Pancreatic Tumor Cells Research Articles

Combining Olaparib and Ascorbic Acid on Nanoparticles to Enhance the Drug Toxic Effects in Pancreatic Cancer.

Pancreatic cancer (PC) shows a very poor response to current treatments. Development of drug resistance is one of the causes of the therapy failure, being PARP1 (poly ADP-ribose polymerase 1) a relevant protein in the resistance mechanism. In this work, we have functionalized calcium phosphate-based nanoparticles (NPs) with Olaparib (OLA, a PARP-1 inhibitor) in combination with ascorbic acid (AA), a pro-oxidative agent, to enhance their individual effects. Amorphous Calcium Phosphate (ACP) NPs were synthesized through a biomimetic approach and then functionalized with OLA and AA (NP-ACP-OLA-AA). After evaluation of the loading capacity and release kinetic, cytotoxicity, cell migration, immunofluorescence, and gene expression assays were performed using pancreatic tumor cell lines. In vivo studies were carried out on tumors derived from the PANC-1 line in NOD SCID gamma (NSG) mice. NP-ACP-OLA-AA was loaded with 13%wt of OLA (75% loading efficiency) and 1% of AA, respectively. The resulting dual nanosystem exhibited a gradual release of OLA and AA, being the latter protected from degradation in solution. This ensured the simultaneous availability of OLA and AA for a longer period, at least, during the entire time of in vitro cell experiments (72 hours). In vitro studies indicated that NP-ACP-OLA-AA showed the best cytotoxic effect outperforming that of the free OLA and a higher genotoxicity and apoptosis-mediated cytotoxic effect in human pancreatic ductal adenocarcinoma cell line. Interestingly, the in vivo assays using immunosuppressed mice with PANC-1-induced tumors revealed that NP-ACP-OLA-AA produced a higher tumor volume reduction (59.1%) compared to free OLA (28.3%) and increased the mice survival. Calcium phosphate NPs, a highly biocompatible and biodegradable system, were an ideal vector for the OLA and AA co-treatment in PC, inducing significant therapeutic benefits relative to free OLA, including cytotoxicity, induction of apoptosis, inhibition of cell migration, tumor growth, and survival.

Ectopic expression of DOCK8 regulates lysosome-mediated pancreatic tumor cell invasion

Amplified lysosome activity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) orchestrated by oncogenic KRAS that mediates tumor growth and metastasis, though the mechanisms underlying this phenomenon remain unclear. Using comparative proteomics, we found that oncogenic KRAS significantly enriches levels of the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 8 (DOCK8) on lysosomes. Surprisingly, DOCK8 is aberrantly expressed in a subset of PDAC, where it promotes cell invasion invitro and invivo. DOCK8 associates with lysosomes and regulates lysosomal morphology and motility, with loss of DOCK8 leading to increased lysosome size. DOCK8 promotes actin polymerization at the surface of lysosomes while also increasing the proteolytic activity of the lysosomal protease cathepsin B. Critically, depletion of DOCK8 significantly reduces cathepsin-dependent extracellular matrix degradation and impairs the invasive capacity of PDAC cells. These findings implicate ectopic expression of DOCK8 as a key driver of KRAS-driven lysosomal regulation and invasion in pancreatic cancer cells.

Black Phosphorus as a Targeting PPAR-γ Agonist to Reverse Chemoresistance in Patient-derived Organoids, Mice, and Pancreatic Tumor Cells.

Black phosphorus (BP) exhibits significant potential for clinical applications. However, further research is necessary to uncover the unknown biological functions of BP and broaden its applications across various fields. This study investigates the potential of BP as a targeting PPAR-γ agonist to overcome chemoresistance in the treatment of pancreatic adenocarcinoma (PAAD) using 2D and 3D cell lines, patient-derived organoids (PDOs), and mouse models. RNA-sequencing analysis shows that BP treatment enriches differentially expressed genes in the PPAR pathway, and molecular modeling predicts the potential docking site between BP and PPAR-γ. Transcriptional activity assays are further to verify the activation of PPAR-γ. BP-activated PPAR-γ inhibits cancer stem cell (CSC) properties and expression of biomarkers such as CD44 and c-Myc, which are involved in chemoresistance. Notably, CD44 overexpression in tumor cells renders them susceptible to BP while insensitive to gemcitabine. This indicates that BP preferentially targets stem-like cells, which exhibit heightened resistance to chemotherapeutic drugs. A combination treatment strategy involving BP and gemcitabine is developed, demonstrating enhanced treatment efficacy of PAAD in both in vitro and in vivo models. Thus, BP serves as a PPAR-γ agonist capable of reversing chemoresistance, establishing it as a potent anti-tumor approach for the treatment of PAAD.

High glucose-upregulated PD-L1 expression through RAS signaling-driven downregulation of PTRH1 leads to suppression of T cell cytotoxic function in tumor environment

BackgroundNearly 80% of patients with pancreatic cancer suffer from glucose intolerance or diabetes. Pancreatic cancer complicated by diabetes has a more immunosuppressive tumor microenvironment (TME) and is associated with a worse prognosis. The relationship between glucose metabolism and programmed cell death-Ligand 1 (PD-L1) is close and complex. It is important to explore the regulation of high glucose on PD-L1 expression in pancreatic cancer and its effect on infiltrating immune effectors in the tumor microenvironment.MethodsDiabetic murine models (C57BL/6) were used to reveal different immune landscape in euglycemic and hyperglycemic pancreatic tumor microenvironment. Bioinformatics, WB, iRIP [Improved RNA Binding Protein (RBP) Immunoprecipitation]-seq were used to confirm the potential regulating role of peptidyl-tRNA hydrolase 1 homolog (PTRH1) on the stability of the PD-L1 mRNA. Postoperative specimens were used to identify the expression of PD-L1 and PTRH1 in pancreatic cancer. Co-culturing T cells with pancreatic cancer cells to explore the immunosuppressive effect of pancreatic tumor cells.ResultsOur results revealed that a high dose of glucose enhanced the stability of the PD-L1 mRNA in pancreatic tumor cells by downregulating PTRH1 through RAS signaling pathway activation following epidermal growth factor receptor (EGFR) stimulation. PTRH1 overexpression significantly suppressed PD-L1 expression in pancreatic cells and improved the proportion and cytotoxic function of CD8+ T cells in the pancreatic TME of diabetic mice.ConclusionsPTRH1, an RBP, plays a key role in the regulation of PD-L1 by high glucose and is closely related to anti-tumor immunity in the pancreatic TME.

Gold Nanoparticle-Based Microfluidic Chips for Capture and Detection of Circulating Tumor Cells.

Liquid biopsy has progressed to its current use to diagnose and monitor cancer. Despite the recent advances in investigating cancer detection and diagnosis strategies, there is still a room for improvements in capturing CTCs. We developed an efficient CTC detection system by integrating gold nanoparticles with a microfluidic platform, which can achieve CTC capture within 120 min. Here, we report our development of a simple and effective way to isolate CTCs using antibodies attached on gold nanoparticles to the surface of a lateral filter array (LFA) microdevice. Our method was optimized using three pancreatic tumor cell lines, enabling the capture with high efficiency (90% ± 3.2%). The platform was further demonstrated for isolating CTCs from patients with metastatic pancreatic cancer. Our method and platform enables the production of functionalized, patterned surfaces that interact with tumor cells, enhancing the selective capture of CTCs for biological assays.

Pharmacological inhibition of IRAK4 kinase activity does not prevent cachexia in mice with pancreatic cancer

AbstractBackgroundInflammation is a hallmark of cachexia; however, effective anti‐inflammatory treatments have not yet been identified. Interleukin‐1 receptor‐associated kinase 4 (IRAK4) is a key signalling node linking interleukin‐1 receptor (IL‐1R) and toll‐like receptor (TLR) activation to the production of multiple proinflammatory cytokines that are elevated in cancer cachexia. The purpose of this work is to evaluate whether pharmacological inhibition of IRAK4 kinase activity with PF‐06426779 could prevent cachexia using a model of pancreatic cancer. The effect of appetite stimulation via the ghrelin receptor agonist anamorelin was also examined as a benchmark of clinically validated mechanisms.MethodsFemale C57Bl/6J mice were given an intraperitoneal injection of KrasG12D; p53R172H; Pdx1‐Cre (KPC) pancreatic tumour cells. PF‐06426779 or anamorelin treatment was initiated at the onset of anorexia. Body weight and food intake were measured throughout the study. Body composition, muscle function (force), and physical activity (treadmill running endurance) were assessed at the end of the study.ResultsChronic treatment with PF‐06426779, at doses covering in vitro IC50 and IC90 at Cmin, did not increase body weight, food intake, and muscle function in the KPC tumour model. In contrast, anamorelin (vs. vehicle) increased food intake (P < 0.01), hindlimb skeletal muscle mass (P < 0.01), and muscle strength (P < 0.05); however, treadmill running endurance was not increased.ConclusionsThese data suggest that inhibition of IRAK4 kinase activity is not sufficient to treat cachexia, at least in pancreatic cancer, and exploration of alternative anti‐inflammatory strategies that increase appetite is required.

Pancreatic Tumor-targeting Stemsome Therapeutics.

Owing to the intrinsic ability of stem cells to target the tumor environment, stem cell membrane-functionalized nanocarriers can target and load active anticancer drugs. In this study, a strategy that focuses on stem cells that self target pancreatic cancer cells was deveolped. In particular, malignant deep tumors such as pancreatic cancer cells, one of the intractable tumors that currently have no successful clinical strategy, are available for targeting and destruction. By gaining the targeting ability of stem cells against pancreatic tumor cells, stem cell membranes can encapsulate nano polylactide-co-glycolide (PLGA) loaded with doxorubicin to target and reduce deep pancreatic tumor tissues. Considering the lack of known target proteins on pancreatic tumor cells, the suggested platform technology could be utilized for targeting any malignant tumors in which surface target receptors are unavailable. This article is protected by copyright. All rights reserved.

Quantitative intravital imaging for real-time monitoring of pancreatic tumor cell hypoxia and stroma in an orthotopic mouse model.

Pancreatic cancer is a lethal disease with few successful treatment options. Recent evidence demonstrates that tumor hypoxia promotes pancreatic tumor invasion, metastasis, and therapy resistance. However, little is known about the complex relationship between hypoxia and the pancreatic tumor microenvironment (TME). In this study, we developed a novel intravital fluorescence microscopy platform with an orthotopic mouse model of pancreatic cancer to study tumor cell hypoxia within the TME in vivo, at cellular resolution, over time. Using a fluorescent BxPC3-DsRed tumor cell line with a hypoxia-response element (HRE)/green fluorescent protein (GFP) reporter, we showed that HRE/GFP is a reliable biomarker of pancreatic tumor hypoxia, responding dynamically and reversibly to changing oxygen concentrations within the TME. We also characterized the spatial relationships between tumor hypoxia, microvasculature, and tumor-associated collagen structures using in vivo second harmonic generation microscopy. This quantitative multimodal imaging platform enables the unprecedented study of hypoxia within the pancreatic TME in vivo.

ATP6AP2 is robustly expressed in pancreatic β cells and neuroendocrine tumors, and plays a role in maintaining cellular viability

ATP6AP2, also known as (pro)renin receptor, has been shown to be expressed in several tissues including pancreatic β cells. Whereas ATP6AP2 plays an important role in regulating insulin secretion in mouse pancreatic β cells, the expression profiles and roles of ATP6AP2 in human pancreatic endocrine cells and neuroendocrine tumor cells remain unclear. Here in this study, we investigated the expression profiles of ATP6AP2 in pancreatic endocrine cells, and found that ATP6AP2 is robustly expressed in pancreatic insulinoma cells as well as in normal β cells. Although ATP6AP2 was also expressed in low-grade neuroendocrine tumors, it was not or faintly detected in intermediate- and high-grade neuroendocrine tumors. Knockdown experiments of the Atp6ap2 gene in rat insulinoma-derived INS-1 cells demonstrated decreased cell viability accompanied by a significant increase in apoptotic cells. Taken together, these findings suggest that ATP6AP2 plays a role in maintaining cellular homeostasis in insulinoma cells, which could lead to possible therapeutic approaches for endocrine tumors.

Active U11 Peptide Luminescent Gold Nanoclusters for Pancreatic Tumor Cell Targeting

The imaging of different intercellular regions is attracting growing interest in the fields of biosensing, drug delivery, and gene therapy for cancer treatment. We present the synthesis of luminescent gold nanoclusters (AuNCs), grafted with a PEGylated U11 peptide derivative, for tumor recognition and their capacity to label the nucleus of pancreatic cancer cells. The short peptide named C3E6U11, used to synthesize and functionalize gold nanoclusters, is composed of a tricysteine sequence with side-chain thiol residues as peptide anchors for the Au nanoclusters together with the recognition peptide domain (U11). The active targeting red- or blue-emitting AuNCs can label pancreatic cancer cells and localize preferentially in the nucleus. The presence of U11 at their surface facilitates their penetration into pancreatic cell nuclei. Finally, in vivo imaging experiments in early embryos and free-swimming juvenile zebrafish are also performed to evaluate their biodistribution.

EZH2 regulates pancreatic cancer cells through E2F1, GLI1, CDK3, and Mcm4

Pancreatic cancer (PC) is one of the most common malignant tumors in digestive tract. To explore the role of epigenetic factor EZH2 in the malignant proliferation of PC, so as to provide effective medical help in PC. Sixty paraffin sections of PC were collected and the expression of EZH2 in PC tissues was detected by immunohistochemical assay. Three normal pancreas tissue samples were used as controls. The regulation of EZH2 gene on proliferation and migration of normal pancreatic cell and PC cell were determined by MTS, colony forming, Ki-67 antibody, scratch and Transwell assays. Through differential gene annotation and differential gene signaling pathway analysis, differentially expressed genes related to cell proliferation were selected and verified by RT-qPCR. EZH2 is mainly expressed in the nuclei of pancreatic tumor cells, but not in normal pancreatic cells. The results of cell function experiments showed that EZH2 overexpression could enhance the proliferation and migration ability of PC cell BXPC-3. Cell proliferation ability increased by 38% compared to the control group. EZH2 knockdown resulted in reduced proliferation and migration ability of cells. Compared with control, proliferation ability of cells reduced by 16%-40%. The results of bioinformatics analysis of transcriptome data and RT-qPCR demonstrated that EZH2 could regulate the expression of E2F1, GLI1, CDK3 and Mcm4 in normal and PC cells. The results revealed that EZH2 might regulate the proliferation of normal pancreatic cell and PC cell through E2F1, GLI1, CDK3 and Mcm4.

Iminosugar-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors as Potential Anti-Pancreatic Cancer Agents.

The nicotinamide phosphoribosyltransferase (NAMPT) is considered a very promising therapeutic target because it is overexpressed in pancreatic cancer. Although many inhibitors have been prepared and tested, clinical trials have shown that NAMPT inhibition may result in severe haematological toxicity. Therefore, the development of conceptually new inhibitors is an important and challenging task. We synthesized ten β-d-iminoribofuranosides bearing various heterocycle-based chains carbon-linked to the anomeric position starting from non-carbohydrate derivatives. They were then submitted to NAMPT inhibition assays, as well as to pancreatic tumor cells viability and intracellular NAD+ depletion evaluation. The biological activity of the compounds was compared to that of the corresponding analogues lacking the carbohydrate unit to assess, for the first time, the contribution of the iminosugar moiety to the properties of these potential antitumor agents.

Sequential therapy with supercharged NK cells with either chemotherapy drug cisplatin or anti-PD-1 antibody decreases the tumor size and significantly enhances the NK function in Hu-BLT mice.

In this study we report that sequential treatment of supercharged NK (sNK) cells with either chemotherapeutic drugs or check-point inhibitors eliminate both poorly differentiated and well differentiated tumors in-vivo in humanized-BLT mice. sNK cells were found to be a unique population of activated NK cells with genetic, proteomic, and functional attributes that are very different from primary untreated or IL-2 treated NK cells. Furthermore, NK-supernatant differentiated or well-differentiated oral or pancreatic tumor cell lines are not susceptible to IL-2 activated primary NK cell-mediated cytotoxicity; however, they are greatly killed by the CDDP and paclitaxel in in-vitro assays. Injection of one dose of sNK cells at 1 million cells per mouse to aggressive CSC-like/poorly differentiated oral tumor bearing mice, followed by an injection of CDDP, inhibited tumor weight and growth, and increased IFN-γ secretion as well as NK cell-mediated cytotoxicity substantially in bone marrow, spleen and peripheral blood derived immune cells. Similarly, the use of check point inhibitor anti-PD-1 antibody increased IFN-γ secretion and NK cell-mediated cytotoxicity, and decreased the tumor burden in-vivo, and tumor growth of resected minimal residual tumors from hu-BLT mice when used sequentially with sNK cells. The addition of anti-PDL1 antibody to poorly differentiated MP2, NK-differentiated MP2 or well-differentiated PL-12 pancreatic tumors had different effects on tumor cells depending on the differentiation status of the tumor cells, since differentiated tumors expressed PD-L1 and were susceptible to NK cell mediated ADCC, whereas poorly differentiated OSCSCs or MP2 did not express PD-L1 and were killed directly by the NK cells. Therefore, the ability to target combinatorially clones of tumors with NK cells and chemotherapeutic drugs or NK cells with checkpoint inhibitors at different stages of tumor differentiation may be crucial for successful eradication and cure of cancer. Furthermore, the success of check point inhibitor PD-L1 may relate to the levels of expression on tumor cells.

Dual IL-6 and CTLA-4 blockade regresses pancreatic tumors in a T cell- and CXCR3-dependent manner.

This study aimed to enhance antitumor immune responses to pancreatic cancer via Ab-based blockade of IL-6 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Mice bearing s.c. or orthotopic pancreatic tumors were treated with blocking Abs to IL‑6 and/or CTLA-4. In both tumor models, dual IL-6 and CTLA-4 blockade significantly inhibited tumor growth. Additional investigations revealed that dual therapy induced an overwhelming infiltration of T cells into the tumor as well as changes in CD4+ T cell subsets. Dual blockade therapy elicited CD4+ T cells to secrete increased IFN-γ in vitro. Likewise, in vitro stimulation of pancreatic tumor cells with IFN-γ profoundly increased tumor cell production of CXCR3-specific chemokines, even in the presence of IL-6. In vivo blockade of CXCR3 prevented orthotopic tumor regression in the presence of the combination treatment, demonstrating a dependence on the CXCR3 axis for antitumor efficacy. Both CD4+ and CD8+ T cells were required for the antitumor activity of this combination therapy, as their in vivo depletion via Abs impaired outcomes. These data represent the first report to our knowledge of IL-6 and CTLA‑4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy.

Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance.

Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Abstract 4561: 3D Reconstructed Pancreas: A model capturing the unique tumor microenvironment and stromal architecture of pancreatic cancer

Abstract As it is rarely discovered early, the prognosis for people with pancreatic cancer is poor, with an average 5-year survival rate of 10% and only 3% for those with metastatic illness. Pancreatic cancer patients have few therapeutic options and innovative therapies are sorely needed to improve treatment of the disease. This is mainly due to the late diagnosis and partially due to the biology of the disease. Capturing the components of the tumor microenvironment, which serve as both a physical barrier and a source of stromal-driven resistance to therapeutics, is one of the issues faced by drug developers searching for agents to combat pancreatic cancer. We have created a 3D model of the pancreatic tumor microenvironment, the Reconstructed Pancreas (r-Pancreas), using Mia PaCa-2, PANC-1, and BxPC-3 pancreatic tumor cell lines embedded in an extracellular matrix (ECM) that was formulated to recapitulate the typical ECM of pancreatic tumors. We have shown that gemcitabine was effective against pancreatic tumor cells cultured in r-Pancreas (IC50=0.4-0.8µM), while 5-fluorouracil was ineffective (IC50 not reached), mimicking clinical response. In nude mice, subcutaneous PANC-1 tumors exhibited similar responses to gemcitabine and 5-fluorocurcil. Treatment with gemcitabine resulted in a Day 63 median ΔT/ΔC of 37% while 5-fluorouracil resulted in a Day 63 median delta ΔT/ΔC of 102%. Incorporating a collagen-rich capsule increases the physiological relevance of the r-Pancreas model because it has been shown that human pancreatic tumors have an outer layer of stiff ECM that functions as a physical barrier and prevents drugs from penetrating the tumor. Recently, we showed that, at least in the Mia PaCa-2 and BxPC-3 tumor cell lines, introducing such a collagen capsule dramatically reduced sensitivity to gemcitabine. Disrupting the collagen capsule with MMP-9 restored the sensitivity of the tumor cells to gemcitabine demonstrating that the capsule provides a physical barrier to drug entry. Additionally, creating a co-culture of primary activated pancreatic stromal cells and pancreatic tumor cell lines produces a more complete 3D model that permits the testing of potential therapeutic agents inside the pancreatic tumor microenvironment. Hence, we have created a 3D model where pancreatic tumor cells were co-cultured with human pancreatic fibroblasts. According to our results, the IC50 values for pancreatic tumor cell lines treated with gemcitabine increased by twofold when co-cultured with activated pancreatic fibroblasts compared to control sets of tumor cells alone (IC50=1-3µM). Together, these data imply that, to be clinically relevant, in vitro models of pancreatic cancer must integrate tumor-specific components of the microenvironment, including the collagen capsule and activated fibroblasts. Citation Format: Arnat Balabyev, Justin D. Phillips, Michael Steffey, Eleanore J. Kirshner, Aayushi Ahlawat, Arlette H. Uihlein, Daniel Saims, Scott Wise, Julia Kirshner. 3D Reconstructed Pancreas: A model capturing the unique tumor microenvironment and stromal architecture of 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 4561.

Abstract 2674: Synergistic combinations of lurbinectedin with irinotecan and ONC212 in pancreatic cancer

Abstract Pancreatic cancer is a devastating disease with a five-year survival rate below 10% according to the American Cancer Society. Novel chemotherapeutics and targeted therapies in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin is a chemotherapeutic synthetic tetrahydroisoquinoline alkaloid that inhibits active transcription by binding to guanine rich sequences in the minor groove of DNA. Lurbinectedin has been shown to reduce oncogenic transcription by stalling and degrading RNA Polymerase II while also inducing single- and double-stranded breaks to DNA, causing subsequent apoptosis. Lurbinectedin received accelerated FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. We recently described a synergistic interaction between lurbinectedin and ONC201/TIC10, a compound that induces the TRAIL pathway, in killing SCLC cell lines associated with activation of p-Chk1 and the integrated stress response. We now demonstrate lurbinectedin’s efficient killing of pancreatic tumor cells as a single agent in PANC-1, BxPC-3, and HPAF-II cell lines, with IC-50s corresponding to sub-nanomolar concentrations. We also demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in synergistic killing of pancreatic tumor cells in vitro. We further demonstrate a combination of lurbinectedin and ONC212, an imipridone, the same class of compounds as ONC201, also results in synergistic killing of pancreatic tumor cells. Cell viability was measured using the CellTiterGlo assay at varying drug concentrations. We hypothesize a combination therapy of lurbinectedin and irinotecan or ONC212 can enhance immune cell killing of pancreatic tumor cells. This is being explored by co-culturing CD8+ T lymphoblast cells with pancreatic tumor cells treated with lurbinectedin, irinotecan, ONC212, and combination, along with molecular mechanisms of cell death and effects on cytokines in the tumor microenvironment. Our results are developing insights regarding molecular mechanisms underlying therapeutic efficacy of a novel combination drug treatment for pancreatic cancer. Citation Format: Tej Tummala, Arielle De La Cruz, Ash Uruchurtu, Nicholas R. Liguori, Abbas E. Abbas, Leiqing Zhang, Christopher G. Azzoli, Lanlan Zhou, Wafik S. El-Deiry. Synergistic combinations of lurbinectedin with irinotecan and ONC212 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 2674.

Novel treatment strategy of targeting epigenetic dysregulation in pancreatic neuroendocrine tumors.

Epigenetic dysregulation is an integral step in the progression of pancreatic neuroendocrine tumors. We hypothesized that tumor suppressor repression by DNA methyltransferase 1 in pancreatic neuroendocrine tumors could be targeted with epigenetic treatment. Resected pancreatic neuroendocrine tumors from 32 patients were stained for DNA methyltransferase 1 and scored. Human (BON1) and murine (STC) pancreatic neuroendocrine tumor cells were treated with DNA methyltransferase 1 inhibitor 5-azacytidine and chemotherapeutic agents 5-fluorouracil and temozolomide. Cell proliferation assay and tumor suppressor gene analysis were performed with qRT-PCR and Clarion S microarray. Tumor measurements were compared in a murine treatment model. High DNA methyltransferase scores were associated with high Ki-67 (6.7% vs 70.6% P < .01), mitotic rate (0.0% vs 31.3%), and grade (20.0% vs 80.4%, P < .01). Treatment with 5-azacytidine and chemotherapy resulted in a reduction of cell proliferation compared to chemotherapy alone in BON1 (77.3% vs 53.1%, P < .001) and STC (73.4% vs 34.2%, P < .001). Treatment with 5-azacytidine and chemotherapy resulted in upregulation of tumor suppressors CDKN1A (7.6 rel. fold, P < .001), BRCA2 (4.3 rel. fold, P < .001), and CDH1 (6.0 rel. fold, P= .026) in BON1 and CDKN1a (14.5 rel. fold, P < .001) and CDH (17.5 rel. fold, P < .001) in STC. In microarray, 5-azacytidine drove global genetic changes in combination treatment. Invivo tumors treated with chemotherapy measured 88.6 ± 19.54 mm3 vs 52.89 ± 10.51 mm3 in those treated with combination therapy (P= .009). Epigenetic dysregulation with DNA methyltransferase 1 is associated with pancreatic neuroendocrine tumors and is a potential targetable strategy. 5-azacytidine and chemotherapy in combination can reduce cell proliferation, upregulate silenced tumor suppressor genes, and decrease invivo tumors in pancreatic neuroendocrine tumors.

Cytotoxic Responses of Pancreatic Cancer Cell Line to Bitter Leaf (Vernonia Amygdalina), Tomato (Solanum Lycopersicum) and Coconut (Cocos Nucifera) Milk Extracts

This work is primarily designed to determine, through cell lines, the cytotoxic responses of pancreatic cancer cell (PANC-1) to reconstituted lyophilized extracts of coconut milk, bitter leaf and tomato with a view of evaluating the anticancer properties of these extract. The cytotoxicity analysis was performed on the extracts using the MTT assay method as previously described on pancreatic tumour (PANC-1) cell lines . The cytotoxicity studies showed concentration and dose-dependent decrease in cell viability of pancreatic cancer cell line (PANC-1) to the three lyophilized extracts of coconut milk, bitter leaf and tomato. Bitter leaf aqueous extract was significantly the most cytotoxic to the cancer cells followed by coconut and lastly tomato extracts. The cytotoxicity of coconut milk compared with tomato and bitter leaf to PANC-1 increased in the order of bitter leaf &gt; coconut milk &gt; tomato.

ScRNA-Seq and imaging mass cytometry analyses unveil iNKT cells-mediated anti-tumor immunity in pancreatic cancer liver metastasis

Invariant natural killer T (iNKT) cells are innate-like T cells that are abundant in liver sinusoids and play a critical role in tumor immunity. However, the role of iNKT cells in pancreatic cancer liver metastasis (PCLM) has not been fully explored. In this study, we employed a hemi-spleen pancreatic tumor cell injection mouse model of PCLM, a model that closely mimics clinical conditions in humans, to explore the role of iNKT cells in PCLM. Activation of iNKT cells with α-galactosylceramide (αGC) markedly increased immune cell infiltration and suppressed PCLM progression. Via single cell RNA sequencing (scRNA-seq) we profiled over 30,000 immune cells from normal liver and PCLM with or without αGC treatment and were able to characterize the global changes of the immune cells in the tumor microenvironment upon αGC treatment, identifying a total of 12 subpopulations. Upon treatment with αGC, scRNA-Seq and flow cytometry analyses revealed increased cytotoxic activity of iNKT/NK cells and skewing CD4 T cells towards a cytotoxic Th1 profile and CD8 T cells towards a cytotoxic profile, characterized by higher proliferation and reduced exhaustion marker PD1 expression. Moreover, αGC treatment excluded tumor associated macrophages. Lastly, imaging mass cytometry analysis uncovered the reduced epithelial to mesenchymal transition related markers and increased active CD4 and CD8 T cells in PCLM with αGC treatment. Overall, our findings uncover the protective function of activated iNKT cells in pancreatic cancer liver metastasis through increased NK and T cell immunity and decreased tumor associated macrophages.