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Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth.Significance:This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

SLC7A11 knockdown in PDAC CAFs does not affect glutamate secretion and is maintained in the presence of oxidative stress.

Australian Pancreatic Cancer Genome Initiative International Cancer Genome Cohort patient cohort characteristics.

SLC7A11 multivariate survival analysis parameters.

SLC7A11 expression in iCAFs, myCAFs and quiescent PSCs.

List of antibodies

The microtubule protein, βIII-tubulin, has been implicated as a prognostic, pro-survival, and chemoresistance factor in some of the most lethal malignancies including pancreatic ductal adenocarcinoma (PDAC). However, precise survival mechanisms controlled by βIII-tubulin in cancer cells are unknown. Here, we report an unexpected role of βIII-tubulin as a brake on extrinsic caspase 8-dependent apoptosis in PDAC. We show that βIII-tubulin knockdown frees death-receptor DR5 to increase its membrane diffusion, clustering, and activation of cell-death. We demonstrate that βIII-ubulin silencing increases sensitivity of PDAC cells to chemotherapeutic and microenvironment-derived extrinsic cell-death signals including TRAIL, TNFα, and FasL. Finally, nanoparticle delivery of βIII-tubulin siRNA to mouse orthotopic PDAC tumours in vivo and human patient-derived PDAC tumour explants ex vivo increases extrinsic apoptosis and reduces tumour progression. Thus, silencing of βIII-tubulin represents an innovative strategy to unleash a suicide signal in PDAC cells and render them sensitive to microenvironment and chemotherapy-derived death signals.

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attributed to the highly fibrotic stroma and complex multi-cellular microenvironment that is difficult to fully recapitulate in pre-clinical models. To fast-track translation of therapies and to inform personalised medicine, we aimed to develop a whole-tissue ex vivo explant model that maintains viability, 3D multicellular architecture, and microenvironmental cues of human pancreatic tumours. Patient-derived surgically-resected PDAC tissue was cut into 1–2 mm explants and cultured on gelatin sponges for 12 days. Immunohistochemistry revealed that human PDAC explants were viable for 12 days and maintained their original tumour, stromal and extracellular matrix architecture. As proof-of-principle, human PDAC explants were treated with Abraxane and we observed different levels of response between patients. PDAC explants were also transfected with polymeric nanoparticles + Cy5-siRNA and we observed abundant cytoplasmic distribution of Cy5-siRNA throughout the PDAC explants. Overall, our novel model retains the 3D architecture of human PDAC and has advantages over standard organoids: presence of functional multi-cellular stroma and fibrosis, and no tissue manipulation, digestion, or artificial propagation of organoids. This provides unprecedented opportunity to study PDAC biology including tumour-stromal interactions and rapidly assess therapeutic response to drive personalised treatment.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and new therapeutic strategies are urgently required. A key drug delivery barrier and promoter of tumor progression is the fibrotic and heterogeneous microenvironment of PDAC which is mainly orchestrated by stromal cancer-associated fibroblasts (CAFs). Thus, any novel therapeutic strategy should not only target PDAC cells but should also aim to inhibit the pro-tumor properties of the PDAC stroma. However, to accurately evaluate the clinical potential of novel PDAC therapeutics, preclinical models are required that closely mimic the heterogeneity and multicellular architecture of human disease. Aim: We aimed to investigate the therapeutic potential of novel functionalized benzopyrans (CRO-05 & CRO-67) in a patient-derived PDAC tumor explant model. Methods: We collected PDAC tumor samples from patients undergoing resection of their primary tumor. We cultured whole tumor explants (1-2mm diameter) on gelatin sponges. This model accurately maintains the multicellular tumor and stromal architecture of human PDAC [Kokkinos et al, Scientific Reports, 11,1941 (2021)]. Tumor explants were treated every 3 days with the racemic compound CRO-05 or its active enantiomer CRO-67 and explants were fixed in paraformaldehyde at day-12. Therapeutic response was assessed by immunohistochemistry for markers of tumor cells (cytokeratin), CAFs (α-Smooth Muscle Actin), cell-proliferation (Bromodeoxyuridine), and cell-death (TUNEL). Results: Treatment of human PDAC explants with CRO-05 resulted in reduced tumor cell frequency in 3/4 patients and in 4/4 patients, reduced CAF frequency, decreased cell-proliferation, and increased cell-death relative to controls. CRO-67 treatment in explants from 4/5 patients led to reduced tumor cell frequency, and in 5/5 patients, reduced CAF frequency, decreased cell-proliferation, and increased cell-death relative to controls. Conclusions: This work identifies the functionalized benzopyrans CRO-05 and CRO-67 as novel dual-cell therapeutics that potently inhibit both PDAC tumor cells and their surrounding CAFs and provides support for their clinical development as novel PDAC therapeutics. Citation Format: John Kokkinos, George Sharbeen, Koroush S. Haghighi, Janet Youkhana, Aparna Raina, Omali Pitiyarachchi, Quach Truong, Daniel Wenholz, Olivier Laczka, Naresh Kumar, David Goldstein, Phoebe A. Phillips. CRO-67 is a novel therapeutic for pancreatic cancer: Implications for tumor and stromal reprogramming [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C073.

Fibroblast growth factor 21 (FGF21) has been suggested as a novel biomarker for cardiovascular disease (CVD), especially in people with high CVD risk. However, it is not known whether FGF21 is a CVD biomarker in an initially healthy cohort. We therefore investigated the relationship of plasma FGF21 levels with measures of subclinical atherosclerosis and cardiovascular events in Multi-Ethnic Study of Atherosclerosis participants without known CVD at baseline.A total of 5788 participants had plasma FGF21 levels measured at the baseline exam (2000-2002). Carotid intima-media thickness (IMT), ankle-brachial index (ABI) and coronary artery calcification (CAC) were measured at baseline. Participants were followed up for incident CVD events over a median period of 14 years.In cross-sectional analyses adjusting for socio-demographic variables, participants with higher FGF21 levels had higher carotid IMT, lower ABI, and higher prevalence of CAC (p 0.001). However, these associations were not significant after simultaneously adjusting for demographic, socioeconomic and lifestyle factors, traditional CVD risk factors, and biomarkers of inflammation and hemostasis. Among 5768 patients with follow-up data, 820 developed incident CVD endpoints. Higher baseline FGF21 levels were not associated with the risk for incident CVD endpoints after adjusting for multiple confounding factors (odds ratio 1.03; 95% confidence interval, 0.94-1.12, per SD increase in ln-transformed FGF21 levels).Although FGF21 has been suggested as a CVD biomarker for people with high CVD risk, our findings do not support a role of FGF21 as a CVD biomarker in those without a history of CVD.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle genesilencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. Significance: this study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC., NHMRC Project Grant; Avner Innovation Grant; NHMRC CDF-I; NHMRC Ideas Grant ; Cancer-Institute NSW ECF/CDFs ; Cancer Institute NSW Innovation Grant; Cancer Institute NSW The Professor Rob Sutherland AO Make a Difference Award; Cancer Australia/Cancer Council; Cancer Australia/Kids Cancer Project; Cure Cancer Australia; Tour de Cure PhD Support Scholarship; Tour de Cure Established Research Grant; Tour de Cure Pioneering Research Grant; UNSW Interlude Grant Scheme; Cancer Research UK Core Funding and Grand Challenge Grants; NHMRC CDF-II; NHMRC Senior Research Fellowship; Suttons, Cancer Council NSW; Avner Grant from PanKind; Australian Pancreatic Cancer Foundation; Translational Cancer Research Network and Australian Postgraduate Award Scholarships; Australian Government Research Training Program Scholarship; UNSW Sydney Scientia PhD Scholarship; Pancreatic Cancer UK Future Leaders Academy; Len Ainsworth Pancreatic Cancer Fellowship

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is attributed to the highly fibrotic stroma and complex multi-cellular microenvironment that is difficult to fully recapitulate in pre-clinical human models. To fast-track translation of therapies and to inform personalised medicine, we aimed to develop a whole-tissue ex vivo explant model that maintains viability, 3D multicellular architecture, and microenvironmental cues present in human pancreatic tumours. Patient-derived surgically-resected PDAC tissue was cut into 2 mm explants, cultured on gelatin sponges, and grown for 12 days. Immunohistochemistry revealed that human PDAC tissue explants were viable for 12 days and maintained their original tumour, stromal and extracellular matrix architecture. As proof-of-principle, human PDAC tissue explants responded to Abraxane® treatment with a 3.7-fold increase in cell-death (p=0.0007). PDAC explants were also transfected with polymeric nanoparticles+Cy5-siRNA and we observed abundant cytoplasmic distribution of nanoparticle+Cy5-siRNA throughout the PDAC explant tissue. Our novel model retains the 3D architecture of human pancreatic tumours and has several advantages over standard organoids: presence of functional multi-cellular stroma and fibrosis and no tissue manipulation, digestion, or artificial propagation of organoids. This provides an unprecedented opportunity to study PDAC biology, tumour-stromal interactions and rapidly assess therapeutic response that could drive personalised treatment for PDAC.

Cancer-Associated Fibroblasts (CAFs) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression, through pro-tumour cross-talk and the generation of fibrosis (physical barrier to drugs). CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumour stroma and its prognostic significance. Herein we show that high expression of SLC7A11 in PDAC tumour stroma (but not tumour cells) is independently prognostic of poorer overall survival. We demonstrate using orthogonal approaches that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis, and that SLC7A11 inhibition significantly decreases their proliferation, reduces their resistance to oxidative stress and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, our paradigm-shifting work demonstrates the need to inhibit SLC7A11 in the PDAC stroma, as genetic ablation of SLC7A11 in PDAC cells alone is not enough to reduce tumour growth. Finally, our work validates that a nano-based gene-silencing drug against SLC7A11, developed by our group, reduces PDAC tumour growth, CAF activation and fibrosis in a mouse model of PDAC.

Background and aimsFibroblast growth factor 21 (FGF21) plays an important role in glucose and lipid metabolism. We have investigated the relationship of plasma FGF21 levels with both prevalent and incident metabolic syndrome (MetS) in participants from the Multi-Ethnic Study of Atherosclerosis (MESA).Methods5783 participants from four major ethnic groups (non-Hispanic white, African American, Hispanic American, and Chinese American) were included in the cross-sectional analysis. Longitudinal analysis involved 3479 participants without MetS at baseline, of whom 1100 participants developed incident MetS over 9.2 years.ResultsElevated FGF21 levels were found in participants with prevalent MetS (median [interquartile range] = 189.4 [114.4-302.1] vs. 123.7 [65.9-210.3] pg/mL, p

Simple Summary Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all major cancers. We now know that a ‘one-size-fits-all’ approach when treating PDAC patients does not work, so research has focused on developing personalised treatment strategies based on the biology of each patient’s tumour. However, most of these strategies ignore the presence of the characteristic fibrotic PDAC stroma which can act like a fortress to protect the tumour from chemotherapy and drive its aggressive nature. In this review, we summarise the role of this ‘fortress’ in protecting PDAC from chemotherapy and highlight the need for pre-clinical models that are informing precision medicine to reflect this hallmark feature of PDAC. We also propose the power of ex vivo whole-tissue culture models that mimic the tumour and its surrounding fortress to inform personalised treatment for PDAC. Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and no significant improvement in patient survival has been seen in the past three decades. Treatment options are limited and selection of chemotherapy in the clinic is usually based on the performance status of a patient rather than the biology of their disease. In recent years, research has attempted to unlock a personalised treatment strategy by identifying actionable molecular targets in tumour cells or using preclinical models to predict the effectiveness of chemotherapy. However, these approaches rely on the biology of PDAC tumour cells only and ignore the importance of the microenvironment and fibrotic stroma. In this review, we highlight the importance of the microenvironment in driving the chemoresistant nature of PDAC and the need for preclinical models to mimic the complex multi-cellular microenvironment of PDAC in the precision medicine pipeline. We discuss the potential for ex vivo whole-tissue culture models to inform precision medicine and their role in developing novel therapeutic strategies that hit both tumour and stromal compartments in PDAC. Thus, we highlight the critical role of the tumour microenvironment that needs to be addressed before a precision medicine program for PDAC can be implemented.

Pancreatic cancer is predicted to be the second leading cause of cancer-related death by 2025. The best chemotherapy only extends survival by an average of 18 weeks. The extensive fibrotic stroma surrounding the tumor curbs therapeutic options as chemotherapy drugs cannot freely penetrate the tumor. RNA interference (RNAi) has emerged as a promising approach to revolutionize cancer treatment. Small interfering RNA (siRNA) can be designed to inhibit the expression of any gene which is important given the high degree of genetic heterogeneity present in pancreatic tumors. Despite the potential of siRNA therapies, there are hurdles limiting their clinical application such as poor transport across biological barriers, limited cellular uptake, degradation, and rapid clearance. Nanotechnology can address these challenges. In fact, the past few decades have seen the conceptualization, design, pre-clinical testing and recent clinical approval of a RNAi nanodrug to treat disease. In this review, we comment on the current state of play of clinical trials evaluating siRNA nanodrugs and review pre-clinical studies investigating the efficacy of siRNA therapeutics in pancreatic cancer. We assess the physiological barriers unique to pancreatic cancer that need to be considered when designing and testing new nanomedicines for this disease.

The metabolic properties of the endocrine fibroblast growth factor 21 (FGF21) have been extensively studied in the past decade. Previous studies have demonstrated the lipid-lowering, anti-inflammatory and anti-oxidant properties of FGF21. FGF21 is mainly secreted in the liver and adipose tissue in response to a range of physiological and pathological stimuli. In animal and in vitro studies, FGF21 has been shown to improve lipid profiles and inhibit key processes in the pathogenesis of atherosclerosis. It exerts its effects on the cardiovascular system via adiponectin dependent and independent mechanisms. However, the signalling pathways by which FGF21 exerts its effects on endothelial cells remains unknown and needs to be further investigated. The elevation of circulating FGF21 levels in cardiovascular disease has also raised questions as to whether FGF21 can be used as a biomarker to predict subclinical atherosclerosis and cardiovascular events. Recent findings from population studies must be validated in independent cohorts before FGF21 can be used as a biomarker in the clinical setting. The anti-atherosclerotic effects of FGF21 have been investigated in two recent clinical trials, where treatment with an FGF21 analog significantly improved the cardiometabolic profile in obese patients with type 2 diabetes. This review will evaluate recent advances that suggest there may be a role for FGF21 in atherosclerosis.