Primary Pancreatic Cancer Cells Research Articles

Abstract 7223: A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma

Abstract KRAS mutations drive over 90% of pancreatic cancers, but KRAS has been proven difficult to target. Downstream of KRAS, MEK inhibition in combination with BCL-XL inhibition is synthetically lethal and an effective treatment for gynecologic, but not colorectal malignancies. Like colorectal cancer, pancreatic cancer often exhibits aberrant HER/EGFR activity. We hypothesized that a triplicate regimen targeting MEK, BCL-XL, and EGFR/HER would be effective for pancreatic cancer. Trametinib (MEK inhibitor), Afatinib (pan-EGFR/HER inhibitor), and Navitoclax (BCL-XL inhibitor); or DT2216 (a BCL-XL/BCL-1 proteolysis targeting chimera, PROTAC) were explored using various combinatorial strategies. These drug combinations were tested using four primary human pancreatic cancer cell lines using live cell imaging (Incucyte) and an in vivo murine model. Group comparisons were analyzed using one-way ANOVAs, and adjusted p-values <0.05 were considered significant. We observed that no single-agent treatments were effective. MEK inhibition plus anti-BCL-XL (Trametinib + Navitoclax or Trametinib + DT2216) demonstrated efficacy in one of the four cell lines. We noted synergistic effects on reduced cell growth and increased cell death when EGFR/HER inhibition was included by the administration of Afatinib. The triplet combination resulted in synergistic effects in three of four cell lines with Navitoclax and four of four cell lines with DT2216 (q<0.05). This effect was confirmed with DT2216 inhibition of BCL-XL in an in-vivo murine model. In conclusion, our study shows that a combinatory strategy targeting MEK, BCL-XL, and pan-EGFR/HER receptor kinase shows significant synergistic effects in pancreatic cancer. Clinical trials that assess the efficacy and safety of this strategy for patients with KRAS-mutant pancreatic cancer are warranted. Our data also suggests that novel KRAS inhibitors as monotherapy are likely to fail. Citation Format: Gerik W. Tushoski-Alemán, Alexandra J. Crespin, Chibeze J. Oguejiofor, Jordan A. McKean, Kelly M. Herremans, Thomas J. George, Carmen J. Allegra, Steven J. Hughes, Song Han. A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7223.

Differential modulation of cellular phenotype and drug sensitivity by extracellular matrix proteins in primary and metastatic pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be the third highest cause of cancer-related deaths in the United States. PDAC is known for its high proportion of stroma, which accounts for 90% of the tumor mass. The stroma is made up of extracellular matrix (ECM) and nonmalignant cells such as inflammatory cells, cancer-associated fibroblasts, and lymphatic and blood vessels. Here, we decoupled the effects of the ECM on PDAC cell lines by culturing cells on surfaces coated with different ECM proteins. Our data show that the primary tumor-derived cell lines have different morphology depending on the ECM proteins on which they are cultured, while metastatic lesion-derived PDAC lines' morphology does not change with respect to the different ECM proteins. Similarly, ECM proteins modulate the proliferation rate and the gemcitabine sensitivity of the primary tumor PDAC cell lines, but not the metastatic PDAC lines. Lastly, transcriptomics analysis of the primary tumor PDAC cells cultured on different ECM proteins reveals the regulation of various pathways, such as cell cycle, cell-adhesion molecules, and focal adhesion, including the regulation of several integrin genes that are essential for ECM recognition.

Comparison of Tumour-Specific Phenotypes in Human Primary and Expandable Pancreatic Cancer Cell Lines.

There is an ongoing need for patient-specific chemotherapy for pancreatic cancer. Tumour cells isolated from human tissues can be used to predict patients' response to chemotherapy. However, the isolation and maintenance of pancreatic cancer cells is challenging because these cells become highly vulnerable after losing the tumour microenvironment. Therefore, we investigated whether the cells retained their original characteristics after lentiviral transfection and expansion. Three human primary pancreatic cancer cell lines were lentivirally transduced to create expandable (Ex) cells which were then compared with primary (Pri) cells. No obvious differences in the morphology or epithelial-mesenchymal transition (EMT) were observed between the primary and expandable cell lines. The two expandable cell lines showed higher proliferation rates in the 2D and 3D models. All three expandable cell lines showed attenuated migratory ability. Differences in gene expression between primary and expandable cell lines were then compared using RNA-Seq data. Potential target drugs were predicted by differentially expressed genes (DEGs), and differentially expressed pathways (DEPs) related to tumour-specific characteristics such as proliferation, migration, EMT, drug resistance, and reactive oxygen species (ROS) were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We found that the two expandable cell lines expressed similar chemosensitivity and redox-regulatory capability to gemcitabine and oxaliplatin in the 2D model as compared to their counterparts. In conclusion, we successfully generated expandable primary pancreatic cancer cell lines using lentiviral transduction. These expandable cells not only retain some tumour-specific biological traits of primary cells but also show an ongoing proliferative capacity, thereby yielding sufficient material for drug response assays, which may provide a patient-specific platform for chemotherapy drug screening.

Priming therapy by targeting enhancer-initiated pathways in patient-derived pancreatic cancer cells.

Systems biology leveraging multi-OMICs technologies, is rapidly advancing development of precision therapies and matching patients to targeted therapies, leading to improved responses. A new pillar of precision oncology lies in the power of chemogenomics to discover drugs that sensitizes malignant cells to other therapies. Here, we test a chemogenomic approach using epigenomic inhibitors (epidrugs) to reset patterns of gene expression driving the malignant behavior of pancreatic tumors. We tested a targeted library of ten epidrugs targeting regulators of enhancers and super-enhancers on reprogramming gene expression networks in seventeen patient-derived primary pancreatic cancer cell cultures (PDPCCs), of both basal and classical subtypes. We subsequently evaluated the ability of these epidrugs to sensitize pancreatic cancer cells to five chemotherapeutic drugs that are clinically used for this malignancy. To comprehend the impact of epidrug priming at the molecular level, we evaluated the effect of each epidrugs at the transcriptomic level of PDPCCs. The activating epidrugs showed a higher number of upregulated genes than the repressive epidrugs (χ2 test p-value <0.01). Furthermore, we developed a classifier using the baseline transcriptome of epidrug-primed-chemosensitized PDPCCs to predict the best epidrug-priming regime to a given chemotherapy. Six signatures with a significant association with the chemosensitization centroid (R≤-0.80; p-value < 0.01) were identified and validated in a subset of PDPCCs. We conclude that targeting enhancer-initiated pathways in patient-derived primary cells, represents a promising approach for developing new therapies for human pancreatic cancer. This work was supported by INCa (Grants number 2018-078 to ND and 2018- 079 to JI), Canceropole PACA (ND), Amidex Foundation (ND), and INSERM (JI).

Abstract 816: Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models

Abstract Purpose: This study aimed to improve the systemic stability and enhance the anticancer activity of gemcitabine (Gem) by modifying Gem with stearic acid to form 4-N-stearoylGem (4NSG). The 4NSG compound was formulated into solid lipid nanoparticles and tested against patient-derived pancreatic cancer (PCa) cell lines and patient-derived xenograft (PDX) mice bearing subcutaneous tumors. Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4-(N)-stearoyl-gemcitabine (4NSG). 4NSG was characterized using high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and elemental analysis. 4NSG was developed into solid lipid nanoparticles (4NSG-SLN) and characterized using a particle size analyzer. Patient-derived primary PCa cells (PPCL-46 and PPCL-68) were treated with GemHCl and 4NSG-SLN. Cytotoxicity, cell migration, and cell-cycle studies were performed to determine the effectiveness of 4NSG-SLN against primary PCa cell lines. We performed antitumor efficacy testing using GemHCl and 4NSG-SLN in PDX mice bearing subcutaneous pancreatic tumors. Results: Analysis of the H-NMR spectra displayed an amide bond at 10.88 ppm, confirming the conjugated bond between the 4-amino group of Gem and stearic acid. The purity of 4NSG was 99.8%. The hydrodynamic diameter (particle size) of 4NSG-SLN was 82±2.3nm, while the blank nanoparticle (SLN) was found to be 35 ± 4.3 nm. The half-maximal inhibitory concentration of 4NSG-SLN-treated PPCL-46 (IC50 =12 ± 2.1µM) and PPCL-68 cultures (IC50 =22 ± 2.6µM) showed higher cytotoxic activity compared with GemHCl-treated PPCL-46 and PPCL-68 cultures respectively (IC50 = 56 ± 2.4 µM, IC50 = 57 ± 1.5 µM, p-value: p&amp;lt;0.001). Our migration study showed 4NSG-SLN treated PPCL-46 cells at 5μΜ concentrations significantly reduced cell mobility towards the wound area with (28 ± 3.5) number of cells migrated compared to GemHCl treatment which showed (120 ± 3.8). The cell-cycle analysis showed that 4NSG-SLN treated PPCL-46 cells at 5μΜ concentration had a higher G1 population (78.25%) than GemHCl (72.75%). 4NSG-SLN may have triggered apoptosis in PPCL-46 cells (2.45%) at 20μΜ in the S and G2/M phases compared to GemHCl treatments which showed a higher cell population of (9.54%).4NSG-SLN treated PDX mice exhibited a two-fold decrease in tumor growth compared to GemHCl-treated PDX mice bearing tumors. Conclusion: This study reveals that 4NSG-SLNs may potentially prolong Gem's systemic circulation, increase its bioavailability, and, most importantly, enhance the therapeutic efficacy of Gem in the treatment of PCa. Citation Format: Esther K. Frimpong, Andriana Inkoom, Nkafu B. Ndemazie, Raviteja Bulusu, Joy C. Okoro, Bo Han, Jose Trevino, Edward Agyare. Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models [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 816.

Abstract 5048: Combatting pancreatic cancer stem cells by novel titanium peroxides nanoparticles combined with X-ray radiation

Abstract Background: Radiotherapy is widely used as the main treatment for multiple malignancies. However, several types of cancers, including pancreatic cancer, show resistance to radiation therapy. The presence of cancer stem cells (CSCs) are the major cause of radiation resistance in pancreatic cancer. Whereas, titanium peroxides nanoparticles (TiOxNPs) which are a modified form of titanium oxide nanoparticles promote enhanced remarkable efficacy of radiation therapy. Therefore, we examined the efficacy of TiOxNPs as radiosensitizers to eradicate pancreatic cancer stem cells. Methods and materials: In vitro, Sphere-forming assay, survival assay, migration, and invasion assay were evaluated after using TiOxNPs prior to radiation exposure to pancreatic cancer cell line. In vivo, tumor-bearing nude mice were injected by TiOxNPs either intratumoral or intravenous one hour prior to radiation treatment, and the tumor volume, body weight, and mice survival was calculated. In addition, proteins-related stemness were measured in vitro and in vivo to evaluate the usage of TiOxNPs as radiosensitizers to pancreatic CSCs. Moreover, we planned to evaluate the mechanism beyond the efficacy of TiOxNPs as radiosensitizers to CSCs by detecting reactive oxygen species (ROS) production, mitochondrial function, and the phosphorylation of some signaling proteins. We found that TiOxNPs combined with ionizing radiation showed anti-cancer effects on radioresistant CSCs both in vitro and in vivo. Results: In vitro, a marked reduction in growth was detected after exposing the TiOxNPs-treated cells to radiation therapy, specifically with a 5Gy dose compared with 2Gy dose. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. In vivo, we first established an aggressive xenograft by injecting MIA PaCa-2 sphere cells into the flank region of BALB/c nude mice, and found that animals treated with combined TiOxNPs and irradiation showed a dramatic reduction in tumor volume and weight compared to the untreated group. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Radiosensitizing TiOxNPs suppressed pancreatic xenograft outgrowth after primary or dissociating sphere MIA PaCa-2 cell implantation. It is assumed that synergy is created by inactivating the AKT signaling pathway and producing unbearable amounts of ROS. Conclusion: Our findings showed that using TiOxNPs in combination with radiation might be a favorable therapeutic approach to eradicate pancreatic CSCs. Citation Format: Mohammed Salah, Hiroki Kawaguchi, Hiroaki Akasaka, Yasuyuki Shimizu, Kenta Morita, Yuya Nishimura, Hikaru Kubota, Tomomi Sogawa, Naritoshi Mukumoto, Chiaki Ogino, Ryohei Sasaki. Combatting pancreatic cancer stem cells by novel titanium peroxides nanoparticles combined with X-ray radiation. [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 5048.

Abstract 3955: Functional evaluation of pancreatic cancer prognostic marker, LXN, utilizing multiomics analysis based on patient-derived cancer model

Abstract Background: Pancreatic cancer is the most dreadful malignant tumor and expected becoming the second cause of death in 2030. Despites of vigorous studies, there were no definitive prognostic biomoleculars marker for pancreatic cancer. Herein, we already established primary pancreatic cancer cell lines from the real patients of pancreatic cancer, using conditionally reprogrammed cell culture (CRC). We tried to find novel prognostic biomarkers with multi-omics analysis of our primary pancreatic cancer cell lines. Methods: In this study, we selected pancreatic cancer prognostic markers using established CRCs and FFPE samples and validated the molecular mechanism of LXN. As the first step, we performed both transcriptome and proteome analysis using RNA sequencing and liquid chromatography-tandem mass spectrometry (LC/MS-MS) with 6 primary pancreatic cancer cell lines established by CRC. After analysis, we mined several candidate markers of prognosis including latexin (LXN) and sialic acid acetylesterease (SIAE). And we validated these markers clinicopathologically using immunohistochemical (IHC) staining of 136 tissues from a different set of pancreatic cancer patients. And we confirmed the knock-down effect of LXN using siLXN in pancreatic cancer cells. Results: We selected 2 highly prognostic markers with LXN and SIAE after transcriptome and proteome analysis. In these genes, high LXN expression group showed longer median overall survival (OS) than low expression group, respectively (14.8 vs. 10.4 months, P-value=0.28), respectively. For the evaluation of IHC, we established 3D organoid model using CRCs and confirmed the correlation between mRNA expression and protein expression with western blotting and IHC. The expression of Akt/mTOR was decreased after siLXN treatment compared to control groups. In order to validation as a prognostic marker, we stained formalin-fixed, paraffin-embedded (FFPE) slide from 136 pancreatic cancer patients. After IHC analysis, LXN high group (H-score &amp;gt; 12) showed significantly longer OS than low LXN group (52 vs. 28 months, P-value = 0.043). Conclusion: In this study, we found LXN as a prognostic marker with inhibition of proliferation utilizing patient-derived cancer model and FFPE samples. Citation Format: Chan Hee Park, Hee Seung Lee, Jin Su Kim, Yoo Keung Tae, Jin Young Lee, Seungmin Bang. Functional evaluation of pancreatic cancer prognostic marker, LXN, utilizing multiomics analysis based on patient-derived 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 3955.

Identification of matrix-remodeling associated 5 as a possible molecular oncotarget of pancreatic cancer

Pancreatic cancer has an extremely poor prognosis. Here we examined expression, potential functions and underlying mechanisms of MXRA5 (matrix remodeling associated 5) in pancreatic cancer. Bioinformatics studies revealed that MXRA5 transcripts are significantly elevated in pancreatic cancer tissues, correlating with the poor overall survival, high T-stage, N1 and pathologic stage of the patients. MXRA5 mRNA and protein expression is significantly elevated in microarray pancreatic cancer tissues and different pancreatic cancer cells. In primary and immortalized (BxPC-3 and PANC-1 lines) pancreatic cancer cells, shRNA-induced MXRA5 silencing or CRISPR/Cas9-mediated MXRA5 knockout suppressed cell survival, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT), while provoking cell apoptosis. Conversely, forced overexpression of MXRA5 further promoted pancreatic cancer cell progression and EMT. Bioinformatics studies and the protein chip analyses revealed that differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in MXRA5-overexpressed primary pancreatic cancer cells were enriched in the PI3K-Akt-mTOR cascade. Indeed, Akt-mTOR activation in primary human pancreatic cancer cells was inhibited by MXRA5 shRNA or knockout, but was augmented following MXRA5 overexpression. In vivo, the growth of MXRA5 KO PANC-1 xenografts was largely inhibited in nude mice. Moreover, intratumoral injection of adeno-associated virus-packed MXRA5 shRNA potently inhibited primary pancreatic cancer cell growth in nude mice. Akt-mTOR activation was also largely inhibited in the MXRA5-depleted pancreatic cancer xenografts. Contrarily MXRA5 overexpression promoted primary pancreatic cancer cell growth in nude mice. Together, overexpressed MXRA5 is important for pancreatic cancer cell growth possibly through promoting EMT and Akt-mTOR activation. MXRA5 could be a potential therapeutic oncotarget for pancreatic cancer.

Primary Human Pancreatic Cancer Cells Cultivation in Microfluidic Hydrogel Microcapsules for Drug Evaluation.

Chemotherapy is an essential postoperative treatment for pancreatic cancer, while due to the lack of effective drug evaluation platforms, the therapeutic outcomes are hampered by tumor heterogeneity among individuals. Here, a novel microfluidic encapsulated and integrated primary pancreatic cancer cells platform is proposed for biomimetic tumor 3D cultivation and clinical drug evaluation. These primary cells are encapsulated into hydrogel microcapsules of carboxymethyl cellulose cores and alginate shells based on a microfluidicelectrospraytechnique. Benefiting from the good monodispersity, stability, and precise dimensional controllability of the technology, the encapsulated cells can proliferate rapidly and spontaneously form 3D tumor spheroids with highly uniform size and good cell viability. By integrating these encapsulated tumor spheroids into a microfluidic chip with concentration gradient channels and culture chambers, dynamic and high-throughput drug evaluation of different chemotherapy regimens could be realized. It is demonstrated that different patient-derived tumor spheroids show different drug sensitivity on-chip, which is significantly consistent with the clinical follow-up study after the operation. The results demonstrate that the microfluidic encapsulated and integrated tumor spheroids platform has great application potential in clinical drug evaluation.

Abstract B034: CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer characterized by late diagnosis, lack of early symptoms and extensive metastasis. One of the foremost reasons for such startling statistics is the presence of a subpopulation of highly plastic stem-like cells within the tumor called cancer stem cells (CSCs). We have previously identified a distinct subset of these CSCs within the invasive front of patient tumors. This subset, called migrating cancer stem cells (miCSCs), is characterized by CD133+CXCR4+ expression and determines the metastatic phenotype of pancreatic cancer. Therefore, targeting CXCR4 may represent a potential therapeutic approach to lower metastatic burden in PDAC. Here, we examined the effect of endogenous human peptides EPI-X4 and other derivatives thereof as CXCR4 antagonist on (i) patient-derived primary pancreatic cancer cells and (ii) tumor-stroma crosstalk by using a dual culture system with pancreatic stellate cells. We established these peptides as novel therapeutic strategy for combating the metastatic activity of pancreatic cancer using combinatorial therapeutic approaches and testing different in vivo delivery system such as peptide fatty-acid (FA) conjugates and silica nanoparticles (Si-NP). Our results show that EPI-X4 as well as its derivatives (e.g., JM#21) strongly inhibited migratory capacity of primary pancreatic cancer cells towards the CXCR4 ligand CXCL12 in vitro. Thereby, JM#21 was identified as the most potent EPI-X4 derivate. Mechanistical analysis by western blot, gene expression and immunofluorescence revealed that JM#21 increased Cadherin-1 expression by suppression of Snail1 via inactivation of SHH pathway. Moreover, JM#21 decreased CXCL12-induced phosphorylation of AKT and IKBa as well as NANOG expression, which further suppressed self-renewal capacity and EMT in the tumor cells. Strikingly, JM#21 sensitized selected cell lines towards gemcitabine and paclitaxel. Furthermore, FA conjugated and Si-NP encapsulated JM#21 restricted miCSCs maintenance which was predominantly regulated via stellate cell secreted CXCL12. In serum conditions, both FA conjugated, and Si-NP encapsulated JM#21 was found to be stable and active, proving as a valuable delivery system for in vivo studies. In conclusion, our study reveals that targeting CXCR4/CXCL12 signaling axis using human endogenous EPI-X4 derivates particularly JM#21 inhibits tumor-stroma crosstalk which is paramount for the propagation and maintenance of miCSC. Particularly, we demonstrate, in both mechanistic and preclinical set up, that these peptides abrogate the metastatic capacity of patient-derived pancreatic cancer cells by selective targeted elimination of miCSCs. Moreover, tumor cells show increased susceptibility towards conventional treatment strategies enforcing EPI-X4 derivate as a novel combinatory therapy to treat metastatic pancreatic cancer. Citation Format: Kanishka Tiwary, Mirja Harms, Bastian Beitzinger, Roman Schmid, Syeda Inaas, Karolin Walter, Alexander Kleger, Mika Lindén, Thomas Seufferlein, Jan Münch, Patrick Christian Hermann. CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B034.

FOLFIRINOX Pharmacodynamic Interactions in 2D and 3D Pancreatic Cancer Cell Cultures

The multi-drug combination regime, FOLFIRINOX, is a standard of care chemotherapeutic therapy for pancreatic cancer patients. However, systematic evaluation of potential pharmacodynamic interactions among multi-drug therapy has not been reported previously. Here, pharmacodynamic interactions of the FOLFIRINOX agents (5-fluorouracil (5-FU), oxaliplatin (Oxa) and SN-38, the active metabolite of irinotecan) were assessed across a panel of primary and established pancreatic cancer cells. Inhibition of cell proliferation was quantified for each drug, alone and in combination, to obtain quantitative, drug-specific interaction parameters and assess the nature of drug interactions. The experimental data were analysed assuming Bliss independent interactions, and nonlinear regression model fitting was conducted in SAS. Estimates of the drug interaction term, psi (ψ), revealed that the Oxa/SN-38 combination appeared synergistic in PANC-1 (ψ = 0.6, 95% CI = 0.4, 0.9) and modestly synergistic, close to additive, in MIAPaCa-2 (ψ = 0.8, 95% CI = 0.6, 1.0) in 2D assays. The triple combination was strongly synergistic in MIAPaCa-2 (ψ = 0.2, 95% CI = 0.1, 0.3) and modestly synergistic/borderline additive in PANC-1 2D (ψ = 0.8, 95% CI = 0.6, 1.0). The triple combination showed antagonistic interactions in the primary PIN-127 and 3D PANC-1 model (ψ > 1). Quantitative pharmacodynamic interactions have not been described for the FOLFIRINOX regimen; this analysis suggests a complex interplay among the three chemotherapeutic agents. Extension of this pharmacodynamic analysis approach to clinical/translational studies of the FOLFIRINOX combination could reveal additional pharmacodynamic interactions and guide further refinement of this regimen to achieve optimal clinical responses.Graphical abstract

Combined Targeting of the Glutathione and Thioredoxin Antioxidant Systems in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is characterized by increased generation of reactive oxygen species that can cause lethal oxidative stress. Here, we evaluated the combined inhibition of the glutathione and thioredoxin antioxidant systems in preclinical models of pancreatic ductal adenocarcinoma, using buthionine sulfoximine (BSO) that targets glutathione synthesis, and auranofin that targets thioredoxin recycling. BSO potentiated the cytotoxicity of auranofin and induced lethal oxidative stress in primary pancreatic cancer cells. As assessed by the cellular thermal shift assay, auranofin engaged with thioredoxin reductase 1 in primary cells at concentrations known to induce cell death. Moreover, we used imaging mass cytometry to map the biodistribution of atomic gold in patient-derived xenografts treated with auranofin, and the drug was readily detectable throughout the epithelial and stromal compartments after treatment with a clinically relevant dose. In conclusion, combinatorial treatment with BSO and auranofin could serve as a potential therapeutic strategy in pancreatic ductal adenocarcinoma.

Abstract 1746: Optimizing Gemcitabine efficacy through chemical modification for the treatment of pancreatic cancer using PDX mouse model

Abstract PURPOSE: Despite the remarkable advances in cancer treatment, the incidence rate of pancreatic cancer (PCa) for decades among African Americans (AA) has been 30% to 70% higher than other racial groups in the United States. Gemcitabine (Gem) remains the first-choice treatment for PCa. It is administered as monotherapy in patients with locally advanced and metastatic PCa; but, drug resistance develops over time. Gem treatment resistance coupled with poor pharmacokinetics (short half-life) has resulted in poor treatment outcomes. The purpose of this study is to enhance the anticancer efficacy and pharmacokinetic profile of Gem through chemical modification of Gem with stearic acid by using mice bearing tumors from AA and White pancreatic cancer patients. METHODS: The Gem was modified by conjugating the 4-amino group of Gem and stearoyl acyl derivative to form 4-(N)-stearoyl-gemcitabine (4NSG). 4NSG was developed into solid lipid nanoparticles (4NSG-SLN) and characterized using nuclear magnetic resonance (NMR), micro-elemental analysis, and high liquid chromatography (HPLC). Patient-derived primary pancreatic cancer cells from AA (PPCL-135 and PPCL-192) and Whites (PPCL-46 and PPCL-68) were used to evaluate the anticancer activity of Gem and 4NSG-SLN. We performed antitumor efficacy testing of Gem and 4NSG-SLN in a patient-derived xenograft (PDX) mouse model harboring pancreatic tumors from the same AA and White patients. RESULTS: The H-NMR spectra displayed an amide bond at 11ppm, confirming the conjugated bond between the 4-amino group of Gem and stearoyl derivative. The half-maximal inhibitory concentration (IC50 =12 ± 2.1µM) of 4NSG-SLN-treated PPCL-46 and (IC50 =22 ± 2.6µM) of 4NSG-SLN-treated PPCL-68 cultures were significantly higher than that of Gem treated PPCL-46 and PPCL-68 cultures respectively (IC50 = 56 ± 2.4 µM, IC50 = 57 ± 1.5 µM, p-value: p&amp;lt;0.001). We found a similar trend of higher growth inhibition of 4NSG-SLN treated PPCL-135 and PPCL-192 cultures (IC50 = 11± 1.3 µM; IC50 = 9 ± 1.1 µM) respectively, P&amp;lt; 0.001 (4NSG-SLN-PPCL-135 vs. Gem-PPCL-135) and P&amp;lt;0.001 (4NSG-SLN-PPCL-192-2 vs Gem-PPCL-192) compared with Gem treated PPCL-135 and PPCL-192 cultures (IC50 = 56 ± 1.8 µM; IC50 =57 ± 2.4 µM) respectively. The anticancer activity of 4NSG-SLN showed an enhanced efficacy in PPCL-46, PPCL-68, PPCL-135, and PPCL-192 treated cultures compared with their corresponding Gem treatments. In vivo studies, 4NSG-SLN treated mice bearing AA tumors showed significant tumor growth inhibition compared with GemHCl treated mice bearing AA tumors. A similar pattern was observed where 4NSG-SLN treated mice bearing White tumors showed significant tumor growth inhibition than GemHCl treated mice bearing White tumors. CONCLUSION: This study reveals that 4NSG-SLN may be a novel approach to significantly enhance the therapeutic efficacy of Gem in the treatment of pancreatic cancer. Citation Format: Andriana Inkoom, Edward Agyare, Nkafu Bechem Ndemazie, Taylor Smith, Bo Han, Jose Trevino Trevino. Optimizing Gemcitabine efficacy through chemical modification for the treatment of pancreatic cancer using PDX mouse model [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 1746.

CYP26A1 Is a Novel Cancer Biomarker of Pancreatic Carcinoma: Evidence from Integration Analysis and In Vitro Experiments.

Background CYP26A1 has been reported in multiple cancers. However, the role of CYP26A1 in pancreatic cancer (PC) has not been explored. Method The public data used for this study was obtained from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Cancer Cell Line Encyclopedia (CCLE) cell lines. CCK8, colony formation, and EdU assay were used to assess the proliferation ability of cancer cells. Transwell and wound healing assays were used to evaluate the invasion and migration ability of cancer cells. qRT-PCR and western blot assays were used to analyze the RNA and protein level of genes. Survival package was used for prognosis analysis. Gene Set Enrichment Analysis (GSEA) was used to identify biological pathway differences between two groups. ssGSEA analysis was used to quantify the immune microenvironment in PC tissue. GDSC and TIDE analyses were used for sensitivity analysis of chemotherapy and immunotherapy. Results Our results showed that CYP26A1 was overexpressed in PC tissue and cell lines. Meanwhile, metastatic PC cell lines tend to have a higher CYP26A1 level compared with the primary PC cell lines based on CCLE data. Moreover, CYP26A1 was associated with worse clinical features. Also, we found that CYP26A1 had a satisfactory efficiency in predicting overall survival, disease-specific survival, and progression-free interval of PC patients, independent of other clinical features. In vitro experiments indicated that CYP26A1 could significantly facilitate the proliferation, invasion, and migration ability of PC cells. GSEA showed that the pathways of angiogenesis, E2F target, MYC target, mTORC signaling, G2M checkpoint, and epithelial-mesenchymal transition were activated in high CYP26A1 patients. Immune infiltration analysis showed that CYP26A1 was positively correlated with macrophages, Th1 cells, and Treg cells, but negatively correlated with Th17 cells. TIDE analysis showed that non_responder patients had a higher CYP26A1 level compared with predicted responder patients of immunotherapy. Drug sensitivity analysis and assay showed that CYP26A1 could increase the chemotherapy sensitivity of gemcitabine. Conclusions In summary, CYP26A1 promotes PC progression and is a novel biomarker of PC, with potential for clinical application.

First-in-class monoclonal antibody (mAb) PBP1510 targeting pancreatic adenocarcinoma upregulated factor (PAUF) for pancreatic cancer (PC) treatment: Preclinical perspectives.

e16274 Background: PAUF has been shown to induce PC cell proliferation, angiogenesis and evasion of immune surveillance, thereby promoting tumour progression and metastasis. PBP1510, a novel humanized IgG1 mAb developed by Prestige Biopharma Limited, Singapore, binds to and neutralizes PAUF with high specificity and affinity. With no available targeted therapies against PAUF, the first in its class, PBP1510, has been granted Orphan Drug Designation by EMA, US FDA and Korea MFDS. Methods: To assess inhibition of migration and invasion of PC in vitro, PC cell lines (CFPAC-1 and BxPC-3) with high expression levels of PAUF, and primary PC cells extracted from patients were treated with PBP1510 or human IgG (negative control). Suitability of cynomolgus monkey as animal model for pharmacokinetic (PK) and toxicity studies was verified by comparing binding affinities of PBP1510 to human and monkey PAUF. PK characteristics and immunogenicity of PBP1510 (up to 20 mg/kg), were examined in single dose studies in 6 monkeys. A 4-week repeated dose toxicity study was also conducted in 32 monkeys with PBP1510 (up to 40 mg/kg). An in vitro tissue cross-reactivity study was also conducted in a full panel of normal human tissues, to assess potential toxicity in humans. Results: PBP1510 suppressed the proliferation of the PC cell lines compared to negative control. The migration and invasion of PBP1510 treated PC cell lines as well as the primary tumour cells from patients was significantly reduced compared to the respective control groups in vitro. Similarity in binding affinity of PBP1510 to human PAUF and monkey PAUF, and amino acid sequence homology between human and monkey PAUF, suggest that monkey is an appropriate animal species for toxicity and PK studies of PBP1510. In monkey studies, a dose-proportional increase in systemic exposure was observed at low and high doses. PBP1510 at doses up to 40 mg/kg was well tolerated by study animals and no notable local or systemic toxicity was observed. Low immunogenic profile of PBP1510 was indicated by an absence of anti-PBP1510 antibodies till up to 57 days (8 doses and 28-day recovery period). These data are used to simulate human PK profile and to derive the starting dose and maximum escalated dose for the upcoming Phase 1 clinical studies. Cross-reactivity results did not reveal non-specific binding of PBP1510 in the human tissues except in the cytoplasm of acinar and duct epithelium in the salivary gland which is not expected to be of major toxicologic significance. Conclusions: The efficacy and safety data presented here, along with the data from in vivo mouse studies demonstrating superior anti-tumour activity of PBP1510 treatment, support further clinical development of PBP1510 as a novel anti-cancer agent to treat PAUF-positive PC. The pivotal step of advancing PBP1510 into initial human studies is now in progress.

Reactive oxygen species-inducing titanium peroxide nanoparticles as promising radiosensitizers for eliminating pancreatic cancer stem cells

BackgroundDespite recent advances in radiotherapy, radioresistance in patients with pancreatic cancer remains a crucial dilemma for clinical treatment. Cancer stem cells (CSCs) represent a major factor in radioresistance. Developing a potent radiosensitizer may be a novel candidate for the eradication of pancreatic CSCs.MethodsCSCs were isolated from MIA PaCa-2 and PANC1 human pancreatic cancer cell lines. Titanium peroxide nanoparticles (TiOxNPs) were synthesized from titanium dioxide nanoparticles (TiO2NPs) and utilized as radiosensitizers when added one hour prior to radiation exposure. The antitumor activity of this novel therapeutic strategy was evaluated against well-established pancreatic CSCs model both in vitro and in vivo.ResultsIt is shown that TiOxNPs combined with ionizing radiation exhibit anti-cancer effects on radioresistant CSCs both in vitro and in vivo. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Notably, radiosensitizing TiOxNPs suppressed the growth of pancreatic xenografts following primary or dissociating sphere MIA PaCa-2 cell implantation. It is inferred that synergy is formed by generating intolerable levels of reactive oxygen species (ROS) and inactivating the AKT signaling pathway.ConclusionsOur data suggested the use of TiOxNPs in combination with radiation may be considered an attractive therapeutic strategy to eliminate pancreatic CSCs.

Lead identification using 3D models of pancreatic cancer.

Recent technological advances have enabled 3D tissue culture models for fast and affordable HTS. We are no longer bound to 2D models for anti-cancer agent discovery, and it is clear that 3D tumor models provide more predictive data for translation of preclinical studies. In a previous study, we validated a microplate 3D spheroid-based technology for its compatibility with HTS automation. Small-scale screens using approved drugs have demonstrated that drug responses tend to differ between 2D and 3D cancer cell proliferation models. Here, we applied this 3D technology to the first ever large-scale screening effort completing HTS on over 150K molecules against primary pancreatic cancer cells. It is the first demonstration that a screening campaign of this magnitude using clinically relevant, ex-vivo 3D pancreatic tumor models established directly from biopsy, can be readily achieved in a fashion like traditional drug screen using 2D cell models. We identified four unique series of compounds with sub micromolar and even low nanomolar potency against a panel of patient derived pancreatic organoids. We also applied the 3D technology to test lead efficacy in autologous cancer associated fibroblasts and found a favorable profile for better efficacy in the cancer over wild type primary cells, an important milestone towards better leads. Importantly, the initial leads have been further validated in across multiple institutes with concordant outcomes. The work presented here represents the genesis of new small molecule leads found using 3D models of primary pancreas tumor cells.

Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway.

Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

Abstract PO-108: Evaluation of antitumor activity of modified-gemcitabine solid-lipid nanoparticle in pancreatic pdx models

Abstract Purpose: Pancreatic cancer (PCa) is one of the most aggressive and devastating malignancies in the United States, with a high mortality rate. Despite the remarkable advances in cancer therapy, there are limited treatment options for PCa patients. Gemcitabine (Gem), a nucleoside analog, remains the preferred choice, either alone or in combination with other chemotherapeutic agents, to treat PCa. While Gem has proven to be effective against PCa in the early stages of treatment, PCa cells resistance to Gem and rapid metabolism of Gem has resulted in poor treatment outcomes. The objective of this study was to address the issues associated with Gem via chemical modification of Gem to 4NSG. This significantly reduced the metabolism and highly sensitized PCa cells to Gem. Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4NSG and characterized using nuclear magnetic resonance (NMR) for) to determine new bond formation, micro-elemental analysis micro-elemental analysis used to ascertain the presence of elemental composition and purity, high liquid chromatography (HPLC) used to determine percent purity of 4NSG. The 4NSG was developed into solid lipid nanoparticles (4NSG-SLN), and the size was determined using a particle size analyzer. Patient-derived primary pancreatic cancer cells (CMZ and G68Ca) and MiaPaCa-2 cells were treated with free Gem and 4NSG-SLN. Percent cell viability was determined using resazurin assay and antitumor efficacy testing of Gem, and 4NSG-SLN was performed in patient-derived xenograft (PDX) mouse models bearing G68Ca PCa. Results: Analysis of the H-NMR spectra displayed an amide bond at 11ppm, confirming the conjugated bond between the 4-amino group of Gem and stearoyl derivative. The half-maximal inhibitory concentration (IC50 =11±1.3µM) of 4NSG-SLN-treated CMZ culture was significantly higher than that of Gem treated CMZ culture (IC50 = 56±2 µM, p&amp;lt;0.001). We found a similar trend of higher growth inhibition of 4NSG-SLN treated G68Ca and MiaPaCa-2 cultures (IC50 (4NSG-SLN-G68Ca) = 12±2.1 µM; IC50(Gem-MiaPaCa-2) = 27±4 µM) respectively. Where p&amp;lt; 0.001 (4NSG-SLN-G68Ca vs. Gem-G68Ca) and p&amp;lt;0.001 (4NSG-SLN-MiaPaCa-2 vs Gem-MiaPaCa-2) compared with Gem treated G68Ca and Mia-PaCa-2 cultures (IC50 (G68Ca) = 68±26 µM; IC50 (Mia-PaCa-2) =54±5 µM) respectively. Put together, the anticancer activity of 4NSG-SLN demonstrated enhanced efficacy in CMZ, G68Ca, and MiaPaCa-2 treated cultures compared with their corresponding Gem treated cultures. For antitumor efficacy testing, 4NSG-SLN demonstrated significant tumor growth inhibition in PDX models harboring G68Ca pancreatic tumors compared with Gem treated with PDX models. Immunohistostaining studies of 4NSG-SLN treated pancreatic tumors significantly reduced vascular endothelial growth factor receptor (VEGFR) expression compared with Gem treated pancreatic tumors. Conclusion: This study demonstrated that 4NSG might be a novel approach to significantly enhance the therapeutic efficacy of Gem in the treatment of PCa. Citation Format: Edward Agyare, Taylor Smith, Andriana Inkoom, Bo Han, Jose Trevino, Nkafu Bechem Ndemazie. Evaluation of antitumor activity of modified-gemcitabine solid-lipid nanoparticle in pancreatic pdx models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-108.

Identification of 2 Chinese Primary Pancreatic Ductal Adenocarcinoma Cancer Cell Lines and Their Phenotypes.

Pancreatic cancer (PC) is one of the most lethal cancers. Currently, the most commonly used PC cell lines were all derived from White individuals, and few models can be used to study in Asian populations. In China, the incidence of PC is increasing every year, highlighting the need to develop new Chinese PC cell lines for cancer research. A total of 203 patients diagnosed with PC, enrolled from January 2012 to December 2017 in our hospital (Department of Hepatobiliary and Pancreatic Surgery), had received surgery. Primary PC patient tumor samples were harvested sterilely from surgery, minced, and digested in collagenase. Cells were cultured in plates precoated with l-polylysine. Two primary PC cell lines, Si-Liang 187 and Si-Liang 188, were established, both of which grew as adherent monolayers and demonstrated distinguished phenotypes. Si-Liang 187 shows the typical mesenchymal pattern, whereas Si-Liang 188 shows epithelial phenotype. Moreover, they carry different genetic backgrounds according to whole exome sequencing. Two new Chinese PC cell lines were established, both of which were characterized and confirmed with high tumorigenicity. They may serve as useful tools for pathogenesis research when evaluating new treatment strategies in Asian patients.