Patient-derived Pancreatic Cancer Xenograft Models Research Articles

Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth.

Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo fatty acid synthesis, and its ACC1 isoform is overexpressed in pancreatic and various other cancers. The activity of many oncogenic signaling molecules, including WNT and Hedgehog (HH), is post-translationally modified by lipidation. Here, we report that inhibition of ACC by a small molecule inhibitor, BAY ACC002, blocked WNT3A lipidation, secretion, and signaling. In pancreatic cancer cells, where WNT and HH are key oncogenic drivers, ACC inhibition simultaneously suppressed WNT and HH signaling, and led to anti-proliferative effects. Treatment with ACC inhibitors blocked tumor growth and converted the poorly differentiated histological phenotype to epithelial phenotype in multiple cell line-based and patient-derived pancreatic cancer xenograft models. Together, our data highlight the potential utility of ACC inhibitors for pancreatic cancer treatment, and provide novel insight into the link between upregulated de novo fatty acid synthesis in cancer cells, protein lipidation, and oncogenic signaling.

Abstract A24: Early-stage pancreatic cancer patient derived xenograft (PDX) models established from endoscopic ultrasound (EUS) fine needle aspiration (FNA) biopsies

Abstract Background: We developed and characterized PDX models generated from FNA endoscopic biopsies at the time of diagnosis of pancreatic ductal adenocarcinoma (PDAC). Our goal was to determine if an FNA-PDX model could be established within a clinically relevant time frame to inform treatment decisions prior to disease recurrence in patients with early stage, localized disease. Methods: Patients presenting with a localized mass in the pancreas and no metastatic disease were considered for EUS/FNA biopsy. Under sonographic guidance FNA biopsies were obtained. On-site cytopathology confirmed a histologic diagnosis of PDAC. The diagnostic FNA specimen was sent to pathology for formalin fixed paraffin embedded tissue block analysis per standard institutional pathologic protocols. Additional endoscopic FNA passes from the primary tumor were obtained during the index procedure by EUS guidance. The cell specimen was washed in cold DMEM and centrifuged. The cell pellet was rapidly transplanted into a subcutaneous pocket within the flank of a single NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD scid gamma, NSG) mouse. Tumor growth was monitored with calipers and engrafted tumors were passaged to subsequent mice when they reached a diameter of 10mm2. Growth kinetics and immunohistochemical staining for human cells with antibody against the human HLA Class 1 ABC antigen were obtained. Treatment groups were established from the F3 passage and randomized for in vivo chemosensitivity testing using FDA-approved chemotherapeutics: gemcitabine, cisplatin, docetaxel, irinotecan and fluorouracil. Results: The warm ischemia time was negligible. The average cold ischemia time, defined as the time from the endoscope to transplantation in the mouse, was 39 minutes. Engraftment was successful in 9 of 24 (37.5%) individual FNA patient specimens. Engraftment however increased with the number of FNA biopsy passes: 3/12 (25%) engraftment from one pass and 6/12 (50%) from two passes. Tumor size and surgical resectability status by CT imaging did not correlate with engraftment success. Procedural variables such as needle gauge and cytopathologic features such as degree of differentiation and percent tumor cellularity also did not correlate with rate of engraftment. PDX tumor morphology was maintained between F0 and F4 mice by H&E and human HLA immunohistochemical staining. The initial engrafted PDX tumors took 12 weeks (average) to grow to a diameter of 5 mm. The rate of engraftment for subsequent passaged mice was faster, averaging 6 weeks for the F1 development and stable for passaged tumors to the F5 generation. As expected, in vivo chemosensitivity was variable between FNA-PDX tumors from different patients as depicted in patient model #048 which was resistant to oxaliplatin, intermediate to docetaxel and fluorouracil but sensitive to cisplatin, irinotecan and gemcitabine. Conclusions: Pancreatic cancer PDX models can be generated from the diagnostic FNA biopsy specimen obtained during a patient's EUS procedure. The rate of engraftment doubles with the number of endoscopic passes suggesting that the amount of biopsy specimen tissue is important for engraftment while cytopathologic features associated with aggressive tumor growth such as tumor cellularity and tumor cell differentiation did not. Chemosensitivity was variable between FNA-PDX models. In patients with early stage disease the FNA-PDX model is stable and results from chemosensitivity assays can be obtained in a clinically meaningful window. Citation Format: Kerrington Smith, Dawn Fischer, Timothy Gardner, Stuart Gordon, Vijayalakshmi Padmanabhan, Thomas Colacchio, Richard Barth, Arief Suriawinata. Early-stage pancreatic cancer patient derived xenograft (PDX) models established from endoscopic ultrasound (EUS) fine needle aspiration (FNA) biopsies. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A24.

Abstract A95: Local iontophoretic delivery of FOLFIRINOX minimizes systemic toxicity for the treatment of pancreatic cancer

Abstract Systemic toxicity and poor delivery of many cytotoxic agents restrict their utility in the treatment of pancreatic cancer. Newer drug regimens, such as FOLFIRINOX, are limited to patients with the best performance status due to substantial toxicity. In an attempt to mitigate systemic toxicity, improve delivery and thus the efficacy of cytotoxic agents, we have developed modalities for the localized iontophoretic delivery of cytotoxic agents to primary pancreatic tumors. Iontophoretic delivery of mitomycin C, dexamethasone, and lidocaine have been successfully implemented for bladder cancer, anterior uveitis, and regional anesthesia. We initially evaluated the local iontophoretic device delivery of gemcitabine (GEM) to orthotopic patient-derived xenograft (PDX) models of pancreatic cancer. Devices were surgically implanted when tumors reached a median volume of 200 mm3, as determined by high-resolution 3D ultrasound. High-performance liquid chromatography (LC) analysis of the tumors directly after a single treatment showed a mean concentration of 229.4±233.0 µg/g for device delivery compared to 19.4±15.9 µg/g for intravenous (IV) delivery. Tumor pharmacokinetics (PK) as measured by area under the curve (AUC) was 384.1 hr*µg/g for device and 30.8 hr*µg/g for IV delivery; plasma GEM AUC was undetectable for device and 65.2 hr*µg/mL for IV delivery. To evaluate the tumor response to device delivery of GEM (DEVGEM), twice weekly treatments were administered for 7 weeks. DEVGEM resulted in significant tumor regression in 7 of 7 mice, outperforming IVGEM and the control arms of IV and device saline. Mice treated with DEVGEM had a mean log2-fold change in tumor volume of -0.8 compared to a mean log2-fold change in tumor volume of 1.1 for IVGEM, 3.0 for IV saline, and 2.6 for device saline groups (p < 0.01). DEVGEM was better tolerated based on greater body weight gain compared to IVGEM with minimal changes in alanine transaminase and lipase. Overall, DEVGEM resulted in tumor regression in 7/7 mice compared to no regression (0/7) in the IVGEM group while maintaining low systemic exposure of GEM. The safe and effective delivery of GEM in the PDX models supported the extension of this delivery modality to the more effective, but highly toxic regimen of FOLFIRINOX. Dose toxicity studies in mice were performed and IV dosing of FOLFIRINOX was determined. A doublet regimen (oxaliplatin (OX)/leucovorin (LV) followed by fluorouracil (FU)/irinotecan(IRI)), which leverages the potentiation of FU activity by LV and possible enhancement of IRI activity by OX, was used for both device and IV delivery. To evaluate the PK of device delivery of FOLFIRINOX, devices were implanted in PDX mice and a single treatment was administered. LC- and inductively coupled plasma-mass spectrometry analysis of the tumors directly after the treatment revealed a 33-fold greater concentration of FU (261.1±321.5 vs 8.0±2.1 µg/g), 31-fold greater concentration of OX (15.6±7.6 vs 0.5±0.3 µg/g), and 3-fold greater concentration of IRI (21.7±9.3 vs 7.2±1.9 µg/g) for device delivery compared to IV FOLFIRINOX. Analysis of plasma, pancreas, liver, and kidney showed significantly lower concentrations of drugs after device delivery compared to IV delivery, suggesting that device delivery of FOLFIRINOX will have minimal systemic toxicity. Long-term therapeutic studies in orthotopic PDX models of pancreatic cancer are ongoing. The local iontophoretic delivery of chemotherapeutics, especially FOLFIRINOX, has the potential to surpass conventional techniques of treatment by driving cytotoxic therapies directly into the tissue. This has potential paradigm shifting implications for the treatment of pancreatic cancer for patients with unresectable non-metastatic disease, palliation of local symptoms, and may be considered as a neoadjuvant modality prior to surgery. Citation Format: James D. Byrne, Mohammed R. Jajja, Allison N. Schorzman, Amanda W. Keeler, Adrian T. O'Neill, James C. Luft, William C. Zamboni, Joseph M. DeSimone, Jen Jen Yeh. Local iontophoretic delivery of FOLFIRINOX minimizes systemic toxicity for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A95.

Abstract A102: Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts

Abstract Rationale: Using orthotopically-grown primary pancreatic cancer xenografts we recently identified an association between hypoxia and spontaneous metastasis formation, and we hypothesized that metastatic potential is conferred by the presence of tumor-initiating cells (TIC). We therefore investigated the relationship between hypoxia, tumor growth and TIC, and examined the effect of the hypoxia-activated prodrug TH-302 and ionizing radiation (IR) on pancreatic TIC and tumor growth. Methods: Eleven patient-derived pancreatic cancer xenograft models were used to examine tumor-initiation potential in relation to the tumor phenotype. To examine the effect of TH-302 and IR on TIC, tumor-bearing mice were treated with 50 mg/kg or 150 mg/kg TH-302, 10Gy of IR, or with the combination of TH-302 and IR. To examine the effect of TH-302 and IR on tumor growth, mice were treated with either TH-302 or IR alone, or with the combination treatment according to a clinically relevant schedule. Animals were treated with 50mg/kg TH-302 injected ip on days 1, 5 and 9 and/or 2Gy irradiation per day on days 2, 3 and 4. Tumor hypoxia was measured by EF5 administration and immunostaining. Results: TIC frequency varied across the patient-derived xenograft models and rapid tumor growth was strongly correlated with high TIC frequency but not hypoxia. Treatment with TH-302 in combination with IR led to a significant reduction in TIC frequency compared with either treatment alone in all models tested. Treatment-induced reduction in TIC frequency was independent of the magnitude of tumor hypoxia. Treatment-induced reduction of tumor growth was only observed in fast-growing hypoxic models. Additionally, TH-302 treatment also induced DNA damage in tumor tissue adjacent to the hypoxic zone which is consistent with the previously demonstrated bystander effect of TH-302. Conclusions: These results suggest that TIC can reside in both the oxic and the hypoxic microenvironment and that the combination of TH-302 and IR may present a novel therapeutic strategy to reduce the frequency of TIC and tumor growth rate of pancreatic tumors. Citation Format: Ines Lohse, Joanna Rasowski, Pinjian Cao, Melania Pintilie, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, Richard P. Hill, David W. Hedley. Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A102.

Abstract B247: The MEK inhibitor BAY 869766 inhibits tumor growth and metastatic spread, prolongs the survival and acts synergistically with standard of care drugs in models of hepatocellular carcinoma and pancreatic cancer.

Abstract Introduction: BAY 869766 (RDEA119) is an orally available, allosteric MEK1/2 inhibitor, targeting the central switch in the MAP kinase pathway. BAY 869766 demonstrates potent in vitro and in vivo activity in different human cancer cell lines and preclinical models. BAY 869766 is currently in clinical phase I/II studies in the indications HCC (hepatocellular carcinoma) and pancreatic cancer (PaCa). In the described preclinical studies BAY 869766 was studied in two orthotopic, syngeneic HCC and PaCa models and five subcutaneous PaCa xenografts as a single agent and combination therapy. In contrast to subcutaneous xenografts, orthotopic syngeneic models provide additional important information about microenvironment-dependent tumor growth, metastatic spread, immune response and survival. Results: To establish a syngeneic, orthotopic HCC model, the rat HCC cell line MH3924a was first evaluated in vitro. BAY 869766 induced antiproliferative effects in MH3924a HCC cells in monotherapy and showed synergistic effects in combination with sorafenib. In the corresponding rat allograft model efficacy of BAY 869766 in mono- and combination therapy with sorafenib was confirmed. Primary tumor growth and metastatic spread were strongly reduced. These effects were accompanied by a reduction of pERK levels, proliferation index and microvessel density. Finally, the median overall survival in combination treatment was increased by 100% in comparison with vehicle treatment. To characterize BAY 869766 in an orthotopic syngenic PaCa model, the Panc02 mouse allograft model was established. BAY 869766 was tested in mono- and combination therapy with Gemcitabine. Especially, combination treatment led to significant lower tumor weight, reduction of metastatic spread and increase of median survival by 135%. To test BAY 869766 in further clinical relevant pancreatic cancer models, five patient-derived pancreatic cancer tumor samples were subcutaneously implanted in mice and subsequently treated with BAY 869766. Significant tumor growth inhibition in these patient-derived pancreatic cancer xenograft models was observed (Treatment/Control ratios from 23,5% − 44%). Conclusion: These preclinical results support the clinical development of the MEK inhibitor BAY 869766 in HCC and pancreatic cancer. Inhibition of Ras-Raf-MEK-Erk-signaling, direct antiproliferative effects on tumor cells, reduction of metastatic spread and anti-angiogenic properties were demonstrated in these studies. In combination with the respective SoC drugs a significant benefit in survival could be achieved in the orthotopic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B247.

Abstract 3117: Expression of Hedgehog pathway molecules in patient-derived pancreatic adenocarcinoma xenograft models

Abstract Introduction. Pancreatic cancer (PC) is a devastating disease characterized by high failure rates of the conventional therapies. Increasing evidence suggest that PC resistance to therapies is due to their intrinsic ability to develop high contents of fibrotic stroma. It was recently shown that Hedgehog (HH) pathway can influence PC sensitivity to gemcitabine by modulating stroma production via paracrine mechanisms in between tumor cells and its stroma environment. Taking advantage that the stroma in engrafted human tumors in nude mice is produced by the host, we investigated the differential expression of HH pathway molecules by human PC cells and associated murine stroma in PC xenograft models. For this purpose we developed a new species-discriminating qRT-PCR methodology and analyzed a comprehensive collection of 27 patient-derived PC xenograft models previously characterized for their chemosensitivity. Methods. RNA was extracted from snap frozen PC xenograft samples. After reverse transcription, the samples were assessed using human and mouse species-specific qPCR assays for Tata Binding Protein (TBP), Sonic (SHH), Indian (IHH) and Desert (DHH) Hedgehog ligands, the Hedgehog receptor PATCH1 (PTCH1) and the G-coupled downstream receptor Smoothened (SMO), and for the Zn finger transcription factors GLI1, GLI2, GLI3. Data from human and mouse TBP assays were used for normalization and as surrogate markers of content of human tumor cells vs murine stroma cells in the models. Results. As measured using TBP, the amount of murine stroma in the PC xenograft models is comparable to that in primary PC tumors and could be as high as 50%. The PC xenograft models with the highest amount of stroma were also most resistant to standard therapies. Among the 27 PC xenograft models the expression of the HH molecules was highly variable. However, we consistently evidenced that the human PC cells preferentially express the HH ligands with much lower or neclectable expression of PTCH1, SMO and GLI transcription factors. SHH and at a lower level IHH were found to be predominant compared to DHH. Of note, tumors cells of few selected xenograft models express however a consistent level of HH receptors and GLI transcription factors. On the opposite, the stroma cells were shown to preferentially express the receptors PTCH1 and SMO as well as GLI transcription factors, but not the HH ligands. In nearly all xenograft models, the transcription factor GLI1 and at lower levels GLI2 were more highly expressed than GLI3. Conclusion. In PC xenograft models, HH ligands are expressed by tumor cells, whereas respective receptors and downstream signaling molecules are predominantly expressed in the murine stroma. This result argues in favor of a predominantly paracrine mechanism of HH in most of the PC xenograft models investigated. The activity of HH pathway inhibitors to modulate the tumor stroma and affect tumor growth will be investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2011-3117