Human Pancreatic Ductal Adenocarcinoma Xenografts Research Articles

Nanotechnology-Based Strategy for Enhancing Therapeutic Efficacy in Pancreatic Cancer: Receptor-Targeted Drug Delivery by Somatostatin Analog

A novel nanotechnology-based drug delivery system (DDS) targeted at pancreatic cancer cells was developed, characterized, and tested. The system consisted of liposomes as carriers, an anticancer drug (paclitaxel) as a chemotherapeutic agent, and a modified synthetic somatostatin analog, 5-pentacarbonyl-octreotide, a ligand for somatostatin receptor 2 (SSTR2), as a targeting moiety for pancreatic cancer. The cellular internalization, cytotoxicity, and antitumor activity of the DDS were tested in vitro using human pancreatic ductal adenocarcinoma (PDAC) cells with different expressions of the targeted SSTR2 receptors, and in vivo on immunodeficient mice bearing human PDAC xenografts. The targeted drug delivery system containing paclitaxel exhibited significantly enhanced cytotoxicity compared to non-targeted DDS, and this efficacy was directly related to the levels of SSTR2 expression. It was found that octreotide-targeted DDS proved exceptionally effective in suppressing the growth of PDAC tumors. This study underscores the potential of octreotide-targeted liposomal delivery systems to enhance the therapeutic outcomes for PDAC compared with non-targeted liposomal DDS and Paclitaxel-Cremophor® EL, suggesting a promising avenue for future cancer therapy innovations.

Allysine-Targeted Molecular MRI Enables Early Prediction of Chemotherapy Response in Pancreatic Cancer.

Neoadjuvant therapy (NAT) is routinely used in pancreatic ductal adenocarcinoma (PDAC), but not all tumors respond to this treatment. Current clinical imaging techniques are not able to precisely evaluate and predict the response to neoadjuvant therapies over several weeks. A strong fibrotic reaction is a hallmark of a positive response, and during fibrogenesis allysine residues are formed on collagen proteins by the action of lysyl oxidases (LOX). Here we report the application of an allysine-targeted molecular magnetic resonance imaging (MRI) probe, MnL3, to provide an early, noninvasive assessment of treatment response in PDAC. Allysine increased 2- to 3-fold after one dose of NAT with FOLFIRINOX in sensitive human PDAC xenografts in mice. Molecular MRI with MnL3 could specifically detect and quantify fibrogenesis in PDAC xenografts. Comparing the MnL3 signal before and 3 days after one dose of FOLFIRINOX predicted subsequent treatment response. The MnL3 tumor signal increased by 70% from day 0 to day 3 in mice that responded to subsequent doses of FOLFIRINOX, while no signal increase was observed in FOLFIRINOX-resistant tumors. This study indicates the promise of allysine-targeted molecular MRI as a noninvasive tool to predict chemotherapy outcomes.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1392.

Anti-proliferative and apoptotic effect of cannabinoids on human pancreatic ductal adenocarcinoma xenograft in BALB/c nude mice model

Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti–tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti–proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.

Use of DNA-alkylating pyrrole-imidazole polyamides for anti-cancer drug sensitivity screening in pancreatic ductal adenocarcinoma.

Activating mutations of the KRAS occurs in >90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, direct pharmacological targeting of the activated KRAS protein has been challenging. We previously reported that KR12, a DNA-alkylating pyrrole-imidazole polyamide designed to recognize the KRAS G12D/V mutation, showed an anti-tumor effect in colorectal cancer. In this study, we evaluated the anti-tumor effect of KR12 in PDAC. KR12 was synthesized by an automated peptide synthesizer PSSM-8 and tested for anti-tumor effect in PDAC mouse models. KR12 inhibited tumor growth in a spontaneous PDAC mouse model, although the anti-tumor activity appeared to be limited in a human PDAC xenograft model. We developed a pyrrole-imidazole polyamide screening process based on the hypothesis that genetic elements otherwise unaffected by KR12 could exert attenuating effects on KRAS-suppression-resistant PDAC. We identified RAD51 as a potential therapeutic target in human PDAC cells. A RAD51 inhibitor showed an inhibitory effect on cell growth and affected the cytotoxic activity of KR12 in PDAC cells. These data suggested that the simultaneous inhibition of RAD51 and mutant KRAS blockage would be an important therapeutic strategy for PDAC.

Abstract 5997: The role of neutrophil extracellular trap-CCDC25-integrin linked kinase complex in pancreatic cancer progression and metastasis

Abstract Neutrophils and neutrophil extracellular traps (NETs) contribute to the hypoxic, immunosuppressive pancreatic ductal adenocarcinoma (PDAC) tumour microenvironment (TME) which promotes treatment resistance, increased invasion and metastasis. While recent studies have demonstrated a critical role of tumour cell CCDC25-ILK complex in promoting NET-DNA-induced tumour metastasis in breast cancer, here we have investigated the presence and function of NETosis-induced CCDC25-ILK signaling in the context of pancreatic cancer. We demonstrate that CCDC25 and ILK are expressed in human PDAC cell lines, patient-derived xenograft (PDX) cell lines and in cell lines derived from the KPCY genetic model of PDAC. Importantly, co-immunoprecipitation analyses showed that endogenous CCDC25 and ILK interact in PDAC cells, and the addition of neutrophil-derived NET-DNA resulted in a greater amount of ILK associated with CCDC25 and in increased wound-induced migration. Time-lapse imaging demonstrated a dramatic effect of inhibition of ILK activity on NET-DNA-induced migration by tumour cells in vitro. In vivo, co-localization of CCDC25 and ILK was observed in tumours and liver metastases from both the KPCY GEMM and the MIA PaCa-2 human PDAC xenograft models that were stained by multispectral immunofluorescence for these markers and analyzed by confocal microscopy. Similarly, both tumours and liver metastases showed the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Collectively, these data identify the neutrophil extracellular trap-induced CCDC25-ILK interaction as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent NET-induced PDAC progression and metastasis. Citation Format: Paul C. McDonald, Wells S. Brown, Zack Gerbec, Shannon Awrey, Joanna Karasinska, David Schaeffer, Daniel Renouf, Ben Stanger, Shoukat Dedhar. The role of neutrophil extracellular trap-CCDC25-integrin linked kinase complex in pancreatic cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5997.

Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs.

microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC.

Abstract 1906: Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer

Abstract Activating mutations in the KRAS oncogene are found in greater than 90% of pancreatic ductal adenocarcinoma (PDAC), yet KRAS remains difficult to inhibit pharmacologically. Small molecule inhibitors of RAF/MEK/ERK (MAPK) or mTOR are first line treatments for several human malignancies, however resistance to these targeted therapies continues to be a major clinical problem. We show in vitro dose dependent inhibition of proliferation and pERK1/2 in human PDAC cell lines and a KRAS-driven genetically engineered mouse model (GEMM) of pancreatic cancer in response to Trametinib, a small molecule MEK-inhibitor (MEKi). Similarly, Trametinib elicited a dose dependent inhibition of the in vivo tumor growth of a subcutaneous human PDAC xenograft, however these tumors rapidly developed resistance and contained areas of increased activation of pAkt(Ser473). We determined that genetic depletion of KRAS using doxycycline-inducible shRNA or small molecule MEKi leads to increased pAkt(Ser473) and activation of mTORC2 by western blot, implicating activation of the Akt/mTORC1/2 pathway may be a mechanism of resistance to MEKi. Interestingly, treatment of PDAC cell lines with Torin1, a small molecule inhibitor of mTORC1/2, results in the increased activation of pERK1/2, suggesting crosstalk and compensation of these two pathways as mechanisms of resistance. The combination of Trametinib and Torin1 leads to the inhibition of both pERK1/2 and pAkt(Ser473) that is accompanied by a dose dependent, synergistic increase in cytotoxicity not observed with either single agent. In human PDAC xenografts and a syngeneic GEMM, combination treatment of Trametinib and Torin1 dramatically reduced tumor burden and increased survival, without serious toxicity. These results demonstrate that combined inhibition of MEK/ERK and mTORC1/2 is a potent and viable therapeutic strategy for PDAC. Citation Format: Wells Brown, Oksana Nemirovsky, Jordan Gillespie, Paul McDonald, Shoukat Dedhar. Combination MEK and mTORC1/2 inhibition overcomes resistance to single agent therapy in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1906.

GRP78 expression and clinical outcomes in pancreatic cancer: A single institution review.

e16767 Background: Pancreatic Ductal Adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers. Poor prognosis can be attributed in part to late detection and chemoresistance. Perioperative chemotherapy with or without radiation is the standard treatment for resectable PDAC, however recurrence rates remain high. GRP78 (HSPA5/BiP) is an endoplasmic reticulum (ER) chaperone protein involved in regulation of the ER stress response that leads to pro-survival pathway activation and chemoresistance in tumor cells. Prior studies of suppression of GRP78 expression in PDAC cell lines led to increased pancreatic cell apoptosis in response to gemcitabine. In vivo studies of human PDAC xenografts showed that tumors with higher GRP78 expression were associated with increased chemoresistance. Methods: We conducted a single institution retrospective review of 53 patients who underwent resection of PDAC from 1/1/2009-7/1/2016. Archived tumor tissue expression of GRP78 was assessed by immunohistochemistry (IHC) and scored as 0-3+. GRP78 IHC scores of 0-1+ were categorized as low and scores of 2-3+ as high. Corresponding clinical data were collected. Adjusted effects of GRP78 expression on the hazard of treatment failure or death were estimated using Cox proportional hazards models. Type III Wald Chi-square p-values are reported for the overall effect in each model. Results: GRP78 expression level demonstrated a significant effect on disease free survival (DFS) in unadjusted analyses (HR 2.00, p = 0.05), as well as after adjusting for performance status (p = 0.03), initial overall stage (p = 0.05), pathological tumor stage (p = 0.02), pathological node stage (p &lt; 0.01), resection margins (p = 0.05), and neoadjuvant therapy (p = 0.05) in separate models. In unadjusted analysis, high GRP78 expression showed a trend towards worsened overall survival (OS), but did not reach statistical significance (HR 1.8, p = 0.11). The effect on OS was significant when adjusting for pathological tumor stage (p = 0.04) and pathological node stage (p = 0.01). (575). Conclusions: Consistent with prior animal studies, our retrospective review revealed that high GRP78 expression was associated with worsened DFS in patients with resectable PDAC. When adjusting for pathological staging, high GRP78 expression had a similar impact on OS. These findings warrant further evaluation of the role of GRP78 in prognosis, response to therapy and as a potential treatment target.

TFEB-mediated lysosomal biogenesis and lysosomal drug sequestration confer resistance to MEK inhibition in pancreatic cancer

Oncogenic KRAS mutations are encountered in more than 90% of pancreatic ductal adenocarcinomas. MEK inhibition has failed to procure any clinical benefits in mutant RAS-driven cancers including pancreatic ductal adenocarcinoma (PDAC). To identify potential resistance mechanisms underlying MEK inhibitor (MEKi) resistance in PDAC, we investigated lysosomal drug accumulation in PDAC models both in vitro and in vivo. Mouse PDAC models and human PDAC cell lines as well as human PDAC xenografts treated with the MEK inhibitor trametinib or refametinib led to an enhanced expression of lysosomal markers and enrichment of lysosomal gene sets. A time-dependent, increase in lysosomal content was observed upon MEK inhibition. Strikingly, there was a strong activation of lysosomal biogenesis in cell lines of the classical compared to the basal-like molecular subtype. Increase in lysosomal content was associated with nuclear translocation of the Transcription Factor EB (TFEB) and upregulation of TFEB target genes. siRNA-mediated depletion of TFEB led to a decreased lysosomal biogenesis upon MEK inhibition and potentiated sensitivity. Using LC-MS, we show accumulation of MEKi in the lysosomes of treated cells. Therefore, MEK inhibition triggers lysosomal biogenesis and subsequent drug sequestration. Combined targeting of MEK and lysosomal function may improve sensitivity to MEK inhibition in PDAC.

The integrin αvβ6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.

Pancreatic ductal adenocarcinoma (PDAC) has a 5‐year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvβ6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of β6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10−8). In two separate cohorts, we showed that over 80% of PDACs expressed αvβ6 protein and that paired metastases retained αvβ6 expression. In vitro, integrin αvβ6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvβ6‐positive human PDAC xenografts and transgenic mice bearing αvβ6‐positive PDAC with the αvβ6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log‐rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase‐3) and suppression of the pro‐tumourigenic microenvironment (suppression of TGFβ signalling, fewer αSMA‐positive myofibroblasts, decreased blood vessel density). These data show that αvβ6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 41: A novel high-throughput microfluidic drug screening platform using pancreatic ductal adenocarcinoma derived organoids

Abstract Pancreatic cancer is one of the deadliest tumors due to the limited treatment options and late diagnosis. Here, we describe a novel high throughput drug screening platform combining a microfluidic based 3D-culture plate, and the recently described pancreatic ductal adenocarcinoma (PDAC) derived organoids. The microfluidic plate is a high throughput microfluidic 3D cell culture platform, supporting physiologically relevant models with a minimal requirement of cell material and enabling a wide range of flow and co-culture options (e.g. with blood vessels). Organoids were derived from human PDAC xenografts and seeded in the microfluidic plate. The low amount of tissue material required (4000 cells per chip) and the high number of replicates on one plate (n=96 on a standard microtiter format plate) renders the microfluidic plate an efficient and cost-effective platform for drug screening and toxicity assays on complex, 3D models. Organoids were exposed to various chemotherapeutic drugs for 72 hours. The viability of the organoids before and after drug treatment is monitored with standard viability assays and subsequently used to generate dose response curves. In conclusion, we showed that the microfluidic plate can be used for high throughput drug screening assays and toxicity screening, and demonstrated its compatibility with human pancreatic PDAC derived organoids. Citation Format: Bart Kramer, Wijnand van Paassen, Luuk de Haan, Henriette Lanz, Jos Joore. A novel high-throughput microfluidic drug screening platform using pancreatic ductal adenocarcinoma derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 41.

IFN Expressing Oncolytic Adenovirus as a Tool to Improve Pancreatic Cancer Therapy

Pancreatic Ductal Adenocarcinoma (PDAC) is the 3rd cause of cancer related death in the US. Aside from surgery, which is not applicable to 80% of patients, there is no highly effective therapy to treat the disease. Due to late diagnosis and advanced disease, PDAC patients live 3–6 months post‐diagnosis and 5‐year survival is 6%. Despite this discouraging scenario, clinical trials testing use of adjuvant therapy combined with IFN‐α (IFN), 5‐FU, CDDP, and radiation improved the 2‐year survival of patients by 20–41%, and the 5‐year survival by 35%. While efficacious, IFN‐therapy drawbacks included high IFN systemic toxicity, increasing patient drop‐out from trials, and low levels of the cytokine in tumors, reducing chemo‐radio sensitization of IFN in tumors. Aiming to perfect this promising therapy, we studied the use of IFN‐expressing oncolytic adenovirus (OAd) vectors in combination with chemoradiation mimicking the aforementioned IFN‐chemoradiation based clinical trials. As IFN can stimulate tumor specific immune response, we first evaluated the effect of a hamster IFN expressing vector (OAd‐hamIFN) in a syngeneic immunocompetent hamster model of PDAC. To analyse the interaction of OAd with chemotherapy and radiation without the interference of the immune system, we later accessed the effect of a human IFN‐expressing vector (OAd‐IFN) in immunodeficient mice bearing human PDAC xenografts. In both studies, OAd vectors included 5/3 fiber modification to enhance infectivity in PDAC cells, over expressed Adenovirus Death Protein (ADP) to potentiate cell oncolysis, and expressed IFN in a replication dependent manner. As PDAC cells upregulate Cox‐2, we used the Cox‐2 promoter in the OAd‐IFN to restrict viral replication to human PDAC cells. Data showed that combinations of 5‐FU, radiation, and 5‐FU + radiation with either OAd‐hamIFN in hamster PDAC cells, or OAd‐IFN in human PDAC cells resulted in highly synergistic and cytotoxic combinations in vitro. Studies in immunocompetent hamsters showed that use of OAd‐hamIFN in combination with therapeutics used in IFN‐therapy improved treatment efficacy, and that use of the virus mimicking the IFN‐therapy was the most efficacious treatment. When analyzing effect of OAd‐IFN in combination with 5‐FU + CDDP in human PDAC cells, combinations were shown to be antagonistic and weakly cytotoxic. However, addition of radiation to treatment (OAd‐IFN + (5‐FU + CDDP) + radiation) overruled chemotherapy antagonism, resulting in highly synergistic and extremely potent treatments in vitro and in mice bearing PDAC xenografts. As radiation was shown to abolish antagonism of chemotherapy to virus in vivo, we tested different radiation protocols in combination with virus. We concluded that the best radiation protocol comprised of infecting tumors before irradiation. In summary, our data strongly supported inclusion of an IFN expressing OAd in treatments mimicking IFN‐therapy. As this therapy is one of the few showing an impact in patient survival, improvement of therapy with the virus might finally result in an potent treatment against PDAC.Support or Funding InformationNIH/NCIP50CA101955, NIH/NCIR01CA094084This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic and Physiologic Imaging Biomarkers of the Tumor Microenvironment Predict Treatment Outcome with Radiation or a Hypoxia-Activated Prodrug in Mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783-92. ©2018 AACR.

Abstract 3483: 1H MRS characterization of the cachetic brain metabolome induced by human pancreatic pancer xenografts

Abstract The uncontrollable extreme weight loss due to cachexia results in a particularly poor quality of life causing profound weakness, listlessness, and an inability to function. A major characteristic of cachexia is accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as ketone bodies and glucose derived from liver keto- and gluconeogenesis with systemic distribution including to the tumor through the bloodstream. Previously, we have reported the initial characterization of a myoblast optical imaging reporter that allowed real-time longitudinal monitoring of the early onset of cancer induced wasting and measured plasma metabolic changes associated with pancreatic ductal adenocarcinoma (PDAC)-induced cachexia. Here, for the first time, we have performed high-resolution quantitative 1H MR spectroscopy (MRS) of brain tissue to characterize the brain metabolome of normal mice and mice with human PDAC xenografts that induce cachexia (Pa04C cells) or are noncachectic (Panc1 cells). Male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (2×106) and in the right hind leg muscle with reporter myoblasts (2×106). Once the mice were sacrificed, brains were harvested, freeze clamped and stored in -80°C until 1H MRS analysis. Dual phase solvent extraction was performed on brain tissues and MR spectra were acquired on 750 MHz (17.6T) spectrometer. As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. For the first time, we found that brains from Pa04C tumor bearing mice exhibited a profound reduction in water soluble metabolites as compared to Panc1 and nontumor bearing normal mice. Significant decreases in neurotransmitters: γ-aminobutyric acid (GABA), N-acetyl aspartate (NAA), and taurine as well as lactate, myo-inositol, phosphocholine, glycerophosphocholine, creatine, formate, and essential amino acids: Leu, ILe, Val, and Phe were observed in brains from cachectic mice compared to noncachectic and healthy mice. Non-cachexia inducing Panc1 tumors also induced a significant decrease of brain GABA, glutamate, aspartate, total choline and tyrosine compared to normal brains. These results provide new insights into profound changes in brain metabolism during cachexia that are likely indicative of compromised CNS function and may be a major contributing factor to the systemic control of cachectic wasting. These data provide strong evidence to support investigating new metabolic interventions to reverse CNS injury and cachexia. and provide new CNS targets for early detection using in vivo MRS techniques. Acknowledgement: This work was supported by NIH R01CA193365, R35CA209960, and P30CA06973. Citation Format: Santosh Kumar Bharti, Paul T. Winnard, Yelena Mironchik, Marie-France Penet, Anirban Maitra, Zaver M. Bhujwalla. 1H MRS characterization of the cachetic brain metabolome induced by human pancreatic pancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3483.

PO-421 A novel high-throughput drug screening platform using pancreatic ductal adenocarcinoma derived organoids in the organoplate®

Introduction Pancreatic cancer is one of the deadliest tumours due to the limited treatment options and late diagnosis. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate, a microfluidic based 3D-culture plate, and the recently described Pancreatic Ductal AdenoCarcinoma (PDAC) derived organoids. Material and methods The OrganoPlate is a high throughput microfluidic 3D cell culture platform, supporting physiologically relevant models with a minimal requirement of cell material and enabling a wide range of flow and co-culture options (e.g. with blood vessels). Organoids were derived from human PDAC xenografts and seeded in the OrganoPlate. The low amount of tissue material required (4000 cells per chip) and the high number of replicates on one plate (n=96 on a standard microtiter format plate) renders the OrganoPlate an efficient and cost-effective platform for drug screening and toxicity assays on complex, 3D models. Results and discussions Organoids were exposed to various chemotherapeutic drugs for 72 hours. The viability of the organoids before and after drug treatment is monitored with standard viability assays and subsequently used to generate dose response curves. Conclusion In conclusion, we showed that the OrganoPlate can be used for high throughput drug screening assays and toxicity screening, and demonstrated its compatibility with human pancreatic PDAC derived organoids.

Thy1-Targeted Microbubbles for Ultrasound Molecular Imaging of Pancreatic Ductal Adenocarcinoma.

Purpose: To engineer a dual human and murine Thy1-binding single-chain-antibody ligand (Thy1-scFv) for contrast microbubble-enhanced ultrasound molecular imaging of pancreatic ductal adenocarcinoma (PDAC).Experimental Design: Thy1-scFv were engineered using yeast-surface-display techniques. Binding to soluble human and murine Thy1 and to Thy1-expressing cells was assessed by flow cytometry. Thy1-scFv was then attached to gas-filled microbubbles to create MBThy1-scFv Thy1 binding of MBThy1-scFv to Thy1-expressing cells was evaluated under flow shear stress conditions in flow-chamber experiments. MBscFv-scrambled and MBNon-targeted were used as negative controls. All microbubble types were tested in both orthotopic human PDAC xenografts and transgenic PDAC mice in vivoResults: Thy1-scFv had a KD of 3.4 ± 0.36 nmol/L for human and 9.2 ± 1.7 nmol/L for murine Thy1 and showed binding to both soluble and cellularly expressed Thy1. MBThy1-scFv was attached to Thy1 with high affinity compared with negative control microbubbles (P < 0.01) as assessed by flow cytometry. Similarly, flow-chamber studies showed significantly (P < 0.01) higher binding of MBThy1-scFv (3.0 ± 0.81 MB/cell) to Thy1-expressing cells than MBscFv-scrambled (0.57 ± 0.53) and MBNon-targeted (0.43 ± 0.53). In vivo ultrasound molecular imaging using MBThy1-scFv demonstrated significantly higher signal (P < 0.01) in both orthotopic (5.32 ± 1.59 a.u.) and transgenic PDAC (5.68 ± 2.5 a.u.) mice compared with chronic pancreatitis (0.84 ± 0.6 a.u.) and normal pancreas (0.67 ± 0.71 a.u.). Ex vivo immunofluorescence confirmed significantly (P < 0.01) increased Thy1 expression in PDAC compared with chronic pancreatitis and normal pancreas tissue.Conclusions: A dual human and murine Thy1-binding scFv was designed to generate contrast microbubbles to allow PDAC detection with ultrasound. Clin Cancer Res; 24(7); 1574-85. ©2018 AACR.

Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumor desmoplasia, which is in part responsible for its aggressiveness. Endothelial cells have been shown to display cellular plasticity in the form of endothelial-to-mesenchymal transition (EndMT) that serves as an important source of fibroblasts in pathological disorders, including cancer. Angiogenic co-receptor, neuropilin-1 (NRP-1) actively binds TGFβ1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFβ1 signaling have been shown to be aberrantly up-regulated in PDAC. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis in human PDAC xenografts. Loss of NRP-1 in HUVECs limited TGFβ1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFβ canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFβ1-induced EndMT and up-regulated TGFβ signaling and expression of pro-fibrotic genes. In vivo, loss of NRP-1 attenuated tumor perfusion and size, accompanied by reduction in EndMT and fibrosis. This study defines a previously unrecognized role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression.

Abstract 3367: Overexpression of neuropilin-1 exacerbates endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumour desmoplasia/fibrosis, which is partially responsible for its aggressive nature. Recently, endothelial cells have been shown to display an extreme form of plasticity that serves as an important source of fibroblasts in several pathological disorders, including cancer. The pro-fibrotic cytokine TGFβ1 promotes fibroblast accumulation via endothelial-to-mesenchymal transition (EndMT), thereby identifying EndMT as a potential therapeutic target against fibrosis. Previous studies have implicated angiogenic co-receptor, neuropilin-1 (NRP-1) also as a co-receptor for TGFβ1 in mediating several oncogenic processes. NRP-1 expression and TGFβ1 signaling have been shown to be up-regulated in PDAC. However, the driving mechanisms linking NRP-1 and EndMT in cancer remain unexplored. We therefore hypothesized that the over-expressed NRP-1 may exacerbate TGFβ1-mediated EndMT as a source of fibrosis in PDAC. Methods: NRP-1 over-expression studies were performed using lentivirus (Applied Biological Materials Inc.) in human umbilical vein endothelial cells (HUVECs), at baseline and after TGFβ1 stimulation (10 ng/mL). Total RNA was extracted using TRIzol® reagent and protein using RIPA buffer (both Invitrogen). Real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green (Bio-Rad) following manufacturer's protocols. Markers of EndMT, fibrosis and TGFβ1 signaling were evaluated by RT-PCR, western blotting, Masson's trichrome staining and immunocytochemistry. Similarly, expression of NRP-1, EndMT and fibrosis markers was assessed in human PDAC xenografts to establish a correlation between NRP-1 levels with EndMT and fibrosis. Results: RT-PCR, western blotting and tube formation assay in HUVECs confirmed successful NRP-1 over-expression. TGFβ1-stimulated HUVECs demonstrated a distinct change from “cobblestone-like endothelial cell morphology” to an enlarged spindle-shaped appearance consistent with a “fibroblast-like morphology”, accompanied by rearrangement of the cytoskeleton. Moreover, over-expression of NRP-1 in HUVECs exacerbated TGFβ1-induced EndMT as demonstrated by loss of endothelial and gain of mesenchymal markers, and was further accompanied by changes in microscopic cellular characteristics consistent with EndMT. NRP-1 over-expression also up-regulated TGFβ1 signaling and expression of pro-fibrotic genes. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis markers at mRNA and protein levels in human PDAC xenografts. Conclusions: Overall, these results define a previously undetermined role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis in tumours and advocate NRP-1 as a potential therapeutic target to reduce tumour fibrosis and PDAC progression. Citation Format: Pratiek N. Matkar, Krishna K. Singh, Gerald J. Prud’homme, David W. Hedley, Howard Leong-Poi. Overexpression of neuropilin-1 exacerbates endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3367.