Human Pancreatic Adenocarcinoma Cell Lines Research Articles

TGF-β-Induced PAUF Plays a Pivotal Role in the Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cell Line Panc-1

TGF-β-Induced PAUF Plays a Pivotal Role in the Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cell Line Panc-1

MUC17 is a potential new prognostic biomarker and promotes pancreatic cancer progression in obstructive jaundice.

Our working group has previously shown that bile acids (BAs) accelerate carcinogenic processes in pancreatic cancer (PC) in which mucin 4 (MUC4) expression has a central role. However, the role of other mucins in PC are less clear, especially in bile-induced cancer progression. The study aim was to investigate expression of MUC17 in BAs- or human serum-treated pancreatic ductal adenocarcinoma (PDAC) cell lines and use different assays with RNA silencing/overexpression to study the role of MUC17 in cancer progression. Protein expression of MUC17 was evaluated in 55 human pancreatic samples by immunohistochemistry, and Kaplan-Meier survival analysis was used to compare survival curves. Expression of MUC17 increased in PDAC patients, especially in obstructive jaundice (OJ) and the elevated MUC17 expression associated with poorer overall survival (10.66±1.99 vs. 15.05±2.03 months; Log rank: 0.0497). Treatment of Capan-1 and AsPC-1 cells with BAs or with human serum obtained from PDAC + OJ patients enhanced the expression of MUC17, as well as the proliferative potential of the cells, whereas knockdown of MUC17 alone or in combination with MUC4 decreased BAs-induced carcinogenic processes. Our results demonstrated that MUC17 has a central role in bile-induced PC progression, and in addition to MUC4, this isoform also can be used as a novel prognostic biomarker.

Evaluation of the anti-tumor activity of histone deacetylase inhibitors (HDAC6) on human pancreatic adenocarcinoma (PANC-1) cell line using a zebrafish xenograft model

Evaluation of the anti-tumor activity of histone deacetylase inhibitors (HDAC6) on human pancreatic adenocarcinoma (PANC-1) cell line using a zebrafish xenograft model

The apoptotic effect of garlic (Allium sativum) derived SEVs on different types of cancer cell lines in vitro.

Small extracellular vesicles (SEVs) are known to have an impact on the physiological conditions of target cells, are a critical component of cell-to-cell communication, and have been implicated in a variety of diseases. Although it has been proposed that edible plant-derived nanoparticles have an effect on communication with mammalian cells, the influence of these nanoparticles on cancer cell development has yet to be explored. In order to characterize small extracellular vesicles obtained from garlic, specific SEV surface markers, antibodies, and size detections were identified using scanning electron microscopy and nanoparticle tracking analysis. Human hepatoma (Hep3B), human neuroblastoma (SH-SY5Y), human pancreatic adenocarcinoma (Panc-1a), human glioblastoma (U87), prostate cancer (PC-3), and human umbilical vein endothelial (HUVEC) cell lines were treated with garlic SEVs to examine their anticancer properties. Annexin V FITC/PI staining for apoptosis, mRNA, and protein expression levels via RT-PCR and ELISA indicated that garlic SEVs triggered apoptosis by activating the intrinsic pathway. Our findings support the idea that SEVs produced from garlic may trigger apoptotic cell death in cancer cells while having no effect on healthy cells. It was discovered that plant SEVs had anti-cancer effects by activating caspase-mediated apoptosis.

Unfolding the role of cell state on stress granule heterogeneity and function in pancreatic cancer.

e16357 Background: A major impediment to cancer therapy is the reality that cancer is a heterogeneous disease. A single tumor can consist of many genotypic or phenotypic profiles, which are marked by degrees of sensitivity to different therapeutics. Identifying and targeting origins of cancer cell heterogeneity is critical for predicting patient outcome and enhancing treatment efficacy. We observe a novel source of cancer cell heterogeneity that involves one of the largest stress-sensing platforms described to date: the stress granule (SG). Stress granules are non-membranous, cytoplasmic organelles that assemble in response to stress, and promote cell survival. Deregulation of SGs has been linked to several pathological conditions, including cancer, neurodegeneration, ischemia, and viral infections. SGs have been shown to promote tumor growth and metastasis, as well as contribute to mechanisms of chemo-resistance. Thus, these signaling hubs are emerging as a potential target in cancer. Methods: To induce SG formation, human pancreatic ductal adenocarcinoma (PDAC) cell lines were treated with sodium arsenite, thapsigargin, or exposed to hypoxic conditions. Immunoflourescent staining of core SG proteins was then conducted. To investigate cell cycle-driven mechanisms of SG heterogeneity, cell cycle stage was identified by expression of a Fluorescent ubiquitination-based cell cycle indicator (Fucci) lentiviral system. To investigate cPLA2’s role in SG formation, the chemical inhibitor Pyrrophenone, an shRNA targeting cPLA2, and an exogenous full-length or cleaved form of cPLA2, were used. In vivo, ES149 cells expressing Luciferase and the Fucci system were orthotopically implanted into C57BL/6 mice. Established tumors were treated every 3 days with Vehicle, Oxaliplatin, Pyrrophenone, or a combination of the latter. Tumor size was tracked via IVIS Spectrum. Fucci flourescence was used to identify cell cycle phase. SGs and cell death were assessed by immunoflourescent staining of tumors. Results: Cancer cells show a high degree of heterogeneity in their production of SGs. Cell cycle phase dictates SG heterogeneity. G2 phase cells have significantly higher levels of SG formation as compared cells in G1/S. This process is regulated by cleavage of the cPLA2 protein. Oxaliplatin administration increases G2 phase content in tumors. Co-treatment with Oxaliplatin and Pyrrophenone significantly decreases SG formation, increases cell death, and leads to tumor regressions in vivo. Conclusions: G2 phase cancer cells develop higher levels of SGs when exposed to stressful stimuli, as compared to G1/S phase counterparts. The cPLA2 protein is cleaved into a catalytically dead form that is present in G1/S phases, but absent in G2 phase. Oxaliplatin treatment in vivo leads to an increase in G2 phase tumor cells. When paired with cPLA2 inhibition, this results in increased cancer cell death and decreased tumor growth.

Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

BackgroundEGFR and/or HER2 expression in pancreatic cancers is correlated with poor prognoses. We generated homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (T-BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors.MethodsEGFR and HER2 T-BsAbs were constructed using the 2 + 2 IgG-[L]-scFv T-BsAbs format bearing two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. A Fab arm exchange strategy was used to generate EGFRxHER2 heterodimeric T-BsAb carrying one Fab specific for EGFR and one for HER2. EGFR and HER2 T-BsAbs were also heterodimerized with a CD33 control T-BsAb to generate ‘tumor-monovalent’ EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence.ResultsThe EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (∼150-fold and ∼6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost.ConclusionEGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging both targets, EGFRxHER2 heterodimeric T-BsAb exhibited potent anti-tumor effects if CDX or PDX were EGFR+HER2+ double-positive with the potential to spare single-positive normal tissue.

Abstract 3336: Regulation of liver X receptor protein stability by novel ligands in pancreatic cancer cells

Abstract Liver X Receptors (LXRs) are nuclear receptors that act as ligand-modulated transcription factors. LXRs are overexpressed in various cancers, including pancreatic cancer, and targeting LXR with small molecule ligands has emerged as a promising therapeutic strategy in cancer. Current small molecule LXR ligands were originally developed for the treatment of atherosclerosis and other metabolic diseases. To identify ligands specifically targeting cancer cells, we screened a focused library of drug-like molecules predicted to bind to LXR using molecular docking. In the screen, novel LXR ligand GAC0001E5 (1E5) exhibited significant anti-proliferative effects in human pancreatic ductal adenocarcinoma (PDAC) cell lines. Functionally, 1E5 is an LXR inverse agonist which decreases LXR target gene expression and a “degrader” of LXR proteins following prolonged treatment. We posit that this novel ligand inhibits PDAC cells by downregulating the expression of LXR target genes. To test this hypothesis, we treated cancer cells with the previously described LXR inverse agonist SR9238 and examined its effect on cancer cell proliferation. Interestingly, treatments with SR9238 had no effect on pancreatic cancer cell proliferation as compared to the novel ligand 1E5 and the 1E5 derivative KD-95, suggesting that LXR inverse agonism was insufficient to inhibit cell proliferation. To determine whether SR9238 has similar effects on LXR protein levels as novel ligands, we performed western analysis of protein expression following ligand treatment. In contrast to the novel ligands 1E5 and KD-95, treatments with SR9238 increased LXR protein levels. Furthermore, time-course studies in cycloheximide treated cells indicated that novel ligands decreased protein stability whereas SR9238 had the opposite effect. Inhibition of proteasomal degradation and autophagy abolished the effects of 1E5 and KD-95 in a cell line-dependent manner. Related to these observations, previously published data revealed that knockdown of LXR expression greatly diminished PDAC cell proliferation. Taken together, these findings indicate that LXR expression is essential in pancreatic cancer cells and suggest that the novel ligands may disrupt its involvement in protein-protein interactions and non-genomic mechanisms. Citation Format: Abhinav Bagchi, Kasuni T. Gamage, Scott R. Gilbertson, Chin-Yo Lin. Regulation of liver X receptor protein stability by novel ligands in pancreatic cancer cells [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 3336.

Cannabigerol Induces Autophagic Cell Death by Inhibiting EGFR-RAS Pathways in Human Pancreatic Ductal Adenocarcinoma Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) is the most frequent infiltrating type of pancreatic cancer. The poor prognosis associated with this cancer is due to the absence of specific biomarkers, aggressiveness, and treatment resistance. PDAC is a deadly malignancy bearing distinct genetic alterations, the most common being those that result in cancer-causing versions of the KRAS gene. Cannabigerol (CBG) is a non-psychomimetic cannabinoid with anti-inflammatory properties. Regarding the anticancer effect of CBG, up to now, there is only limited evidence in human cancers. To fill this gap, we investigated the effects of CBG on the PDAC cell lines, PANC-1 and MIAPaCa-2. The effect of CBG activity on cell viability, cell death, and EGFR-RAS-associated signaling was investigated. Moreover, the potential synergistic effect of CBG in combination with gemcitabine (GEM) and paclitaxel (PTX) was investigated. MTT was applied to investigate the effect of CBG on PDAC cell line viabilities. Annexin-V and Acridine orange staining, followed by cytofluorimetric analysis and Western blotting, were used to evaluate CBG's effect on cell death. The modulation of EGFR-RAS-associated pathways was determined by Western blot analysis and a Milliplex multiplex assay. Moreover, by employing the MTT data and SynergyFinder Plus software analysis, the effect of the combination of CBG and chemotherapeutic drugs was determined.

Indole Diketopiperazine Alkaloids from the Marine Sediment-Derived Fungus Aspergillus chevalieri against Pancreatic Ductal Adenocarcinoma.

A new prenylated indole diketopiperazine alkaloid, rubrumline P (1), was isolated along with six more analogues and characterized from the fermentation culture of a marine sediment-derived fungus, Aspergillus chevalieri, collected at a depth of 15 m near the lighthouse in Dahab, Red Sea, Egypt. In the current study, a bioassay-guided fractionation allowed for the identification of an active fraction displaying significant cytotoxic activity against the human pancreatic adenocarcinoma cell line PANC-1 from the EtOAc extract of the investigated fungus compared to the standard paclitaxel. The structures of the isolated compounds from the active fraction were established using 1D/2D NMR spectroscopy and mass spectrometry, together with comparisons with the literature. The absolute configuration of the obtained indole diketopiperazines was established based on single-crystal X-ray diffraction analyses of rubrumline I (2) and comparisons of optical rotations and NMR data, as well as on biogenetic considerations. Genome sequencing indicated the formation of prenyltransferases, which was subsequently confirmed by the isolation of mono-, di-, tri-, and tetraprenylated compounds. Compounds rubrumline P (1) and neoechinulin D (4) confirmed preferential cytotoxic activity against PANC-1 cancer cells with IC50 values of 25.8 and 23.4 µM, respectively. Although the underlying mechanism-of-action remains elusive in this study, cell cycle analysis indicated a slight increase in the sub-G1 peak after treatment with compounds 1 and 4.

Temoporfin-Conjugated Upconversion Nanoparticles for NIR-Induced Photodynamic Therapy: Studies with Pancreatic Adenocarcinoma Cells In Vitro and In Vivo.

Upconverting nanoparticles are interesting materials that have the potential for use in many applications ranging from solar energy harvesting to biosensing, light-triggered drug delivery, and photodynamic therapy (PDT). One of the main requirements for the particles is their surface modification, in our case using poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) and temoporfin (THPC) photosensitizer to ensure the colloidal and chemical stability of the particles in aqueous media and the formation of singlet oxygen after NIR irradiation, respectively. Codoping of Fe2+, Yb3+, and Er3+ ions in the NaYF4 host induced upconversion emission of particles in the red region, which is dominant for achieving direct excitation of THPC. Novel monodisperse PMVEMA-coated upconversion NaYF4:Yb3+,Er3+,Fe2+ nanoparticles (UCNPs) with chemically bonded THPC were found to efficiently transfer energy and generate singlet oxygen. The cytotoxicity of the UCNPs was determined in the human pancreatic adenocarcinoma cell lines Capan-2, PANC-01, and PA-TU-8902. In vitro data demonstrated enhanced uptake of UCNP@PMVEMA-THPC particles by rat INS-1E insulinoma cells, followed by significant cell destruction after excitation with a 980 nm laser. Intratumoral administration of these nanoconjugates into a mouse model of human pancreatic adenocarcinoma caused extensive necrosis at the tumor site, followed by tumor suppression after NIR-induced PDT. In vitro and in vivo results thus suggest that this nanoconjugate is a promising candidate for NIR-induced PDT of cancer.

Evaluation of the Antitumor Effects of Platinum-Based [Pt(η1-C2H4-OR)(DMSO)(phen)]+ (R = Me, Et) Cationic Organometallic Complexes on Chemoresistant Pancreatic Cancer Cell Lines.

Pancreatic cancer is one of the most lethal malignancies with an increasing incidence and a high mortality rate, due to its rapid progression, invasiveness, and resistance to anticancer therapies. In this work, we evaluated the antiproliferative and antimigratory activities of the two organometallic compounds, [Pt(η1-C2H4-OMe)(DMSO)(phen)]Cl (1) and [Pt(η1-C2H4-OEt)(DMSO)(phen)]Cl (2), on three human pancreatic ductal adenocarcinoma cell lines with different sensitivity to cisplatin (Mia PaCa-2, PANC-1, and YAPC). The two cationic analogues showed superimposable antiproliferative effects on the tested cells, without significant differences depending on alkyl chain length (Me or Et). On the other hand, they demonstrated to be more effective than cisplatin, especially on YAPC cancer cells. For the interesting cytotoxic activity observed on YAPC, further biological assays were performed, on this cancer cell line, to evaluate the apoptotic and antimetastatic properties of the considered platinum compounds (1 and 2). The cytotoxicity of 1 and 2 compounds appeared to be related to their intracellular accumulation, which was much faster than that of cisplatin. Both 1 and 2 compounds significantly induced apoptosis and cell cycle arrest, with a high accumulation of sub-G1 phase cells, compared to cisplatin. Moreover, phenanthroline-containing complexes caused a rapid loss of mitochondria membrane potential, ΔΨM, if compared to cisplatin, probably due to their cationic and lipophilic properties. On 3D tumor spheroids, 1 and 2 significantly reduced migrated area more than cisplatin, confirming an antimetastatic ability.

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.

Hyperthermia Influences the Secretion Signature of Tumor Cells and Affects Endothelial Cell Sprouting.

Tumors are a highly heterogeneous mass of tissue showing distinct therapy responses. In particular, the therapeutic outcome of tumor hyperthermia treatments has been inconsistent, presumably due to tumor versus endothelial cell cross-talks related to the treatment temperature and the tumor tissue environment. Here, we investigated the impact of the average or strong hyperthermic treatment (43 °C or 47 °C for 1 h) of the human pancreatic adenocarcinoma cell line (PANC-1 and BxPC-3) on endothelial cells (HUVECs) under post-treatment normoxic or hypoxic conditions. Immediately after the hyperthermia treatment, the distinct repression of secreted pro-angiogenic factors (e.g., VEGF, PDGF-AA, PDGF-BB, M-CSF), intracellular HIF-1α and the enhanced phosphorylation of ERK1/2 in tumor cells were detectable (particularly for strong hyperthermia, 2D cell monolayers). Notably, there was a significant increase in endothelial sprouting when 3D self-organized pancreatic cancer cells were treated with strong hyperthermia and the post-treatment conditions were hypoxic. Interestingly, for the used treatment temperatures, the intracellular HIF-1α accumulation in tumor cells seems to play a role in MAPK/ERK activation and mediator secretion (e.g., VEGF, PDGF-AA, Angiopoietin-2), as shown by inhibition experiments. Taken together, the hyperthermia of pancreatic adenocarcinoma cells in vitro impacts endothelial cells under defined environmental conditions (cell-to-cell contact, oxygen status, treatment temperature), whereby HIF-1α and VEGF secretion play a role in a complex context. Our observations could be exploited for the hyperthermic treatment of pancreatic cancer in the future.

Relative Biological Effectiveness (RBE) of [64Cu]Cu and [177Lu]Lu-NOTA-panitumumab F (ab')2 radioimmunotherapeutic agents vs. γ-radiation for decreasing the clonogenic survival in vitro of human pancreatic ductal adenocarcinoma (PDAC) cells

IntroductionOur objective was to compare [64Cu]Cu-NOTA-panitumumab F(ab')2 and [177Lu]Lu-NOTA-panitumumab F(ab')2 radioimmunotherapy (RIT) agents for decreasing the clonogenic survival fraction (SF) in vitro of EGFR-positive human pancreatic ductal adenocarcinoma (PDAC) cell lines and estimate the relative biological effectiveness (RBE) vs. γ-radiation (XRT). MethodsEGFR-positive PDAC cell lines (AsPC-1, PANC-1, MIAPaCa-2, Capan-1) and EGFR-knockout PANC-1 EGFR KO cells were treated in vitro for 18 h with (0–19.65 MBq; 72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 or XRT (0–8 Gy) followed by clonogenic assay. The SF was determined after culturing single treated cells for 14 d. Cell fractionation studies were performed for cells incubated with 1 MBq (72 nmols/L) of [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 for 1, 4, or 24 h to estimate the time-integrated activity (Ã) on the cell surface, cytoplasm, nucleus and medium. Radiation absorbed doses in the nucleus were calculated by multiplying à by S-factors calculated by Monte Carlo N Particle (MCNP) modeling using monolayer cell culture geometry. The SF of PDAC cells was plotted vs. dose and fitted to a linear quadratic model to estimate the dose required to decrease the SF to 0.1 (D10). The D10 for RIT agents were compared to XRT to estimate the RBE. DNA double-strand breaks (DSBs) caused by [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 continuous exposure for 5 h or 20 h were probed by immunofluorescence for γ-H2AX. Relative EGFR expression of PDAC cells was assessed by flow cytometry (scored + to +++) and cell doubling times for untreated cells were determined. ResultsThe D10 for [64Cu]Cu-NOTA-panitumumab F(ab′)2 ranged from 9.1 Gy (PANC-1) to 39.9 Gy (Capan-1). The D10 for [177Lu]Lu-NOTA-panitumumab F(ab′)2 ranged from 11.7 Gy (AsPC-1) to 170.8 Gy (Capan-1). The D10 for XRT ranged from 2.5 Gy (Capan-1) to 6.7 Gy (PANC-1 EGFR KO). D10 values were not correlated with EGFR expression over a relatively narrow range (++ to +++) or with cell doubling times. Based on D10 values, PANC-1 EGFR KO cells were 1.6-fold less sensitive than PANC-1 cells to [64Cu]Cu-NOTA-panitumumab F(ab′)2 and 1.9-fold less sensitive to [177Lu]Lu-NOTA-panitumumab F(ab′)2. The RBE for [64Cu]Cu-NOTA-panitumumab F(ab′)2 ranged from 0.06 for Capan-1 cells to 0.45 for PANC-1 cells. The RBE for [177Lu]Lu-NOTA-panitumumab F(ab′)2 ranged from 0.015 for Capan-1 cells to 0.28 for AsPC-1 cells. DNA DSBs were detected in PDAC cells exposed to [64Cu]Cu-NOTA-panitumumab F(ab′)2 or [177Lu]Lu-NOTA-panitumumab F(ab′)2 but were not correlated with the SF of the cells. ConclusionsWe conclude that at the same dose delivered to the cell nucleus [64Cu]Cu-NOTA-panitumumab F(ab′)2 and [177Lu]Lu-NOTA-panitumumab F(ab′)2 were less radiobiologically effective than XRT for decreasing the SF of human PDAC cells, but [64Cu]Cu-NOTA-panitumumab F(ab′)2 was more cytotoxic than [177Lu]Lu-NOTA-panitumumab F(ab′)2 except for AsPC-1 cells which were more sensitive to [177Lu]Lu-NOTA-panitumumab F(ab′)2. Advances in knowledge and implications for patient careThis study demonstrates that higher radiation doses may be required for RIT than XRT to achieve radiobiologically equivalent effects when used to treat PDAC.

Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids

Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1SMAD4 (-/-) cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-β pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1SMAD4 (-/-) cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION-MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION-MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.

Cytotoxic, apoptotic, and necrotic effects of silver nanoparticles biosynthesized using Origanum majorana extract

application. Anticancer activity of Origanum majorana extract and silver nanoparticles was investigated by MTT assay using human pancreatic adenocarcinoma cell lines (Capan-1), mouse normal fibroblast cell lines (L929), and human colon adenocarcinoma cell line (Caco-2). AgNPs@Om exhibited the excellent cytotoxic effect on Capan-1 cell lines with the cell viability of 29.17% and 25.70%, 22.49%, 21.16% at 1.0, 0.5, 0.25, 0.125 μg/mL respectively. However, the extract was determined to show moderate activity. A considerable fall in the viable cell number in Caco-2 cell lines was observed after the treatment of AgNPs@Om. The viable cells of Caco-2 cell lines were detected as 32.0% at 1.0 μg/mL with the treatment of nanoparticles. Yet, the viable Caco-2 cells were determined as 48.2% with the treatment of O. majorana extract. Both extract and nanoparticles had no impact on the viability of non-tumor cells (L929). Moreover, nanoparticles induced apoptosis. Consequently, AgNPs@Om may be a promising anticancer drug candidate.

Synergistic effects of agonists and two-pore-domain potassium channels on secretory responses of human pancreatic duct cells Capan-1

Mechanisms of synergistic agonist stimulation and modulation of the electrochemical driving force for anion secretion are still not fully explored in human pancreatic duct epithelial cells. The first objective of this study was therefore to test whether combined agonist stimulation augments anion transport responses in the Capan-1 monolayer model of human pancreatic duct epithelium. The second objective was to test the influence of H+,K+-ATPase inhibition on anion transport in Capan-1 monolayers. The third objective was to analyze the expression and function of K+ channels in Capan-1, which could support anion secretion and cooperate with H+,K+-ATPases in pH and potassium homeostasis. The human pancreatic adenocarcinoma cell line Capan-1 was cultured conventionally or as polarized monolayers that were analyzed by Ussing chamber electrophysiological recordings. Single-cell intracellular calcium was assayed with Fura-2. mRNA isolated from Capan-1 was analyzed by use of the nCounter assay or RT-PCR. Protein expression was assessed by immunofluorescence and western blot analyses. Combined stimulation with different physiological agonists enhanced anion transport responses compared to single agonist stimulation. The responsiveness of Capan-1 cells to histamine was also revealed in these experiments. The H+,K+-ATPase inhibitor omeprazole reduced carbachol- and riluzole-induced anion transport responses. Transcript analyses revealed abundant TASK-2, TWIK-1, TWIK-2, TASK-5, KCa3.1, and KCNQ1 mRNA expression. KCNE1 mRNA and TREK-1, TREK-2, TASK-2, and KCNQ1 protein expression were also shown. This study shows that the Capan-1 model recapitulates key physiological aspects of a bicarbonate-secreting epithelium and constitutes a valuable model for functional studies on human pancreatic duct epithelium.

Abstract C022: Heterodimeric bispecific antibodies for highly specific treatment of EGFR(+)HER2(+) pancreatic ductal adenocarcinoma

Abstract Background: EGFR and HER2 are highly expressed in many pancreatic cancers which strongly correlate with poorer prognoses. We tested homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors. Methods: We used the 2+2 IgG(L)-scFv format to create BsAbs, previously shown to have exceptional anti-tumor properties compared to other structural platforms. These BsAbs utilize two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. Anti-EGFR was based on the Cetuximab and anti-HER2 on the Trastuzumab amino acid sequences. Using a ‘knob-into-hole’ strategy, we generated an EGFRxHER2 heterodimeric BsAb bearing one Fab specific for EGFR and one for HER2. T cells were tagged with luciferase for tracking by bioluminescence for in vivo trafficking studies. Results: The EGFRxEGFR, HER2xHER2, and EGFRxHER2 BsAbs demonstrate high avidity (KD: 2pM to 300 pM) and T cell-mediated cytotoxicity against several human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They mediate potent T cell-mediated antitumor effects against subcutaneous PDAC xenografts and were highly efficient in driving human polyclonal T cells into tumors as measured by bioluminescence. To examine the importance of anti-tumor valency in antitumor effects, EGFR and HER2 BsAbs were heterodimerized with a CD33 control BsAb to generate EGFRxCD33 and HER2xCD33 BsAbs. Both EGFRxCD33 and HER2xCD33 BsAbs showed modest reductions in target avidity by surface plasmon resonance (2.7-fold and 12.6-fold, respectively), cell binding strength by FACS (8.0-fold and 63.6-fold) and cytotoxicity (7.7-fold and 47.2-fold) compared to their homodimeric counterparts and, remarkably, showed no therapeutic efficacy against SW1990 cell line-derived and patient-derived PDAC xenografts, correlating with reduced tumor-infiltrating bioluminescent T cells, when compared to EGFRxHER2 BsAb. When tested against SW1990 with CRISPR-mediated target KOs, only HER2xHER2 BsAb treatment could eliminate EGFR-KO tumors, while EGFRxEGFR and EGFRxHER2 BsAbs were ineffective. Conversely, in HER2-KO SW1990 tumors, while EGFRxEGFR BsAb demonstrated strong tumor control, HER2xHER2 and EGFRxHER2 BsAbs demonstrated only weak activity. Conclusion: EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. HER2 was overall more effective as a target when compared to EGFR for pancreatic cancer in vivo. Bivalent Fab binding to tumor was critical for robust anti-tumor effect in vivo even when in vitro differences of monovalent BsAb were less obvious. EGFRxHER2 heterodimeric BsAb exhibited potent antitumor efficacy against EGFR(+)HER2(+) double positive PDAC xenografts, but not single-positive tumors, and could offer a strategy to spare healthy single-positive tissues to reduce ‘on target/off tumor’ side effects. Citation Format: Alan W. Long, Brian Santich, Hongfen Guo, Nai-Kong Cheung. Heterodimeric bispecific antibodies for highly specific treatment of EGFR(+)HER2(+) pancreatic ductal adenocarcinoma [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 C022.

Abstract B030: The quadruplex-binding compound QN-302 targets the S100P gene in PDAC

Abstract The compound QN-302, a tetra-substituted naphthalene diimide derivative has been previously disclosed to have single-digit nM anti-proliferative activity in a panel of human pancreatic ductal adenocarcinoma (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It also has significant anti-tumor activity in the MIA-PACA2 xenograft model for PDAC as well as in the more demanding KPC model. Its novel mode of action involves the targeting of quadruplex (G4)-forming motifs in promoter regions of cancer-associated genes, where their prevalence is over-represented compared to other genes. The presence of a promoter G4 motif, stabilized by a ligand, presents an effective block to transcription. Transcriptome analyses of QN-302 in MIA-PACA2 cells has validated this concept, with the disclosure of a pattern of down-regulated genes involved in cancer-associated pathways and carrying G4 motifs in their promoters. We have also previously shown using RT-PCR and Western blots that a sub-set of these genes and their gene products are down-regulated in the MIA-PACA2 xenograft model following QN-302 treatment. We have now examined cellular changes in the expression of the 229 gene set associated with PDAC and compared these with data from cells and xenografts treated with QN-302. We find that expression of the key gene S100P, a prominent and well-authenticated marker of PDAC disease and progression, is highly down-regulated in QN-302 treated cells. The S100P gene contains 31 putative G4 motifs, including a classic G4 site in the S100P promoter, just upstream of the transcription start site. Transcriptome analyses have also been performed on tumor material from human patients, comprising a panel of poorly differentiated as well as more highly differentiated tumors, and has been compared with normal pancreas expression data. The S100P gene is statistically significantly highly over-expressed (especially in the poorly differentiated tumors), giving added confidence to the concept that the S100P gene is a therapeutic target and may also be useful as a marker of QN-302 response in future clinical use. We also find that the anti-tumor effect of QN-302 treatment in PDAC xenografts results in a high level of apoptosis, which is consistent with earlier reports that S100P in tumors binds to and suppresses p53 activity. The suppression of S100P transcription by QN-302 would thus be expected to remove this roadblock to apoptosis and cell death, in accordance with our observations. QN-302 is bio-available at therapeutic doses and is well tolerated at these levels in these animal models. It is being developed for clinical evaluation by Qualigen Therapeutics Inc and is currently at the pre-IND stage. Citation Format: Ahmed Ahmed, William Greenhalf, Tariq Arshad, Stephen S. Neidle. The quadruplex-binding compound QN-302 targets the S100P gene in PDAC [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 B030.

Expression of CASC8 RNA in Human Pancreatic Cancer Cell Lines

A lot of long non-coding RNAs (lncRNAs) are expressed in human cells in a number of transcripts of different lengths and composition of exons. In case of cancer-associated lncRNAs, an actual task is to determine their specific isoforms, since each transcript can perform its own function in carcinogenesis and might have a unique expression profile in various types of tumors. For the first time, we analyzed the expression of CASC8 lncRNA in human pancreatic ductal adenocarcinoma cell lines and found an abundant isoform that was previously considered as the minor one in this type of cancer. We also revealed extremely high expression levels of all CASC8 transcripts in MIA PaCa-2 cells and, conversely, the lack of this lncRNA in PANC-1. This allows to use them as convenient models for further in vitro studies.