warranted.

Beta-cell death and dysfunction drives hyperglycaemia in organ donors.

Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death. In pancreas donors after brain death, we compared clinical and biochemical data in 'insulin-treated' and 'not insulin-treated donors' (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated. Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)]. In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients.

Evaluation of the Impact of a New Cooling Cell Processor System on Islet Cell Isolation Facility.

Standard cell therapy equipment, including the gold standard cell processor to purify human islets for clinical transplantation, is rarely refrigerated, potentially exposing cells to elevated temperatures during the centrifugation step. Custom cooling systems have a direct benefit on human islet viability and function. The current study was designed to test the effectiveness of a newly developed, readily available cooled cell processor system requiring minimal modifications and to evaluate its impact on human cell viability and the GMP cleanroom environment.The cooler system, a mechanically refrigerated heat exchanger set at -30 °C was used to deliver cooled medical grade dry air to the cell processor bowl through a hole drilled in the centrifuge cover. With the limited availability of pancreas donors in Qatar, system validation was done with continuous density gradient purification of pooled human bone marrow buffy coat. Sterility, turbulence, and particle count were measured in class C and class B clean room environments. No turbulence developed around the cooled cell processor, and no excess 0.5 µm and 5 µm airborne particulates were generated as per cleanroom GMP standards. At the beginning and end of the collection steps, the temperature rose respectively to 21.50 °C ± 0.34 °C and 21.93 °C ± 0.20 °C in the non-cooled cell processor and to only 10.9 °C ± 0.17 °C and 11.16 °C ± 0.35 °C in the cooled- cell processor (p <0.05). The cooled cell processor led to both improved recovery (98%) of the mononuclear cell fraction and viability (100% ± 2%) post-processing. The new cooling system effectively reduces the heat produced by the cell processor while having no particulate impact on the GMP clean room environment. The cooled cell processor described here is an inexpensive ($16,000 without including taxes, customs clearance, and transportation) and minimally invasive method to provide robust cooling. Currently, this technology in the GMP cell therapy facilityis being applied to human islet cell isolation and transplantation for the clinical program.

European Society for Organ Transplantation (ESOT) Consensus Statement on the Role of Pancreas Machine Perfusion to Increase the Donor Pool for Beta Cell Replacement Therapy.

The advent of Machine Perfusion (MP) as a superior form of preservation and assessment for cold storage of both high-risk kidney's and the liver presents opportunities in the field of beta-cell replacement. It is yet unknown whether such techniques, when applied to the pancreas, can increase the pool of suitable donor organs as well as ameliorating the effects of ischemia incurred during the retrieval process. Recent experimental models of pancreatic MP appear promising. Applications of MP to the pancreas, needs refinement regarding perfusion protocols and organ viability assessment criteria. To address the "Role of pancreas machine perfusion to increase the donor pool for beta cell replacement," the European Society for Organ Transplantation (ESOT) assembled a dedicated working group comprising of experts to review literature pertaining to the role of MP as a method of improving donor pancreas quality as well as quantity available for transplant, and to develop guidelines founded on evidence-based reviews in experimental and clinical settings. These were subsequently refined during the Consensus Conference when this took place in Prague.

Islet transplantation: overcoming the organ shortage

BackgroundType 1 diabetes mellitus (T1D) is a condition resulting from autoimmune destruction of pancreatic β cells, leading patients to require lifelong insulin therapy, which, most often, does not avoid the most common complications of this disease. Transplantation of isolated pancreatic islets from heart-beating organ donors is a promising alternative treatment for T1D, however, this approach is severely limited by the shortage of pancreata maintained under adequate conditions.MethodsIn order to analyze whether and how this problem could be overcome, we undertook a retrospective study from January 2007 to January 2010, evaluating the profile of brain-dead human pancreas donors offered to our Cell and Molecular Therapy NUCEL Center (www.usp.br/nucel) and the basis for organ refusal.ResultsDuring this time period, 558 pancreata were offered by the São Paulo State Transplantation Central, 512 of which were refused and 46 were accepted for islet isolation and transplantation. Due to the elevated number of refused organs, we decided to analyze the main reasons for refusal in order to evaluate the possibility of improving the organ acceptance rate. The data indicate that hyperglycemia, technical issues, age, positive serology and hyperamylasemia are the top five main causes for declination of a pancreas offer.ConclusionsThis study underlines the main reasons to decline a pancreas offer in Sao Paulo—Brazil and provides some guidance to ameliorate the rate of eligible pancreas donors, aiming at improving the islet isolation and transplantation outcome.Trial registration: Protocol CAPPesq number 0742/02/CONEP 9230.

Pancreas Rejection in the Artificial Intelligence Era: New Tool for Signal Patients at Risk.

Pancreas transplantation is currently the only treatment that can re-establish normal endocrine pancreatic function. Despite all efforts, pancreas allograft survival and rejection remain major clinical problems. The purpose of this study was to identify features that could signal patients at risk of pancreas allograft rejection. We collected 74 features from 79 patients who underwent simultaneous pancreas-kidney transplantation (SPK) and used two widely-applicable classification methods, the Naive Bayesian Classifier and Support Vector Machine, to build predictive models. We used the area under the receiver operating characteristic curve and classification accuracy to evaluate the predictive performance via leave-one-out cross-validation. Rejection events were identified in 13 SPK patients (17.8%). In feature selection approach, it was possible to identify 10 features, namely: previous treatment for diabetes mellitus with long-term Insulin (U/I/day), type of dialysis (peritoneal dialysis, hemodialysis, or pre-emptive), de novo DSA, vPRA_Pre-Transplant (%), donor blood glucose, pancreas donor risk index (pDRI), recipient height, dialysis time (days), warm ischemia (minutes), recipient of intensive care (days). The results showed that the Naive Bayes and Support Vector Machine classifiers prediction performed very well, with an AUROC and classification accuracy of 0.97 and 0.87, respectively, in the first model and 0.96 and 0.94 in the second model. Our results indicated that it is feasible to develop successful classifiers for the prediction of graft rejection. The Naive Bayesian generated nomogram can be used for rejection probability prediction, thus supporting clinical decision making.

Noninvasive evaluation of donor and native pancreases following simultaneous pancreas-kidney transplantation using positron emission tomography/computed tomography.

It is crucial to develop practical and noninvasive methods to assess the functional beta-cell mass in a donor pancreas, in which monitoring and precise evaluation is challenging. A patient with type 1 diabetes underwent noninvasive imaging following simultaneous kidney-pancreas transplantation with positron emission tomography/computed tomography (PET/CT) using an exendin-based probe, [18 F]FB(ePEG12)12-exendin-4. Following transplantation, PET imaging with [18 F]FB(ePEG12)12-exendin-4 revealed simultaneous and distinct accumulations in the donor and native pancreases. The pancreases were outlined at a reasonable distance from the surrounding organs using [18 F]FB(ePEG12)12-exendin-4 whole-body maximum intensity projection and axial PET images. At 1 and 2 h after [18 F]FB(ePEG12)12-exendin-4 administration, the mean standardized uptake values were 2.96 and 3.08, respectively, in the donor pancreas and 1.97 and 2.25, respectively, in the native pancreas. [18 F]FB(ePEG12)12-exendin-4 positron emission tomography imaging allowed repeatable and quantitative assessment of beta-cell mass following simultaneous kidney-pancreas transplantation.

Pasireotide Versus Octreotide in Preventing Complications After Simultaneous Pancreas-Kidney Transplantation.

In elective pancreatic surgery, somatostatin-analogues pasireotide and octreotide are variably used to reduce postoperative complications, but knowledge on their role in pancreas transplantation is limited. This study compared pasireotide and octreotide for their association with complications after simultaneous pancreas-kidney transplantation (SPK). This retrospective study included consecutive patients undergoing SPK's from July 2013 to July 2022. Between July 2013 and April 2020, octreotide was administered 0.1mg s.c. once daily and between May 2020 and July 2022 pasireotide was administered 0.9mg twice daily, both until third postoperative day. Complications within 90days postoperatively were collected, and reoperation rate and Comprehensive Complication index (CCI) ≥ 33.7 (morbidity equal to one reoperation) were used as primary outcomes. Of the 213 patients undergoing SPK, 150 patients received octreotide and 63 pasireotide. Baseline characteristics were comparable. Reoperation rate was 25.3% (n = 38) and 17.5% (n = 11) (p = 0.213) and rate of CCI ≥ 33.7 was 40.7% (n = 61) and 30.2% (n = 19) (p = 0.148) in octreotide and pasireotide groups, respectively. When adjusted with donor BMI, pancreas donor risk index, and donor sex, receiving pasireotide translated into OR 0.49 (95% CI: 0.25-0.96 p = 0.037) for CCI ≥ 33.7. Pasireotide was independently associated with lower postoperative morbidity within 90days of SPK compared to octreotide.

Utilization of the Pancreas From Donors With an Extremely High Pancreas Donor Risk Index: Report of the National Registry of Pancreas Transplantation.

Pancreas transplants from expanded criteria donors are performed widely in Japan because there is a shortage of brain-dead donors. However, the effectiveness of this strategy is unknown. We retrospectively studied 371 pancreas transplants to evaluate the possibility of pancreas transplantation from expanded criteria donors by the Pancreas Donor Risk Index (PDRI). Patients were divided into five groups according to quintiles of PDRI values (Q1-Q5). The 1-year pancreas graft survival rates were 94.5% for Q1, 91.9% for Q2, 90.5% for Q3, 89.3% for Q4, and 79.6% for Q5, and were significantly lower with a lower PDRI (p = 0.04). A multivariate analysis showed that the PDRI, donor hemoglobin A1c values, and pancreas transplantation alone significantly predicted 1-year pancreas graft survival (all p < 0.05). Spline curve analysis showed that the PDRI was incrementally associated with an increased risk of 1-year graft failure. In the group with a PDRI ≥ 2.87, 8/56 patients had graft failures within 1month, and all were due to graft thrombosis. The PDRI is a prognostic factor related to the 1-year graft survival rate. However, pancreas transplantation from high-PDRI donors shows acceptable results and could be an alternative when the donor pool is insufficient.

Abstract IA19: Oncogenic KRAS and the regulation of the pancreatic cancer microenvironment.

Abstract Pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, is characterized by almost universal presence of oncogenic KRAS. A key characteristic of pancreatic cancer is the abundant accumulation of tumor microenvironment, rich in infiltrating immune cells and an extensive fibroblast population. Pancreatic cancer is preceded by precursor lesions known as Pancreatic Intraepithelial Neoplasia (PanIN); PanIN formation is accompanied by the formation of a pre-TME. Analysis of donor pancreas from healthy individual revealed that PanIN is common in human samples, and accompanied by pre-TME accumulation in human samples. We sought to determine whether oncogenic KRAS drives accumulation and maintenance of the pre-TME. For this purpose, we utilized the iKRAS mouse model of pancreatic cancer, which allows tissue-specific, inducible and reversible activation of KRASG12D, the most common mutant form in human pancreatic cancer. Activation of oncogenic KRAS in iKRAS mice leads to PanIN formation. We observed that fibroblast activation occurs upon KRAS activation and precedes the formation of overt lesions. Further, epithelial oncogenic KRAS is required to maintain fibroblast reprogramming in PanIN. To comprehensively understand changes in fibroblasts, we used single cell RNA sequencing, and observed that the fibroblast transcriptional program is extrinsically regulated by epithelial oncogenic KRAS. The reprogramming process can be modeled in vivo using conditioned media from pancreatic cancer cells expressing oncogenic KRAS, and is mediated by JAK/STAT3 signaling in fibroblasts. We then sought to determine whether the extrinsic effects of oncogenic KRAS induced reprogramming of fibroblasts at distal sites. We chose to study the lung, which is a common site of metastasis for pancreatic cancer. Our data show that lung fibroblasts are activated following expression of oncogenic KRAS in the pancreas; fibroblast activation is accompanied by phospho-Stat3 signaling, a readout of the JAK/Stat3 pathway, and precedes metastasis outgrowth in the pancreas. Thus, oncogenic KRAS extrinsically reprograms the local and systemic environments, possibly facilitating metastatic spread of pancreatic cancer. Citation Format: Marina Pasca di Magliano. Oncogenic KRAS and the regulation of the pancreatic cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA19.

Single molecule spatial transcriptomics demonstrates spatial heterogeneity of pre-miRNA and classifies β-cells in type 1 diabetes.

Recently developed spatial transcriptomics approaches combine RNA sequencing (RNA-Seq) with cell localization to reveal the spatial heterogeneity of transcriptomes in tissue samples. However, the interrogation of transcriptomic profiles in subcellular locations has not been reported. Here we propose a quantitative approach to measure the spatial distribution of single-cell transcriptomes, and we use this technique to study pre-miRNA expression in single β-cells obtained from human pancreatic tissues. A multi-dimensional quantitative model was established to describe the spatial distribution of RNAs as “features”, including RNA expression, location, and degree of clustering/dispersion. These features were analyzed using statistical distribution modeling and supervised machine learning to enable the classification of β-cells from the pancreas of non-diabetic control organ donors, autoantibody-positive (AAb+) organ donors, and organ donors with type 1 diabetes (T1D). This approach provided a quantitative evaluation of the distinctive features contributing to the classification and phenotyping and showed that expression levels of pre-miR-155 in β cells differ significantly among the groups, with more pre-miR-155 in the nucleus than cytoplasm of β-cells in the T1D and AAb+ samples compared to β-cells from control donors (p<0.0001). Interestingly, pre-miRNA distribution directly correlated with β-cell phenotype and T1D disease status. Results show higher degree of clustering for nuclear pre-miR-155 at short distances and greater amount of aggregation at the nuclear boundary for T1D samples (p<0.0001). These findings suggest that the spatial heterogeneity of the transcriptome of β-cells can classify these cells into different pathological conditions. Taken together, data from this study reveal fundamental mechanisms associated with RNA distribution and the β-cell phenotype during the pathogenesis of T1D. This approach could lead to the discovery of important subcellular spatial transcriptomic features that may have clinical relevance in stratifying T1D phenotypes.

Comparison of long-term outcomes in simultaneous pancreas-kidney transplant versus simultaneous deceased donor pancreas and living donor kidney transplant

Simultaneous deceased donor pancreas and living donor kidney transplant (SPLK) has certain advantages over conventional simultaneous pancreas-kidney transplant (SPK) and may be beneficial for overcoming the paucity of organs needed for diabetic patients requiring transplant. We compared the clinical outcomes of patients who underwent either SPK (n = 149) or SPLK (n = 46) in terms of pre- and post-transplantation variables, development of de novo DSA, occurrence of biopsy-proven acute rejection (BPAR), and graft survival rates. There were no significant differences in the baseline characteristics between the SPK and SPLK groups except for the shorter cold ischemic time of kidney grafts, shorter duration of diabetes, older age of pancreas graft-donors, and younger age of kidney graft-donors in the SPLK group. Our results showed that the death-censored pancreas graft survival rate was lower in the SPLK group. In addition, the incidence of BPAR of the pancreas graft was higher in the SPLK group. There was no significant difference in the presence of de novo DSA and the rates of kidney graft failure, kidney BPAR, and mortality. Our results show that SPLK can be considered an alternative option for SPK although higher incidences of BPAR and graft failure of pancreas after SPLK need to be overcome.

Decellularization of Human Pancreatic Fragments with Pronounced Signs of Structural Changes

A significant lack of donor organs restricts the opportunity to obtain tissue-specific scaffolds for tissue-engineering technologies. One of the acceptable solutions is the development of decellularization protocols for a human donor pancreas unsuitable for transplantation. A protocol of obtaining a biocompatible tissue-specific scaffold from decellularized fragments with pronounced human pancreas lipomatosis signs with preserved basic fibrillary proteins of a pancreatic tissue extracellular matrix was developed. The scaffold supports the adhesion and proliferation of human adipose derived stem cell (hADSCs) and prolongs the viability and insulin-producing function of pancreatic islets. Experiments conducted allow for the reliance on the prospects of using the donor pancreas unsuitable for transplantation in the technologies of tissue engineering and regenerative medicine, including the development of a tissue equivalent of a pancreas.