Pancreatic Adenocarcinoma Cells Research Articles

CT109-SN-38, a Novel Antibody-drug Conjugate with Dual Specificity for CEACAM5 and 6, Elicits Potent Killing of Pancreatic Cancer Cells.

CEACAM5 and CEACAM6 are glycosylphosphatidylinositol (GPI)- linked members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which are frequently upregulated in epithelial cancers where they contribute to invasion, metastasis, immune evasion, and resistance to anoikis. CT109 is a novel antibody with dual specificity to both CEACAM5 and 6. In this study, we aimed to perform the preclinical characterization of CT109 and antibody- drug conjugate (ADCs) derivatives of CT109, focusing on CT109-SN-38. CT109's cognate epitope was characterized by scanning mutagenesis. CT109 specificity and internalization kinetics were assessed by immunoblot and flow cytometry, respectively. Cognate antigen expression prevalence in colorectal cancer and normal tissue arrays was determined by immunohistochemistry. CT109 conjugations were generated by the reaction of reduced CT109 cysteines with maleimide-functionalized payload linkers. In vitro cytotoxic activity of CT109 ADCs was characterized on antigen-positive and negative pancreatic ductal adenocarcinoma cell (PDAC) lines using a luminometric viability assay. In vivo efficacy of CT109-SN-38 was assessed on a PDAC tumor xenograft model at 10 and 25 mg/kg concentrations. CT109 was shown to bind a glycoepitope centered on N309. CT109 is internalized in the CEACAM5+/CEACAM6+ double-positive PDAC line, BxPC-3, with a t1/2 of 2.3 hours. CT109 ADCs elicit a dose and antigen-dependent cytotoxic effect, with CT109-SN-38 exhibiting an IC50 value of 21 nM in BxPC-3 cells. In a BxPC-3 tumor xenograft model, CT109-SN-38 reduced tumor growth and induced regression in 3/10 mice at a concentration 25 mg/kg. These data suggest that further preclinical and clinical development of CT109-SN-38 is warranted.

LncRNA HULC augments high glucose-associated pancreatic cancer progression and drug resistance by enhancing YAP activity and autophagy.

One of the confounding factors in pancreatic cancer (PC) pathogenesis is hyperglycemia. The molecular mechanism by which high glucose (HG) influences PC severity is poorly understood. Our investigation delved into the impact of lncRNA highly upregulated in liver cancer (HULC) and its interaction with yes-associated protein (YAP) in regulating the fate of pancreatic ductal adenocarcinoma cells (PDAC) under HG-induced conditions. PDAC cells were cultured under normal or HG conditions. We thereafter measured the effect of HG on the viability of PDAC cells, their migration potential and drug resistance properties. The lncRNAs putatively dysregulated in PC and diabetes were shortlisted by bioinformatics analysis followed by wet lab validation of function. HG led to enhanced proliferation and drug refractoriness in PDAC cells. HULC was identified as one of the major deregulated lncRNAs following bioinformatics analysis. HULC was found to regulate the expression of the potent transcriptional regulator - YAP through selective histone modifications at the YAP promoter. siRNA-mediated ablation of HULC resulted in a concurrent decrease in YAP transcriptional activity. Importantly, HULC and YAP were found to co-operatively regulate the cellular homeostatic process autophagy, thus inculcating drug resistance and proliferative potential in PDAC cells. Moreover, inhibition of autophagy or YAP led to a decrease in HULC levels, suggesting the existence of an inter-regulatory feedback loop. We observed that HG triggers aggressive properties in PDAC cells. Mechanistically, up-regulation of lncRNA HULC resulted in activation of YAP and differential regulation of autophagy coupled to increased proliferation of PDAC cells. Inhibition of HULC and YAP may represent a novel therapeutic strategy for PDAC. Furthermore, this study portrays the intricate molecular interplay between HULC, YAP and autophagy in PDAC pathogenesis.

TRNA-derived RNA fragment, tRF-18-8R6546D2, promotes pancreatic adenocarcinoma progression by directly targeting ASCL2

Pancreatic adenocarcinoma (PAAD) is a life-threatening cancer. Exploring new diagnosis and treatment targets helps improve its prognosis. tRNA-derived small non-coding RNAs (tsRNAs) are a novel type of gene expression regulators and their dysregulation is closely related to many human cancers. Yet the expression and functions of tsRNAs in PAAD are not well understood. Our study used RNA sequencing to identify tsRNA expression profiles in PAAD cells cultured in no or high glucose media and found tRF-18-8R6546D2 was an uncharacterized tsRNA, which has significantly high expression in PAAD cells and tissues. Clinically, tRF-18-8R6546D2 is linked to poor prognosis in PAAD patients and can be used to distinguish them from healthy populations. Functionally, in vitro and vivo, tRF-18-8R6546D2 over-expression promoted PAAD cell proliferation, migration and invasion, inhibited apoptosis, whereas tRF-18-8R6546D2 knock-down showed opposite effects. Mechanistically, tRF-18-8R6546D2 promoted PAAD malignancy partly by directly silencing ASCL2 and further regulating its downstream genes such as MYC and CASP3. These findings show that tRF-18-8R6546D2 is a novel oncogenic factor and can be a promising diagnostic or prognostic biomarker and therapeutic target for PAAD.

Elucidation of the Gemcitabine Transporters of Escherichia coli K-12 and Gamma-Proteobacteria Linked to Gemcitabine-Related Chemoresistance.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine), a widely used anticancer drug, is considered a gold standard in treating aggressive pancreatic cancers. Gamma-proteobacteria that colonize the pancreatic tumors contribute to chemoresistance against gemcitabine by metabolizing the drug to a less active and deaminated form. The gemcitabine transporters of these bacteria are unknown to date. Furthermore, there is no complete knowledge of the gemcitabine transporters in Escherichia coli or any other related proteobacteria. In this study, we investigate the complement of gemcitabine transporters in E. coli K-12 and two common chemoresistance-related bacteria (Klebsiella pneumoniae and Citrobacter freundii). We found that E. coli K-12 has two high-affinity gemcitabine transporters with distinct specificity properties, namely, NupC and NupG, whereas the gemcitabine transporters of C. freundii and K. pneumoniae include the NupC and NupG orthologs, functionally indistinguishable from their counterparts, and, in K. pneumoniae, one additional NupC variant, designated KpNupC2. All these bacterial transporters have a higher affinity for gemcitabine than their human counterparts. The highest affinity (KM 2.5-3.0 μΜ) is exhibited by NupGs of the bacteria-specific nucleoside-H+ symporter (NHS) family followed by NupCs (KM 10-13 μΜ) of the concentrative nucleoside transporter (CNT) family, 15-100 times higher than the affinities reported for the human gemcitabine transporter hENT1/SLC29A1, which is primarily associated with gemcitabine uptake in the pancreatic adenocarcinoma cells. Our results offer a basis for further insight into the role of specific bacteria in drug availability within tumors and for understanding the structure-function differences of bacterial and human drug transporters.

A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells.

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

RECK/GPR124-driven WNT signaling in pancreatic and gastric cancer cells.

RECK has been described to modulate extracellular matrix components through negative regulation of MMP activities. Recently, RECK was demonstrated to bind to an orphan G protein-coupled receptor GPR124 to mediate WNT7 signaling in nontumor contexts. Here, we attempted to clarify the role of RECK in driving WNT signaling in cancer cells. RECK and GPR124 formed a complex in 293T cells, and when both were expressed, WNT signaling was significantly enhanced in a WNT7-dependent manner. This cooperation was abolished when RECK mutants unable to bind to GPR124 were transduced. RECK stimulated the growth of KRAS-mutated pancreatic ductal adenocarcinoma (PDAC) cells with increased sensitivity to WNT inhibitor in a GPR124-dependent manner. A gastric cancer cell line SH10TC endogenously expresses both RECK and GPR124 under regular culture conditions. In this cell line, inhibited cell growth and WNT signaling as well as increased apoptosis in the GPR124 depletion was dominantly found over those in the RECK deletion. These findings suggest that RECK promotes tumor cell growth by positively modulating WNT signaling through GPR124. This study proposes that the RECK/GPR124 complex might be a good therapeutic target in PDAC and gastric cancer.

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.

14-3-3σ downregulation sensitizes pancreatic cancer to carbon ions by suppressing the homologous recombination repair pathway.

This study explored the role of 14-3-3σ in carbon ion-irradiated pancreatic adenocarcinoma (PAAD) cells and xenografts and clarified the underlying mechanism. The clinical significance of 14-3-3σ in patients with PAAD was explored using publicly available databases. 14-3-3σ was silenced or overexpressed and combined with carbon ions to measure cell proliferation, cell cycle, and DNA damage repair. Immunoblotting and immunofluorescence (IF) assays were used to determine the underlying mechanisms of 14-3-3σ toward carbon ion radioresistance. We used the BALB/c mice to evaluate the biological behavior of 14-3-3σ in combination with carbon ions. Bioinformatic analysis revealed that PAAD expressed higher 14-3-3σ than normal pancreatic tissues; its overexpression was related to invasive clinicopathological features and a worse prognosis. Knockdown or overexpression of 14-3-3σ demonstrated that 14-3-3σ promoted the survival of PAAD cells after carbon ion irradiation. And 14-3-3σ was upregulated in PAAD cells during DNA damage (carbon ion irradiation, DNA damaging agent) and promotes cell recovery. We found that 14-3-3σ resulted in carbon ion radioresistance by promoting RPA2 and RAD51 accumulation in the nucleus in PAAD cells, thereby increasing homologous recombination repair (HRR) efficiency. Blocking the HR pathway consistently reduced 14-3-3σ overexpression-induced carbon ion radioresistance in PAAD cells. The enhanced radiosensitivity of 14-3-3σ depletion on carbon ion irradiation was also demonstrated in vivo. Altogether, 14-3-3σ functions in tumor progression and can be a potential target for developing biomarkers and treatment strategies for PAAD along with incorporating carbon ion irradiation.

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.

Ciprofloxacin is a novel anti-ferroptotic antibiotic

Cancer patients undergoing chemotherapy are susceptible to various bacterial infections, necessitating prompt and precise antimicrobial treatment with antibiotics. Ciprofloxacin is a clinically utilized broad-spectrum antimicrobial agent known for its robust antiseptic activity. While ferroptosis, an oxidative form of cell death, has garnered attention as a promising avenue in cancer therapy, the potential impact of ciprofloxacin on the anticancer effects of ferroptosis remains unclear. This study seeks to investigate the potential influence of antibiotics on ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. Here, we report a previously unrecognized role of ciprofloxacin in inhibiting ferroptosis in human PDAC cells. Mechanistically, ciprofloxacin suppresses erastin-induced endoplasmic reticulum (ER) stress through the activating transcription factor 6 (ATF6) and ER to nucleus signaling 1 (ERN1) pathway. Excessive ER stress activation can trigger glutathione peroxidase 4 (GPX4) degradation through autophagic mechanisms. In contrast, ciprofloxacin enhances the protein stability of GPX4, a crucial regulator that suppresses ferroptosis by inhibiting lipid peroxidation. Thus, our study demonstrates the anti-ferroptotic role of ciprofloxacin, highlighting the importance of careful consideration when contemplating the combination of ciprofloxacin with specific ferroptosis inducers in PDAC patients.

A phase Ia/Ib, non-randomized, open-label, dose escalation and expansion trial of the B7-H6/CD3 T-cell engager BI 765049 with or without ezabenlimab in Asian patients with advanced solid tumors.

TPS2669 Background: Bispecific T-cell engagers represent a promising therapeutic approach for patients with solid tumors; however, these agents require an appropriate target antigen. B7-H6 is an encouraging target as it is expressed on multiple solid tumors but shows only limited expression on normal tissue (1). BI 765049 is a novel immunoglobulin G (IgG)-like T-cell engager designed to simultaneously bind B7-H6-expressing tumor cells and CD3 on T-cells resulting in the formation of a cytolytic synapse that triggers apoptosis of the tumor cells. Methods: NCT06091930 is a Phase I, non-randomized, open-label, multi-center, dose escalation and expansion trial that aims to assess the safety and tolerability of BI 765049 alone or in combination with programmed cell death protein 1 inhibitor ezabenlimab, in patients with advanced or unresectable gastric cancer, colorectal cancer (CRC), pancreatic ductal adenocarcinoma, hepatocellular cancer, head and neck squamous cell carcinoma, or non-small cell lung cancer. Patients must have progressed on, or be ineligible for, standard therapies. Key inclusion criteria include confirmed B7-H6 expression (central pathology review) except in CRC; ≥1 evaluable target lesion outside of the central nervous system (Response Evaluation Criteria in Solid Tumors [RECIST] v1.1); and Eastern Cooperative Oncology Group performance status 0/1. Patients with prior B7-H6-targeted treatment are ineligible. The study is divided into four parts: dose escalation and expansion of BI 765049 monotherapy (Parts I and II, respectively), and in combination with ezabenlimab (Parts III and IV, respectively). BI 765049 dose escalation will be guided by a Bayesian logistic regression model with overdose control. Treatment will continue until progressive disease or discontinuation for other reasons, or for up to 36 months. The primary endpoint for Parts I and III is the occurrence of dose-limiting toxicities; and for Parts II and IV it is objective response (OR; determined by the investigator; RECIST v1.1). The secondary endpoints for Parts I and III are pharmacokinetic (PK) parameters and OR (RECIST v1.1); and for Parts II and IV secondary endpoints are progression-free survival, duration of response, disease control, PK measurements, and OR (immunotherapy RECIST [iRECIST]). 1. Zhang W, et al. Clin Cancer Res 2022;28(23):5190–5201. Clinical trial information: NCT06091930 .

A novel MRI-based tumor-targeting theragnostic agent: Magnetoelectric nanoparticles in an in vivo murine KRAS/P53 pancreatic flank tumor model.

3075 Background: Magnetoelectric nanoparticles (MENPs) are a novel material with a magnetoelectric (ME) property that allows for conversion of an external magnetic field to a local electric field (EF) that can be exploited to generate a Coulomb force to target and electroporate the relatively non-polarized tumor cell walls. We hypothesized that their local EF and superparamagnetic cores can be exploited as an MRI-based anti-cancer theragnostic agent for solid tumors. Methods: 35 immunocompetent C57BL/6 mice were inoculated with KPC961 pancreatic adenocarcinoma cells in their flank. The 27 mice with the largest tumors at 4 weeks were randomized into 5 cohorts based on treatment: 300 µg of MENP (300; n=6), 600 µg of MENP (600; n=6), 300 µg of low-ME coefficient MENP (LM; n=5), 300 µg of MENP without M1 MRI (NM; MRI field control, n=5), and normal saline (NS; MENP causal effect control; n=5). The 300, 600, LM, and NS cohorts had T2 MRI maps (M0) on a 7T MRI. NM had T2 weighted MRIs at M0. All mice had a neodymium magnet placed over their tumors for 12 hours after tail vein injections (300 µl for all mice) for targeting. The 300, 600, LM, and NS cohorts then underwent MRI mapping (M1) after magnet removal (No M1 MRI for NM). MRI mapping was repeated 5 days after (M2). NM had a T2 weighted MRIs at M2. M1 and M2 data were used to assess relaxation time (contrast effect) and tumor volume from M0, respectively. Student t and ANOVA tests were used to compare differences for tumor volume and relaxation time data and one-sided Fisher’s exact test was used for complete response (CR) data. Results: The median M0 tumor volume was 109.6 mm3 for all mice, and there were no differences between any cohort ( p = 0.978; Table). The 300 and 600 MENP cohorts demonstrated a 102.7% reduction in volume (M2/M0; 56.7% vs. 159.4% for NS and NM controls, p = <0.001) and a 7.2% T2 negative contrast effect (M1/M0; 94.2% vs. 101.3% for NS, p = 0.046) compared to controls. Six mice in 300, 600, and LM (2 each) achieved a clinical CR (between 1 to 2 weeks after M1) vs. none in the NS and NM control cohorts ( p = 0.0418), without any recurrences until end of study (6 weeks after M2). Conclusions: This is the first report demonstrating the theragnostic effects of MENPs having both T2 contrast and locally ablative therapy properties as an in vivo tumor targeting agent. These results confirm prior pilot study and point towards irreversible electroporation as the likely mechanism of action (MOA) since the antitumor effect is not seen within the NM cohort. Additional studies characterizing the MOA, dose-response relationships, and immunomodulatory effects are needed to better understand their full clinical potential.[Table: see text]

Dextran-based nanoparticles with 5-FU-conjugated polymethacrylate segments for drug delivery: Synthesis of amphiphilic graft copolymers by mechanochemical solid-state polymerization and characterization

Dextran (Dx) is a biodegradable and biocompatible polysaccharide, thus promising as a drug delivery carrier for tumor therapy. Herein, we applied mechanical energy to a high molecular weight Dx to control its molecular weight and simultaneously generate mechanoradicals. The solid-state polymerization of methacrylate- or methacrylamide derivatives initiated with Dx mechanoradicals showed polymer conversion of >95%, yielding Dx-based graft copolymers with molecular weights of approximately 30,000 g mol−1. The Dx-based graft copolymers with hydrophobic segments formed nanoparticles with a particle size of 25–35 nm in an aqueous solution. The anti-pancreatic tumor drug 5-fluorouracil (5-FU) was covalently conjugated onto the hydrophobic segments of the amphiphilic Dx, and the nanoparticles were also prepared. The drug release profile from 5-FU-conjugated nanoparticles corresponded well to the Korsmeyer-Peppas model applied to drug release from matrix substrates, and was also immensely predicted by the Logistic and Gompertz curves. The 5-FU-conjugated nanoparticles showed cytotoxicity against the pancreatic adenocarcinoma cell lines (BxPC-3) that were not significantly inferior to the 5-FU positive group. Furthermore, the fluorescein-labeled nanoparticles internalized into BxPC-3 within 6 h and actively migrated into the cytosol. These results suggest that Dx-based graft copolymers with hydrophobic segments might be used to enhance therapeutic activity.

Partial Acquisition of Spectral Projections Accelerates Four-dimensional Spectral-spatial EPR Imaging for Mouse Tumor Models: A Feasibility Study.

Our study aimed to accelerate the acquisition of four-dimensional (4D) spectral-spatial electron paramagnetic resonance (EPR) imaging for mouse tumor models. This advancement in EPR imaging should reduce the acquisition time of spectroscopic mapping while reducing quality degradation for mouse tumor models. EPR spectra under magnetic field gradients, called spectral projections, were partially measured. Additional spectral projections were later computationally synthesized from the measured spectral projections. Four-dimensional spectral-spatial images were reconstructed from the post-processed spectral projections using the algebraic reconstruction technique (ART) and assessed in terms of their image qualities. We applied this approach to a sample solution and a mouse Hs766T xenograft model of human-derived pancreatic ductal adenocarcinoma cells to demonstrate the feasibility of our concept. The nitroxyl radical imaging agent 2H,15N-DCP was exogenously infused into the mouse xenograft model. The computation code of 4D spectral-spatial imaging was tested with numerically generated spectral projections. In the linewidth mapping of the sample solution, we achieved a relative standard uncertainty (standard deviation/| mean |) of 0.76 μT/45.38 μT = 0.017 on the peak-to-peak first-derivative EPR linewidth. The qualities of the linewidth maps and the effect of computational synthesis of spectral projections were examined. Finally, we obtained the three-dimensional linewidth map of 2H,15N-DCP in a Hs766T tumor-bearing leg in vivo. We achieved a 46.7% reduction in the acquisition time of 4D spectral-spatial EPR imaging without significantly degrading the image quality. A combination of ART and partial acquisition in three-dimensional raster magnetic field gradient settings in orthogonal coordinates is a novel approach. Our approach to 4D spectral-spatial EPR imaging can be applied to any subject, especially for samples with less variation in one direction.

The bacterial metabolite, lithocholic acid, has antineoplastic effects in pancreatic adenocarcinoma

Lithocholic acid (LCA) is a secondary bile acid. LCA enters the circulation after bacterial synthesis in the gastrointestinal tract, reaches distantly located cancer cells, and influences their behavior. LCA was considered carcinogenic, but recent studies demonstrated that LCA has antitumor effects. We assessed the possible role of LCA in pancreatic adenocarcinoma. At the serum reference concentration, LCA induced a multi-pronged antineoplastic program in pancreatic adenocarcinoma cells. LCA inhibited cancer cell proliferation and induced mesenchymal-to-epithelial (MET) transition that reduced cell invasion capacity. LCA induced oxidative/nitrosative stress by decreasing the expression of nuclear factor, erythroid 2-like 2 (NRF2) and inducing inducible nitric oxide synthase (iNOS). The oxidative/nitrosative stress increased protein nitration and lipid peroxidation. Suppression of oxidative stress by glutathione (GSH) or pegylated catalase (pegCAT) blunted LCA-induced MET. Antioxidant genes were overexpressed in pancreatic adenocarcinoma and decreased antioxidant levels correlated with better survival of pancreatic adenocarcinoma patients. Furthermore, LCA treatment decreased the proportions of cancer stem cells. Finally, LCA induced total and ATP-linked mitochondrial oxidation and fatty acid oxidation. LCA exerted effects through the farnesoid X receptor (FXR), vitamin D receptor (VDR), and constitutive androstane receptor (CAR). LCA did not interfere with cytostatic agents used in the chemotherapy of pancreatic adenocarcinoma. Taken together, LCA is a non-toxic compound and has antineoplastic effects in pancreatic adenocarcinoma.

Nanotechnology-Based Strategy for Enhancing Therapeutic Efficacy in Pancreatic Cancer: Receptor-Targeted Drug Delivery by Somatostatin Analog

A novel nanotechnology-based drug delivery system (DDS) targeted at pancreatic cancer cells was developed, characterized, and tested. The system consisted of liposomes as carriers, an anticancer drug (paclitaxel) as a chemotherapeutic agent, and a modified synthetic somatostatin analog, 5-pentacarbonyl-octreotide, a ligand for somatostatin receptor 2 (SSTR2), as a targeting moiety for pancreatic cancer. The cellular internalization, cytotoxicity, and antitumor activity of the DDS were tested in vitro using human pancreatic ductal adenocarcinoma (PDAC) cells with different expressions of the targeted SSTR2 receptors, and in vivo on immunodeficient mice bearing human PDAC xenografts. The targeted drug delivery system containing paclitaxel exhibited significantly enhanced cytotoxicity compared to non-targeted DDS, and this efficacy was directly related to the levels of SSTR2 expression. It was found that octreotide-targeted DDS proved exceptionally effective in suppressing the growth of PDAC tumors. This study underscores the potential of octreotide-targeted liposomal delivery systems to enhance the therapeutic outcomes for PDAC compared with non-targeted liposomal DDS and Paclitaxel-Cremophor® EL, suggesting a promising avenue for future cancer therapy innovations.

Impact of heparanase-2 (Hpa2) on cancer and inflammation: Advances and paradigms.

HPSE2, the gene-encoding heparanase 2 (Hpa2), is mutated in urofacial syndrome (UFS), a rare autosomal recessive congenital disease attributed to peripheral neuropathy. Hpa2 lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase (Hpa1), yet it exhibits a high affinity toward HS, thereby inhibiting Hpa1 enzymatic activity. Hpa2 regulates selected genes that promote normal differentiation, tissue homeostasis, and endoplasmic reticulum (ER) stress, resulting in antitumor, antiangiogenic, and anti-inflammatory effects. Importantly, stress conditions induce the expression of Hpa2, thus establishing a feedback loop, where Hpa2 enhances ER stress which, in turn, induces Hpa2 expression. In most cases, cancer patients who retain high levels of Hpa2 survive longer than patients bearing Hpa2-low tumors. Experimentally, overexpression of Hpa2 attenuates the growth of tumor xenografts, whereas Hpa2 gene silencing results in aggressive tumors. Studies applying conditional Hpa2 knockout (cHpa2-KO) mice revealed an essential involvement of Hpa2 contributed by the host in protecting against cancer and inflammation. This was best reflected by the distorted morphology of the Hpa2-null pancreas, including massive infiltration of immune cells, acinar to adipocyte trans-differentiation, and acinar to ductal metaplasia. Moreover, orthotopic inoculation of pancreatic ductal adenocarcinoma (PDAC) cells into the pancreas of Hpa2-null vs. wild-type mice yielded tumors that were by far more aggressive. Likewise, intravenous inoculation of cancer cells into cHpa2-KO mice resulted in a dramatically increased lung colonization reflecting the involvement of Hpa2 in restricting the formation of a premetastatic niche. Elucidating Hpa2 structure-activity-relationships is expected to support the development of Hpa2-based therapies against cancer and inflammation.

Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating Tcell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased Tcell activation. Our data reveal an immune role for CLDN18 in orchestrating Tcell infiltration and shaping the tumor immune contexture.

Paris saponin VII induces Caspase-3/GSDME-dependent pyroptosis in pancreatic ductal adenocarcinoma cells by activating ROS/Bax signaling

ObjectiveParidis Rhizoma (Chonglou in Chinese), a traditional Chinese herbal medicine, has been shown have strong anti-tumor effects. Paris saponin VII (PSVII), an active constituent isolated from Paridis Rhizoma, was demonstrated to significantly suppress the proliferation of BxPC-3 cells in our previous study. Here, we aimed to elucidate the anti-pancreatic ductal adenocarcinoma (PDAC) effect of PSVII and the underlying mechanism. MethodsCell viability was determined by CCK-8, colony formation, and cell migration assays. Cell apoptosis and reactive oxygen species (ROS) production were measured by flow cytometry with annexin V/propidine iodide (Annexin V/PI) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), respectively. Pyroptosis was evaluated by morphological features, Hoechst 33342/PI staining assay, and release of lactate dehydrogenase (LDH). JC-1 fluorescent dye was employed to measure mitochondrial membrane potential. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to determine the levels of proteins or mRNAs. The effect in vivo was assessed by a xenograft tumor model. ResultsPSVII inhibited the viability of PDAC cells (BxPC-3, PANC-1, and Capan-2 cells) and induced gasdermin E (GSDME) cleavage, as well as the simultaneous cleavage of Caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). Knockdown of GSDME shifted PSVII-induced pyroptosis to apoptosis. Additionally, the effect of PSVII was significantly attenuated by Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-FMK), on the induction of GSDME-dependent pyroptosis. PSVII also elevated intracellular ROS accumulation and stimulated Bax and Caspase-3/GSDME to conduct pyroptosis in PDAC cells. The ROS scavenger N-acetyl cysteine (NAC) suppressed the release of LDH and inhibited Caspase-9, Caspase-3, and GSDME cleavage in PDAC cells, ultimately reversing PSVII-induced pyroptosis. Furthermore, in a xenograft tumor model, PSVII markedly suppressed the growth of PDAC tumors and induced pyroptosis. ConclusionThese results demonstrated that PSVII exerts therapeutic effects through Caspase-3/GSDME-dependent pyroptosis and may constitute a novel strategy for preventing chemotherapeutic resistance in patients with PDAC in the future.

Culture Shock! Investigating the Importance of Physiological Tumour-Like Conditions on Cultured Cancer Cells

The tumour microenvironment (TME) is complex and dynamic, characterized by poor vascularization, limited nutrient availability, hypoxia, and an acidic pH. This environment plays a critical role in cancer progression, driving hallmark changes in cancer cell metabolism, morphology, proliferation, and motility. Although mammalian cell culture is a foundational tool for investigating cancer cell biology in vitro, standard cell culture practices fall short of replicating the in vivo conditions of tumours. Recently, ‘physiologic’ cell culture media that closely resemble human plasma (e.g., Plasmax, HPLM) have been developed, along with more frequent adoption of physiological oxygen conditions (2-8% O2) rather than standard atmosphere (~18% O2). Although an improvement, these conditions are not representative of all in vivo cellular environments, particularly that of the TME. Considering the tight regulatory capacity of cancer cells to sense and respond to changes in metabolic availability, we hypothesized that alterations in nutrient and oxygen availability, even if within the physiological range, may subtly rewire the metabolism in cultured cancer cells. This may necessitate further refinement of cell-specific physiologic culture conditions to better maintain the in vivo biology of cells. In this study, we directly address this hypothesis by describing the development of a tumour microenvironment-like medium (TMEM) based on metabolomic profiling of murine pancreatic ductal adenocarcinoma tumour interstitial fluid from relevant literature. We found that murine pancreatic ductal adenocarcinoma (KPCY) cells cultured under TME-like conditions (TMEM, pH 7.0, 1.5% O2) exhibited increased glucose uptake and lactate production rates compared to those cultured under healthy plasma-like conditions (Mouse Plasma-like Medium, pH 7.4, 5% O2), despite lower initial glucose concentrations in TMEM (2.3 mM vs. 4.4 mM in Mouse Plasma-like Medium). Moreover, cell proliferation rates were lower in TMEM, suggesting an overall reduction in ATP consumption fueling cell division. Seahorse Extracellular Flux Analysis is being performed to determine effects on cellular bioenergetics. Metabolomic amino acid uptake using LCMS is also being performed to further assess the effect of TMEM on nutrient utilization. Using RNA-sequencing, we showed that culture in TMEM increased expression of genes associated with angiogenesis, amino acids biosynthesis, cell migration, and endothelial-to-mesenchymal transition. Migration assays confirmed increased motility in KPCY cells cultured in TMEM. Taken together, these results demonstrate that growth in a medium designed to simulate the TME alters KPCY cell biology in ways that are relevant to their pathology, which highlights the significance of maintaining TME-like conditions in culture. This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant from JAS and a NSERC Canadian Graduate Student (Masters) Grant awarded to GLG. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.