Tumour–stroma interactions in an organotypic culture model of pancreatic cancer

PhDPancreatic cancer is characterised by an intense fibrotic, or desmoplastic, stroma, which contributes to tumour progression. Three-dimensional in vitro culture models incorporating this non-tumour component may more closely recapitulate the complex in vivo situation. The aim of my project was to develop a physiologically relevant, three-dimensional organotypic culture model of pancreatic cancer to study the tumour-stroma interactions and its modulation by novel therapeutic agents. Cancer cells cultured on top of collagen/Matrigel gels, embedded with or without stromal cells (hTERT immortalised PS1 stellate cells or MRC5 fibroblasts), differentiated into luminal structures, exhibiting a central apoptotic core with a proliferating peripheral rim and apicobasal polarity. Stromal cells induced a reduction in total tumour cell number, which was associated with a decrease in E-cadherin expression, upregulated β-catenin expression and translocation of ezrin from the apical to the basal aspect of cancer cells, where it was associated with invasive activity. Subsequently, this organotypic model was raised to an air-liquid interface to study the direct and indirect effects of all-trans retinoic acid (ATRA), which rendered stellate cells back to their quiescent phenotype. Indirect effects of quiescent stellate cells on pancreatic cancer cells included changes in proliferation (decrease), apoptosis (increase), invasion (decrease), Wnt/β-catenin signalling (decrease) and an altered morphology. The Wnt/β-catenin signalling 6 perturbations were mediated by restoration of sFRP4 (secreted frizzled-related protein 4) secretion by quiescent stellate cells, resulting in reduced cancer cell invasion (reporter and invasion assays). All such observations could be validated in human pancreatic cancer tissue samples. Taken together, pancreatic organotypic culture offers a reproducible, in vitro three-dimensional culture model, which allows the study of tumour-stroma interactions in a physiologically relevant system. For treatment of pancreatic cancer, a tumour characterised by a poor response to conventional chemotherapeutic drugs, targeting the tumour-stroma cross-talk with agents such as ATRA offers an exciting novel therapeutic strategy.European Association of Cancer Researc

Phase I clinical trial repurposing all-trans retinoic acid as a stromal targeting agent for pancreatic cancer

Abstract: Pre-clinical models have shown that targeting pancreatic stellate cells with all-trans-retinoic-acid (ATRA) reprograms pancreatic stroma to suppress pancreatic ductal adenocarcinoma (PDAC) growth. Here, in a phase Ib, dose escalation and expansion, trial for patients with advanced, unresectable PDAC (n = 27), ATRA is re-purposed as a stromal-targeting agent in combination with gemcitabine-nab-paclitaxel chemotherapy using a two-step adaptive continual re-assessment method trial design. The maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D, primary outcome) is the FDA/EMEA approved dose of gemcitabine-nab-paclitaxel along-with ATRA (45 mg/m2 orally, days 1–15/cycle). Dose limiting toxicity (DLT) is grade 4 thrombocytopenia (n = 2). Secondary outcomes show no detriment to ATRA pharmacokinetics.. Median overall survival for RP2D treated evaluable population, is 11.7 months (95%CI 8.6–15.7 m, n = 15, locally advanced (2) and metastatic (13)). Exploratory pharmacodynamics studies including changes in diffusion-weighted (DW)-MRI measured apparent diffusion coefficient after one cycle, and, modulation of cycle-specific serum pentraxin 3 levels over various cycles indicate stromal modulation. Baseline stromal-specific retinoid transport protein (FABP5, CRABP2) expression may be predicitve of response. Re-purposing ATRA as a stromal-targeting agent with gemcitabine-nab-paclitaxel is safe and tolerable. This combination will be evaluated in a phase II randomized controlled trial for locally advanced PDAC. Clinical trial numbers: EudraCT: 2015-002662-23; NCT03307148. Trial acronym: STARPAC

HNF4A and GATA6 loss reveals therapeutically actionable subtypes in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer

Background and aims: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress and novel therapeutic response in PC to develop a biomarker driven therapeutic strategy targeting DDR and replication stress in PC. Methods: We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient derived xenografts and human PC organoids. Results: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum (P < 0.001) and PARP inhibitor therapy (P < 0.001) in vitro and in vivo. We generated a novel signature of replication stress with which predicts response to ATR (P < 0.018) and WEE1 inhibitor (P < 0.029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < 0.001) but not associated with DDR deficiency. Conclusions: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy

Host phenotypic adversity independently predicts progression to resection in neoadjuvantly-treated pancreatic cancer

Background: Neoadjuvant treatment (NAT) offers a modest survival benefit to the otherwise dismal outcomes of pancreatic ductal adenocarcinoma (PDAC), however methods for assessing treatment response and disease trajectory during NAT are limited. Cachexia and sarcopenia are hallmark features of PDAC and defining host phenotypic adversity is crucial in understanding the tumour-host relationship and its influence on clinical outcomes. We sought to determine the relationship between body composition, systemic inflammation, and outcomes in NAT PDAC to lay foundations for improving assessment of treatment response. Materials and Methods: This is a retrospective, observational study with an intention-to-treat design, analysing resected (n = 109) and non-resected (n = 117) NAT PDAC (total n = 226) from the tertiary West of Scotland Pancreatic Unit, Glasgow Royal Infirmary. Patients were included if they had resectable or borderline resectable disease and received NAT. Patients were excluded if they had locally advanced or metastatic disease. Body composition was determined by single-slice L3 cross-sectional measurements. Results: Patients with an adverse phenotype defined by change in body composition before and after treatment were significantly less likely to progress to resection (40.9% vs 68.8%, p < 0.001). Logistic regression confirms the independence of this effect (OR 3.79, 95% CI 1.99-7.22, p < 0.001). Similarly, there was a significant trend towards the development of or the persistence of systemic inflammation in the non-resected group (CRP rise or persistent elevation 51.4% vs 29.9%, p = 0.008). Patients treated with Folfirinox who required dose reduction or who did not tolerate six cycles received relatively higher doses of irinotecan, 5-fluorouracil (bolus) and 5-fluorouracil (infusion) per unit muscle mass (7.3mg/cm2⋅m-2 vs 6.9mg/cm2⋅m-2, p = 0.010; 16.2mg/cm2⋅m-2 vs 14.7mg/cm2⋅m-2, p = 0.002; 96.5mg/cm2⋅m-2 vs 91.1mg/cm2⋅m-2, p = 0.026). Conclusions: Host phenotypic adversity during NAT presents a potentially valuable addition to a limited arsenal of tools to aid decision making in resectable and borderline resectable PDAC. Early assessment of body composition trend during chemotherapy may present an opportunity to switch the host phenotype from adverse to favourable in some patients, or highlight the futility of continuing aggressive treatment in others. Chemotherapeutic prescribing by body surface area overlooks crucial differences in lean body mass and may be exposing patients to avoidable and burdensome toxicities

Exploring the biology of cancer-associated fibroblasts in pancreatic cancer

No abstract available

Molecular Subtyping and Precision Medicine for Pancreatic Cancer

Substantial progress in recent years has dramatically increased our knowledge of the molecular basis of cancer, revealing new potential therapeutic targets and paving the way for effective personalised medicine for the treatment of many tumour types. However, pancreatic cancer has been lagging behind in this success and continues to be one of the most lethal solid malignancies. Its molecular heterogeneity and the unselected design of the majority of clinical trials to date can in part explain the reason for our failure to make a significant change in the survival outcomes for patients with pancreatic cancer. A changing paradigm in drug development is required to validate the new molecular taxonomy and to rapidly translate preclinical discovery into clinical trials. Here, we review the molecular subtyping of pancreatic cancer, the challenges in identifying effective treatment regimens according to defined low-prevalence molecular subgroups and we illustrate a new model of translational therapeutic development that was established in the U.K. (Precision-Panc) as a potentially effective solution to improve outcomes for patients with pancreatic cancer