Human Pancreatic Cancer Cell Lines Research Articles

Abstract 1594: Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response

Abstract Background: While T-cell-based immunotherapy emerged as a promising tool for cancer management, clinical trials and translational studies revealed that cancer cells develop resistance by modulating tumor microenvironment and cell-intrinsic mechanisms. Pancreatic cancer (PC), consisting of dense stroma, immunosuppressive environment, and aberrant mucin expression, has a dismal survival rate and responds poorly to immunotherapies. MUC4, a member of the mucin family, has been reported to block lymphokine-activated killer cells and induce apoptosis of cytotoxic T-cells, suggesting its possible role in immune modulation. However, how the T-cell secretome influences MUC4 expression in PC and its role in modulating the T-cell response is poorly investigated. Method: The scRNA-seq data analysis and primary T-cell conditioned media (CM) were utilized to investigate the MUC4 and T-cell crosstalk. T-cell CM-treated murine PC cell line (KCT-3266) was analyzed by RNA-seq analysis followed by MUC4 silencing studies to investigate its role in evading T-cell response. The subcutaneous murine model was utilized to investigate the influence of MUC4 KO on immune cells and cytotoxic cell infiltration. The RNA isolated from tumor tissues from KrasG12D/+, Trp53R127H/+, Pdx-1-Cre (KPC), and Muc4 knockout (KPMC) murine models were used for PanCancer immune profiling. Results: The scRNA-seq analysis suggests that intratumoral T-cells positively correlate with MUC4 expression in PC patients. The activated T-cell CM consisting of predominantly IL-2, IFN-γ, and TNF-α cytokines, significantly induce the expression of MUC4 transcriptionally and translationally in both human (SW1990 and COLO357) and murine (KCT-3248 and KCT3266) PC cell lines. RNA-seq analysis from T-cell CM-treated cancer cells revealed that T-cell secretome induced the pathways related to cancer cell death. The CRISPR knockout of MUC4 in PC cells showed increased expression of cleaved caspase-3 after T-cell CM treatment, indicating that MUC4 plays a protective role against the T-cell CM-mediated killing. Subcutaneous implantation of MUC4 proficient and deficient PC cells on the flanks of C57BL/6 immunocompetent mice demonstrated a significantly higher infiltration of CD3 positive and cytotoxic T-cell infiltration in the MUC4 deficient tumors, resulting in significantly lower tumor weight. The PanCancer immune profiling also showed that depletion of Muc4 increases the T-cells and cytotoxic T-cell signaling scores in the KPMC tumor tissues compared to KPC. Conclusion: T-cell secretome induces MUC4 expression in pancreatic cancer cells, and the increased MUC4 expression reduces T-cell infiltration and protects PC cells against T-cell-mediated killing. Citation Format: Xiaoqi Li, Imran Khan, Rachel Kehrberg, Zahraa Wajih Alsafwani, Rakesh Bhatia, Sushil Kumar, Surinder K. Batra. Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response [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 1594.

Mucin 1 and venous thrombosis in tumor-bearing mice and patients with cancer

IntroductionMucins released from epithelial tumors have been proposed to play a role in cancer-associated thrombosis. Mucin1 (MUC1) is a transmembrane mucin that is overexpressed in a variety of human malignancies, including breast and pancreatic cancer. We analyzed the association of MUC1 and venous thrombosis in a mouse tumor model and in patients with cancer. Materials and methodsWe used a human pancreatic cancer cell line HPAF-II that expresses a high level of MUC1. We grew HPAF-II tumors in the pancreas of Crl:NU-Foxn1nu male mice. MUC1 in plasma and extracellular vesicles (EVs) isolated from plasma was measured using an enzyme-linked immunosorbent assay. MUC1 in EVs and venous thrombi from tumor-bearing mice was assessed by western blotting. We measured MUC1 in plasma from healthy controls and patients with stomach, colorectal or pancreatic cancer with or without venous thromboembolism. Results and discussionMUC1 was detected in the plasma of mice bearing HPAF-II tumors and was associated with EVs. MUC1 was present in venous thrombi from mice bearing HFAP-II tumors. Recombinant MUC1 did not induce platelet aggregation. Levels of MUC1 were higher in patients with pancreatic cancer compared with healthy controls. In contrast to the mouse model, MUC1 was present in EV-free plasma in samples from healthy controls and patients with cancer. There was no significant difference in the levels of MUC1 in cancer patients with or without VTE. Our data did not find any evidence that MUC1 contributed to VTE in patients with cancer.

Gluing GAP to RAS Mutants: A New Approach to an Old Problem in Cancer Drug Development.

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.

NDRG1 overcomes resistance to immunotherapy of pancreatic ductal adenocarcinoma through inhibiting ATG9A-dependent degradation of MHC-1

AimsPancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is resistant to immune checkpoint blockade (ICB) therapies. Emerging evidence suggests that NDRG1 may be an important target for the development of new therapies for PDAC. Herein, we investigated the novel roles of NDRG1 and Combretastatin A-4 (CA-4) in the treatment of PDAC ICB resistance. MethodsEnrichment of MHC class I was detected by RNA sequence and verified by RT-qPCR and immunoblotting in NDRG1-knockdown human pancreatic cancer cell lines. The protein degradation mode was found by stimulation with various inhibitors, and the autophagy degradation pathway was found by immunoprecipitation and immunolocalization. The roles of NDRG1 and MHC-I in immunotherapy were investigated by orthotopic solid tumors, histology, immunohistochemistry, multiplex immunofluorescence staining and flow cytometry. ResultsHere, we identified a previously undescribed role of NDRG1 in activating major histocompatibility complex class 1 (MHC-1) expression in pancreatic ductal adenocarcinoma (PDAC) cells through lysosomal-autophagy-dependent degradation. In mouse models of PDAC, either tumor cell overexpression or pharmacologic activation of NDRG1 leads to MHC-1 upregulation in tumor cells, which in turn promotes the infiltration and activity of CD8 + T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. Moreover, combination therapy of CA-4 and ICB overcomes the drug resistance of pancreatic cancer to ICB therapy. In PDAC patients, NDRG1 expression correlates with high MHC-1 expression and better survival. ConclusionOur results reveal NDRG1 in PDAC cancer cells as a tumor suppressor and suggest that pharmaceutically targeting NDRG1 is a promising way to overcome pancreatic cancer resistance to immunotherapy and provides a potential therapeutic strategy for the treatment of pancreatic cancer patients.

Inferring Drug Set and Identifying the Mechanism of Drugs for PC3.

Drug repurposing is a strategy for discovering new applications of existing drugs for use in various diseases. Despite the use of structured networks in drug research, it is still unclear how drugs interact with one another or with genes. Prostate adenocarcinoma is the second leading cause of cancer mortality in the United States, with an estimated incidence of 288,300 new cases and 34,700 deaths in 2023. In our study, we used integrative information from genes, pathways, and drugs for machine learning methods such as clustering, feature selection, and enrichment pathway analysis. We investigated how drugs affect drugs and how drugs affect genes in human pancreatic cancer cell lines that were derived from bone metastases of grade IV prostate cancer. Finally, we identified significant drug interactions within or between clusters, such as estradiol-rosiglitazone, estradiol-diclofenac, troglitazone-rosiglitazone, celecoxib-rofecoxib, celecoxib-diclofenac, and sodium phenylbutyrate-valproic acid.

Precise Non-invasive Imaging Mouse Model of Pancreatic Cancer: Very Narrow Band-width Laser Fluorescence Excitation of Green Fluorescent Protein Provides Ultra-bright Tumor Images With no Skin Autofluorescence.

Pancreatic cancer is a recalcitrant disease with 5-year survival of only 12%. Improved mouse models of pancreatic cancer are critical for discovery of effective therapeutics. Orthotopic mouse nude-mouse models of pancreatic cancer were established with the human pancreatic-cancer cell line Panc-1 expressing green fluorescent protein (GFP) by transplanting tumor fragments into the pancreas, using the procedure of surgical orthotopic implantation (SOI). Four weeks after establishment of the orthotopic models, the mice were imaged with the Analytik Jena UVP Biospectrum Advanced with a very-narrow-band-width excitation at 487 nm and peak emission at 513 nm. Non-invasive fluorescence imaging of the mice implanted with Panc-1-GFP showed a very bright tumor in the area of the pancreas and peritoneal cavity. The skin background autofluorescence was absent. When a laparotomy was performed on the mouse for open imaging, the tumor on the pancreas was clearly imaged. There was very clear concordance of the non-invasive image and the image obtained during laparotomy. A precise orthotopic mouse model of pancreatic cancer was developed in which there was high concordance between non-invasive and invasive fluorescence imaging due to the ultra-bright signal and ultra-low background using very-narrow-band-width laser fluorescence excitation. This model can be used for high-throughput in vivo screening for improved therapeutics for pancreatic cancer.

Synergistic anti-tumor activity, reduced pERK, and immuno-stimulatory cytokine profiles with 5-FU or ONC212 plus KRAS G12D inhibitor MRTX1133 in CRC and pancreatic cancer cells independent of G12D mutation.

KRAS mutations occur in ~40-50% of mCRC and are associated with aggressive disease that is refractory to anti-EGFR therapies. Pancreatic cancer harbors ~90% KRAS driver gene mutation frequency. Small molecules targeting KRAS G12C gained FDA approval for KRAS G12C-mutated NSCLC. ONC212, a fluorinated imipridone with nM anti-cancer activity has preclinical efficacy against pancreatic cancer and other malignancies. MRTX1133, identified as a noncovalent selective KRAS G12D inhibitor that suppresses G12D signaling by binding to the switch II pocket thereby inhibiting protein-protein interactions. We investigated cell viability, drug synergies, pERK suppression and cytokine, chemokine or growth factor alterations following treatment with 5-Fluorouracil (5-FU) or ONC212 plus MRTX1133 in 6 human CRC and 4 human pancreatic cancer cell lines. IC50 sensitivities ranged from 7 to 12 µM for 5-FU, 0.2-0.8 µM for ONC212, and > 100 nM to > 5,000 nM for MRTX1133 (G12D N = 4: LS513 > 100, HPAF-II > 1,000, SNUC2B > 5,000, PANC-1 > 5,000). For non-G12D, the range of IC50 for MRTX1133 was > 1,000 to > 5,000 nM for CRC lines with G12V, G13D, or WT KRAS (N = 7). Synergies between MRTX1133 plus 5-FU or ONC212 were observed regardless of KRAS G12D mutation with combination indices of < 0.5 indicating strong synergy. Observed synergies were greater with MRTX1133 plus ONC212 compared to MRTX1133 plus 5-FU. pERK was suppressed with mutant but not wild-type KRAS at nM MRTX1133 doses. Immunostimulatory profiles included reduction in IL8/CXCL8, MICA, Angiopoietin 2, VEGF and TNF-alpha and increase in IL-18/IL-1F4 with MRTX treatments and combinations. Our studies reveal preclinical activity of MRTX1133 alone or synergies when combined with 5-FU or ONC212 against mCRC and pancreatic cancer cells regardless of KRAS G12D mutation. The results suggest that KRAS G12V and KRAS G13D should be further considered in clinical trials including combination therapies involving MRTX1133 and 5-FU or ONC212.

Characterization and Evaluation of Nano-niosomes Encapsulating Docetaxel against Human Breast, Pancreatic, and Pulmonary Adenocarcinoma Cancer Cell Lines.

Docetaxel (DXL) is an antineoplastic agent for cancer treatment, the therapeutic efficiency of which is limited due to low solubility, hydrophobicity, and tissue specificity. In this study, nano-niosomes were introduced for improving therapeutic index of DXL. In this experimental study, two nano-niosomes were synthesized using Span 20® and Span 80® and a thin film hydration method with DXL loading (DXL-Span20 and DXL-Span80). Characterization, in-vitro cytotoxicity and bioavailability of the nano-niosomes was also evaluated via in-vivo experiments. DXL-Span20 and DXL-Span80 have vesicles size in a range of 84-90 nm and negative zeta potentials. DXL entrapment efficiencies were obtained as 69.6 and 74.0% for DXL-Span20 and DXL-Span80, respectively; with an in-vitro sustained release patterns. Cytotoxicity assays were performed against MDA-MB-231, Calu-6, and AsPC-1 cell lines, and the results indicated that DXL loading into nano-niosomes led to decrement in values of half-maximal inhibitory concentration (IC50) at least 2.5 times and at most 6.5 times, compared to free DXL. Moreover, the rat blood bioavailability of DXL after intraperitoneal administration and the pharmacokinetic parameters indicated higher DXL plasma level and the higher effectiveness of DXL-Span80 compared to DXL-Span20. Carrying DXL by the nano-niosomes led to enhanced cytotoxicity (and lower IC50 values) and higher efficacy with enhanced pharmacokinetic parameters.

DT-13 inhibits the proliferation of pancreatic cancer by inducing apoptosis via AMPK-mTOR signaling

Background/ObjectiveDT-13, the principal active component of Mysidium shortscapes from the Liliaceae family, has garnered substantial interest in cancer therapy owing to its potential anticancer properties. This study investigated the effects of DT-13 on the proliferation and apoptosis of human pancreatic cancer cell lines and aimed to elucidate the underlying mechanisms. MethodsPANC1 and CFPAC1 cells were exposed to DT-13 and their proliferation was assessed using RTCA and clone formation assays. Apoptotic protein expression was analyzed by western blotting, and apoptotic cells were identified by flow cytometry. RNA was extracted from DT-13 treated and untreated PANC1 cells for RNA sequencing. Differentially expressed genes were identified and subjected to GO bioprocess, KEGG pathway analysis, and western blotting. Finally, to evaluate tumor growth, CFPAC1 cells were subcutaneously injected into BALB/c nude mice. ResultsDT-13 inhibited proliferation and induced apoptosis of PANC1 and CFPAC1 cells by activating the AMPK/mTOR pathway and suppressing p70 S6K. Moreover, DT-13 hindered the growth of CFPAC1 xenograft tumors in nude mice. ConclusionsDT-13 effectively inhibited the growth of human pancreatic cancer cells.

Phytochemicals in Pancreatic Cancer Treatment: A Machine Learning Study.

The discovery of new strategies and novel therapeutic agents is crucial to improving the current treatment methods and increasing the efficacy of cancer therapy. Phytochemicals, naturally occurring bioactive constituents derived from plants, have great potential in preventing and treating various diseases, including cancer. This study reviewed 74 literature studies published between 2006 and 2022 that conducted in vitro cytotoxicity and cell apoptosis analyses of the different concentrations of phytochemicals and their combinations with conventional drugs or supplementary phytochemicals on human pancreatic cell lines. From 34 plant-derived phytochemicals on 20 human pancreatic cancer cell lines, a total of 11 input and 2 output variables have been used to construct the data set that contained 2161 different instances. The machine learning approach has been implemented using random forest for regression, whereas association rule mining has been used to determine the effects of individual phytochemicals. The random forest models developed are generally good, indicating that the phytochemical type, its concentration, and the type of cell line are the most important descriptors for predicting the cell viability. However, for predicting cell apoptosis the primary phytochemical type is the most significant descriptor . Among the studied phytochemicals, catechin and indole-3-carbinol were found to be non-cytotoxic at all concentrations irrespective of the treatment time. On the other hand, berbamine and resveratrol were strongly cytotoxic with cell viabilities of less than 40% at a concentration range between 10 and 100 μM and above 100 μM, respectively, which brings them forward as potential therapeutic agents in the treatment of pancreatic cancer.

Abstract C034: First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors

Abstract Background: ATRN-119 is an oral and highly selective nanomolar inhibitor of Ataxia Telangiectasia and Rad3- related (ATR) kinase, an enzyme that is activated by replication stress and DNA damage and prevents the collapse of DNA replication forks into double strand breaks. ATRN-119 is being studied in advanced solid tumors with dysregulated components of the DNA damage response (DDR) based on the hypothesis that these tumors will be more susceptible to ATR inhibition. Single agent ATRN-119 demonstrates increased cytotoxicity in vitro against a broad spectrum of human colon, breast, pancreatic, sarcoma, ovarian and prostate cancer cell lines that harbor genomic alterations of DDR genes. In vivo, ATRN-119 has demonstrated tumor growth inhibition in human castration-resistant prostate cancer and human colon cancer cell line-derived xenografts (CDX) mice models, and in BRCA mutant high grade serous ovarian cancer patient derived xenografts (PDXs). Trial design: This is a first-in-human Phase 1/2a open-label study (NCT04905914) to evaluate safety, tolerability, pharmacokinetics (PK), and preliminary e!cacy of oral daily ATRN-119 in subjects with advanced solid tumors, conducted in 2 parts. Part 1 consists of a 3+3 dose escalation design at up to 6 dose levels to evaluate the tolerability, PK, dose limiting toxicity (DLT) and maximum tolerated dose (MTD) of repeated daily doses of ATRN-119. Enrollment in Part 1 is ongoing. Subjects must be ≥12 years old with advanced solid tumor, have documented DDR mutations, or advanced Merkel cell carcinoma, must have failed at least one prior standard treatment, have ECOG performance status of 0 or 1, with a life expectancy &amp;gt; 3 months. Primary objective is safety/tolerability and secondary objective is efficacy. After determination of the MTD or maximum feasible dose (MFD) in Part 1, Part 2 will enroll up to 30 additional subjects. The trial is currently recruiting across 4 locations in the United States. Clinical trial identification: ClinicalTrials.gov Identifier: NCT04905914 Citation Format: Fiona Simpkins, Reva Schneider, Amit Mahipal, Patricia LoRusso, Crystal Miller, Eric Brown, Mike Carleton, Joachim Gullbo, Nadeem Q Mirza. First in Human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C034.

Abstract C167: An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition

Abstract Background: Irinotecan is a topoisomerase 1 inhibitor pro-drug used to treat gastrointestinal (GI) cancers including as first line for colorectal cancer (CRC) as part of the FOLFIRI plus monoclonal antibody (mAb) regimen, and for pancreatic cancer in the FOLFIRINOX regimen. However, irinotecan causes severe and dose-limiting side effects, including diarrhea and neutropenia. Starpharma has developed a highly optimized polylysine dendrimer enhanced (DEP) nanoparticle conjugate of the irinotecan active metabolite, SN38 (DEP SN38 or DEP irinotecan). DEP SN38 avoids the need for liver conversion of irinotecan to SN38 and achieves preferential tumor targeting. DEP SN38 is in phase 2 clinical investigation as monotherapy or combination therapy in patients with solid tumors (EudraCT no: 2019-001318-40). SN38 is known to enhance the efficacy of immune checkpoint blockade (ICB) via effects on immune cells in the tumor microenvironment. Here, we evaluated the anti-tumor effects of DEP SN38 alone or in combination with either ICB (anti- programmed cell death-1 [PD1] mAb) or poly adenosine diphosphate (ADP) ribose polymerase (PARP) inhibition (olaparib) in mouse models of GI cancer. Methods: HT29 (human CRC) and CAPAN-1 (human pancreatic cancer) cell line xenografts were established subcutaneously in BALB/c nude or NOD-scid-Gamma immunodeficient mice, respectively. Three doses of DEP SN38 (at 5-10mg/kg SN38) or irinotecan (35-80 mg/kg) were administered intravenously (IV) on days 1, 8, 15. Olaparib (50 mg/kg) was delivered by daily IV injection for 5 days/week over 3 weeks. DEP SN38 (IV on days 1, 8, 15 at 18-35 mg/kg SN38) or irinotecan (75 mg/kg) plus rat anti-PD1 mAb therapy or control mAb (intraperitoneally, 100 mg/kg then 50 mg/kg on days 5, 8, 12) was evaluated in both MC38 (C57BL/6) and CT26 (BALB/c) allograft models of CRC. Toleration of drug was bodyweight loss ≤15%. Results: DEP SN38 treatment of HT29 or CAPAN-1 xenografts led to tumor regressions, prolonged control of tumor growth and improved survival. In contrast, conventional irinotecan had minimal effect on tumor growth versus vehicle control. DEP SN38 was well tolerated by mice with minimal bodyweight loss. Administration of anti-PD1 mAb alone confirmed the known modest sensitivity of MC38, and resistance of CT26 syngeneic tumors, to ICB. DEP SN38 plus anti-PD1 mAb, enhanced anti-tumor responses in the MC38 model beyond those seen for each agent alone and also resulted in tumor eradication and prolonged survival. In the CT26 model, anti-PD1 mAb was also augmented by addition of DEP SN38 but not irinotecan alone, despite resistance of CT26 tumors to anti-PD1 mAb alone. The HT29 CRC model was resistant to Olaparib alone. In contrast, anti-tumor effects and prolonged survival were observed with olaparib plus DEP SN38. Conclusions: DEP SN38 demonstrated superior efficacy compared to irinotecan when used in models of GI cancer. The anti-tumor efficacy of both ICB and PARP inhibition was augmented by DEP SN38 in models of CRC, and these combinations should be investigated clinically. Citation Format: Benjamin J Blyth, Brian D Kelly, Michael Giannis, Anne Cargill, Aynaz Seta, Graham P Heery, Anthony Eglezos, Cameron N Johnstone, Jeremy R A Paull. An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C167.

Wild Chaga (Inonotus obliquus) modulates inflammation, neural cell survival and inhibits proliferation of cancer cells

Background and AimInonotus obliquus (Ach. Ex. Pers), commonly known as Chaga, is a parasitic fungus that grows on birch trees. Indigenous North Americans and Northern Eurasians have used Chaga tea as a folk medicine for centuries, and there are currently significant interests in commercializing Chaga products, obtained either from conks (sclerotia) collected from nature or from laboratory cultured mycelia. The cultured mycelia's identity to date is based solely on chemical profiling and the morphologic characteristics; and genome identity are often overlooked and unreported in the literature. The aims of this study include: 1. To authenticate mycelium isolates derived from a Chaga sclerotium; 2. To determine Chaga sclerotium's bioactivities unreported previously. Experimental proceduresIdentification of Chaga conk was carried out by chemical analyses, culture characteristics, and genome identity. The Chaga extracts’ bioactivities were evaluated via effects on cytokine and reactive oxygen species production on normal and cancer cell survival and proliferation. Results and ConclusionThe features of this mycelium isolate conformed to I. obliquus mycology, with > 99 % DNA sequence identity to the known GenBank Chaga Ribosomal Internal Spacer (ITS) sequence. Water extracts of Chaga were effective in protecting against oxidative stress in mouse neuroblastoma-spinal motor neuron NSC-34 cells and inhibited the proliferation of human brain glioblastoma DBTRG-05MG and pancreatic ductal adenocarcinoma PANC-1 cancer cell lines in vitro. In addition, the water extract potently stimulated inflammatory cytokines release from human blood cells, consistent with the biological response modifier features of mushroom beta- glucan polysaccharides.

Tetrahydrobenzothiophene derivatives ameliorate Mia PaCa-2 cell progression and induces apoptosis via inhibiting EGFR2 tyrosine kinase signal

A series of new thiophene analogues with acarbonitrile-basedmoiety were designed and synthesized via structural optimization. The conjugates were assessed for their in-vitro cytotoxic activity against a human pancreatic cancer cell line (Mia PaCa-2) and among them compound 5b showed IC50 value of 13.37 ± 2.37 μM. The compounds 5b (20 µM & 25 µM) and 7c (30 & 35 µM) also showed reduced clonogenicity, enhanced ROS and decreased mitochondrial membrane potential in Mia PaCa-2 cells. Treatment with these compounds also increased apoptotic population as evident with the double staining assay. Among the evaluated series, compounds 5b, 5g, 7c, and 9a attained a greater inhibitory potency than first generation’s reversible EGFR inhibitor, Gefitinib. EGFR2 enzyme inhibitory studies revealed that 5b efficiently and arbitrarily suppressed the development of EGFR2 dependent cells and inhibited the enzymatic activity with an IC50 value of 0.68 µM; interestingly, the most effective molecule 5b with N-methyl piperazine substitution, has 1.29-fold greater potency than well-known EGFR inhibitor Gefitinib and increased Gefitinib's anti-growth impact with 2.04 folds greater against Mia PaCa-2. The in-vitro studies were validated with in-silico docking studies wherein compounds 5b and 7c exhibited binding energies of −8.2 and −7.4 Kcal/mol respectively. The present study reveals that tetrahydrobenzothiophene based analogues could be a promising lead for the evolution of potent chemo preventives over pancreatic cancer.

Biodegradable nanocarrier of gemcitabine and tocopherol succinate synergistically ameliorates anti-proliferative response in MIA PaCa-2 cells

Gemcitabine (GEM) is an important chemotherapeutic agent used alone or in combination with other anticancer agents for the treatment of various solid tumors. In this study, the potential of a dietary supplement, α-tocopherol succinate (TOS) was investigated in combination with GEM by utilizing human serum albumin-based nanoparticles (HSA NPs). The developed nanoparticles were characterized using DLS, SEM and FTIR and evaluated in a panel of cell lines to inspect cytotoxic efficacy. The ratio metric selected combination of the NPs was further investigated in human pancreatic cancer cell line (MIA PaCa-2 cells) to assess the cellular death mechanism via a myriad of biochemical and bio-analytical assays including nuclear morphometric analysis by DAPI staining, ROS generation, MMP loss, intracellular calcium release, in vitro clonogenic assay, cell migration assay, cell cycle analysis, immunocytochemical staining followed by western blotting, Annexin V-FITC and cellular uptake studies. The desolvation-crosslinking method was used to prepare the NPs. The average size of TOS-HSA NPs and GEM-HSA NPs was found to be 189.47 ± 5 nm and 143.42 ± 7.4 nm, respectively. In combination, the developed nanoparticles exhibited synergism by enhancing cytotoxicity in a fixed molar ratio. The selected combination also significantly triggered ROS generation and mitochondrial destabilization, alleviated cell migration potential and clonogenic cell survival in MIA PaCa-2 cells. Further, cell cycle analysis, Annexin-V FITC assay and caspase-3 activation, up regulation of Bax and down regulation of Bcl-2 protein confirmed the occurrence of apoptotic event coupled with the G0/G1 phase arrest. Nanocarriers based this combination also offered approximately 14-folds dose reduction of GEM. Overall, the combined administration of TOS-HSA NPs and GEM-HSA NPs showed synergistic cytotoxicity accompanied with dose reduction of the gemcitabine. These encouraging findings could have implication in designing micronutrient based-combination therapy with gemcitabine and demands further investigation.

Successful In Situ Targeting of Pancreatic Tumors in a Novel Orthotopic Porcine Model Using Histotripsy.

New therapeutic strategies and paradigms are direly needed to treat pancreatic cancer. The absence of a suitable pre-clinical animal model of pancreatic cancer is a major limitation to biomedical device and therapeutic development. Traditionally, pigs have proven to be ideal models, especially in the context of designing human-sized instruments, perfecting surgical techniques and optimizing clinical procedures for use in humans. However, pig studies have typically focused on healthy tissue assessments and are limited to general safety evaluations because of the inability to effectively model human tumors. Here, we establish an orthotopic porcine model of human pancreatic cancer using RAG2/IL2RG double-knockout immunocompromised pigs and treat the tumors ex vivo and in vivo with histotripsy. Using these animals, we describe the successful engraftment of Panc-1 human pancreatic cancer cell line tumors and characterize their development. To illustrate the utility of these animals for therapeutic development, we determine for the first time, the successful targeting of in situ pancreatic tumors using histotripsy. Treatment with histotripsy resulted in partial ablation in vivo and reduction in collagen content in both in vivo tumor in pig pancreas and ex vivo patient tumor. This study presents a first step toward establishing histotripsy as a non-invasive treatment method for pancreatic cancer and exposes some of the challenges of ultrasound guidance for histotripsy ablation in the pancreas. Simultaneously, we introduce a highly robust model of pancreatic cancer in a large mammal model that could be used to evaluate a variety biomedical devices and therapeutic strategies.

Conditioned media of tonsil derived mesenchymal stem cells shows different rates of cytotoxicity on solid cancer cells

Aims: Mesenchymal stem cells (MSCs) are the apple of the eye of cancer studies. It was indicated that the secreted factors, especially released by MSCs, have tumoral or anti-tumoral effects on tumor progression. MSCs obtained from different sources show different anti-tumoral effects, while MSCs originating from the same source also show different tumoral effects in different cancer cells. Here, we investigated the anti-tumor effects of soluble factors secreted from palatine tonsil MSCs (TMSC) as a new source of MSC on human lung carcinoma (A549) and pancreatic cancer (PANC-1) cell lines.&#x0D; Methods: Conditioned medium (CM) was obtained from TMSCs isolated from palatine tonsil tissue, and the cytotoxic effect of CM on the growth of A549 and PANC-1 in a dose-dependent manner was demonstrated by MTT analysis. In addition, the function of CM treatment on the cell cycle status of cancer cells and the apoptosis process were investigated through cell cycle analysis with propidium iodide (PI) and Annexin-V/PI detection method by flow cytometry analysis, respectively. &#x0D; Results: We demonstrated that TMSC-CM treatment significantly decreased the viability of A549 and PANC-1 cell lines in a dose-dependent manner post-48 hours. In addition, CM treatment differentially induced the apoptosis on A549 and PANC-1 cells and also, caused G2/M arrest in the cells.&#x0D; Conclusion: In light of these findings, our study is the first to show that TMSC-CM has an anti-tumoral effect by stimulating apoptosis on A549 and PANC-1 cells. These findings reveal that the usage of CM has a cell-free cellular therapeutic potential.

Cell surface GRP78-directed CAR-T cells are effective at treating human pancreatic cancer in preclinical models

Pancreatic cancer is a highly lethal solid malignancy with limited treatment options. Chimeric antigen receptor T (CAR-T) cell therapy has been successfully applied to treat hematological malignancies, but faces many challenges in solid tumors. One major challenge is the shortage of tumor-selective targets. Cell surface GRP78 (csGRP78) is highly expressed on various solid cancer cells including pancreatic cancer, but not normal cells, providing a potential target for CAR-T cell therapy in pancreatic cancer. Here, we demonstrated that csGRP78-directed CAR-T (GRP78-CAR-T) cells effectively killed the human pancreatic cancer cell lines Bxpc-3-luc, Aspc-1-luc and MIA PaCa-2-luc, and pancreatic cancer stem-like cells derived from Aspc-1-luc cells and MIA PaCa-2-luc cells in vitro by a luciferase-based cytotoxicity assay. Importantly, we showed that GRP78-CAR-T cells efficiently homed to and infiltrated Aspc-1-luc cell-derived xenografts and significantly inhibited pancreatic tumor growth in vivo by performing mouse xenograft experiments. Interestingly, we found that gemcitabine treatment increased csGRP78 expression in gemcitabine-resistant MIA PaCa-2-luc cells, and the coapplication of gemcitabine with GRP78-CAR-T cells led to a robust cytotoxic effect on these cells in vitro. Taken together, our study demonstrates that csGRP78-directed CAR-T cells, alone or in combination with chemotherapy, selectively and efficiently target csGRP78-expressing pancreatic cancer cells to suppress pancreatic tumor growth.

A novel triptolide analog downregulates NF-κB and induces mitochondrial apoptosis pathways in human pancreatic cancer.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5 -year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, (E)-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and used human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo. Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 hr and ~8,000 DEGs at 12 hr. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. Thus, CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.

Unveiling the complexation mechanism of phloretin with Sulfobutylether–β–cyclodextrin (Captisol®) and its impact on anticancer activity

The incorporation of phloretin (PHL), a dihydrochalcone flavonoid, in functional foods is usually hampered by its low aqueous solubility. In this regard, we investigated the impact of complexation of PHL with sulfobutylether-β-cyclodextrin (SBE-β-CD, Captisol®) to overcome its limitation. The phase solubility studies of PHL with different β-CDs derivatives represents AL-type of curve where, SBE-β-CD demonstrated highest apparent stability constant (Ks: 15,856 M−1) indicating its strong affinity to form host-guest assembly. Solid-state characterizations (including SEM, FT-IR, PXRD, DSC, TGA) provided direct evidences of inclusion complex (IC) formation. The detailed molecular interaction between the host–guest assembly was performed using 1H NMR and 2D-NOESY revealed that protons of aromatic phenyl ring of PHL (guest) molecule exhibit direct correlation with the protons lying in the inner cavity of SBE-β-CD (host). Furthermore, the anticancer potential of PHL/SBE-β-CD-IC was examined using cytotoxicity studies, caspase 3/7 activation assay, reactive oxygen species (ROS) generation and disturbance in mitochondrial membrane potential (MMP) in lung carcinoma cell line (A549) and human pancreatic cancer cell line (MiaPaCa–2). The in-vitro cytotoxicity assay on both cell lines demonstrated higher antiproliferative activity of IC as compared to free PHL in concentration dependent manner. In addition, it was found that PHL/SBE-β-CD-IC induces apoptosis via activation of caspase 3/7, reactive oxygen species generation and inducing depolarization of mitochondrial membrane potential in both the cell lines (MiaPaCa–2 and A549). Overall, the study provides a promising approach to enhance the solubility and bioactivity of PHL utilizing SBE-β-CD inclusion complexation.