Tumor and stromal reaction under drug treatment in EUS-derived xenografts of human pancreatic adenocarcinoma

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is unique for its high stromal components play important role for tumor progression and therapeutic resistance. However, relevant preclinical models that recapitulate these tumor microenvironment are lacking and how the differences in the microenvironment affect cancer cell behavior are poorly understood. Here, we investigated the effects of tumor microenvironment on cancer cells behavior and therapeutic response. METHODS: Previously established two behaviorally distinct patient-derived tumor xenografts (PDTX) models (moderate: PAC006 and poorly differentiated: PAC010) were re-implanted in Nude mice. Tumor volume, histology, immuno-histochemical, RNA sequencing and other molecular technique were used to characterize the models and study the effect of two classes of drugs (Gemcitabine (nucleoside analog) and Acriflavine (HIF-inhibitor)). RESULTS: -Models- The models recapitulated the histologic, genetic and biological characteristics of the corresponding primary tumors except the replacement of human stroma with mice stroma. The growth-rate of the tumors was significantly different between both models (cell doubling time, PAC006: 5.8 days vs PAC010: 3.6 days, p=0.005). Rapid tumor growth was associated with poor tumor differentiation that may reflect the ability of our PDTX models to mimic the cellular and non-cellular features of the parental tumor. At the molecular level, the poorly differentiated model showed increased Ki-67 staining and higher phosphorylation levels of AKT, ERK and NF-kB65. In addition, RT-qPCR, NGS and protein expression showed significant up-regulation of mesenchymal markers in tumor cells (e.g. VIM, SNAI2, HIF1A and TGF-β1) and growth factors and cytokines in mice stroma. RNA sequencing analysis from tumor cells and stroma demonstrated activation of pathways related to tumor progression and aggressiveness in PAC010 compared to PAC006. Gemcitabine treatment resulted in a shrinking of the tumor and reduction of proliferation in both models. Interestingly, gemcitabine treatment also significantly enhanced the expression of mesenchymal marker (e.g. VIM, SNAI2, SPARC, ZEB1), particularly in the well-moderately differentiated tumor model. Acriflavine had little effect on tumor growth in both models. Tumor implantation, treatment procedure, growth and sample collection: Stored tumor tissue was implanted into a first group of 4 mice, after establishment of growth tumor tissue in these mice the tumors were used for expansion in a new generation of mice. After the tumor reach a volume of 100-200 mm3, these mice were randomly divided into three groups with eight mice in each group; A) the control group that was treated with vehicle (0.9% NaCl) and the experimental groups was treated intraperitoneally for up to 28 days with B) Gemcitabine (50 mg/kg) injected twice a week. C) Acriflavine: schedule four doses of acriflavine were injected daily with a fixed dose of 5 mg/kg Monday to Thursday and single dose of 10 mg/kg on Friday for the weekend. Prior to actual study, a toxicity study for ACF was carried out. The body weight and tumor size were measured thrice a week (Monday, Wednesday and Friday). The tumor volume was calculated using the formula: Tumor length (mm) x Tumor width (mm) x Tumor depth (mm) x (3.14/6) = Tumor volume (mm³). Tumors were harvested as soon as their volume reached 1000 – 1500  mm3. The tissue samples from each tumor were weighed, photographed and stored for histological analysis and molecular profiling.