Murine Model Of Pancreatic Cancer Research Articles

Abstract 5775: GLIS3 drives a neural-like malignant state enriched after neoadjuvant treatment in pancreatic cancer

Abstract Pancreatic cancer is a challenging disease which lacks a robust precision oncology framework. Prior work has demonstrated that transcriptional subtypes in pancreatic cancer can stratify patients by response to chemotherapy and overall survival. These include classical and basal-like as the predominant subtypes in untreated disease, as well as a transdifferentiated neural-like malignant subtype enriched after standard-of-care chemotherapy. Nonetheless, our understanding of transcriptional subtypes remains critically limited by genetic and culture-based confounders in preclinical models. Furthermore, most murine pancreatic cancer models do not fully reflect the classical vs. basal-like divide observed in humans, preventing faithful in vivo investigation. Collectively, these challenges have hindered the study of and development of therapeutic strategies against these transcriptional subtypes. To enable systematic study of the classical, basal-like and neural-like subtypes, we developed isogenic KP (KrasG12D/+;Trp53FL/FL) organoids and leveraged CRISPR activation to endogenously overexpress state-specific transcription factors. Our data revealed that Gata6 and dNTrp63 drove classical and basal-like identity, respectively, supporting prior studies. Furthermore, through in silico approaches, we identified candidate drivers of the neural-like state, including Glis3, a zinc finger protein that mediates neuroendocrine fate during pancreatic development. We confirmed that Glis3 overexpression results in neural-like transdifferentiation in cancerous organoids through RNA-seq and proteomics, and that GLIS3 knockout abrogates neural-like identity in human cell lines. To study their malignant potential in vivo, we performed orthotopic transplants into murine pancreata, and observed that Glis3 and dNTrp63 accelerated tumor formation and progression, corresponding to clinical outcomes observed in patients. These models may therefore provide a means for in vivo interrogation of subtype biology and vulnerabilities. Finally, to model and predict therapeutic sensitivities, we performed ex vivo treatment assays with chemotherapy and emerging KRASG12D inhibitors. A selection assay showed that basal-like and neural-like organoids were starkly enriched by multi-agent chemotherapy, while classical organoids were depleted. Finally, we learned that the neural-like subtype exhibits differential sensitivity to KRASG12D inhibition through an RTK-associated mechanism. Taken together, we provide a foundational system for interrogating subtype-specific vulnerabilities in pancreatic cancer. Citation Format: Jimmy A. Guo, Jennifer Su, Carina Shiau, Ananya Jambhale, Annan Yang, Westley Wu, Junning Wang, Connor Hennessey, Patrick Yu, Brendan Parent, Giselle Uribe, Julien Dilly, Laleh Abbassi, Qijia Yu, Arnav Mehta, David Ting, Brian Wolpin, William Freed-Pastor, Joseph Mancias, Tyler Jacks, William L. Hwang, Andrew J. Aguirre. GLIS3 drives a neural-like malignant state enriched after neoadjuvant treatment in pancreatic cancer. [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 5775.

Abstract 5191: Modeling age-related changes in the tumor microenvironment in an orthotopic immunocompetent murine pancreatic cancer model - a novel approach

Abstract Pancreatic cancer is the third leading cause of cancer-related deaths in the U.S. and is most frequently diagnosed between the ages of 65-74 years. Only 11.5% of patients are expected to survive 5 years after diagnosis. There is extensive evidence that tumor microenvironment (TME) plays a critical role in cancer progression and metastasis. Despite the fact that most patients are diagnosed over the age of 60, most preclinical murine tumor models utilize young, healthy, adolescent mice, failing to account for structural and/or functional changes in stroma and immune cells that occur in tissues over time and contribute to tumor progression. Marked differences in lifespan between mice and humans preclude natural development of many age-related physiologic changes in mice that occur in humans over the course of decades. We performed a pilot study to test our hypothesis that KPC pancreatic tumors grown orthotopically in the pancreas of genetically engineered, C57BL/6-Tg (LMNA*G608G) HClns/J mice, a model of Hutchinson-Gilford progeria syndrome with features of accelerated aging, would have altered tumor growth in comparison to tumors in age-matched wild type controls. We found that tumor growth in LMNA mice outpaced that of WT mice. Quantitative digital image analysis showed significantly reduced intratumoral infiltration of CD45+ leukocytes in LMNA mice in comparison to WT (p=0.0098). There was a significant reduction in tumor-infiltrating CD3+ T cells (p=0.038) and F4/80+ macrophages (p=0.0102). Our data supports the hypothesis that functional, age-related, immune and/or stromal changes in the TME impact pancreatic tumor growth and progression. Innovative strategies that better model age-related changes in the TME may better recapitulate the aggressive course of pancreatic cancer in aged patients. Citation Format: Morgan M. Green, Tara Fujimoto, Cullen Taniguchi, Natalie Fowlkes. Modeling age-related changes in the tumor microenvironment in an orthotopic immunocompetent murine pancreatic cancer model - a novel approach. [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 5191.

Abstract 2675: Vactosertib combined treatment with T1-44, a PRMT5 activity inhibitor, improves the survival and inhibits tumor invasion of murine pancreatic cancer model

Abstract Pancreatic cancer is the most lethal type of cancer and the third leading cause of cancer death with the lowest 5-year survival rate. Heterogeneity, difficulty in diagnosis, and rapid metastatic progression are the causes of high mortality in pancreatic cancer. Recent studies have shown that Protein arginine methyltransferase 5 (PRMT5) is overexpressed in pancreatic cancers, and these patients have a worse prognosis. Therefore, PRMT5 as an anti-cancer target has gained considerable interest. In this study, we investigated whether inhibition of PRMT5 activity was synergistic with blockade of TGF-β1 signaling, which plays an important role in the construction of the desmoplastic matrix and invasiveness in pancreatic cancer and induces therapeutic vulnerability. Compared with T1-44, a selective inhibitor of PRMT5 activity, the combination of T1-44 with the TGF-β1 signaling inhibitor vactosertib significantly reduced tumor size and surrounding tissue invasion and significantly improved long-term survival. RNA sequencing analysis of mouse tumors revealed that the combination of T1-44 and vactosertib significantly altered the expression of genes involved in cancer progression, such as cell migration, extracellular matrix, and apoptotic processes. These data demonstrate that the combination therapy of T1-44 with vactosertib is synergistic for pancreatic cancer, suggesting that this novel combination therapy has value in the treatment strategy of patients with pancreatic cancer. Citation Format: Eunji Hong, Sujin Park, Seok Hee Park, Nick B. La Thangue, Seong-Jin Kim. Vactosertib combined treatment with T1-44, a PRMT5 activity inhibitor, improves the survival and inhibits tumor invasion of murine pancreatic cancer model [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 2675.

Pressure-Enabled Drug Delivery (PEDD) of a class C TLR9 agonist in combination with checkpoint inhibitor therapy in a murine pancreatic cancer model.

729 Background: Systemic immunotherapy has had limited clinical benefit in pancreatic ductal adenocarcinoma (PDAC). This is thought to be due to its desmoplastic immunosuppressive tumor microenvironment in addition to high intratumoral pressures that limit drug delivery. Recent pre-clinical cancer models and early-phase clinical trials have demonstrated the efficacy of toll-like receptor 9 agonists (TLR9a), including the synthetic CpG oligonucleotide SD101, to stimulate innate and adaptive immune cells and eliminate suppressive myeloid cells. We hypothesized that Pressure Enabled Drug Delivery (PEDD) via Pancreatic Retrograde Venous Infusion (PRVI) of a TLR9a would improve responsiveness to systemic anti-programmed death receptor-1 (PD-1) checkpoint inhibitor therapy in a murine orthotopic PDAC model. Methods: Murine PDAC (KPC4580P) tumors were implanted into the pancreatic tails of C57BL/6J mice and treated 8 days after implantation. Mice were assigned to PRVI saline (pSAL, n=9), systemic anti-PD1 100 mcg/mouse on Day 0, 2 and 4 (sAPD1, n = 6), 30mcg PRVI TLR9a (pTLR9a, n=9), 30mcg systemic TLR9a (sTLR9a, n=7) on Day 0, and combination 30mcg PRVI TLR9a on Day 0 and systemic anti-PD1 100 mcg/mouse on Day 0, 2 and 4 (COMBO, n =9). Fluorescently-labeled TLR9a (radiant efficiency [RE]) was measured on day 1. Blood and tumors were collected at necropsy 12 days after infusion. Results: All mice survived to necropsy. Site of tumor fluorescence measurements revealed higher intensity fluorescence in pTLR9a compared to sTLR9a (7.5x10^5 vs. 2.4x10^5 RE, p= 0.048). Significantly lower MDSCs in the COMBO vs. pSAL are shown. Tumor weights were significantly lower in the COMBO group compared to pSAL (400 vs. 964 mg, p=0.003), and were also lower, although not significantly, than in the pTLR9a (400 vs. 518mg, p=0.50), and sAPD1 (400 vs. 645 mg, p = 0.70) groups. Conclusions: PEDD of TLR9a by PRVI with systemic anti-PD-1 demonstrated improved PDAC tumor control. These results support study of this combination therapy in PDAC patients and expansion of ongoing PEDD clinical trials. [Table: see text]

Effects of Gemcitabine/Nab-Paclitaxel and DMAPT in PDAC Cachexia in vivo and in vitro

Cachexia is the involuntary wasting of skeletal muscle and adipose tissue, affecting over 80% of patients with pancreatic ductal adenocarcinoma (PDAC). Gemcitabine and nab-paclitaxel (GemNP) combination are commonly given as first-line treatments for PDAC. Data from our lab showed GemNP reduced tumor growth and prevented muscle and fat wasting. Dimethylaminoparthenolide (DMAPT), a small molecule inhibitor, prevented muscle wasting and prolonged survival in a genetic murine model of breast cancer. It has yet to determine if GemNP in combination with DMAPT can improve indices of cachexia and overall survival. Therefore, I hypothesize that GemNP in combination with DMAPT will improve indices of cachexia and overall survival in a murine model of pancreatic cancer. Male 10-week-old C57BL/6J mice underwent orthotopic injection of 5x104 KPC cells or SHAM surgery. Mice were randomly assigned to 1 of 4 groups (N=10/group): SHAM+Vehicle, KPC+Vehicle, KPC+GemNP, KPC+GemNP+DMAPT. Gemcitabine (120mg/kg) and Nab-paclitaxel (10mg/kg) were injected intraperitoneally starting on day 4 and continued every 6 days. DMAPT (100mg/kg) was administered by gavage Monday-Friday. To determine the role of GemNP and DMAPT in vitro, KPC cells were treated with GemNP and/or DMAPT and cell viability evaluated. Additionally, we treated myotubes with GemNP with/without DMAPT to assess myotube diameter in an established KPC conditioned media. In KPC mice, GemNP (24.4days) increased survival compared to Vehicle (16.8days, p=.0007). However, the combination GemNP+DMAPT did not extend survival over GemNP alone (25.4days, p=.693). Tumor mass was similar between all groups (p=0.411). Interestingly, at the time of sacrifice, all KPC treated mice independent of treatment had similar reduction in adipose tissue and muscle mass compared to SHAM. In conclusion, the addition of DMAPT to GemNP did not extend survival over GemNP alone in an aggressive pancreatic tumor cell line. Future studies should determine if less aggressive tumor cell lines might benefit from GemNP and DMAPT.

TGFβ-derived immune modulatory vaccine: targeting the immunosuppressive and fibrotic tumor microenvironment in a murine model of pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is associated with very poor survival, making it the third and fourth leading cause of all cancer-related deaths in the USA and European Union, respectively. The...

Abstract A052: Systematic dissection of transcriptional states in pancreatic cancer

Abstract Transcriptional states in pancreatic cancer can stratify patients by response to chemotherapy and clinical outcomes. These include the classical and basal-like states as well as a newly identified neural-like progenitor (NRP) state, which we have previously found to be enriched in primary patient tumors treated with neoadjuvant chemotherapy and radiotherapy. While several transcription factor drivers of classical and basal-like identity have been described, key regulators of the NRP state are unknown. Through in silico approaches, we identified candidate transcription factors of the NRP state, including GLIS3, a Krüppel-like zinc finger protein that mediates neuroendocrine fate during pancreatic development and differentiation of human embryonic stem cells into posterior neural progenitor cells. Our understanding of biologic and clinically-relevant attributes of transcriptional cell states remains limited by state-specific biases in various preclinical models. Existing human cell lines maintained as two-dimensional cultures tend to preferentially represent the basal-like state, whereas human three-dimensional organoid models grown in standard culture conditions best reflect the classical state. These phenotypes are therefore impacted by culture conditions as well as underlying genetic features. Furthermore, most murine pancreatic cancer models do not fully reflect the classical vs. basal-like state heterogeneity observed in humans. To enable systematic study of the classical, basal-like and NRP phenotypes, we developed isogenic KP (KrasG12D/+;Trp53FL/FL) murine organoids with a germline dCas9-VPR system to enable facile overexpression of state-specific transcription factors through CRISPR activation approaches. Quantitative PCR, RNA-sequencing, and proteomics confirmed Gata6, deltaN Trp63, and Glis3 as drivers of classical, basal-like, and NRP identity, respectively. DeltaN Trp63 organoids were further differentiated by loss of luminal morphology. Pairwise comparisons of global transcriptional alterations suggest the greatest similarities between the Gata6- and Glis3-overexpressed models, which is consistent with enhanced associations between classical and NRP states in patient tumors. Finally, although basal-like and NRP states are associated with poorer response to multi-agent chemotherapy, state-specific therapeutic sensitivities to other treatments remain incompletely defined. We therefore performed drug sensitivity assays with a panel of targeted therapies and unveiled state-specific sensitivities. These data were corroborated by drug sensitivity profiling of human patient-derived organoids and cell lines. Taken together, these results suggest a framework for defining cell state-specific vulnerabilities that may aid in stratifying and treating pancreatic cancer patients with new therapies. Citation Format: Jimmy A. Guo, Jennifer Su, Ananya Jambhale, Julien Dilly, Connor J. Hennessey, Carina Shiau, Patrick Yu, Steven Wang, Junning Wang, Laleh Abbassi, James Neiswender, Tate Bertea, Annan Yang, Qijia Yu, Peter Westcott, Jason Schenkel, Daniel Y. Kim, Hannah I. Hoffman, Grissel Cervantes Jaramillo, Giselle A. Uribe, Westley W. Wu, Arnav Mehta, David Ting, Julian A. Pacheco, Amy Deik, Clary Clish, Francisca Vazquez, Brian Wolpin, Aviv Regev, William A. Freed-Pastor, Joseph D. Mancias, Tyler Jacks, William L. Hwang, Andrew J. Aguirre. Systematic dissection of transcriptional states in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A052.

Abstract A032: Targeting pancreatic cancer senescence with ImmunoPET

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths for men and women, with five-year overall survival rates around 10 percent. Despite years of cancer research and new immunotherapy solutions, pancreatic cancer is still mainly treated by chemotherapies, starting with gemcitabine. Many cytostatic drug combinations have been shown, in a wide range of cancers, to induce senescence, a persistent, anti-apoptotic process characterized almost exclusively by beta galactosidase activity. Studies inducing senescence with the combination of trametinib and palbociclib (TP) have linked several tumorigenic and pro-metastatic factors in murine pancreatic cancer models to the senescence-associated secretory profile (SASP), giving rise to new antigen targets during the induction of senescence. Accordingly, senescence has recently been elevated as a new hallmark of cancer. There is thus a critical need for antigen-based tools to noninvasively identify markers of senescence in vivo. Senescence targets comprise of shed antigens (VEGF and IL-6) in the context of senescence-inducing therapy with trametinib and palbociclib (TP), when membrane bounds targets such as urokinase plasminogen activated receptor (uPAR) are potentially enriched. We show that VEGF and IL-6 targeting antibodies labeled with zirconium-89 deferoxamine (89Zr-DFO) bind cells treated with TP though in vivo, marked reduction in tumor targeting is seen. Preloading and targeting antigen shed are being actively considered as alternative imaging approaches. For membrane bound antigens associated with senescence, here we report the latest work developing uPAR immunoPET agents. First, we have shown that attachment of 89Zr-DFO preserves the binding function of both murine (muPAR) and human uPAR (huPAR) antibodies with high cross-reactivity of the murine uPAR (muPAR) antibody with huPAR. Cell uptake was increased two-fold in murine KPC cells with senescence-inducing TP treatment. In vivo, accumulation in subcutaneously implanted murine KPC pancreatic tumors was observed with TP-treated mice; however lower liver and blood activity was imaged by PET/CT and confirmed by terminal biodistribution at 144h. Human uPAR-targeting antibody in nude mice bearing flank MiaPaCa2 tumors showed strong antibody uptake by 144 h, with increased tumor uptake for mice undergoing TP therapy. Preloading studies at 10 µg, 40 µg, and 100µg at 4h prior to injection of 40µg 89Zr-DFO-huPAR showed improved tumor uptake kinetics with the highest preloading level, though overall tumor uptake was decreased at the higher radiolabeled antibody dose used, thus more optimization is needed. The development and optimization of 89Zr-DFO-uPAR antibodies for human and murine targets provides a superior means of identifying pancreatic cancer and pancreatic cancer senescence than that afforded by shed antigens. Future directions include further dose optimization and testing uptake in other pancreatic models, senescence-inducing drug combinations, and possible antibody-conjugated senolytic or endoradiotherapy strategies. Citation Format: Edwin C. Pratt, Riccardo Mezzadra, Tara Viray, Spencer Kaminsky, Scott W. Lowe, Jason S. Lewis. Targeting pancreatic cancer senescence with ImmunoPET [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A032.

IDO1 Is a Therapeutic Target for Pancreatic Cancer-Associated Depression.

Metabolites of tryptophan degradation are known to alter mood. Their effects have only been superficially examined in the context of pancreatic cancer. Herein, we study the role of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme important in the conversion of tryptophan to kynurenine, in a murine model of pancreatic cancer-associated depression. Behavioral tests (open field, forced swim, tail suspension, and elevated plus maze) and biochemical assays (LC-MS metabolomics) were used to characterize a depressive-phenotype in tumor-bearing mice (relative to non-tumor-bearing mice). In addition, we determine whether pharmacologic blockade of IDO1 affects mood in tumor-bearing mice. Immunocompetent mice bearing orthotopic pancreatic tumors exhibit depressive-like behavior relative to non-tumor-bearing mice. Pancreatic tumors strongly express IDO1. Consequently, serum kynurenine levels in tumor-bearing mice are elevated relative to non-tumor-bearing mice. Tumor-bearing mice treated with epacadostat, an IDO1 inhibitor, exhibited improved mood relative to mice receiving vehicle. There was a 95% reduction in serum kynurenine levels in mice receiving epacadostat relative to mice treated with vehicle. As confirmatory evidence of on-target activity, tumors of mice treated with epacadostat exhibited a compensatory increase in IDO1 protein levels. Escitalopram, an approved antidepressant, was ineffective at improving mood in tumor-bearing mice as measured by behavioral assays and did not affect kynurenine levels. Neither epacadostat, nor escitalopram, affected overall survival relative to vehicle. Mice with pancreatic cancer exhibit depressive-like behavior. Epacadostat was effective as an antidepressant for pancreatic cancer-associated depression in mice. These data offer a rationale to consider IDO1 inhibition as a therapeutic strategy to mitigate depressive symptoms in patients with pancreatic cancer.

Cytokine-Induced Killer Cell Immunotherapy Combined With Gemcitabine Reduces Systemic Metastasis in Pancreatic Cancer: An Analysis Using Preclinical Adjuvant Therapy-Mimicking Pancreatic Cancer Xenograft Model.

To evaluate the efficacy and safety of cytokine-induced killer (CIK) cell therapy in pancreatic cancer. An orthotopic murine model of pancreatic cancer and adjuvant therapy-mimicking xenograft murine model that underwent splenectomy was created. Eighty mice were randomized into four groups: the control, gemcitabine alone, CIK alone, and CIK with gemcitabine groups. The tumor growth was monitored using bioluminescence imaging once weekly. In the orthotopic murine model, the treatment groups showed a significantly longer survival than the control group (median: not reached vs 125.0 days; 95% confidence interval, 119.87-130.13; P = 0.04); however, the overall survival did not differ significantly among the treatment groups (P = 0.779). The metastatic recurrence rate and overall survival were also not significantly different among the groups in the adjuvant therapy-mimicking xenograft murine model (P = 0.497). However, the CIK and gemcitabine combination suppressed the metastatic recurrence effectively, with recurrence-free survival being significantly longer in the CIK with gemcitabine group than in the control group (median, 54 days; 95% confidence interval, 25.00-102.00; P = 0.013). The combination of CIK and gemcitabine suppressed systemic metastatic recurrence, with promising efficacy and good tolerability in an adjuvant setting of pancreatic cancer.

An Innovative Approach to Tissue Processing and Cell Sorting of Fixed Cells for Subsequent Single-Cell RNA Sequencing.

Although single-cell RNA sequencing (scRNA-seq) is currently the gold standard for the analysis of cell-specific expression profiles, the options for processing, staining, and preserving fresh cells remain very limited. Immediate and correct tissue processing is a critical determinant of scRNA-seq success. One major limitation is the restricted compatibility of fixation approaches, which must not destabilize or alter antibody labeling or RNA content or interfere with cell integrity. An additional limitation is the availability of expensive, high-demand cell-sorting equipment to exclude debris and dead or unwanted cells before proceeding with sample sequencing. The goal of this study was to develop a method that allows cells to be fixed and stored prior to FACS sorting for scRNA-seq without compromising the quality of the results. Finally, the challenge of preserving as many living cells as possible during tissue processing is another crucial issue addressed in this study. Our study focused on pancreatic ductal adenocarcinoma samples, where the number of live cells is rather limited, as in many other tumor tissues. Harsh tissue dissociation methods and sample preparation for analysis can negatively affect cell viability. Using the murine pancreatic cancer model Pan02, we evaluated the semi-automated mechanical/enzymatic digestion of solid tumors by gentleMACS Dissociator and compared it with mechanical dissociation of the same tissue. Moreover, we investigated a type of cell fixation that is successful in preserving cell RNA integrity yet compatible with FACS and subsequent scRNA-sequencing. Our protocol allows tissue to be dissociated and stained in one day and proceeds to cell sorting and scRNA-seq later, which is a great advantage for processing clinical patient material.

Transforming Growth Factor-Beta Signaling in Cancer-Induced Cachexia: From Molecular Pathways to the Clinics.

Cachexia is a metabolic syndrome consisting of massive loss of muscle mass and function that has a severe impact on the quality of life and survival of cancer patients. Up to 20% of lung cancer patients and up to 80% of pancreatic cancer patients are diagnosed with cachexia, leading to death in 20% of them. The main drivers of cachexia are cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), macrophage inhibitory cytokine 1 (MIC-1/GDF15) and transforming growth factor-beta (TGF-β). Besides its double-edged role as a tumor suppressor and activator, TGF-β causes muscle loss through myostatin-based signaling, involved in the reduction in protein synthesis and enhanced protein degradation. Additionally, TGF-β induces inhibin and activin, causing weight loss and muscle depletion, while MIC-1/GDF15, a member of the TGF-β superfamily, leads to anorexia and so, indirectly, to muscle wasting, acting on the hypothalamus center. Against this background, the blockade of TGF-β is tested as a potential mechanism to revert cachexia, and antibodies against TGF-β reduced weight and muscle loss in murine models of pancreatic cancer. This article reviews the role of the TGF-β pathway and to a minor extent of other molecules including microRNA in cancer onset and progression with a special focus on their involvement in cachexia, to enlighten whether TGF-β and such other players could be potential targets for therapy.

Antihypertension Nanoblockers Increase Intratumoral Perfusion of Sequential Cytotoxic Nanoparticles to Enhance Chemotherapy Efficacy against Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), one of the worst prognosis types of tumors, is characterized by dense extracellular matrix, which compresses tumor vessels and forms a physical barrier to inhibit therapeutic drug penetration and efficacy. Herein, losartan, an antihypertension agent, is applied as a tumor stroma modulator and developed into a nanosystem. A series of lipophilic losartan prodrugs are constructed by esterification of the hydroxyl group on losartan to fatty acids. Based on the self‐assembly ability and hydrodynamic diameter, the losartan‐linoleic acid conjugate is selected for further investigation. To improve the stability in vivo, nanoassemblies are refined with PEGylation to form losartan nanoblocker (Los NB), and administered via intravenous injection for experiments. On murine models of pancreatic cancer, Los NB shows a greater ability to remodel the tumor microenvironment than free losartan, including stromal depletion, vessel perfusion increase, and hypoxia relief. Furthermore, Los NB pretreatment remarkably enhances the accumulation and penetration of 7‐ethyl‐10‐hydroxycamptothecin (SN38)‐loaded nanodrugs (SN38 NPs) in tumor tissues. Expectedly, overall therapeutic efficacy of SN38 NPs is significantly enhanced after Los NB pretreatment. Since losartan is one of the most commonly used antihypertension agents, this study may provide a potential for clinical transformation in stroma‐rich PDAC treatment.

CDK7 inhibition augments response to multidrug chemotherapy in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance remain as severe hurtles towards improved prognosis. Thus, new therapeutic targets and innovative strategies are urgently needed.MethodsIn this study, we used the KPC mouse model-derived PDAC cell line TB32047 to perform kinome-wide CRISPR-Cas9 loss-of-function screening. Next-generation sequencing and MAGeCK-VISPR analysis were performed to identify candidate genes. We then conducted cell viability, clonogenic, and apoptosis assays and evaluated the synergistic therapeutic effects of cyclin-dependent kinase 7 (CDK7) depletion or inhibition with gemcitabine (GEM) and paclitaxel (PTX) in a murine orthotopic pancreatic cancer model. For mechanistic studies, we performed genome enrichment analysis (GSEA) and Western blotting to identify and verify the pathways that render PDAC sensitive to GEM/PTX therapy.ResultsWe identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, CDK7 ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction, and DNA damage at least predominantly through the STAT3-MCL1-CHK1 axis. Furthermore, THZ1 synergized with GEM and PTX in vitro and in vivo, resulting in enhanced antitumor effects.ConclusionsOur findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and suggest new strategies for overcoming chemoresistance in pancreatic cancer.

Mechanisms of pancreatic tumor suppression mediated by Xiang-lian pill: An integrated in silico exploration and experimental validation

Ethnopharmacological relevanceXiang-lian pill, consisting of Coptis chinensis Franch. coprocessed with Tetradium ruticarpum (A.Juss.) T.G.Hartley (Yu-huang-lian) and Aucklandia lappa DC. (Mu-xiang), is traditionally used to relieve fever, abdominal pain, and gastrointestinal inflammatory symptoms observed in patients with malignancies of the gastrointestinal tract. Each of the three herbs contained in Xiang-lian pill has been indicated to have anticancer effects on a variety of cancers, but its effects on pancreatic cancer remain unexplored. The main extracts of these herbs have anti-pancreatic cancer effects, but the comprehensive mechanism of this compound prescription of Xiang-lian pill in pancreatic cancer remains to be revealed. Aim of the studyTo explore the main active ingredients, potential anti-pancreatic cancer targets, and related mechanisms of the Xiang-lian pill and to determine its therapeutic value in vivo. Materials and methodsNetwork pharmacology and bioinformatics analysis were applied to screen the potential effective ingredients and key targets. Liquid/gas-mass spectrometry was performed for ingredients validation. Molecular docking and the cellular thermal shift assay were performed to test the binding efficiency between ingredients and targets. A murine pancreatic cancer model was established and administered different doses of the Xiang-lian pill. Hematoxylin-eosin staining was used for histopathological observation. Immunohistochemistry and immunoblotting were conducted for target validation. In vitro studies (cell viability and clonogenicity assays) were conducted to investigate the impact of three main ingredients in Xiang-lian pill on pancreatic cancer cells. PTGS2 overexpression was performed to reversely confirm the antitumor mechanisms of rutaecarpine as a specific PTGS2 inhibitor. ResultsXiang-lian pill suppressed pancreatic cancer growth in the dose range of 0.78-2.34g/kg with no significant toxicity. Sixteen potentially active ingredients and 26 corresponding therapeutic targets for pancreatic cancer were identified. PTGS2, PTGS1, KCNH2, PRSS1, and HSP90AA1 were the top 5 significant genes targeted by the Xiang-lian pill. Evodiamine, rutaecarpine and stigmasterol bound to PTGS2 and PTGS1 with different affinities and inhibited pancreatic cancer cell proliferation. The PTGS2-associated metabolic pathway MEK/ERK was downregulated by rutaecarpine in vitro and the Xiang-lian pill in vivo. ConclusionsXiang-lian pill mainly regulates inflammation, apoptosis, metastasis, and metabolism to exert an antitumor effect. The main active ingredients in Xiang-lian pill exhibit antitumor roles through directly binding to key targets in pancreatic cancer. PTGS2 mediated MEK/ERK inhibition by rutaecarpine represents a key therapeutic mechanism of Xiang-lian pill.

Orthotopic and Heterotopic Murine Models of Pancreatic Cancer Exhibit Different Immunological Microenvironments and Different Responses to Immunotherapy

For decades, tumor-bearing murine models established using tumor cell lines have been the most commonly used models to study human cancers. Even though there are several studies reported that implant sites caused disparities in tumor behaviors, few of them illuminated the positional effect on immunotherapy. Herein, we describe surgical techniques for a novel orthotopic implantation of syngeneic pancreatic ductal adenocarcinoma (PDAC) tissue slices. This method has a high success modeling rate and stable growth kinetics, which makes it useful for testing novel therapeutics. Pathological examination indicated that the orthotopic tumor displayed poor vascularization, desmoplastic stromal reaction, and a highly immunosuppressive tumor microenvironment. This unique microenvironment resulted in limited response to PD1/CTLA4 blockade therapy and anti-MUC1 (αMUC1) CAR-T transfer treatment. To reverse the suppressive tumor microenvironment, we developed gene modified T-cells bearing a chimeric receptor in which activating receptor NKG2D fused to intracellular domains of 4-1BB and CD3ζ (NKG2D CAR). The NKG2D CAR-T cells target myeloid-derived suppressor cells (MDSCs), which overexpress Rae1 (NKG2D ligands) within the TME. Results indicated that NKG2D CAR-T cells eliminated MDSCs and improved antitumor activity of subsequently infused CAR-T cells. Moreover, we generated a bicistronic CAR-T, including αMUC1 CAR and NKG2D CAR separated by a P2A element. Treatment with the dual targeted bicistronic CAR-T cells also resulted in prolonged survival of orthotopic model mice. In summary, this study describes construction of a novel orthotopic PDAC model through implantation of tissue slices and discusses resistance to immunotherapy from the perspective of a PDAC microenvironment. Based on the obtained results, it is evident that elimination MDSCs by NKG2D CAR could rescue the impaired CAR-T cell activity.

Toll-like receptor 7/8 agonist R848 alters the immune tumor microenvironment and enhances SBRT-induced antitumor efficacy in murine models of pancreatic cancer

BackgroundStereotactic body radiotherapy (SBRT) has been increasingly used as adjuvant therapy in pancreatic ductal adenocarcinoma (PDAC), and induces immunogenic cell death, which leads to the release of tumor antigen and...

Abstract 916: Dec2, a circadian rhythm gene is necessary for disseminated tumor cell survival in a novel murine model of pancreatic cancer dormancy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a proclivity for early metastasis. Resection remains the only chance for long term survival though most patients die from metastatic recurrence. While most patients recur within the first two years, many develop recurrence much later indicating they harbor dormant disseminated tumor cells (dDTCs). Currently there are no murine models which recapitulate this disease pattern and as a result pancreatic cancer dormancy is poorly understood. We sought to better understand PDAC dormancy using a novel murine model of human pancreatic cancer from which we investigate the genetic and molecular contributions to dormancy. A model of resected PDAC was developed using murine PDAC cells expressing luciferase/mCherry orthotopically implanted into FVB hosts. Primary tumors are resected after 4 weeks and mice followed for recurrence. dDTCs’ are harvested from the livers of mice without evidence of recurrence by fluorescence activated cell sorting (FACS) and subjected to transcriptomic profiling using single cell RNA sequencing. Dec2’s contribution to dormancy was evaluated by overexpression and knockout using CRISPR. Overall survival in the mouse model mirrored 41,552 stage I, II PDAC patients from the National Cancer Database with similar frequency and location of recurrence. One third of mice exhibited latent recurrence with median survival of 568 days before succumbing to recurrence. These mice were used to study dDTCs. Transcriptomic profiling of FACS sorted dDTCs revealed a distinct transcriptome from primary tumors and early recurrences, indicating cell plasticity. dDTCs exhibited decreased proliferation markers and upregulated genes involved in immune modulation, cell stemness, linoleic acid (LA) metabolism, and Dec2. dDTCs were resistant to chemotherapy. An in vitro model of dormancy was constructed in which LA treatment inhibited cell proliferation and increased Dec2. Dec2 overexpression resulted in cell quiescence, and knockout increased apoptosis with LA exposure. Dec2-KO cells implanted in the murine model dramatically improved survival compared to Dec-WT cells (Dec2-WT median survival 30 days, Dec2-KO median survival was not reached, p=0.013). Livers of mice in the resection model where Dec2-KO cells were used had decreased dDTC burden with fewer mice showing detectable luciferase gDNA (p<0.01) indicating that loss of Dec2 reduced survival of dDTC’s. This is the first murine model of pancreatic cancer dormancy that recapitulates outcomes of resected PDAC patients. These studies reveal pancreatic cancer dormancy is characterized by a distinct, plastic cellular state characterized by chemotherapy resistance. Dec2 appears required for dormancy, its absence leads to disseminated tumor cell apoptosis. This suggests Dec2 may be a therapeutic target in pancreatic cancer dormancy. Citation Format: Anthony S. Casabianca, Chris Harris, Crissy Dudgeon, Darren R. Carpizo. Dec2, a circadian rhythm gene is necessary for disseminated tumor cell survival in a novel murine model of pancreatic cancer dormancy [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 916.

Abstract 6145: Increased PD-L1 expression levels were observed on both tumor cells and macrophages by tocilizumab plus gemcitabine/nab-paclitaxel treatment in gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer patients

Abstract Background: Blockade of interlukine-6 (IL-6) and programmed death-1-ligand 1 (PD-L1) limits tumour progression in murine models of pancreatic cancer. When PD-L1 expression of metastatic pancreatic cancer (MPC) is increased under the influence of gemcitabine exposure plus IL-6 inhibition, the spectrum of benefit from immune checkpoint blockade may expand to MPC patients with combined blockade of IL-6 and PD-L1 plus gemcitabine-based chemotherapy. We sought to determine PD-L1 expression of liver metastasis during cycle 1 of a phase I study, tocilizumab (TCZ) plus gemcitabine/nab-paclitaxel (GN) in patient with GN-refractory MPC. Methods: This phase 1 study enrolled 10 patients with MPC that had progressed after GN. All patients experienced cycle 1 of TCZ plus GN, TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. Biopsy of liver metastasis was performed before Cycle 1 and at the end of Cycle 1. Tumour microenvironment including PD-L1 expression were evaluated in paired-biopsied specimen from 7 patients by multicolour immunofluorescence staining. Tumour cell and PD-L1 expression were labelled by anti-cytokeratin 19 antibody and anti-PD-L1 antibody clone SP263, respectively. Cell numbers per millimetre squared (cell density) and the percentage of PD-L1 positive cell in tumor area were calculated according to cell types including tumour cell as cytokeratin 19 positive cell and macrophage as CD68 positive cell. Results: Cell density and the percentage of PD-L1 positive tumor cell were 29/mm2 and 1.1% in mean at baseline and tended to be increased up to 142/mm2 (P = 0.11) and 5.2% (P = 0.06) at the end of cycle 1. Means of PD-L1 positive macrophages were 28/mm2 and 6.9% at baseline and were increased to 69/mm2 (P = 0.01) and 14% (P = 0.08). When region of interest was selected from tumor stroma, differences of PD-L1 positive macrophage during cycle 1 was also maintained in cell density (P = 0.02) and the percentage of PD-L1 positive cell (P = 0.06). Conclusion: TCZ+GN induced an increase of PD-L1 expression levels on tumor cell and macrophages. Citation Format: Kyoko Yamaguchi, Etsuko Fujii, Shuichi Mitsunaga, Noriaki Sawada, Masafumi Ikeda, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Mayu Makikawa, Kazunori Aoki, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Atsukiko Kato, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Increased PD-L1 expression levels were observed on both tumor cells and macrophages by tocilizumab plus gemcitabine/nab-paclitaxel treatment in gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer patients [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 6145.

Abstract 3591: First in class drug ZB131 shows efficacy in cholangiocarcinoma models

Abstract Cholangiocarcinoma (CCA) is a lethal cancer with a high unmet need. The survival rate is low (5-year survival <20%), most non-palliative patients relapse within two years, and treatments in the advanced setting are limited. While FGFR2 and IDH1 inhibitors are effective in patients with select genomic alterations, they address only a small fraction (<20%) of biliary tract cancers. We now present the discovery and characterization of ZB131, a first in class humanized monoclonal antibody targeted to cancer specific plectin (CSP). CSP is a pro-tumorigenic protein exclusively expressed on the cancer cell surface. Previously, we demonstrated remarkable tumor regressions in preclinical murine models of pancreatic and ovarian cancer using a tool anti-CSP murine antibody. Here, we show that intra- and extra-hepatic CCA are 92% and 86% positive for membranous plectin staining by IHC, respectively, suggesting that anti-CSP therapy could be broadly effective against CCA. Indeed, in CCA cell lines in vitro, ZB131 (0.4nM binding affinity) arrests cell cycle, inhibits cell migration, and triggers intracellular ROS accumulation. In addition, both ZB131 and its murine version significantly suppressed tumor growth in 83% of the mice in a xenograft model and caused complete regression in the remaining 17%. Previous data from our group and others suggesting that CSP plays a role in chemoresistance led us to evaluate the efficacy of ZB131 in combination with gemcitabine. In vitro, we treated CSP-positive WITT cells with combinations of 0-500nM gemcitabine plus 0-1000nM ZB131 and found pockets of synergy or additivity. In a CCA xenograft model, the combination of 1mg/kg ZB131 and 100mg/kg gemcitabine showed 91% suppression in tumor growth compared with 74% in gemcitabine alone and 77% in huIgG plus gemcitabine at day 32. Collectively, our results suggest that ZB131 renders CCA tumors more chemosensitive and enhances the antitumor effects of gemcitabine. In contrast to the standard-of-care CCA treatment regimen, ZB131 showed remarkably low toxicity, likely because the antibody bypasses any non-malignant cells where plectin remains strictly cytoplasmic. Fc effector functional studies revealed a lack of antibody-dependent cell-mediated and complement-dependent cytotoxicity. Hemolysis and cytokine release studies suggested that ZB131 is compatible with human blood and unlikely to cause adverse cytokine release. Furthermore, in GLP toxicology studies, no signs of toxicity were observed at the highest dose evaluated of 100mg/kg/week, providing a >30-fold safety margin from the estimated human efficacious dose of 3mg/kg/week. In summary, ZB131 is an exciting new avenue for the treatment of CCA and other CSP-positive cancers with an excellent safety profile and strong antitumor activity in vitro and in murine models. Phase I clinical trials of ZB131 have been initiated. Citation Format: Samantha M. Perez, Randall Burton, Denise Krawitz, Christopher Sheth, Pina Cardarelli, Molly Owens, Brian Murphy, Matthew J. Reilley, Lindsey T. Brinton, Kimberly A. Kelly. First in class drug ZB131 shows efficacy in cholangiocarcinoma models [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 3591.