Design and Evaluation of RNA Aptamer\u2013Mediated Delivery of C/EBP\u03b2 siRNA for Oncological Therapy

Resistance to standard therapy remains a challenge in treatment of pancreatic ductal adenocarcinoma (PDAC) resulting in need for novel therapeutic strategies. Anti-VEGF therapy with r84 delays PDAC progression; however, chronic hypoxia ultimately results in transition to a mesenchymal phenotype and rapid progression at a later timepoint. COX-2 inhibition with apricoxib, a novel antagonist in Phase II trials, reverses EMT in PDAC cells. We investigate the relationship between COX-2 and VEGF production in PDAC cell lines to evaluate the efficacy of a combination strategy in preclinical models of PDAC. In vitro, changes in VEGF production by PDAC cells following apricoxib were assessed by ELISA at baseline and following forced induction of EMT. The effect of r84, apricoxib, or combination on tumor growth and metastatic incidence was determined in SCID mice with established orthotopic pancreatic xenografts. Improvement of survival and reduction in tumor burden was assessed in a murine genetic model of PDAC using p48-Cre; KrasG12D;Cdkn2alox/lox mice, tissue was collected at 4 weeks of therapy with control antibody, r84 or apricoxib for analysis. PDAC cell lines grown in the presence of TGF-β and collagen demonstrated increased Zeb1 and decreased ECAD expression by Western blot. Treatment with clinically-achievable doses of apricoxib reversed this change in phenotype. High expression of COX-2 correlated with high levels of VEGF in conditioned media in human PDAC cell lines. VEGF production was initially sensitive to COX-2 inhibition, with complete depletion of VEGF in the first 6 hours, but VEGF levels returned to baseline by 16 hours and continued to rise throughout the first 72 hours post apricoxib. Upon repeat dosing at 24 hours, VEGF production had become independent of COX-2 and no changes were observed. Similar results were seen following induction of EMT, however, baseline VEGF production was dramatically increased, and while there was an initial decrease after apricoxib, production remained more than 2-fold higher than in cells under normal culture conditions. In vivo, apricoxib and r84 had minimal effect on primary tumor growth as single agents; however each agent resulted in a reduction of metastatic incidence. Combination therapy reduced primary tumor size and virtually eliminated metastases. Overall survival was significantly improved with apricoxib as a single agent. Apricoxib treated animals also demonstrated a delay in progression of disease and decreased collagen deposition in the microenvironment compared to r84, which increased collagen deposition.We conclude that COX-2 inhibition by apricoxib delays progression of PDAC and results in mesenchymal to epithelial transition. Combination with anti-VEGF therapy results in potent antitumor and antimetastatic effects in our models and warrants further evaluation as a strategy to augment chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3819. doi:1538-7445.AM2012-3819

BackgroundSystemic immunotherapy has had limited clinical benefit in pancreatic ductal adenocarcinoma. This is thought to be due to its desmoplastic immunosuppressive tumor microenvironment in addition to high intratumoral pressures that limit drug delivery. Recent preclinical cancer models and early-phase clinical trials have demonstrated the potential of toll-like receptor 9 agonists, including the synthetic CpG oligonucleotide SD-101, to stimulate a wide range of immune cells and eliminate suppressive myeloid cells. We hypothesized that Pressure-Enabled Drug Delivery via Pancreatic Retrograde Venous Infusion of toll-like receptor 9 agonist would improve responsiveness to systemic anti-programmed death receptor-1 checkpoint inhibitor therapy in a murine orthotopic pancreatic ductal adenocarcinoma model. MethodsMurine pancreatic ductal adenocarcinoma (KPC4580P) tumors were implanted into the pancreatic tails of C57BL/6J mice and treated 8 days after implantation. Mice were assigned to one of the following treatment groups: Pancreatic Retrograde Venous Infusion delivery of saline, Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist, systemic anti-programmed death receptor-1, systemic toll-like receptor 9 agonist, or the combination of Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist and systemic anti-programmed death receptor-1 (Combo). Fluorescently labeled toll-like receptor 9 agonist (radiant efficiency) was used to measure uptake of the drug on day 1. Changes in tumor burden were evaluated by necropsy at 2 different time points, 7 and 10 days after toll-like receptor 9 agonist treatment. Blood and tumors were collected at necropsy 10 days after toll-like receptor 9 agonist treatment for flow cytometric analysis of tumor-infiltrating leukocytes and plasma cytokines. ResultsAll mice analyzed survived to necropsy. Site of tumor fluorescence measurements revealed 3-fold higher intensity fluorescence in Pancreatic Retrograde Venous Infusion delivery of toll-like receptor 9 agonist compared to systemic toll-like receptor 9 agonist mice. Tumor weights were significantly lower in the Combo group compared to Pancreatic Retrograde Venous Infusion delivery of saline. Flow cytometry of the Combo group demonstrated significantly increased overall T-cell number, specifically CD4+ T-cells, and a trend toward increased CD8+ T-cells. Cytokine analysis showed significantly decreased IL-6 and CXCL1. ConclusionPressure-Enabled Drug Delivery of toll-like receptor 9 agonist by Pancreatic Retrograde Venous Infusion with systemic anti-programmed death receptor-1 demonstrated improved pancreatic ductal adenocarcinoma tumor control in a murine pancreatic ductal adenocarcinoma model. These results support study of this combination therapy in pancreatic ductal adenocarcinoma patients and expansion of ongoing Pressure-Enabled Drug Delivery clinical trials.

CD137 agonist-based combination immunotherapy enhances activated, effector memory T cells and prolongs survival in pancreatic adenocarcinoma.

Bispecific T cell engager (BiTE®) antibody constructs are designed to redirect T cells to induce lysis of tumor cells through simultaneous binding to CD3 on T cells and to a tumor associated antigen. BiTE® antibody constructs have previously been shown to be effective at depleting blood and tissue targets in B cell malignancies, suggesting a therapeutic potential for BiTE® antibody constructs in solid tumors. Pancreatic ductal adenocarcinoma (PDA) is a devastating malignant disease with a dismal prognosis even with currently available therapies. The tumor differentiation antigen mesothelin (MSLN) is highly expressed in over 80% of pancreatic tumors. Expression of MSLN is restricted in normal tissue implicating MSLN as an attractive target for BiTE® targeted therapy in PDA. In this study, we report the preclinical characterization of an anti-MSLN/CD3 BiTE® antibody construct with half-life extension (MSLN HLE BiTE®) in xenograft models of PDA. Previously the MSLN HLE BiTE® was found to bind CD3 and MSLN with low nanomolar affinity and have low picomolar cytotoxic activity against MSLN-positive cells in vitro. Moreover, the MSLN HLE BiTE® can mediate redirected lysis of cancer cell lines resistant to chemotherapy. Our study examines the efficacy of the highly potent and specific MSLN HLE BiTE® antibody construct in vivo by utilizing the orthotopic implantation of human AsPC-1 luciferase expressing PDA cells in immunocompromised NOD-Prkdcscid IL2rgnull (NSG) mice. We found that mice receiving the MSLN HLE BiTE® antibody construct survived significantly longer and had significantly reduced tumor burden comparing to control mice. Moreover, tumor bearing mice were given fluorescently labeled human T cells to measure T cell localization. Using live animal fluorescent imaging, we found mice treated with the MLSN HLE BiTE® antibody construct had significantly increased T cell localization and retention to the PDA tumor area compared to control. Mice receiving orthotopic implantation of MSLN knock out human AsPC-1 tumors treated with the MSLN HLE BiTE® did not show increased survival, reduction of tumor burden or increased T cell localization, confirming the BiTE® specificity for the tumor target MSLN. Furthermore, using the KPC mouse model of PDA, which develops cancer spontaneously upon conditional pancreatic expression of KRASG12D and TP53R172H mutations, we tested if a mouse-specific surrogate MSLN HLE BiTE® antibody construct could localize to a naturally formed dense tumor microenvironment. We found that mice treated with a murine MSLN HLE BiTE® labeled with CW800 demonstrated strong localization of the MSLN HLE BiTE® through the dense microenvironment to the PDA tumor. Taken together, these results suggest that a MSLN HLE BiTE® may be a unique targeted immunotherapy for PDA patients. Citation Format: Noelle R. Jurcak, MacKenzie Zarecki, Fei Lee, Noah Rozich, Stephen Muth, Elizabeth Jaffee, Sabine Stienen, Julie Bailis, Lei Zheng. Evaluation of mesothelin BiTE® antibody constructs in models of pancreatic ductal adenocarcinoma [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 1561.

Pancreatic Ductal Adenocarcinoma (PDAC) remains remarkably lethal, with a 5-year survival rate of less than 12%. Due to its widespread resistance to conventional therapy, PDAC is expected to be the second most common cause of cancer-related death in the United States by 2030. One of the major impediments to therapeutic failure in PDAC is the presence of an immunosuppressive tumor microenvironment (TME). We examined the function of Mitogen-activated protein kinases (MAPKs) upstream member, MAP4K4, in the PDAC-TME and tested the efficacies of MAP4K4 inhibitor and immunotherapy to overcome PDAC tumor burden. The role of MAP4K4 in regulating innate and adaptive components of PDAC-TME was determined in vivo using a preclinical immunocompetent murine model (i.e., in KPC) of PADC. The genes related to T cell dysfunctions were determined by the RT2 PCR array. Flow cytometry analyses were performed to determine the immune checkpoint proteins and cytotoxic cytokines in T cells. The therapeutic efficacy of MAP4K4 inhibitor and agonists of 4-1BB and OX40 was determined in the murine model of PDAC. The IHC analyses showed that overexpression of MAP4K4 increases tumor infiltration of macrophages and neutrophils and decreases T cells in the murine model of PDAC. Furthermore, treating PDAC mice with MAP4K4 pharmacological inhibitor GNE-495 decreased tumor-infiltrating macrophages and neutrophils and increased T cell counts. However, the GNE-495 treatment increased tumor-infiltrating T cells and was not associated with increased cytotoxicity of T cells. The RT2 PCR array analysis showed that treatment of GNE-495 decreased 4-1BB gene expression in peripheral and tumor-infiltrating T cells. The combined therapy of GNE-495 and 4-1BB agonistic mAb showed noticeable tumor regression and increased survival in the preclinical model of PDAC. Our study suggests that MAP4K4 regulates innate and adaptive immune cells in pancreatic-TME, and pharmacological inhibition of MAP4K4 decreases tumor macrophages and neutrophil counts. Moreover, the rationalized approach of combining a MAP4K4 inhibitor and a 4-1BB agonist mAb induced a T cell-mediated antitumor response, and this combination could serve as a viable treatment for PDAC. Citation Format: Sunil Singh, Sandeep Kumar, Saket Jha, Harsh Vyas, Piush Srivastava, Rakesh S. Nair, Basabi Rana, Ajay Rana. Targeting the oncogenic MAP4K4 regulatory axis in pancreatic cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1609.

Background: Pancreatic ductal adenocarcinoma (PDAC) is resistant to immunotherapy such as immune checkpoint inhibitors. One of the major resistance mechanisms is attributed to myeloid cells as an immunosuppressive element within the stroma of PDAC. It has been reported that focal adhesion kinase inhibitor (FAKi) can suppress immunosuppressive myeloid cells such as tumor associated macrophages and myeloid derived suppressor cells (MDSC), consequently sensitizing tumor to anti-PD-1 antibody therapy in mouse models of PDAC. Our group has previously shown that targeting the stroma via PEGPH20 enhanced the anti-tumor activity of the whole cell vaccine (GVAX) by targeting CXCR4-expressing myeloid cells. Here we are testing the hypothesis that PEGPH20 can synergize with FAKi by targeting myeloid cells in PDAC. Experimental Design: An established murine pancreatic tumor model of hepatic metastases treated with and without anti-PD-1 therapy was used to assess the synergy and immune-modulating effect of FAKi and stromal degradation of hyaluronan via PEGPH20. Results: FAKi and combination of FAKi with anti-PD-1 extends survival in the mouse metastasis model of PDAC. Adding PEGPH20 to the combination of FAKi and anti-PD-1 antibody significantly prolonged survival in this model. Comparing to the combination of FAKi and anti-PD-1 antibody, adding PEGPH20 significantly decreased the number of CXCR4-expressing myeloid cells in the tumor microenvironment (TME) of PDAC and consequently led to an increase in the amount of CCR7+ central memory T cells. Additionally, the amount of G-MDSCs, inflammatory resident monocytes and PD-L1 expressing myeloid cells in the TME of PDAC, was also decreased in PDAC treated with the triple combination of PEGPH20, FAKi and anti-PD-1 antibody compared to the dual combination of FAKi and anti-PD-1 antibody. Conclusion: Stromal degradation of hyaluronan via PEGPH20 in combination with FAKi and anti-PD-1 antibody further depletes immunosuppressive cells in the TME including G-MDSCs, inflammatory resident monocytes and PD-L1 expressing myeloid cells, and appears to target the CXCR4 pathway through PEGPH20. These findings support testing the combination of FAKi and anti-PD-1 antibody with agents targeting CXCR4 directly or indirectly by PEGPH20 in human PDAC. Citation Format: Arsen Osipov, Jianxin Wang, Shiqi Li, David Choi, Mackenzie Henderson, Jonathan Pachter, Jisook Lee, Dan Maneval, Lei Zheng, Alex Blair. PEGylated recombinant human hyaluronidase, PEGPH20, significantly enhances the anti-tumor activity of the combination of focal adhesion kinase Inhibitor and anti-PD-1 antibody by targeting CXCR4-expressing myeloid cells in a murine model of PDAC [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 1588.

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.

Effective therapies are needed to enhance the long-term survival of patients with pancreatic ductal adenocarcinoma (PDAC), which is the fourth leading cause of cancer-related deaths in the United States and eighth worldwide. The mere 5-year survival rate of 5% is the lowest of all cancers and is due in part to late diagnosis and resistance to conventional therapy. Therefore, it is critical to identify molecular vulnerabilities in PDAC for developing targeted therapies. Activating point mutations in the small GTPase, K-Ras are present in 90% of PDAC cases. However, successful strategies that exploit the reliance of PDAC on mutant K-Ras have not been developed. Recent studies have shown that PDAC cell lines demonstrate a pronounced dependence on autophagy and that oncogenic Ras activates autophagy to maintain tumorigenesis. However, the mechanism by which oncogenic Ras induces autophagy is poorly understood. Notably, Tank Binding Kinase 1 (TBK1) operates downstream of the Ras effector, RalB to directly activate AKT pro-survival signaling, independent of mTOR and PI3K. In addition to supporting oncogenic transformation in cancer cells, TBK1 is a crucial component in antibacterial autophagy. This selective form of autophagy requires TBK1 to activate autophagic cargo receptors, p62 and Optineurin for enhanced autophagic clearance. Furthermore, mouse embryonic fibroblasts (MEFs) harvested from mice expressing mutant TBK1 show a block in autophagy, implicating TBK1 as a mediator of non-selective autophagy in addition to xenophagy. Therefore, we hypothesize that TBK1 is the Ras effector driving autophagy in pancreatic cancers to support tumorigenic growth. We assessed the effect of pharmacological inhibition of TBK1 with a derivative of 6-aminopyrazolopyrimidine (Compound II) in human pancreatic cancer cell lines and in a pre-clinical model of PDAC. Inhibition of TBK1 with Compound II substantially reduced cell viability in cancer cells with diverse oncogenotypes. Initial studies in a genetic mouse model of PDAC (p48-Cre; LSL-KrasG12D; Cdkn2alox/lox, KIC) treated with Compound II, show a reduction in tumor burden. Moreover, Compound II reduced the activity of AKT in tumor tissues and decreased disease progression as determined by histology and immunohistochemistry for amylase, a marker of normal acinar tissue. Further studies will be performed to examine the relative level of active autophagy in animals treated with Compound II. Additionally, the contribution of TBK1 to the autophagic pathway and development and progression of PDAC is being assessed by crossing TBK1 mutant animals with KIC mice. These results will further our understanding of Ras signaling in pancreatic cancer and are critical for exploring a new avenue of targeted therapy. Citation Format: Victoria H. Burton, Yi-Hung Ou, Jason E. Toombs, Michael A. White, Rolf A. Brekken. TBK1 as a novel mediator of K-Ras driven pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A04. doi: 10.1158/1557-3125.RASONC14-A04

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1H2O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water [2H2O (D2O, also referred to as ‘heavy water’)]. Here, using diverse panels of cultured human malignant melanoma and pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2O-induced phenotypic anti-proliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential RT-qPCR array analysis revealed early modulation of stress response gene expression elicited by D2O (90%; ≤6 h) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (< 6 h) onset of D2O-induced MAP-kinase signaling (p-JNK) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP) attributable to deuteration-induced alteration of hydrogen bonding triggering proteotoxic stress. Next, we tested the chemotherapeutic efficacy of D2O-based drinking water supplementation in bioluminescent murine models of malignancy (A375-luc melanoma and BxPC-3-luc orthotopic PDAC xenografts in SCID mice). Time course of systemic deuteration (30% D2O in drinking water fed continuously for up to 21 days) was established using time-resolved whole-body proton magnetic resonance imaging (MRI) and isotope-ratio mass spectrometry (IR-MS)-based plasma (D/H)-analysis. In both models, D2O supplementation significantly suppressed tumor growth and metastasis with downregulated expression of PCNA/Ki67 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration by oral administration of D2O, associated with induction of cellular ER stress, reduces tumor burden and metastasis in murine models of human malignant melanoma and PDAC. Citation Format: Jana Jandova, Jean-Philippe Galons, David L. Dettman, Georg T. Wondrak. Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6306.

Photodynamic therapy (PDT) is a light-based anticancer therapy that can induce tumor necrosis and/or apoptosis. Two important factors contributing to the efficacy of PDT are the concentration of the photosensitizer in the tumor tissue and its preferential accumulation in the tumor tissue compared to that in normal tissues. In this study, we investigated the use of optical imaging for monitoring whole-body bio-distribution of the fluorescent (660 nm) photosensitizer Bremachlorin in vivo, in a murine pancreatic ductal adenocarcinoma (PDAC) model. Moreover, we non-invasively, examined the induction of tumor necrosis after PDT treatment using near-infrared fluorescent imaging of the necrosis avid cyanine dye IRDye®-800CW Carboxylate. Using whole-body fluorescence imaging, we observed that Bremachlorin preferentially accumulated in pancreatic tumors. Furthermore, in a longitudinal study we showed that 3 hours after Bremachlorin administration, the fluorescent tumor signal reached its maximum. In addition, the tumor-to-background ratio at all-time points was approximately 1.4. Ex vivo, at 6 hours after Bremachlorin administration, the tumor-to-muscle or -normal pancreas ratio exhibited a greater difference than it did at 24 hours, suggesting that, in terms of efficacy, 6 hours after Bremachlorin administration was an effective time point for PDT treatment of PDAC. In vivo administration of the near infrared fluorescence agent IRDye®-800CW Carboxylate showed that PDT, 6 hours after administration of Bremachlorin, selectively induced necrosis in the tumor tissues, which was subsequently confirmed histologically. In conclusion, by using in vivo fluorescence imaging, we could non-invasively and longitudinally monitor, the whole-body distribution of Bremachlorin. Furthermore, we successfully used IRDye®-800CW Carboxylate, a near-infrared fluorescent necrosis avid agent, to image PDT-induced necrotic cell death as a measure of therapeutic efficacy. This study showed how fluorescence can be applied for optimizing, and assessing the efficacy of, PDT.

To understand T cell immune surveillance in pancreatic ductal adenocarcinoma (PDA), our laboratory has studied the KPC genetically engineered mouse model of this disease. Unexpectedly, we found that effector T cells are scarce in both preinvasive and invasive lesions, whereas immune suppressive cells, including tumor-associated macrophages, immature myeloid cells, and regulatory T cells, are prominent at even the earliest stages of neoplasia and persist through invasive cancer. These findings are consistent with the immune infiltrate observed in human PDA in which a marked macrophage and myeloid cell infiltration is associated with few effector T cells. Because suppressive cells of the immune system appear early during pancreatic tumorigenesis, we hypothesize that the absence of effector T cell immune surveillance in PDA may be more consistent with “immunological ignorance” than “immunoediting.” In this scenario, PDA cells may be particularly sensitive to T cell killing because of a lack of T cell selective pressure in vivo. To explore this hypothesis, we have evaluated a CD40 agonist antibody for its ability to reverse immune suppression and activate the immune system. In patients with metastatic pancreatic cancer, we found that a CD40 agonist antibody in combination with gemcitabine led to major tumor regressions in a fraction of patients. To understand the mechanism of this effect, we studied two murine models of PDA: (1) a transplant model in which PDA tumor cell lines derived from KPC mice are implanted into syngeneic littermates and (2) the spontaneous KPC model of PDA. We have found that PDA tumors implanted subcutaneously into non-KPC mice grow readily, but upon treatment with an agonist CD40 mAb and gemcitabine, established PDA tumors undergo complete regression associated with a robust T cell infiltrate. Systemic T cell depletion abrogated this regression indicating a T-cell dependent effect. In contrast, when KPC mice bearing spontaneously arising PDA tumors were treated with an agonist CD40 mAb and gemcitabine, a fraction of tumors regressed but in the absence of tumor-infiltrating T cells (mirroring the clinical trial). To determine whether T cells in KPC animals were globally tolerized, KPC mice bearing a spontaneous tumor were challenged with PDA cells subcutaneously. As seen in non-KPC mice, subcutaneously implanted tumors grew exponentially and were found to lack infiltrating T cells. However, when treated with an agonist CD40 mAb and gemcitabine, these implanted tumors exhibited a marked T-cell infiltration leading to regression, suggesting that effector T cells can target PDA in the KPC model. Moreover, treatment of KPC mice bearing both implanted and subcutaneously arising tumors produced an impressive influx of T cells into the primary pancreatic tumors of KPC mice, a finding that was not observed when KPC mice were activated with CD40 mAb and gemcitabine in the absence of the PDA subcutaneous implant. Thus, tumor-reactive T cells in KPC mice are not clonally deleted or globally tolerant to PDA. These data suggest that elements of the tumor microenvironment may mediate local immune suppression that can be overcome by immune priming outside the tumor microenvironment. Our findings have important implications for the design of novel immunotherapy trials in pancreatic cancer, and highlight the opportunity to develop vaccines and other T cell therapies for this disease. Citation Format: Gregory L. Beatty, Rafael Winograd, Rebecca A. Evans, Robert H. Vonderheide. Immune surveillance and CD40 therapy of pancreatic cancer in mice and humans. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr IA9.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC) remain the leading causes of cancer-related mortality, with poor outcomes. This study comprehensively evaluated the mechanisms of polymeric micelles co-encapsulating cyclopamine (CPA) and paclitaxel (PTX) (M-CPA/PTX, ONP-001) and the effectiveness against HCC and PDAC. The dual-drug delivery system targets embryonic signaling pathways, such as Hedgehog (Hh), which drives tumor progression and resistance. Methods: CPA and PTX were encapsulated in micelles (∼55 nm in size) to improve bioavailability and tumor concentrations. Preclinical experiments included in vitro assays for cytotoxicity, tumorsphere, and colony formation in both cancer cell lines. In vivo models included orthotopic Kras* cell implanted PDAC and spontaneous HCC in transgenic LAP-tTA/TRE-MYC mice. The antitumor efficacy was examined by intravenously injection of ONP-001 at 2.5, 5.0, and 7.5 mg/drug equivalent/kg thrice weekly for 4 weeks, as well as combination regimens with irreversible electroporation (IRE) and an immune checkpoint inhibitor. Survival, tumor burden, immune modulation, and cytokine profiles were analyzed using Kaplan-Meier survival curves, bioluminescence imaging, and multiplex cytokine assays. Results: In the PDAC model, ONP-001 significantly extended median survival at 2.5 mg equivalent drug/kg (p<0.01), 5.0 mg/kg (p<0.0001), and 7.5 mg/kg (p<0.05) compared untreated control. ONP-001 administered at 7.5 mg/kg displayed shorter median survival than 5.0 mg/kg suggesting that the optimal regimen tested was 5 mg/kg. ONP-001 reprogrammed tumor stroma, and reduced cancer stem cell (CSC)-associated tumorsphere formation. Combination of ONP-001 with IRE and anti-PD-1 further enhanced survival and immune infiltration, overcoming PDAC’s immunosuppressive microenvironment. In HCC, ONP-001 significantly inhibited tumor growth and colony formation. Tumor cytokine profiling revealed significant reductions in markers associated with CSC maintenance (e.g., IL-15, IL-33) and tumor progression, highlighting an anti-inflammatory effect on the tumor microenvironment. Studies in the transgenic HCC model also confirmed significantly greater antitumor efficacy for ONP-001, with no observed hepatotoxicity, compared to sorafenib treatment. Conclusion: ONP-001 was an effective therapeutic strategy for PDAC and HCC, targeting CSCs, modulating the stroma, and enhancing immunotherapy, at 5.0 mg/kg, TIW, with enhanced benefits when in combination regimens. Ongoing studies will further characterize the pharmacokinetics, tumor uptake and biodistribution of both drugs. The data supports further development of ONP-001 for potential future clinical development. Citation Format: Qiu-Xu Teng, Guodong Zhang, Lu Dai, Defeng Deng, Ping Pan, Diana S-L Chow, Guorong Ma, Peiying Yang, Chun Li. Development and comprehensive preclinical evaluation of dual-drug polymeric micelles ONP-001 for PDAC and HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3141.

Background: The majority of patients with localized pancreatic ductal adenocarcinoma (PDAC) die from metastatic disease, typically of the liver, despite a margin-negative (R0) resection. Therefore, these patients likely harbor occult metastatic disease in the liver at the time of surgery. In this study, we evaluated the role of resident liver macrophages in suppressing the progression of hepatic micrometastases using a murine model of micrometastatic PDAC with patient-derived xenografts (PDXs). Methods and Results: Low-passage, patient-derived KRAS-mutant tumor cells expressing firefly luciferase were injected into the spleens of athymic nude mice resulting in liver metastases, followed by splenectomy to remove the primary tumor. Hepatic tumor burden and metastatic growth kinetics were evaluated by bioluminescent imaging on post-injection days 1, 2, 3, 7, and weekly thereafter. Each of the PDX tumors exhibited a decline in tumor burden over the initial days after injection followed by a period of quiescence with a reproducible, PDX-specific time to proliferative outgrowth ranging from 15 days to greater than 200 days. To assess the role of apoptosis in initial tumor cell clearance and suppression of outgrowth, mouse liver preparations were analyzed for expression of cleaved caspase-3 and cleaved PARP in tumor cells using flow cytometry. There was low expression of both apoptosis markers, suggesting that apoptosis is not a major cell-clearance mechanism. Athymic nude mice lack cell-mediated immunity but have functioning innate immunity. Because there is a large population of resident macrophages in the liver, we hypothesized that macrophages play an important role in the clearance and suppression of hepatic PDAC metastases. To test this, hepatic metastasis outgrowth was assessed following macrophage ablation with liposomal clodronate treatment of mice 48 hours prior to tumor cell injection. Following macrophage ablation, there was a trend toward less robust initial tumor cell clearance and a significantly decreased time to proliferative outgrowth compared with control (13 days vs 26 days, p=0.039). H&E sections demonstrated an abundance of hepatic macrophages surrounding tumor cells in the control group while this response was absent in the clodronate group. Splenic tumor cell injections were repeated using athymic nude mice vs NOD scid gamma (NSG) mice which lack both cell-mediated immunity as well as functional macrophages, dendritic cells, and NK cells. Initial tumor cell clearance was significantly reduced in the NSG mice vs nude mice (PDX 608: 35.6% vs 90.1% clearance at seven days, p<0.001; PDX 366: 40.4% vs 76.3% clearance at seven days, p=0.024). Average relative hepatic bioluminescence at 21 days was significantly increased for PDX 608 in the NSG mice (14.7 vs 0.355, p=0.014) and there was a trend toward increase for PDX 366 (3.07 vs 0.025, p=0.065). Conclusions: In a preclinical model of hepatic micrometastatic PDAC, resident liver macrophages are implicated in the initial clearance and the suppression of proliferation of tumor cells. Further investigation of the interaction of resident hepatic macrophages and micrometastatic PDAC cells may lead to novel strategies for therapy. Citation Format: Alex D. Michaels, Timothy E. Newhook, James M. Lindberg, Sara J. Adair, Sarbajeet Nagdas, Matthew G. Mullen, Edward B. Stelow, J. Thomas Parsons, Todd W. Bauer. The role of resident liver macrophages in suppressing the progression of hepatic micrometastases from pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C19.

Introduction: We and others have shown that pancreatic stellate cells (PSCs) in the tumor-associated stroma of PDAC promote tumor progression and resistance to therapy but the precise mechanisms are unclear. We investigated the role of PSC-derived DKK3, a member of the Dickkopf family of glycoproteins, in PDAC progression, metastasis and response to chemotherapy. Methods: We evaluated expression of DKK3 in human PDAC tissue and cell lines, human PSCs and in a genetically engineered mouse model (GEMM) of PDAC. The paracrine and autocrine effects of DKK3 on PDAC and PSCs were examined by treatment with exogenous DKK3 and gain- and loss of function assays for proliferation, migration, invasion, and gemcitabine-induced apoptosis. The effects of DKK3 on PDAC progression and metastasis were determined by shRNA neutralization and genetic ablation in orthotopic xenograft models and the KPC autochthonous model of PDAC. We developed novel monoclonal antibodies (mAbs) against DKK3 and tested their ability to neutralize DKK3 and prolong survival in mouse models of PDAC. Results: DKK3 was expressed at 4.5 times higher levels in human PDAC by Affymetrix profiling compared to normal pancreas and was present in 99% (118/119) of samples on a tissue microarray with moderate to high expression in 59%. In a GEMM of PDAC, DKK3 appeared early with preneoplastic PanIN lesions with increased expression in invasive carcinoma. DKK3 was strongly expressed by PSCs with minimal to no expression in PDAC cells and knockdown by shRNA reduced PSC proliferation and migration by 60% and 84% compared to controls (p<0.001). Treatment of Panc1 and BxPC3 cells with DKK3 stimulated migration and invasion by 100-300% (p<0.001) and proliferation of DKK3-silenced Panc1 cells was decreased by 80% (p<0.001). Overexpression of DKK3 in L3.6pl cells increased colony formation in gemcitabine by >90% (p<0.001) with 65% reduction in apoptosis (p<0.01), indicating that DKK3 contributes to PDAC resistance to chemotherapy. When we ablated DKK3 in KPC mice by breeding with DKK3-knockout mice, tumor growth was inhibited and survival increased by 45% (p=0.0002). In addition, fewer PanIN lesions developed in DKK3-null mice suggesting that DKK3 may contribute to the early developmental stages of PDAC. DKK3 neutralizing mAbs abrogated DKK3-mediated induction of PDAC cell migration, invasion and resistance to gemcitabine in vitro. Furthermore, treatment with DKK3 mAb significantly inhibited primary tumor growth, reduced peritoneal metastases and prolonged survival in an orthotopic model of PDAC by 43% compared to control mAb (p=0.005; HR 0.24, 95% CI 0.01-0.30). Conclusions: These data are the first report, to our knowledge, of a tumor-promoting function of DKK3 and our results suggest that neutralization of DKK3 may be an effective approach as a primary treatment for PDAC and to enhance responsiveness to chemotherapy. Citation Format: Liran Zhou, Hongmei Husted, Todd Moore, Mason Lu, Defeng Deng, Yan Liu, Vijaya Ramachandran, Thiruvengadam Arumugam, Baoan Ji, Huamin Wang, Jeffrey E. Lee, Craig D. Logsdon, Rosa F. Hwang. Targeting stromal-derived Dickkopf-3 (DKK3) for the treatment of pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2017-2961

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a dismal 5-year survival rate of <13%. We have discovered that desmoglein-2 (DSG2), a cell surface protein, is highly expressed on PDAC cells. In silico data indicate that elevated levels of DSG2 is associated with PDAC progression and poor patient survival. Immunohistochemistry (IHC) analysis of a tissue microarray of 302 patient samples showed that DSG2 was expressed by >90% of PDAC patients. We hypothesized that DSG2 is an under-appreciated contributor to PDAC progression and that DSG2 can be targeted. The main aims of this study were to: 1) investigate the role of DSG2 in PDAC cell function in vitro, 2) investigate the molecular biology underpinning the role of DSG2 in PDAC, and 3) to use murine PDAC tumor models to evaluate the contribution of DSG2 to cancer progression in vivo. Our in vitro data showed that reducing the expression of DSG2 (via small interfering RNA (siRNA)) significantly decreased PDAC cell proliferation, migration, and invasion. Moreover, signaling analysis of PDAC cells with or without DSG2 (via siRNA) using a reverse-phase protein array and cytokine array revealed that DSG2 modulates pro-tumorigenic and pro-metastatic cellular pathways, through reducing the effects of certain proteins. Particular alterations were observed in pathways involving gene regulation (e.g., beta-catenin), migration (e.g., epidermal growth factor receptor and integrin-beta1), and cytokine release (e.g., serpine-1). Next, in an orthotopic xenograft mouse model, BxPC-3 PDAC cells with a stable knockdown of DSG2 (via short-hairpin RNA) were injected into NSG (NOD scid gamma) mice, where we observed a significant reduction in tumor burden and liver metastasis when compared to control mice. IHC staining of tumor sections revealed that the DSG2 knockdown tumors contained reduced levels of collagen, tumor vasculature and cancer associated fibroblasts. Thus, suggesting that DSG2 also has a modulatory effect on the tumor microenvironment (TME). A similar reduction in tumor burden, cancer progression and changes to the TME were observed when a neutralizing anti-DSG2 monoclonal antibody was administered intraperitoneally twice a week at 5mg/kg to BxPC-3 tumor bearing mice. Finally, to investigate the immune cell landscape, we used an orthotopic syngeneic mouse model, whereby Dsg2 was knocked out of KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mouse PDAC cells via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This mouse model also showed that loss of Dsg2 resulted in a significant reduction in tumor burden, cancer cell metastasis and changes to the TME. In summary, we have identified a potential new target for the treatment of PDAC, namely DSG2, and future experiments are pursuing clinical opportunities to improve PDAC patient survival. Citation Format: Charlie B Ffrench, Kay K Myo Min, Mark DeNichilo, Michaelia P Cockshell, Emma L Dorward, Emma J Thompson, Michael Ortiz, Michael S Samuel, Savio George Barreto, Claudine S Bonder. Desmoglein-2 is a regulator of pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A073.