Gemcitabine Exposure Research Articles

GFPT2 expression is induced by gemcitabine administration and enhances invasion by activating the hexosamine biosynthetic pathway in pancreatic cancer

Our previous studies revealed a novel link between gemcitabine (GEM) chemotherapy and elevated glutamine-fructose-6-phosphate transaminase 2 (GFPT2) expression in pancreatic cancer (PaCa) cells. GFPT2 is a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP). HBP can enhance metastatic potential by regulating epithelial-mesenchymal transition (EMT). The aim of this study was to further evaluate the effect of chemotherapy-induced GFPT2 expression on metastatic potential. GFPT2 expression was evaluated in a mouse xenograft model following GEM exposure and in clinical specimens of patients after chemotherapy using immunohistochemical analysis. The roles of GFPT2 in HBP activation, downstream pathways, and cellular functions in PaCa cells with regulated GFPT2 expression were investigated. GEM exposure increased GFPT2 expression in tumors resected from a mouse xenograft model and in patients treated with neoadjuvant chemotherapy (NAC). GFPT2 expression was correlated with post-operative liver metastasis after NAC. Its expression activated the HBP, promoting migration and invasion. Treatment with HBP inhibitors reversed these effects. Additionally, GFPT2 upregulated ZEB1 and vimentin expression and downregulated E-cadherin expression. GEM induction upregulated GFPT2 expression. Elevated GFPT2 levels promoted invasion by activating the HBP, suggesting the potential role of this mechanism in promoting chemotherapy-induced metastasis.

A phase 2a safety run-in and preliminary efficacy study of liposomal gemcitabine (FF-10832) in combination with pembrolizumab in patients with advanced solid tumors.

2615 Background: FF-10832 (FF832) [liposomal gemcitabine (GEM)] has demonstrated superior activity preclinically compared to GEM via preferential tumor accumulation & induction of antitumor immune responses. Further enhanced activity has been shown in combination with immune checkpoint inhibitors. We evaluated the tolerability & preliminary efficacy of FF832 in combination with the PD-1 antibody pembrolizumab (PEM) in a Phase 2a safety run-in study in patients (pts) with advanced solid tumors. Methods: Pts received 200 mg PEM followed by 40 mg/m2 FF832 on Day 1 of a 21-day cycle to validate the recommended Phase 2 dose (RP2D) for combination therapy; treatment was continued until disease progression or unacceptable toxicity. Response was assessed by RECIST 1.1 every 2 cycles. Tumor PD-L1 expression, mutational burden, and modulation of circulating immune cells were assessed, & population PK modeling performed. Results: Twelve pts [NSCLC (6), urothelial cancer, UC (4), renal cell carcinoma (2); 6M/6F; median age, 69 (42-82) & median # prior therapies, 5 (1-7); prior GEM (5), prior PEM (9)] received a median of 2 (1-8) cycles FF832+PEM. Median time on study was 6.1 (1.1–23.7) weeks. FF832+PEM was well-tolerated. Common AEs related to FF832 were Gr≤2 fatigue (50%) with 1 Gr 3, anemia (33%) with 2 Gr 3, & Gr ≤2 decreased appetite, diarrhea, ↑AST, ↑AlkPhos, muscular weakness, nausea, and pyrexia (25% each). Common AEs related to FF832+PEM were Gr≤2 fatigue (33%) & nausea (25%). Three pts had Gr≤2 infusion reactions with the first FF832 infusion; all resolved & were successfully rechallenged. FF832 dose was reduced to 30 mg/m2 after Cycle 1 in 3 pts due to Gr 3 rash (1), Gr 2 fatigue (1), & one DLT of Gr 3 malaise, pain, and arthralgia. Of 9 pts evaluable for response, one achieved an unconfirmed PR after one cycle (UC, prior GEM/PEM, 42%↓ in target lesions). Five pts had a best response of SD with 2 maintained for 6-8 cycles. Median PFS was 6 weeks (95%CI: 3.1–NR); median OS was 23.3 weeks (95%CI: 4–NR). An extended plasma t1/2 (~30 hours) & exposures consistent with FF832 monotherapy at the RP2D were observed. As with FF832 monotherapy, multi-log decreases were observed in circulating Ki67+ Tregs relative to total CD4+ cells while CD8+ cells increased, suggesting FF832+PEM could enhance shifts to a more immunocompetent tumor microenvironment. Conclusions: The safety and preliminary efficacy of FF832+PEM was demonstrated in heavily pre-treated pts with solid tumors whose disease progressed on prior GEM and/or PEM. Continuous GEM exposure from FF832 along with immune checkpoint blockade may improve antitumor activity. Evaluation of FF832 at the RP2D/schedule of 40 m/gm2 Q 21 days alone and in combination with PEM is ongoing in a randomized expansion study in pts with metastatic NSCLC and UC with prior disease progression on PD-1/L1 therapy. Clinical trial information: NCT05318573 .

The combination of gemcitabine plus an anti-FGFR inhibitor can have a synergistic antitumor effect on FGF-activating cholangiocarcinoma

Anti-FGFR treatment for cholangiocarcinoma (CCA) with fibroblast growth factor receptor (FGFR) alteration is a promising treatment option. Since the antitumor mechanisms of anti-FGFR inhibitors and conventional cytotoxic drugs differ, synergistic effects can be possible. This study aimed to evaluate the efficacy of the combined administration of gemcitabine (GEM) and pemigatinib in CCA cells with FGFR2 alterations. To simulate the treatment for patients with 3 kinds of CCA, chemonaïve CCA with activation of the FGF pathway, chemo-resistant CCA with activation of the FGF pathway, and CCA without FGF pathway activation (as controls), we evaluated 3 different CCA cell lines, CCLP-1 (with a FGFR2 fusion mutation), CCLP-GR (GEM-resistant cells established from CCLP-1), and HuCCT1 (without FGFR mutations). There was no significant difference between CCLP-1 and HuCCT1 in GEM suspensibility (IC50 = 19.3, 22.6 mg/dl, p = 0.1187), and the drug sensitivity to pemigatinib did not differ between CCLP-1 and CCLP-GR (IC50 = 7.18,7.60 nM, p = 0.3089). Interestingly, only CCLP-1 showed a synergistic effect with combination therapy consisting of GEM plus pemigatinib in vitro and in vivo. In a comparison of the reaction to GEM exposure, only CCLP-1 cells showed an increase in the activation of downstream proteins in the FGF pathway, especially FRS2 and ERK. In association with this reaction, cell cycle and mitosis were increased with GEM exposure in CCLP-1, but HuCCT1/CCLP-GR did not show this reaction. Our results suggested that combination therapy with GEM plus pemigatinib is a promising treatment for chemonaïve patients with CCA with activation of the FGF pathway.

Oncopig bladder cancer cells recapitulate human bladder cancer treatment responses in vitro.

Bladder cancer is a common neoplasia of the urinary tract that holds the highest cost of lifelong treatment per patient, highlighting the need for a continuous search for new therapies for the disease. Current bladder cancer models are either imperfect in their ability to translate results to clinical practice (mouse models), or rare and not inducible (canine models). Swine models are an attractive alternative to model the disease due to their similarities with humans on several levels. The Oncopig Cancer Model has been shown to develop tumors that closely resemble human tumors. However, urothelial carcinoma has not yet been studied in this platform. We aimed to develop novel Oncopig bladder cancer cell line (BCCL) and investigate whether these urothelial swine cells mimic human bladder cancer cell line (5637 and T24) treatment-responses to cisplatin, doxorubicin, and gemcitabine in vitro. Results demonstrated consistent treatment responses between Oncopig and human cells in most concentrations tested (p>0.05). Overall, Oncopig cells were more predictive of T24 than 5637 cell therapeutic responses. Microarray analysis also demonstrated similar alterations in expression of apoptotic (GADD45B and TP53INP1) and cytoskeleton-related genes (ZMYM6 and RND1) following gemcitabine exposure between 5637 (human) and Oncopig BCCL cells, indicating apoptosis may be triggered through similar signaling pathways. Molecular docking results indicated that swine and humans had similar Dg values between the chemotherapeutics and their target proteins. Taken together, these results suggest the Oncopig could be an attractive animal to model urothelial carcinoma due to similarities in in vitro therapeutic responses compared to human cells.

Expression of fibroblast growth factor receptor 1 correlates inversely with the efficacy of single-agent fibroblast growth factor receptor-specific inhibitors in pancreatic cancer.

Elevated fibroblast growth factor receptor (FGFR) activity correlates with pancreatic adenocarcinoma (PDAC) progression and poor prognosis. However, its potential as a therapeutic target remains largely unexplored. The mechanisms of action and therapeutic effects of selective pan-FGFR inhibitors (pan-FGFRi) were explored using in vitro and in vivo PDAC models ranging from gemcitabine-sensitive to highly gemcitabine-resistant (GemR). Gain-/loss-of-function investigations were employed to define the role of individual FGFRs in cell proliferation, migration, and treatment response and resistance. The pan-FGFRi NVP-BGJ398 significantly inhibited cell proliferation, migration, and invasion, and downregulated key cell survival- and invasiveness markers in multiple PDAC cell lines. Gemcitabine is a standard-of-care for PDAC, but development of resistance to gemcitabine (GemR) compromises its efficacy. Acquired GemR was modelled experimentally by developing highly GemR cells using escalating gemcitabine exposure in vitro and in vivo. FGFRi treatment inhibited GemR cell proliferation, migration, GemR marker expression, and tumour progression. FGFR2 or FGFR3 loss-of-function by shRNA knockdown failed to decrease cell growth, whereas FGFR1 knockdown was lethal. FGFR1 overexpression promoted cell migration more than proliferation, and reduced FGFRi-mediated inhibition of proliferation and migration. Single-agent FGFRi suppressed the viability and growth of multiple patient-derived xenografts inversely with respect to FGFR1 expression, underscoring the influence of FGFR1-dependent tumour responses to FGFRi. Importantly, secondary data analysis showed that PDAC tumours expressed FGFR1 at lower levels than in normal pancreas tissue. Single-agent FGFR inhibitors mediate selective, molecularly-targeted suppression of PDAC proliferation, and their effects are greatest in PDAC tumours expressing low-to-moderate levels of FGFR1.

Ameliorative Role of Grape Seed Extract (Vitis Vinifera) on Gemcitabine- Induced Testicular Damage in Rabbits

Spermatogenesis is a highly conserved and regulated process and it is sensitive to fluctuations in the physical and chemical environment. Gemcitabine is a novel antimetabolic anticancer drug used frequently in the treatment of many cancers. Gemcitabine may disrupt spermatogenesis by targeting various testicular cell types. Grape seed extract is a natural product, recently identified as one of the most powerful antioxidants. In addition, it has been reported to exert anticarcinogenic effects. This current work aimed to study the effect of gemcitabine on the testicular tissue of local adult rabbits and to evaluate the possible protective role of Grape seed extract. A experimental study was performed on twenty seven adult male local rabbits, were divided into three groups, nine rabbit in each group as following: group (A): animals of control group received normal saline injection, group (B): animals of treated group received therapeutic dose of gemcitabine alone in a dose of 25 mg/kg body weight intraperitoneally once per week for six successive weeks and group (C): served as protective group and was concomitantly treated with grape seeds extract by oral gavages in a dose of 250 mg/kg body weight and gemcitabine in a dose of 25 mg/kg body weight intraperitoneally once per week for six successive weeks. The results of the present investigation showed that gemcitabine toxicity produced significant structural changes in the testis of group B, gemcitabine treated group, in the form of multiple distortion of the seminiferous tubules, with cellular disorganization, testicular tubular cell degeneration, necrosis, tubular atrophy, desquamation and sloughing of the seminiferous epithelium which was accumulated in the centre of the seminiferous tubules in the form of degenerated tissue. Many seminiferous tubules showed widely spaced spermatogenic cells. Karyopyknosis, disappearance of interstitial cells of Leydig and necrotic Sertoli cells, progressive tubular and interstitial testicular damage together with spermatogenic arrest was an initial necrotic change induced by gemcitabine exposure. Disorganization of spermatogenic cells, an interruption in spermatogenesis process and the seminiferous tubules are devoid of sperms was also showed in this study. Sections from the testes of animals received therapeutic dose of gemcitabine and treated by grape seed extract, group (C) were showing partial preservation of the normal structure of the seminiferous tubules. Most of them more or less resembled the normal structure. It could be concluded that gemcitabine caused marked alterations in the structure of the seminiferous tubules of the rabbit testis that may be minimized by administration of grape seed extract. Our findings simply revealed that grape seeds extract treatment significantly inhibits testicular cell apoptosis, which suggests that apoptosis may be one of the underlying mechanisms by which grape seeds extract protects against gemcitabine induced defective spermatogenesis.

Safety, tolerability, and preliminary efficacy of TAR-200 in patients with intermediate risk non–muscle-invasive bladder cancer: A phase 1 study.

505 Background: TAR-200 is a novel intravesical drug delivery system designed to provide a continuous, slow release of gemcitabine within the bladder. Prolonged gemcitabine exposure over days, instead of hours, such as with current standard intravesical installations, may achieve more efficient and effective tumor response. We report on the safety and tolerability of TAR-200 in patients with non-muscle–invasive bladder cancer (NMIBC). Methods: In this phase 1b, open-label, prospective study, patients with a papillary recurrence after prior histologically proven, intermediate risk (IR)-NMIBC received two 1-week TAR-200 dosing cycles over a 4- to 6-week period. The study used a marker lesion/ablation design. Cystoscopy was performed to assess for recurrent papillary disease and for complete transurethral resection of the residual bladder tumor. The primary outcome was safety of TAR-200. Secondary outcomes were tolerability, pharmacokinetics, preliminary efficacy, and immunohistochemistry. Results: In total, 12 patients received TAR-200 treatment. Insertion and removal of TAR-200 was uneventful. No TAR-200-related serious adverse events (AEs) occurred. Four patients had no TAR-200-associated AEs; the remainder had varying degrees of AEs (all grade ≤2 [CTCAEv4.0]), mainly consisting of low-grade urinary urgency, urinary frequency, and dysuria, with no delay in the treatment schedule. Two patients refused a second dosing cycle due to urinary urgency and frequency. Plasma gemcitabine concentrations remained below the lower limit of detection. Five of 12 patients (42%) had complete response (CR); of these, 4 had a pathologic CR and 1 had CR based on visual assessment (with no biopsy available for pathologic assessment). Conclusions: In this small, phase 1 series, TAR-200 appears to be safe and well tolerated in patients with IR-NMIBC. Clinical trial information: NCT02720367 .

Intra-arterial gemcitabine vs IV gemcitabine PK substudy in patients with locally advanced pancreatic cancer.

728 Background: Localized dual-balloon-mediated, intra-arterial delivery of Gemcitabine (IAG) directly into tumors/tissue can lead to higher local drug potency1. Furthermore, IAG may lead to decreased systemic drug concentration and associated side effects. In patients with Locally Advanced Pancreatic Cancer (LAPC), this approach is currently being tested in a contemporary Phase III clinical trial, TIGeR-PaC. Herein we report the results of a 10-patient PK analysis sub-study within TIGeR-PaC. Methods: We analyzed a total of 10 subjects from 5 sites who received intra-arterial gemcitabine with the RenovoCath dual balloon catheter at 1000mg/m2 over 20 minutes. The target treatment artery was either the celiac axis (n=5) or the superior mesenteric artery (n=5). Samples were drawn at T = -5, 10, 15, 20, 30, 40, 60, and 90 minutes from the onset of infusion. At each timepoint, we collected 2mL of blood in heparinized tubing containing 25 mcg/mL of tetrahydrouridine. The blood samples were then centrifuged collect, freeze, and ship the plasma to a reference lab to perform a gemcitabine assay. The PK parameters for a single dose was estimated for Cmax, AUC, clearance, and distribution volume based on the terminal phase. These values are compared to historical data for intravenous gemcitabine (IVG) infusion at 1000mg/m2 over 30 minutes2. Results: At this time, data has been analyzed for 4 of 8 collected samples. We expect to complete collection and analysis of all 10 samples by November of 2022. We show results available so far; historical values for IVG are given as a reference point. The results suggest that IAG is associated with decreased systemic exposure of gemcitabine compared to IVG. Conclusions: Localized dual-balloon-mediated, intra-arterial delivery of gemcitabine can lead to decreased systemic levels of gemcitabine. This approach increased local potency and may be beneficial in decreasing gemcitabine-related systemic side effects. 1. Farsad K, et al. 04:21 PM Abstract No. 392 . J Vasc Intervent Radiol 2019;30(3):S172. doi:10.1016/j.jvir.2018.12.467. 2. Chow ECY, Zirkelbach JF. Clinical Pharmacology and Biopharmaceutics Review(s) {209604Orig1s000}. FDA: Center for Drug Evaluation and Research. Clinical trial information: NCT03257033 . [Table: see text]

Gemcitabine alters sialic acid binding of the glycocalyx and induces inflammatory cytokine production in cultured endothelial cells

Gemcitabine (GEM) is an anticancer drug inhibiting DNA synthesis. Glomerular thrombotic microangiopathy (TMA) has been reported as an adverse effect. However, the precise mechanism of GEM-induced endothelial injury remains unknown. Cultured human umbilical vein endothelial cells (HUVECs) in the confluent phase were exposed to GEM (5–100 μM) for 48 h and evaluated cell viability and morphology, lectin binding concerning sialic acid of endothelial glycocalyx (GCX), and immunofluorescent staining of platelet–endothelial cell adhesion molecule (PECAM) and vascular endothelial growth factor receptor 2 (VEGFR2). The mRNA expression of α2,6-sialyltransferase (ST6Gal1), sialidase (neuraminidase-1: NEU-1), and interleukin (IL)-1β and IL-6 was also evaluated. GEM exposure at 5 μM induced cellular shrinkage and intercellular dissociation, accompanied by slight attenuation of PECAM and VEGFR2 immunostaining, although cell viability was still preserved. At this concentration, lectin binding showed a reduction of terminal sialic acids in endothelial GCX, probably associated with reduced ST6Gal1 mRNA expression. IL-1β and IL-6 mRNA expression was significantly increased after GEM exposure. GEM reduced terminal sialic acids in endothelial GCX through mRNA suppression of ST6Gal1 and induced inflammatory cytokine production in HUVECs. This phenomenon could be associated with the mechanism of GEM-induced TMA.

MiR-222-3p-containing macrophage-derived extracellular vesicles confer gemcitabine resistance via TSC1-mediated mTOR/AKT/PI3K pathway in pancreatic cancer.

Gemcitabine resistance limits the efficacy of chemotherapy and maintains a challenge for treatment outcomes. Therefore, we aimed to clarify the downstream mechanisms underlying the role of miR-222-3p delivered by M2 macrophage-derived extracellular vesicles (M2 MDEs) in the chemoresistance of pancreatic cancer (PCa). We separated the mouse macrophages and polarized them to M2 phenotypes, from which the EVs were derived. miR-222-3p was highly expressed in M2 MDEs. M2 MDEs were internalized by PCa cells. miR-222-3p overexpressing M2 MDEs were treated with gemcitabine and co-cultured with PCa cells for in vitro experiments. Co-culture with M2 MDEs enriched with miR-222-3p suppressed the sensitivity to gemcitabine, accompanied by diminished apoptosis and promoted proliferation. Furthermore, the M2 MDEs and PCa cells were injected to mice with gemcitabine exposure for in vivo substantiation. The delivery of miR-222-3p inhibitor by M2 MDEs suppressed tumor growth and elevated sensitivity of cancer cells to gemcitabine. Moreover, miR-222-3p was indicated to target and suppress TSC1 expression, while miR-222-3p activated the PI3K/AKT/mTOR pathway. Together, miR-222-3p-containing M2 MDEs enhance chemoresistance in PCa through TSC1 inhibition and activation of the PI3K/AKT/mTOR pathway.

Abstract CT565: Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer

Abstract Background: Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway inhibition may overcome chemoresistance of metastatic pancreatic cancer (MPC) refractory to gemcitabine + nab-paclitaxel (GN) due to suppression of cancer associated fibroblast (CAF) and increase of drug infiltration in tumor. We sought to determine the safety, recommended dose of tocilizumab (TCZ), an IL-6 receptor monoclonal antibody, and biological correlates of tumor shrinkage in patients with GN-refractory MPC. Methods: This phase 1, dose finding trial was conducted following preclinical study of IL-6 signaling blockade in genetically engineered mouse model of pancreatic cancer (pGEMM) and patients-derived CAF, enrolled 10 patients with MPC that had progressed after GN. During the dose-finding part to determine dose-limiting toxicity (DLT), patients initially received TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. The subsequent dose-expansion part used the TCZ dose identified in the dose-finding study. Before Cycle 1 and at the end of Cycle 1, biopsy of liver metastasis was performed 3 to 5 hours after levofloxacin (LVXF) administration to measure LVFX infiltration in tumor using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). Results: In pGEMM tumor, mouse IL-6 receptor antibody plus GN led pathological response. The growth of CAFs from patients with pancreatic cancer was inhibited by TCZ. In this phase I study, no DLTs occurred and the recommended dosage of TCZ was determined to be 8 mg/kg. Treatment-emergent adverse events (TEAEs) occurred in 80% of patients (Grade ≥3 in 60%) with decreased neutrophil count (n=5, 7 events) being the most common TEAE. Disease control rate was 80·0% (95%CI: 44·4-97·5) and tumor reduction during cycle 1 was observed in 4 patients who were defined as responder. In paired-biopsied samples, decrease of phosphorylated (p) STAT3 expression in tumor was observed in 7 of 8 patients (88%). Responder-related biological activities were increase of cleaved PARP expression of tumor nuclei (P = 0.01), decrease of proliferative CAF (P = 0.08), and increase of LVFX infiltration in tumor (P = 0.04). Decrease of pSTAT3 expression (P = 0.02) was favor to increase of LVFX infiltration against increase of gamma-H2AX, an index of gemcitabine exposure (P = 0.20). Conclusion: TCZ+GN-rechallenge had a manageable safety profile and showed preliminary activity via inhibition of CAF and gain of intratumoral drug infiltration in MPC. Citation Format: Shuichi Mitsunaga, Masafumi Ikeda, Kazunori Aoki, Kyoko Yamaguchi, Noriaki Sawada, Etsuko Fujii, Masanobu Nishidate, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Atsuhiko Kato, Mayu Makikawa, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Clinical and translational results of a phase I study, tocilizumab plus gemcitabine/nab-paclitaxel in patient with gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT565.

Abstract 6145: Increased PD-L1 expression levels were observed on both tumor cells and macrophages by tocilizumab plus gemcitabine/nab-paclitaxel treatment in gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer patients

Abstract Background: Blockade of interlukine-6 (IL-6) and programmed death-1-ligand 1 (PD-L1) limits tumour progression in murine models of pancreatic cancer. When PD-L1 expression of metastatic pancreatic cancer (MPC) is increased under the influence of gemcitabine exposure plus IL-6 inhibition, the spectrum of benefit from immune checkpoint blockade may expand to MPC patients with combined blockade of IL-6 and PD-L1 plus gemcitabine-based chemotherapy. We sought to determine PD-L1 expression of liver metastasis during cycle 1 of a phase I study, tocilizumab (TCZ) plus gemcitabine/nab-paclitaxel (GN) in patient with GN-refractory MPC. Methods: This phase 1 study enrolled 10 patients with MPC that had progressed after GN. All patients experienced cycle 1 of TCZ plus GN, TCZ 8 mg/kg on Day 1 and nab-paclitaxel 100 mg/m2 + gemcitabine 750 mg/m2 on Days 2, 9, and 16. Biopsy of liver metastasis was performed before Cycle 1 and at the end of Cycle 1. Tumour microenvironment including PD-L1 expression were evaluated in paired-biopsied specimen from 7 patients by multicolour immunofluorescence staining. Tumour cell and PD-L1 expression were labelled by anti-cytokeratin 19 antibody and anti-PD-L1 antibody clone SP263, respectively. Cell numbers per millimetre squared (cell density) and the percentage of PD-L1 positive cell in tumor area were calculated according to cell types including tumour cell as cytokeratin 19 positive cell and macrophage as CD68 positive cell. Results: Cell density and the percentage of PD-L1 positive tumor cell were 29/mm2 and 1.1% in mean at baseline and tended to be increased up to 142/mm2 (P = 0.11) and 5.2% (P = 0.06) at the end of cycle 1. Means of PD-L1 positive macrophages were 28/mm2 and 6.9% at baseline and were increased to 69/mm2 (P = 0.01) and 14% (P = 0.08). When region of interest was selected from tumor stroma, differences of PD-L1 positive macrophage during cycle 1 was also maintained in cell density (P = 0.02) and the percentage of PD-L1 positive cell (P = 0.06). Conclusion: TCZ+GN induced an increase of PD-L1 expression levels on tumor cell and macrophages. Citation Format: Kyoko Yamaguchi, Etsuko Fujii, Shuichi Mitsunaga, Noriaki Sawada, Masafumi Ikeda, Takashi Fujitomo, Hideaki Mizuno, Yoko Kayukawa, Mayu Makikawa, Kazunori Aoki, Hiroshi Imaoka, Mitsuhito Sasaki, Kazuo Watanabe, Atsukiko Kato, Hiroyuki Tsunoda, Kimio Terao, Atsushi Ochiai. Increased PD-L1 expression levels were observed on both tumor cells and macrophages by tocilizumab plus gemcitabine/nab-paclitaxel treatment in gemcitabine/nab-paclitaxel-refractory metastatic pancreatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6145.

Modelling an evaluation of the efficacy and safety of gemcitabine combined with platinum in the treatment of non-small cell lung cancer.

Gemcitabine combined with platinum is currently the recommended first-line chemotherapy for advanced non-small cell lung cancer (NSCLC). This study aimed to quantitatively compare the efficacy and safety of gemcitabine-platinum combinations in the treatment of advanced NSCLC under different dosing regimens based on extensive literature data. The PubMed and Cochrane Library databases were systematically searched for clinical trials in patients with NSCLC treated with a gemcitabine-platinum regimen. A parametric survival function was used to analyse the time course of overall survival (OS). The objective response rate (ORR) and the incidence of grade 3/4 adverse events were summarized using the random-effects model of a single-arm meta-analysis. The study included 63 arms from 47 publications, with a total sample size of 4344 patients for analysis. The model revealed that East Asians has a better survival benefit than non-East Asians, with a median OS of 16.4 (95% CI: 14.3-19.0) and 9.9 (95% CI: 8.1-12.4) months, respectively. Moreover, the OS of patients that underwent a 6-cycle treatment was significantly longer than those that had a 4-cycle treatment in non-East Asians, with a median OS of 10.2 (95% CI: 9.5-11.1) and 8.4 (95% CI: 7.7-9.3) months, respectively. However, the incidence of neutropenia, nausea and vomiting also increased after 6 cycles of treatment. When the dose of gemcitabine increased from 1000mg/m2 to 1250mg/m2 , the median OS was extended by approximately 1month, but the incidence of grade 3/4 adverse reactions did not increase. Race is an important factor affecting OS in the treatment of advanced NSCLC, which should be considered when conducting international multicentre clinical trials. Additionally, this study found that the OS increased with an increase in gemcitabine exposure, so it is necessary to construct an exposure-response model to obtain the best benefit-risk ratio for patients.

Abstract P071: Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability

Abstract Background: Pancreatic adenocarcinoma, or PDAC, is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine, a nucleoside analog, is a primary standard of care in pancreatic cancer. In addition to its normative cytotoxic function, evidence suggests that this chemotherapy drug also harnesses immunomodulatory capabilities in the form of increasing human leukocyte antigen (HLA) class I expression in lung, breast, colon, and cholangiocarcinoma cells. HLA class I is a complex (alpha heavy chain and beta 2-microglobulin light chain) located at the surface of nearly all cells where it presents peptides, including cancer-associated peptides, to cytotoxic T cells. Recognition of these peptides as atypical leads to T-cell mediated lysis of the presenting cell. Subsequently, understanding the ability of gemcitabine and the mechanisms by which it influences the HLA class I complex are of great importance. Methods: To investigate the effect of gemcitabine treatment on HLA class I expression in pancreatic cancer, alterations in HLA class I protein levels were monitored via western blot analysis, flow cytometry, and quantitative polymerase chain reaction (qPCR) in three pancreatic cancer cell lines. Changes in surface stability of the HLA class I complex were evaluated through brefeldin A (BFA) assays at the 72-hour time point. For western blot and flow cytometry experiments, impacts on the alpha heavy chain were further assessed for all three types of alpha heavy chains (HLA-A, HLA-B/C) at 72 and 96 hours. In qPCR experiments, alterations were analyzed for the HLA-A, HLA-B, and beta 2-microglobulin transcripts after a 48-hour gemcitabine exposure period. Results: Administration of gemcitabine to pancreatic cancer cell lines (S2-013, Capan-1, PANC-1) increased total protein levels of both HLA class I constituents (alpha heavy chain and beta-2-microglobulin light chain). All PDAC cell lines evaluated demonstrated enhanced surface expression of HLA-A2 and HLA-B/C with maximal increases of 3 and 2.5-fold respectively, as indicated by flow cytometry. Our qPCR analysis of the Capan-1 and S2-013 cell lines revealed increases in the levels of HLA-A, HLA-B, and beta 2-microglobulin transcripts (3-12-fold). BFA assays suggested that gemcitabine treatment also enhances stability of the surface HLA class I in the S2-013 and PANC-1 cell lines. Conclusion: In summary, gemcitabine exhibits an immunomodulatory ability to stimulate expression of HLA class I in pancreatic cancer cells. We have demonstrated that this increase in HLA class I is seen at the mRNA, total protein, surface, and stability level. Characterizing the ability of gemcitabine to influence the HLA class I complex and defining the mechanisms by which it does so will increase the potential for identification of suitable immunotherapies, including novel peptide-based cancer vaccines with enhanced treatment efficacy for PDAC patients. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P071.

Cytoplasmic RRM1 activation as an acute response to gemcitabine treatment is involved in drug resistance of pancreatic cancer cells.

RRM1 is functionally associated with DNA replication and DNA damage repair. However, the biological activity of RRM1 in pancreatic cancer remains undetermined. To determine relationships between RRM1 expression and the prognosis of pancreatic cancer, and to explore RRM1 function in cancer biology, we investigated RRM1 expression levels in 121 pancreatic cancer patients by immunohistochemical staining and performed in vitro experiments to analyze the functional consequences of RRM1 expression. Patients with high RRM1 expression had significantly poorer clinical outcomes (overall survival; p = 0.006, disease-free survival; p = 0.0491). In particular, high RRM1 expression was also associated with poorer overall survival on adjuvant chemotherapy (p = 0.008). We found that RRM1 expression was increased 24 hours after exposure to gemcitabine and could be suppressed by histone acetyltransferase inhibition. RRM1 activation in response to gemcitabine exposure was induced mainly in the cytoplasm and cytoplasmic RRM1 activation was related to cancer cell viability. In contrast, cancer cells lacking cytoplasmic RRM1 activation were confirmed to show severe DNA damage. RRM1 inhibition with specific siRNA or hydroxyurea enhanced the cytotoxic effects of gemcitabine for pancreatic cancer cells. Cytoplasmic RRM1 activation is involved in biological processes related to drug resistance in response to gemcitabine exposure and could be a potential target for pancreatic cancer treatment.

Biologically optimized schedule of gemcitabine and nab-paclitaxel regimen in metastatic pancreatic adenocarcinoma.

TPS4168 Background: Metastatic pancreatic adenocarcinoma has a poor prognosis, and improvements in therapy have been challenging. Alongside efforts in developing novel agents, there is a need to optimize and maximize the benefit of currently approved drugs. Gemcitabine + nab-paclitaxel is a frequently used regimen for pancreatic adenocarcinoma. Nab-paclitaxel is albumin–bound chemotherapy; hence the role of albumin uptake is critical for its effect. Caveolae are small membrane invaginations essential for transendothelial albumin uptake. Cav-1 is the principal structural component of caveolae. Williams and colleagues have published a series of preclinical studies demonstrating that tumor cell-specific Cav-1 expression directly correlates with albumin and albumin-bound chemotherapy uptake and subsequent apoptotic response in tumor cells. In vitro studies showed that exposure of pancreatic cancer cells to Gemcitabine resulted in up-regulation of Cav-1 peaking 48 hours after gemcitabine exposure. This Cav-1 up-regulation correlated with increased temporal albumin cellular uptake. In addition, Williams and colleagues noted that exposure of pancreatic cancer cell lines to Gemcitabine resulted in a time–specific re-entry of cells into the G2/M phase (nab-paclitaxel cytotoxicity phase) between 48-60 hours after gemcitabine treatment. Collectively this data suggest that infusing nab-paclitaxel after 48 hours of gemcitabine infusion would be optimal for both increased uptake as well as increased susceptible tumor cells. We had previously shown this effect on multiple cell lines as well as mouse models. Methods: This is a phase II trial; patients will receive a standard of care chemotherapy regimen consisting of FDA-approved Gemcitabine + nab-paclitaxel with modification of the schedule to deliver nab-paclitaxel 48 hours (2 days) after gemcitabine infusions. The primary endpoint is ORR, with a null hypothesis of 20% vs. a target of 35%. Employing a 2-stage design (minimax) and assuming 80% power and a 0.05 significance level, a total of 53 patients will be required. In the first stage, if at least 7/31 patients respond to therapy, an additional 22 patients will be added for a total of 53 patients. The study will be terminated early if ≤ six patients respond in the first stage. Observation of response in at least 16/53 patients would be required to warrant further investigation of this infusion schedule of combination therapy. The secondary endpoints include the safety of the regimen schedule, Relative dose intensity, disease control rate, PFS, and OS. The trial opened to enrollment in June 2020 and is accepting patients. Clinical trial information: NCT04115163.

Enhanced gemcitabine cytotoxicity with knockdown of multidrug resistance protein genes in human cholangiocarcinoma cell lines.

Cholangiocarcinoma (CCA) is an often fatal primary cancer of the liver that tends to be resistant to chemotherapy. Multidrug resistance proteins (MRPs) contribute to the chemoresistance of these tumors. The objectives of the study were to document MRP expression profiles in two representative human intrahepatic and extrahepatic CCA cells lines (HuCCT1 and KMBC, respectively) and gemcitabine-induced cytotoxicity prior to and following MRP knockdown. Multidrug resistance protein mRNA and protein expression were documented by real-time reverse transcription-polymerase chain reaction and western blots, respectively. MRP knockdown was achieved with lentivirus small hairpin RNA constructs. Prior to gemcitabine exposure, MRP1, MRP2, MRP4, MRP5, and MRP6 mRNA were expressed in HuCCT1 cells and MRP1, MRP3, MRP4, and MRP5 in KMBC cells. Following gemcitabine exposure, MRP5 and MRP6 expressions were significantly upregulated in HuCCT1 cells and MRP5 in KMBC cells. In HuCCT1 cells, although MRP5 knockdown had no effect, MRP6 knockdown significantly increased gemcitabine-induced cytotoxicity. In KMBC cells, MRP5 knockdown significantly increased gemcitabine cytotoxicity. Inhibition of MRP6 expression in intra-hepatic and MRP5 in extra-hepatic should be explored as potential treatments for CCA in humans.

Pharmacokinetics of gemcitabine and its amino acid ester prodrug following intravenous and oral administrations in mice

Gemcitabine is an intravenously administered anti-cancer nucleoside analogue. Systemic exposure following oral administration of gemcitabine is limited by extensive first-pass metabolism via cytidine deaminase (CDA) and potentially by saturation of nucleoside transporter-mediated intestinal uptake. An amino acid ester prodrug of gemcitabine, 5′-l-valyl-gemcitabine (V-Gem), was previously shown to be a substrate of the intestinally expressed peptide transporter 1 (PEPT1) and stable against CDA-mediated metabolism. However, preliminary studies did not evaluate the in vivo oral performance of V-Gem as compared to parent drug. In the present study, we evaluated the pharmacokinetics and in vivo oral absorption of gemcitabine and V-Gem following intravenous and oral administrations in mice. These studies revealed that V-Gem undergoes rapid systemic elimination (half-life < 1 min) and has a low oral bioavailability (<1%). Most importantly, the systemic exposure of gemcitabine was not different following oral administration of equimolar doses of gemcitabine (gemcitabine bioavailability of 18.3%) and V-Gem (gemcitabine bioavailability of 16.7%). Single-pass intestinal perfusions with portal blood sampling in mice revealed that V-Gem undergoes extensive activation in intestinal epithelial cells and that gemcitabine undergoes first-pass metabolism in intestinal epithelial cells. Thus, formulation of gemcitabine as the prodrug V-Gem does not increase systemic gemcitabine exposure following oral dosing, due, in part, to the instability of V-Gem in intestinal epithelial cells.

Gemcitabine Exposure Induces Epigenomic Remodeling in Pancreatic Cancer Cells during Resistance Development

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths in the United States with a devastating overall five‐year survival rate of less than five percent. Chemotherapy is a mainstay for PDAC treatment, with Gemcitabine (Gem) as a part of a first‐line regimen. Although Gem has shown clinical benefit, it possesses limited control over cancer progression, due to the major obstacle of chemoresistance. Consequently, there is an urgent need to further improve our understanding of molecular mechanisms underlying resistance development to identify innovative therapeutic targets. Our laboratory is focused on the use of epigenomic drugs as novel therapeutic approaches. Our experimental design involves developing chemoresistance in patient derived PDAC cells to identify epigenomic alterations and gene expression changes that can participate in the initiation and establishment of resistance to chemotherapeutic agents. Identifying such modifications can provide valid druggable targets to enhance the effect or reverse resistance to anti‐cancer drugs. In the present study, we induced resistance to Gem on a naïve patient‐derived PDAC cell line and evaluated epigenomic landscape remodeling through changes of histone post‐translational modifications. In addition, we characterized phenotypic changes in response to Gem exposure. Using the Incucyte® imaging system, we determined that exposure to increasing concentrations of Gem induced resistance on naïve patient‐derived PDAC cells. Gem resistant cells showed a reduced basal proliferation rate and apoptotic response compared to parental cells, as measured by activation of caspases 3 and 7. Additionally, cells developed morphological changes towards an epithelial state, coincident with the increase of Keratin 17/19 and E‐Cadherin. Moreover, treated cells demonstrated reduced sensitivity to the well‐known DNA damaging agents cisplatin and 5‐Fluorouracil. Evaluation of histone post‐translational modifications on these cells revealed reduction in the global amount of repressive marks (H3K9Me1, 2 and 3; H3K27Me2 and 3), as well as an increase of the activating histone mark H3K27Ac, associated with super enhancers. Furthermore, immunofluorescence‐based microscopy showed re‐distribution of the H3K9Me1 and H3K27Me3 repressive marks from the nuclear periphery to the whole nucleus, suggesting epigenomic remodeling of pancreatic cancer cells in response to the development of Gem resistance. In summary, these data demonstrate that Gem treatment induces chromatin remodeling and that targeting key epigenomic pathways may hold promise for novel strategies toward re‐sensitizing or preventing resistance of pancreatic cancer to chemotherapeutic agents.

Novel drug combination nanoparticles exhibit enhanced plasma exposure and dose-responsive effects on eliminating breast cancer lung metastasis.

Early diagnosis along with new drugs targeted to cancer receptors and immunocheckpoints have improved breast cancer survival. However, full remission remains elusive for metastatic breast cancer due to dose-limiting toxicities of heavily used, highly potent drug combinations such as gemcitabine and paclitaxel. Therefore, novel strategies that lower the effective dose and improve safety margins could enhance the effect of these drug combinations. To this end, we developed and evaluated a novel drug combination of gemcitabine and paclitaxel (GT). Leveraging a simple and scalable drug-combination nanoparticle platform (DcNP), we successfully prepared an injectable GT combination in DcNP (GT DcNP). Compared to a Cremophor EL/ethanol assisted drug suspension in buffer (CrEL), GT DcNP exhibits about 56-fold and 8.6-fold increases in plasma drug exposure (area under the curve, AUC) and apparent half-life of gemcitabine respectively, and a 2.9-fold increase of AUC for paclitaxel. Using 4T1 as a syngeneic model for breast cancer metastasis, we found that a single GT (20/2 mg/kg) dose in DcNP nearly eliminated colonization in the lungs. This effect was not achievable by a CrEL drug combination at a 5-fold higher dose (i.e., 100/10 mg/kg GT). A dose-response study indicates that GT DcNP provided a therapeutic index of ~15.8. Collectively, these data suggest that GT DcNP could be effective against advancing metastatic breast cancer with a margin of safety. As the DcNP formulation is intentionally designed to be simple, scalable, and long-acting, it may be suitable for clinical development to find effective treatment against metastatic breast cancer.