Tigecycline Enhances Sensitivity to FOLFIRINOX and Gemcitabine/Nab-Paclitaxel in In Vitro Models of Pancreatic Ductal Adenocarcinoma

Tigecycline Enhances Sensitivity to FOLFIRINOX and Gemcitabine/Nab-Paclitaxel in In Vitro Models of Pancreatic Ductal Adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tigecycline Enhances Sensitivity to FOLFIRINOX and Gemcitabine/Nab-Paclitaxel in In Vitro Models of Pancreatic Ductal Adenocarcinoma Jana Sabová, Lenka Varinská, Lukáš Urban, Peter Gál, Matúš Čoma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9084399/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies and remains highly resistant to systemic chemotherapy. Tigecycline, a glycylcycline antibiotic that inhibits mitochondrial translation, has recently attracted attention as a potential candidate for drug repurposing in oncology because of its proposed chemosensitizing effects. In this study, we investigated the cytotoxic and chemosensitizing effects of tigecycline in combination with the clinically used PDAC chemotherapy regimens FOLFIRINOX and gemcitabine plus nab-paclitaxel (Gem-Pac). Experiments were performed using both 2D monolayer cultures and 3D spheroid models of four PDAC cell lines (PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2). Cell viability and proliferation were assessed using MTS, BrdU incorporation, and CellTiter-Glo® 3D assays, and spheroid growth dynamics were quantified by imaging-based area measurements after 3 days of treatment. Tigecycline showed limited cytotoxicity as a single agent but moderately enhanced the antiproliferative effects of FOLFIRINOX in a cell-line-dependent manner, while a more pronounced and consistent chemosensitizing effect was observed in combination with Gem-Pac, particularly in PaTu-8902 and PANC-1 models. These effects were reproducible across independent assays and were most evident in 3D spheroids, where tigecycline markedly suppressed chemotherapy-induced spheroid growth. Overall, these findings demonstrate that tigecycline can enhance the efficacy of clinically relevant PDAC chemotherapy regimens in vitro and support its further preclinical evaluation as a potential adjunct to existing PDAC treatment strategies. PDAC gemcitabine nab-paclitaxel chemoresistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Pancreatic ductal adenocarcinoma (PDAC) represents the predominant form of pancreatic cancer, accounting for approximately 90% of cases, and is characterized by aggressive behavior and poor clinical outcomes. The five-year survival rate remains below 10%, making PDAC one of the leading causes of cancer-related mortality in Western countries. Early stages of the disease are often clinically silent, frequently resulting in delayed diagnosis. Consequently, most patients present with locally advanced tumors, vascular involvement, distant metastases, or unresectable disease, with only 15–20% of patients eligible for potentially curative treatments such as radical surgery combined with chemotherapy [ 1 ]. For decades, the nucleoside analogue gemcitabine served as the standard therapy for unresectable PDAC, although its impact on overall survival (OS) was limited [ 2 ]. A major advancement in treatment occurred with the introduction of the FOLFIRINOX regimen, which combines folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin, and has been shown to significantly improve survival in patients with metastatic PDAC, achieving a median OS of 11.1 months compared to 6.8 months for gemcitabine monotherapy [ 3 ]. In 2013, the Metastatic Pancreatic Adenocarcinoma Trial demonstrated that nab-paclitaxel plus gemcitabine also conferred a survival benefit, with median OS of 8.5 months versus 6.7 months for gemcitabine monotherapy [ 4 ]. Despite these advances, both FOLFIRINOX and gemcitabine–nab-paclitaxel regimens are associated with substantial toxicity, requiring careful patient selection. Moreover, real-clinical outcomes often fail to reproduce the survival rates reported in controlled phase III trials [ 5 ]. Tigecycline, a broad-spectrum glycylcycline antibiotic originally developed to overcome tetracycline resistance in bacterial infections [ 6 ], has recently attracted attention beyond its antimicrobial role due to its ability to inhibit mitochondrial translation and disrupt cancer cell metabolism, positioning it as a promising candidate for oncologic repurposing [ 7 ]. Numerous studies have demonstrated that tigecycline exerts potent antitumor effects across a range of malignancies, including oral squamous cell carcinoma, melanoma, glioma, hepatocellular carcinoma, and colorectal carcinoma [ 8 – 12 ]. In PDAC, tigecycline has been shown to suppress cell proliferation, migration, and invasion by inducing cell cycle arrest and inhibiting epithelial–mesenchymal transition, partly through downregulation of CCNE2, and to enhance the chemosensitivity of PDAC cells to gemcitabine [ 13 ]. Given the limited progress in PDAC therapy, combining novel agents with established regimens represents a promising strategy to improve outcomes. Building on prior evidence that tigecycline enhances the activity of gemcitabine, we investigated its effects in combination with the standard regimens Gem-Pac and FOLFIRINOX using complementary 2D monolayer and 3D spheroid in in vitro PDAC models. 2 Methods 2.1 Cell lines and culture conditions The human PDAC cell lines PANC-1, MIAPaCa-2, and PaTu-8902 were kindly provided by prof. Libor Vítek (Institute of Medical Biochemistry and Laboratory Diagnostics, Charles University, Prague, Czech Republic). Capan-2 cell line was purchased from ATCC (CRL-1469; Manassas, VA, USA). Capan-2 cells were cultured in McCoy’s 5A medium (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS; Cytiva, Marlborough, MA, USA) and 1% penicillin/streptomycin (ATB; Biochrom, Berlin, Germany) at 37°C in a humidified atmosphere containing 5% CO 2 . MIAPaCa-2, PaTu-8902, and PANC-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Cytiva, Marlborough, MA, USA) supplemented with 10% FBS and 1% ATB penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 . 2.2 Spheroid preparation Spheroids were generated in round-bottom 96-well plates using medium supplemented with methylcellulose (MC). To prepare the MC stock solution, 6 g of MC powder (Sigma-Aldrich, St. Louis, MO, USA) were dispersed in 250 mL of basal medium preheated to 60°C and mixed for 20 min. Subsequently, 250 mL of room-temperature basal medium containing 20% FBS was added to a final volume of 500 mL, and the suspension was mixed overnight at 4°C. The stock was aliquoted and clarified by centrifugation (5,000 × g, 2 h, room temperature). Only the clear, highly viscous supernatant (≈ 90–95% of the stock) was used for the spheroid assay. For spheroid formation, the working medium comprised 20% MC stock and 80% culture medium, yielding a final MC concentration of 0.24%. For each well, 5 x 10 2 cells (PANC-1 or PaTu-8902) were suspended in the MC-containing medium, seeded into ultra-low attachment 96-well round-bottom plates (Corning Inc., Corning, NY, USA), then cultured for 72 h under standard conditions (37°C, 5% CO₂) to allow spheroid formation. Plates were removed from the incubator daily for imaging to monitor spheroid formation. 2.3 FOLFIRINOX, Gem-pac, and tigecycline treatment preparation Tigecycline (Tygacil; Pfizer, New York, NY, USA), gemcitabine, irinotecan, 5-fluorouracil (5-FU), and leucovorin (all from Merck, Darmstadt, Germany), nab-paclitaxel (Abraxane; Bristol-Myers Squibb Pharma, New York, NY, USA), and oxaliplatin (Tocris Bioscience, Bristol, UK) were used in this study. Stock solutions were prepared in dimethyl sulfoxide (DMSO) or purified water, according to solubility, and stored at − 80°C until further use. For combination studies, clinically relevant molar ratios were applied. The FOLFIRINOX mixture was prepared at 1.00 irinotecan : 80.95 5-FU : 0.80 oxaliplatin : 1.07 leucovorin, while the Gemcitabine–Paclitaxel (Gem-Pac) regimen was prepared at 1.00 gemcitabine : 0.04 paclitaxel, reflecting dose equivalents used in patient treatment regimens [ 4 , 14 ]. Tigecycline was used at a concentration of 10 µg/mL, selected based on MTS assay results (Fig. 1 ), which indicated measurable effects in several, though not all, PDAC cell lines. This concentration was chosen as a biologically relevant dose that allowed assessment of potential synergistic interactions with standard chemotherapeutic regimens, and it is consistent with previously reported effective ranges in pancreatic cancer models [ 13 ]. Cell viability of PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2 cell lines following 72-hour exposure to increasing concentrations of tigecycline (250 ng/mL–200 µg/mL) was assessed using the MTS assay. Data represent mean ± SD from three independent experiments 2.4 MTS-assay Cell viability was assessed using the MTS assay (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt), which is reduced by cellular dehydrogenases to a soluble formazan product. Pancreatic cancer cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and incubated for 24 h under standard conditions (37°C, 5% CO₂). Cells were then exposed to FOLFIRINOX or Gem-Pac regimens (each tested independently), tigecycline alone, or tigecycline in combination with FOLFIRINOX or Gem-Pac, at the indicated concentration ranges. After 72 h of treatment, MTS reagent was added directly to the wells according to the manufacturer’s instructions, and plates were incubated for an additional 2 h. Absorbance corresponding to the amount of soluble formazan was measured at 490 nm using a Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Cell viability was expressed as a percentage relative to untreated control cells. 2.5 BrdU-assay The antiproliferative effects of chemotherapeutic treatments on studied cell lines were assessed using a BrdU colorimetric assay (Roche Diagnostics GmbH, Mannheim, Germany). Pancreatic cancer cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and allowed to attach for 24 h at 37°C in 5% CO₂. Cells were then treated with FOLFIRINOX or Gem-Pac regimens (each tested independently), tigecycline alone, or tigecycline in combination with FOLFIRINOX or Gem-Pac, at the indicated concentration ranges. BrdU labeling solution (1:300) was added approximately 12 h prior to the end of the 72 h treatment period to allow incorporation into newly synthesized DNA. Cells were subsequently fixed, DNA was denatured, and incorporated BrdU was detected with a peroxidase-conjugated anti-BrdU antibody according to the manufacturer’s instructions. Color development was achieved using TMB substrate and stopped with 1 M H₂SO₄, producing a yellow endpoint. Absorbance was recorded at 450 nm with a Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Cell proliferation was expressed as a percentage relative to untreated control cells. 2.6 Cell viability assay To evaluate treatment effects on 3D tumor growth, the CellTiter-Glo® 3D Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used according to the manufacturer’s instructions. Briefly, prepared spheroids were treated for 72 h with FOLFIRINOX, Gem-Pac regimens (each regimen tested separately), tigecycline alone, or tigecycline in combination with FOLFIRINOX or Gem-Pac, at the indicated concentrations. After treatment, an equal volume of CellTiter-Glo® 3D reagent was added directly to each well. Plates were shaken for 5 min to facilitate spheroid disruption and ATP release and incubated at room temperature for 25 min to stabilize the luminescent signal. Luminescence was measured using a Cytation™ 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA). Spheroids viability was expressed as a percentage relative to untreated control spheroids. 2.7 Microscopical Analysis of Spheroids Phase-contrast images of spheroids were acquired using a Cytation imaging reader (BioTek Instruments) at defined time points (day 3 and day 6) and analyzed using the open-source software Fiji through a semi-automated macro script generated with the Macro Recorder function. Images were converted to 8-bit grayscale, and brightness/contrast was adjusted uniformly across all samples. Spheroid regions of interest (ROIs) were segmented using the Otsu dark thresholding method to define the total spheroid area. Noise reduction (Despeckle) and gap filling (Fill Holes) were applied automatically within the macro. Particle analysis was performed using the Analyze Particles tool (size = 11 000–∞ px²; circularity = 0.1–1.0) while excluding objects touching image borders. The measured pixel area was converted to µm² according to the microscope calibration (1 pixel = 1.61 µm). For each spheroid, the fold change (Fc) in area was calculated as relative change between day 3 and day 6 (FcArea = Area₆ / Area₃). This approach allowed us to eliminate the influence of initial size differences and objectively compare the treatment effects between groups. 2.8 Statistical analysis Statistical analyses were performed using GraphPad Prism (version 9.0.0, La Jolla, CA, USA). Differences between treatments were evaluated by two-way analysis of variance (ANOVA) followed by multiple comparisons within each row to assess the effect of tigecycline addition on responses to each chemotherapy regimen and concentration. Results are presented as mean ± standard deviation (SD) from at least three independent experiments, each performed in triplicate. A p-value < 0.05 was considered statistically significant. 3 Results 3.1 Effect of tigecycline on cell viability determined by MTS assay To assess the impact of tigecycline on the cytotoxicity of standard PDAC chemotherapy regimens, four pancreatic cancer cell lines (PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2) were treated with FOLFIRINOX (Fig. 2 a) or Gem-Pac (Fig. 2 b), either alone or in combination with tigecycline, for 72 hours. Treatment with FOLFIRINOX alone resulted in a reduction of cell viability only at higher concentrations across all tested cell lines, indicating limited sensitivity to this regimen under standard conditions. Similarly, Gem-Pac treatment displayed only modest cytotoxicity, with a significant reduction in viability observed primarily in Capan-2 cells, while PaTu-8902, PANC-1, and MIAPaCa-2 cells remained largely resistant. The addition of tigecycline markedly enhanced the cytotoxic response to both regimens. In combination with FOLFIRINOX, tigecycline significantly decreased cell viability at multiple concentrations in all tested lines. When combined with Gem-Pac, tigecycline converted otherwise chemoresistant cell lines (PaTu-8902, PANC-1, and MIAPaCa-2) into more chemosensitive phenotypes, leading to a substantial loss of viability. Notably, tigecycline alone resulted in a significant loss of viability in PaTu-8902 and Capan-2 cells, and this effect was further intensified when combined with either FOLFIRINOX or Gem-Pac. Cell viability of PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2 cells was assessed by MTS assay after 72 h of treatment with (A) FOLFIRINOX or (B) Gem-Pac, either alone (yellow) or in combination with tigecycline (blue). Data represent mean ± SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001). 3.2 Effect of tigecycline on cell proliferation determined by BrdU assay To further validate the antiproliferative effect of tigecycline in combination with standard chemotherapeutic regimens, BrdU incorporation assays were performed under identical treatment conditions as the MTS assay. PDAC cell lines were exposed to FOLFIRINOX (Fig. 3 a) or Gem-Pac (Fig. 3 b), either alone or in combination with tigecycline, for a total of 72 hours, with BrdU labeling added during the final 12 hours of incubation. Consistent with viability measurements, treatment with FOLFIRINOX resulted in measurable, dose-dependent reduction in proliferation in MIAPaCa-2 and Capan-2 cells, particularly at higher concentrations. In contrast, PaTu-8902 and PANC-1 cells demonstrated relative resistance, showing minimal proliferation decrease even at elevated doses. Tigecycline alone had minimal effect on DNA synthesis across all cell lines; however, when combined with FOLFIRINOX, a further reduction in proliferation was observed—most noticeably in PaTu-8902 and MIAPaCa-2 cells. In PANC-1 and Capan-2 cells, this enhancement remained modest. Under Gem-Pac treatment, PaTu-8902, PANC-1, and MIAPaCa-2 cells displayed substantial sensitivity, with reduced proliferation observed even at the lowest tested concentrations, while Capan-2 cells followed a more gradual dose-dependent response. The addition of tigecycline further reduced BrdU incorporation across all Gem-Pac–treated groups; however, similar to the FOLFIRINOX condition, this effect represented a visible trend rather than a consistently statistically significant enhancement. BrdU incorporation was measured after 72 hours in pancreatic cancer cell lines (PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2) treated with FOLFIRINOX (A) or Gem-Pac (B), either alone (yellow) or in combination with tigecycline (blue). Data represent mean ± SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001). 3.3 Viability of PDAC spheroids assessed by CellTiter-Glo assay To further assess treatment efficacy under conditions that better approximate the in vivo tumor architecture, 3D spheroid models were generated from PDAC cell lines. Compact and stable spheroids were successfully generated from PANC-1 and PaTu-8902 cells. In contrast, Capan-2 and MIAPaCa-2 cells produced only loose aggregates that disintegrated upon manipulation, preventing their use in subsequent analysis (data not shown). Cell viability within the spheroids was assessed using the CellTiter-Glo® 3D assay following 72 h of exposure to FOLFIRINOX (Fig. 4 a) or Gem-Pac (Fig. 4 b) with or without tigecycline. FOLFIRINOX treatment induced a reduction in viability only in PANC-1 spheroids, and this effect became apparent only at higher concentrations, whereas PaTu-8902 spheroids remained largely resistant across the tested range. Tigecycline alone resulted in a modest decrease in viability in both cell models; however, this reduction was observed as a trend and did not reach statistical significance. When combined with FOLFIRINOX, tigecycline produced a significant viability decline in PaTu-8902 spheroids, while the effect in PANC-1 remained limited and did not differ substantially from FOLFIRINOX alone. In contrast, Gem-Pac treatment reduced viability in PaTu-8902 spheroids in dose-dependent manner, PANC-1 spheroids demonstrated a more resistant response profile. Importantly, co-treatment with tigecycline markedly potentiated the effects of Gem-Pac, resulting in a substantial decrease in viability across all tested doses. These findings indicate that tigecycline exerts a synergistic effect with Gem-Pac, particularly in metabolically active 3D PDAC models, while its impact in combination with FOLFIRINOX remains comparatively modest. Spheroids derived from PaTu-8902 and PANC-1 cells were treated with FOLFIRINOX (A) or Gem-Pac (B), either alone (yellow) or in combination with tigecycline (green). Viability was measured after 72 hours using the CellTiter-Glo luminescence assay. Data represent mean ± SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001). 3.4 Effect of FOLFIRINOX and Gem-Pac treatments on spheroid growth dynamics To assess growth dynamics in 3D models, spheroids derived from PaTu-8902 and PANC-1 were exposed to FOLFIRINOX (Fig. 5 a) or Gem-Pac (Fig. 5 b) treatment, either alone or in combination with tigecycline. Changes in spheroid size were monitored over time, and the fold change in spheroid area between day 3 and day 6 was calculated to quantify treatment-induced alterations in spheroid size. Representative images of PaTu-8902 and PANC-1 spheroids treated with increasing concentrations of FOLFIRINOX or Gem-Pac, in the presence or absence of tigecycline, are presented in Figs. 6 and Fig. 7 , illustrating treatment-induced changes in spheroid morphology and growth dynamics. Under FOLFIRINOX treatment, PaTu-8902 spheroids showed a dose-dependent reduction in expansion, while PANC-1 spheroids remained largely unresponsive, indicating resistance across most tested concentrations. Tigecycline alone resulted in spheroid growth reduction in both cell lines reaching statistical significance in PaTu-8902 spheroids. Notably, combining tigecycline with FOLFIRINOX further suppressed spheroid growth, with a significant effect observed only in PaTu-8902 spheroids, whereas PANC-1 spheroids again showed limited sensitivity. Similarly, Gem-Pac treatment displayed a dose-dependent inhibition of spheroid growth in PaTu-8902 cells, while PANC-1 spheroids maintained high growth rates, indicating chemoresistance. Remarkably, co-treatment with tigecycline profoundly impaired PaTu-8902 spheroid expansion, effectively halting growth even at the lowest concentrations of Gem-Pac. Together, these results confirm that tigecycline exerts a measurable chemosensitizing effect in 3D PDAC models, most prominently in PaTu-8902 spheroids and particularly under Gem-Pac treatment. 4 Discussion In the present study, we provide the first systematic evaluation of tigecycline in combination with the two most widely used chemotherapy regimens for PDAC, FOLFIRINOX and Gem-Pac. Using complementary 2D monolayer and 3D spheroid PDAC models, we demonstrate that tigecycline consistently increases chemosensitivity and enhances the efficacy of both standard treatments. Our results also indicate that PDAC cell lines that were otherwise refractory to conventional chemotherapy displayed a marked improvement in treatment response upon co-treatment with tigecycline, highlighting its potential role as a chemosensitizing adjunct in resistant disease settings. When comparing responses across tested PDAC cell lines, distinct sensitivity profiles to standard chemotherapies were evident. Consistent with previous reports [ 15 , 16 ], Capan-2 and MIAPaCa-2 cells displayed greater sensitivity to Gem-Pac treatment, whereas PaTu-8902 and PANC-1 cells exhibited pronounced chemoresistance to both regimens. Importantly, tigecycline enhanced the efficacy of both regimens, with the most substantial improvement observed in the Gem-Pac–treated PaTu-8902 cells, even at lower concentrations of chemotherapy. These response patterns closely reflect differences in epithelial–mesenchymal transition (EMT) status and metabolic phenotype among the cell lines [ 17 ]. Capan-2 cells retain a predominantly epithelial-like state, characterized by higher E-cadherin expression and lower levels of mesenchymal markers such as vimentin, ZEB1, and SNAIL, which has been associated with increased susceptibility to cytotoxic therapies [ 18 ]. In contrast, PaTu-8902 and PANC-1 exhibit a mesenchymal, EMT-enriched phenotype, with elevated expression of vimentin, N-cadherin, and EMT transcription factors, accompanied by enhanced migratory capacity, resistance to apoptosis, and increased metabolic flexibility [ 19 ]. EMT has been linked to mitochondrial reprogramming and a shift toward oxidative phosphorylation–dependent energy production, enabling tumor cells to adapt to nutrient deprivation and chemotherapeutic stress [ 20 ]. These mesenchymal, metabolically adaptable cells also display stronger interactions with cancer-associated fibroblasts within the desmoplastic tumor microenvironment, further promoting therapy resistance [ 21 ]. The ability of tigecycline to inhibit mitochondrial translation may therefore preferentially disrupt this EMT-associated metabolic dependency, rendering otherwise chemoresistant PDAC cells more vulnerable to standard chemotherapy. Thus, the observed chemosensitizing effect of tigecycline is mechanistically consistent with the EMT-driven metabolic state of resistant PDAC cell lines, providing a functional link between tumor cell plasticity, mitochondrial metabolism, and treatment response. Across assays, combination treatment generally reduced cell viability/proliferation, minor discrepancies were noted between metabolic and proliferation-based readouts. These differences likely reflect the distinct cellular processes each assay measures: MTS primarily captures mitochondrial activity [ 22 ], while BrdU incorporation directly indicates DNA synthesis [ 23 ], and CellTiter-Glo quantifies ATP levels as a measure of metabolic viability in 3D systems [ 24 ]. The observed variations may therefore arise from metabolic reprogramming induced by tigecycline [ 25 ], which can reduce mitochondrial function [ 26 ] prior to detectable decreases in proliferation or viability. The results obtained from 3D spheroid models provide additional insight into the potential of tigecycline as a chemosensitizing agent under more physiologically relevant conditions. Compared to conventional 2D cultures, spheroids better mimic the tumor microenvironment, including nutrient gradients, hypoxia, and cellular heterogeneity—factors known to drive drug resistance in PDAC [ 27 ]. In these 3D systems, FOLFIRINOX and Gem-Pac alone exhibited only modest efficacy on PANC-1 cells, while PaTu-8902 spheroids showed a stronger cytotoxic response. Importantly, co-treatment with tigecycline substantially enhanced the cytotoxic effects of FOLFIRNOX and Gem-Pac, leading to a pronounced decrease in PaTu-8902 viability and practically complete suppression of spheroid growth even at lower concentrations of chemotherapy. The observed chemosensitizing effects of tigecycline may stem from its interference with mitochondrial metabolism, potentially limiting oxidative phosphorylation, reducing energy production, and thereby weakening the metabolic adaptability of PDAC cells—an effect more evident in 3D spheroid models that more closely mimic in vivo tumor conditions. Although preliminary, this metabolic vulnerability represents a plausible mechanistic basis for the observed chemosensitization and warrants further detailed investigation. Several limitations should be acknowledged. The present study focused on in vitro systems, which do not fully capture the complexity of tumor–stroma interactions, drug pharmacokinetics, or immune modulation in vivo [ 28 ]. Future work should therefore aim to validate these findings in animal models of PDAC, incorporating pharmacodynamic and metabolic profiling to clarify how tigecycline modulates tumor energy metabolism in the context of combination therapy. Such preclinical studies will be essential to to determine an appropriate therapeutic window in vivo that maximizes chemosensitization while minimizing systemic toxicity arising from combination therapy, potentially improving therapeutic outcomes in this highly aggressive and chemoresistant malignancy. 5 Conclusion This study demonstrates that tigecycline markedly enhances the efficacy of Gem-Pac in PDAC, with pronounced chemosensitization observed particularly in chemoresistant PaTu-8902 models. Enhancement in combination with FOLFIRINOX was cell-line dependent and less pronounced. In 3D spheroid systems that more closely recapitulate the in vivo tumor microenvironment, co-treatment with tigecycline led to marked reductions in viability and spheroid growth, reinforcing its potential as a chemosensitizing agent. Although preliminary, these results pave the way for future preclinical studies aimed at validating tigecycline usage as a strategy to enhance chemosensitivity in PDAC. Declarations Conflicts of Interest The authors declare no competing interest. Funding This work was supported by Fellowships for Excellent Researchers R2, funded by the European Union NextGenerationEU through the Recovery and Resilience Plan for Slovakia under project no. 09I03-03-V04-00074. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by J.S., L.V., L.U. and M.Č. The first draft of the manuscript was written by P.G. and M.Č. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors are grateful to Martina Hájiková for her excellent technical assistance in the cell culture laboratory. Data availability No datasets were generated or analyzed during the current study. References Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer Statistics, 2021. 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Anticancer Res. 2020;40(7):3659–67. doi: 10.21873/anticanres.14355 . Zhao B, Qin C, Li Z, Wang Y, Li T, Cao H, et al. Multidrug resistance genes screening of pancreatic ductal adenocarcinoma based on sensitivity profile to chemotherapeutic drugs. Cancer Cell Int. 2022;22(1):374. doi: 10.1186/s12935-022-02785-7 . Allen-Coyle TJ, Niu J, Welsch E, Conlon NT, Garner W, Clynes M, et al. FOLFIRINOX Pharmacodynamic Interactions in 2D and 3D Pancreatic Cancer Cell Cultures. AAPS J. 2022;24(6):108. doi: 10.1208/s12248-022-00752-8 . Novak S, Kolar M, Szabo A, Vernerova Z, Lacina L, Strnad H, et al. Desmoplastic Crosstalk in Pancreatic Ductal Adenocarcinoma Is Reflected by Different Responses of Panc-1, MIAPaCa-2, PaTu-8902, and CAPAN-2 Cell Lines to Cancer-associated/Normal Fibroblasts. Cancer Genomics Proteomics. 2021;18(3):221–43. doi: 10.21873/cgp.20254 . Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature. 2015;527(7579):525–30. doi: 10.1038/nature16064 . Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL, et al. Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer. Cancer Res. 2009;69(14):5820–8. doi: 10.1158/0008-5472.CAN-08-2819 . Liu C, Li C, Liu Y. The role of metabolic reprogramming in pancreatic cancer chemoresistance. Front Pharmacol. 2022;13:1108776. doi: 10.3389/fphar.2022.1108776 . Bulle A, Lim KH. Beyond just a tight fortress: contribution of stroma to epithelial-mesenchymal transition in pancreatic cancer. Signal Transduct Target Ther. 2020;5(1):249. doi: 10.1038/s41392-020-00341-1 . Malich G, Markovic B, Winder C. The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology. 1997;124(3):179–92. doi: 10.1016/s0300-483x(97)00151-0 . Yu J, Wang Z, Wang Y. BrdU Incorporation Assay to Analyze the Entry into S Phase. Methods Mol Biol. 2022;2579:209–26. doi: 10.1007/978-1-0716-2736-5_16 . Idrees A, Chiono V, Ciardelli G, Shah S, Viebahn R, Zhang X, et al. Validation of in vitro assays in three-dimensional human dermal constructs. Int J Artif Organs. 2018;41(11):779–88. doi: 10.1177/0391398818775519 . Messner M, Schmitt S, Ardelt MA, Frohlich T, Muller M, Pein H, et al. Metabolic implication of tigecycline as an efficacious second-line treatment for sorafenib-resistant hepatocellular carcinoma. FASEB J. 2020;34(9):11860–82. doi: 10.1096/fj.202001128R . Vandecasteele SJ, Seneca S, Smet J, Reynders M, De Ceulaer J, Vanlander AV, et al. Tigecycline-induced inhibition of mitochondrial DNA translation may cause lethal mitochondrial dysfunction in humans. Clin Microbiol Infect. 2018;24(4):431 e1- e3. doi: 10.1016/j.cmi.2017.08.018 . Fontoura JC, Viezzer C, Dos Santos FG, Ligabue RA, Weinlich R, Puga RD, et al. Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance. Mater Sci Eng C Mater Biol Appl. 2020;107:110264. doi: 10.1016/j.msec.2019.110264 . Saeidnia S, Manayi A, Abdollahi M. From in vitro Experiments to in vivo and Clinical Studies; Pros and Cons. Curr Drug Discov Technol. 2015;12(4):218–24. doi: 10.2174/1570163813666160114093140 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 14 Apr, 2026 Reviews received at journal 14 Apr, 2026 Reviews received at journal 27 Mar, 2026 Reviewers agreed at journal 26 Mar, 2026 Reviewers agreed at journal 25 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers invited by journal 13 Mar, 2026 Editor assigned by journal 11 Mar, 2026 Submission checks completed at journal 11 Mar, 2026 First submitted to journal 10 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9084399","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":606365786,"identity":"a4bdbcc1-2ff7-4dc8-99d6-a07ea48713cf","order_by":0,"name":"Jana Sabová","email":"","orcid":"","institution":"Pavol Jozef Šafárik University","correspondingAuthor":false,"prefix":"","firstName":"Jana","middleName":"","lastName":"Sabová","suffix":""},{"id":606365787,"identity":"0d9f6d7b-c8fe-49ef-8452-521a92514260","order_by":1,"name":"Lenka Varinská","email":"","orcid":"","institution":"Pavol Jozef Šafárik University","correspondingAuthor":false,"prefix":"","firstName":"Lenka","middleName":"","lastName":"Varinská","suffix":""},{"id":606365788,"identity":"af4927de-63b3-41b5-b90b-65e6c9b7be15","order_by":2,"name":"Lukáš Urban","email":"","orcid":"","institution":"Pavol Jozef Šafárik University","correspondingAuthor":false,"prefix":"","firstName":"Lukáš","middleName":"","lastName":"Urban","suffix":""},{"id":606365789,"identity":"c37676cb-6950-48ef-b82b-5f876af0aa37","order_by":3,"name":"Peter Gál","email":"","orcid":"","institution":"Pavol Jozef Šafárik University","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Gál","suffix":""},{"id":606365790,"identity":"693f2d29-b42a-4048-a74b-eaa3a5e9895f","order_by":4,"name":"Matúš Čoma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYHAD5gMMDDwMEExAJYzBlkCyFh4DKE1Ag277+WOSPxjs5HTbz3yTLpCpY5DvOYBfi9mZZDZpHoZkY7MzudukZ/AcZjA420BAywGgFgaGA4nbDgC18PAcYDDgJ+Aws/OP2YAOA2o5/+YZUAvQYf2EtNxIZpPgAWm5kQN0IQ8wMAg67MZjY2seA6Bfbjwztgb6hcfgzAFCDkt8ePNHhZ2c2fnkh7cLe+rk5HsSCLgMDKAxwszYQ0REogBmhh+kaRgFo2AUjIKRAQDMAzxo46FJIQAAAABJRU5ErkJggg==","orcid":"","institution":"Pavol Jozef Šafárik University","correspondingAuthor":true,"prefix":"","firstName":"Matúš","middleName":"","lastName":"Čoma","suffix":""}],"badges":[],"createdAt":"2026-03-10 13:09:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9084399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9084399/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104876886,"identity":"f0e7e467-db16-479a-bec5-23d232fc36be","added_by":"auto","created_at":"2026-03-18 08:43:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1327172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of tigecycline on PDAC cells viability.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/0deeb0016db736e1a1ddefd6.jpg"},{"id":104876788,"identity":"5496dfb4-dad3-4e8c-a579-7bb0cd652073","added_by":"auto","created_at":"2026-03-18 08:43:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3868250,"visible":true,"origin":"","legend":"\u003cp\u003eMTS assay of PDAC cell lines treated with FOLFIRINOX or Gem-Pac ± tigecycline.\u003c/p\u003e\n\u003cp\u003eCell viability of PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2 cells was assessed by MTS assay after 72 h of treatment with (A) FOLFIRINOX or (B) Gem-Pac, either alone (yellow) or in combination with tigecycline (blue). Data represent mean ± SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ***p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/b43750d797048e9131e567da.jpg"},{"id":104876896,"identity":"f1373821-6637-464d-9716-67beb348de73","added_by":"auto","created_at":"2026-03-18 08:44:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3390870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBrdU assay of PDAC cell proliferation following treatment with FOLFIRINOX or Gem-Pac ± tigecycline.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrdU incorporation was measured after 72 hours in pancreatic cancer cell lines (PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2) treated with FOLFIRINOX (A) or Gem-Pac (B), either alone (yellow) or in combination with tigecycline (blue). Data represent mean ± SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ***p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/7c5c395674e3aa5cbd5cf9c4.jpg"},{"id":104876688,"identity":"e9ad0525-c4b4-4370-b553-2b54c66cc245","added_by":"auto","created_at":"2026-03-18 08:43:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1941256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellTiter-Glo viability assay of PDAC spheroids treated with FOLFIRINOX or Gem-Pac, with or without tigecycline.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpheroids derived from PaTu-8902 and PANC-1 cells were treated with FOLFIRINOX (A) or Gem-Pac (B), either alone (yellow) or in combination with tigecycline (green). Viability was measured after 72 hours using the CellTiter-Glo luminescence assay. Data represent mean ± SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ***p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/f337532e90772f0e40ae8ec5.jpg"},{"id":104876678,"identity":"a2bbd71c-cc9f-4ee7-a90f-df3eaaedec3c","added_by":"auto","created_at":"2026-03-18 08:43:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2091017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpheroid growth dynamics of PDAC spheroids treated with FOLFIRINOX or Gem-Pac in the presence or absence of tigecycline.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpheroid area was quantified as fold change in growth between day 3 and day 6 in PaTu-8902 and PANC-1 spheroids treated with FOLFIRINOX (A) or Gem-Pac (B), either alone (yellow) or in combination with tigecycline (green). Data represent mean ± SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ***p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/951c023428ceffb2424263c0.jpg"},{"id":104876888,"identity":"3b23060b-b93a-433d-9b0a-799f5c5275a8","added_by":"auto","created_at":"2026-03-18 08:43:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4438738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of PDAC spheroids treated with FOLFIRINOX in the presence or absence of tigecycline.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) PaTu-8902 and (b) PANC-1 spheroids cultured in 96-well ultra-low attachment plates and treated with increasing concentrations of FOLFIRINOX (1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, or 1 µM), either alone or in combination with tigecycline (10 µg/mL). Images were captured on days 3 and 6 to monitor treatment-induced changes in spheroid morphology and growth dynamics. Scale bar: 1000 µm.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/c037ffd84814885709e85c1b.jpg"},{"id":104876906,"identity":"87ef08f5-720f-4f73-84a7-c7d1f47c1ffd","added_by":"auto","created_at":"2026-03-18 08:44:04","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4027773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative images of PDAC spheroids treated with gemcitabine plus nab-paclitaxel (Gem-Pac) in the presence or absence of tigecycline.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) PaTu-8902 and (b) PANC-1 spheroids cultured in 96-well ultra-low attachment plates and treated with increasing concentrations of Gem-Pac (10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 5 µM, 10 µM, 50 µM, or 100 µM), either alone or in combination with tigecycline (10 µg/mL). Images were acquired on days 3 and 6 to evaluate treatment-induced changes in spheroid morphology and growth dynamics. Scale bar: 1000 µm.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/7a87b5a5d966d81efe3308ec.jpg"},{"id":104876992,"identity":"9ff55b88-0363-4f57-a84f-7401b8371044","added_by":"auto","created_at":"2026-03-18 08:44:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21982903,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9084399/v1/d08b2bfd-9dfd-4538-a3c1-9ca6b93b7cb1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tigecycline Enhances Sensitivity to FOLFIRINOX and Gemcitabine/Nab-Paclitaxel in In Vitro Models of Pancreatic Ductal Adenocarcinoma","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) represents the predominant form of pancreatic cancer, accounting for approximately 90% of cases, and is characterized by aggressive behavior and poor clinical outcomes. The five-year survival rate remains below 10%, making PDAC one of the leading causes of cancer-related mortality in Western countries. Early stages of the disease are often clinically silent, frequently resulting in delayed diagnosis. Consequently, most patients present with locally advanced tumors, vascular involvement, distant metastases, or unresectable disease, with only 15\u0026ndash;20% of patients eligible for potentially curative treatments such as radical surgery combined with chemotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor decades, the nucleoside analogue gemcitabine served as the standard therapy for unresectable PDAC, although its impact on overall survival (OS) was limited [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A major advancement in treatment occurred with the introduction of the FOLFIRINOX regimen, which combines folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin, and has been shown to significantly improve survival in patients with metastatic PDAC, achieving a median OS of 11.1 months compared to 6.8 months for gemcitabine monotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In 2013, the Metastatic Pancreatic Adenocarcinoma Trial demonstrated that nab-paclitaxel plus gemcitabine also conferred a survival benefit, with median OS of 8.5 months versus 6.7 months for gemcitabine monotherapy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite these advances, both FOLFIRINOX and gemcitabine\u0026ndash;nab-paclitaxel regimens are associated with substantial toxicity, requiring careful patient selection. Moreover, real-clinical outcomes often fail to reproduce the survival rates reported in controlled phase III trials [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTigecycline, a broad-spectrum glycylcycline antibiotic originally developed to overcome tetracycline resistance in bacterial infections [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], has recently attracted attention beyond its antimicrobial role due to its ability to inhibit mitochondrial translation and disrupt cancer cell metabolism, positioning it as a promising candidate for oncologic repurposing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Numerous studies have demonstrated that tigecycline exerts potent antitumor effects across a range of malignancies, including oral squamous cell carcinoma, melanoma, glioma, hepatocellular carcinoma, and colorectal carcinoma [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In PDAC, tigecycline has been shown to suppress cell proliferation, migration, and invasion by inducing cell cycle arrest and inhibiting epithelial\u0026ndash;mesenchymal transition, partly through downregulation of CCNE2, and to enhance the chemosensitivity of PDAC cells to gemcitabine [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the limited progress in PDAC therapy, combining novel agents with established regimens represents a promising strategy to improve outcomes. Building on prior evidence that tigecycline enhances the activity of gemcitabine, we investigated its effects in combination with the standard regimens Gem-Pac and FOLFIRINOX using complementary 2D monolayer and 3D spheroid in \u003cem\u003ein vitro\u003c/em\u003e PDAC models.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell lines and culture conditions\u003c/h2\u003e \u003cp\u003eThe human PDAC cell lines PANC-1, MIAPaCa-2, and PaTu-8902 were kindly provided by prof. Libor V\u0026iacute;tek (Institute of Medical Biochemistry and Laboratory Diagnostics, Charles University, Prague, Czech Republic). Capan-2 cell line was purchased from ATCC (CRL-1469; Manassas, VA, USA). Capan-2 cells were cultured in McCoy\u0026rsquo;s 5A medium (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS; Cytiva, Marlborough, MA, USA) and 1% penicillin/streptomycin (ATB; Biochrom, Berlin, Germany) at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. MIAPaCa-2, PaTu-8902, and PANC-1 cells were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; Cytiva, Marlborough, MA, USA) supplemented with 10% FBS and 1% ATB penicillin/streptomycin at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Spheroid preparation\u003c/h2\u003e \u003cp\u003eSpheroids were generated in round-bottom 96-well plates using medium supplemented with methylcellulose (MC). To prepare the MC stock solution, 6 g of MC powder (Sigma-Aldrich, St. Louis, MO, USA) were dispersed in 250 mL of basal medium preheated to 60\u0026deg;C and mixed for 20 min. Subsequently, 250 mL of room-temperature basal medium containing 20% FBS was added to a final volume of 500 mL, and the suspension was mixed overnight at 4\u0026deg;C. The stock was aliquoted and clarified by centrifugation (5,000 \u0026times; g, 2 h, room temperature). Only the clear, highly viscous supernatant (\u0026asymp;\u0026thinsp;90\u0026ndash;95% of the stock) was used for the spheroid assay. For spheroid formation, the working medium comprised 20% MC stock and 80% culture medium, yielding a final MC concentration of 0.24%. For each well, 5 x 10\u003csup\u003e2\u003c/sup\u003e cells (PANC-1 or PaTu-8902) were suspended in the MC-containing medium, seeded into ultra-low attachment 96-well round-bottom plates (Corning Inc., Corning, NY, USA), then cultured for 72 h under standard conditions (37\u0026deg;C, 5% CO₂) to allow spheroid formation. Plates were removed from the incubator daily for imaging to monitor spheroid formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 FOLFIRINOX, Gem-pac, and tigecycline treatment preparation\u003c/h2\u003e \u003cp\u003eTigecycline (Tygacil; Pfizer, New York, NY, USA), gemcitabine, irinotecan, 5-fluorouracil (5-FU), and leucovorin (all from Merck, Darmstadt, Germany), nab-paclitaxel (Abraxane; Bristol-Myers Squibb Pharma, New York, NY, USA), and oxaliplatin (Tocris Bioscience, Bristol, UK) were used in this study. Stock solutions were prepared in dimethyl sulfoxide (DMSO) or purified water, according to solubility, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e \u003cp\u003eFor combination studies, clinically relevant molar ratios were applied. The FOLFIRINOX mixture was prepared at 1.00 irinotecan : 80.95 5-FU : 0.80 oxaliplatin : 1.07 leucovorin, while the Gemcitabine\u0026ndash;Paclitaxel (Gem-Pac) regimen was prepared at 1.00 gemcitabine : 0.04 paclitaxel, reflecting dose equivalents used in patient treatment regimens [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Tigecycline was used at a concentration of 10 \u0026micro;g/mL, selected based on MTS assay results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which indicated measurable effects in several, though not all, PDAC cell lines. This concentration was chosen as a biologically relevant dose that allowed assessment of potential synergistic interactions with standard chemotherapeutic regimens, and it is consistent with previously reported effective ranges in pancreatic cancer models [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell viability of PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2 cell lines following 72-hour exposure to increasing concentrations of tigecycline (250 ng/mL\u0026ndash;200 \u0026micro;g/mL) was assessed using the MTS assay. Data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 MTS-assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the MTS assay (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt), which is reduced by cellular dehydrogenases to a soluble formazan product. Pancreatic cancer cells were seeded in 96-well plates at a density of 5 \u0026times; 10\u0026sup3; cells per well and incubated for 24 h under standard conditions (37\u0026deg;C, 5% CO₂). Cells were then exposed to FOLFIRINOX or Gem-Pac regimens (each tested independently), tigecycline alone, or tigecycline in combination with FOLFIRINOX or Gem-Pac, at the indicated concentration ranges.\u003c/p\u003e \u003cp\u003eAfter 72 h of treatment, MTS reagent was added directly to the wells according to the manufacturer\u0026rsquo;s instructions, and plates were incubated for an additional 2 h. Absorbance corresponding to the amount of soluble formazan was measured at 490 nm using a Cytation\u0026trade; 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Cell viability was expressed as a percentage relative to untreated control cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 BrdU-assay\u003c/h2\u003e \u003cp\u003eThe antiproliferative effects of chemotherapeutic treatments on studied cell lines were assessed using a BrdU colorimetric assay (Roche Diagnostics GmbH, Mannheim, Germany). Pancreatic cancer cells were seeded into 96-well plates at a density of 5 \u0026times; 10\u0026sup3; cells per well and allowed to attach for 24 h at 37\u0026deg;C in 5% CO₂. Cells were then treated with FOLFIRINOX or Gem-Pac regimens (each tested independently), tigecycline alone, or tigecycline in combination with FOLFIRINOX or Gem-Pac, at the indicated concentration ranges. BrdU labeling solution (1:300) was added approximately 12 h prior to the end of the 72 h treatment period to allow incorporation into newly synthesized DNA. Cells were subsequently fixed, DNA was denatured, and incorporated BrdU was detected with a peroxidase-conjugated anti-BrdU antibody according to the manufacturer\u0026rsquo;s instructions. Color development was achieved using TMB substrate and stopped with 1 M H₂SO₄, producing a yellow endpoint. Absorbance was recorded at 450 nm with a Cytation\u0026trade; 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Cell proliferation was expressed as a percentage relative to untreated control cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell viability assay\u003c/h2\u003e \u003cp\u003eTo evaluate treatment effects on 3D tumor growth, the CellTiter-Glo\u0026reg; 3D Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used according to the manufacturer\u0026rsquo;s instructions. Briefly, prepared spheroids were treated for 72 h with FOLFIRINOX, Gem-Pac regimens (each regimen tested separately), tigecycline alone, or tigecycline in combination with FOLFIRINOX or Gem-Pac, at the indicated concentrations. After treatment, an equal volume of CellTiter-Glo\u0026reg; 3D reagent was added directly to each well. Plates were shaken for 5 min to facilitate spheroid disruption and ATP release and incubated at room temperature for 25 min to stabilize the luminescent signal. Luminescence was measured using a Cytation\u0026trade; 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA). Spheroids viability was expressed as a percentage relative to untreated control spheroids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Microscopical Analysis of Spheroids\u003c/h2\u003e \u003cp\u003ePhase-contrast images of spheroids were acquired using a Cytation imaging reader (BioTek Instruments) at defined time points (day 3 and day 6) and analyzed using the open-source software Fiji through a semi-automated macro script generated with the Macro Recorder function. Images were converted to 8-bit grayscale, and brightness/contrast was adjusted uniformly across all samples. Spheroid regions of interest (ROIs) were segmented using the Otsu dark thresholding method to define the total spheroid area. Noise reduction (Despeckle) and gap filling (Fill Holes) were applied automatically within the macro. Particle analysis was performed using the Analyze Particles tool (size\u0026thinsp;=\u0026thinsp;11 000\u0026ndash;\u0026infin; px\u0026sup2;; circularity\u0026thinsp;=\u0026thinsp;0.1\u0026ndash;1.0) while excluding objects touching image borders. The measured pixel area was converted to \u0026micro;m\u0026sup2; according to the microscope calibration (1 pixel\u0026thinsp;=\u0026thinsp;1.61 \u0026micro;m). For each spheroid, the fold change (Fc) in area was calculated as relative change between day 3 and day 6 (FcArea\u0026thinsp;=\u0026thinsp;Area₆ / Area₃). This approach allowed us to eliminate the influence of initial size differences and objectively compare the treatment effects between groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism (version 9.0.0, La Jolla, CA, USA). Differences between treatments were evaluated by two-way analysis of variance (ANOVA) followed by multiple comparisons within each row to assess the effect of tigecycline addition on responses to each chemotherapy regimen and concentration. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three independent experiments, each performed in triplicate. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of tigecycline on cell viability determined by MTS assay\u003c/h2\u003e \u003cp\u003eTo assess the impact of tigecycline on the cytotoxicity of standard PDAC chemotherapy regimens, four pancreatic cancer cell lines (PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2) were treated with FOLFIRINOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) or Gem-Pac (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), either alone or in combination with tigecycline, for 72 hours.\u003c/p\u003e \u003cp\u003eTreatment with FOLFIRINOX alone resulted in a reduction of cell viability only at higher concentrations across all tested cell lines, indicating limited sensitivity to this regimen under standard conditions. Similarly, Gem-Pac treatment displayed only modest cytotoxicity, with a significant reduction in viability observed primarily in Capan-2 cells, while PaTu-8902, PANC-1, and MIAPaCa-2 cells remained largely resistant.\u003c/p\u003e \u003cp\u003eThe addition of tigecycline markedly enhanced the cytotoxic response to both regimens. In combination with FOLFIRINOX, tigecycline significantly decreased cell viability at multiple concentrations in all tested lines. When combined with Gem-Pac, tigecycline converted otherwise chemoresistant cell lines (PaTu-8902, PANC-1, and MIAPaCa-2) into more chemosensitive phenotypes, leading to a substantial loss of viability. Notably, tigecycline alone resulted in a significant loss of viability in PaTu-8902 and Capan-2 cells, and this effect was further intensified when combined with either FOLFIRINOX or Gem-Pac.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell viability of PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2 cells was assessed by MTS assay after 72 h of treatment with (A) FOLFIRINOX or (B) Gem-Pac, either alone (yellow) or in combination with tigecycline (blue). Data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of tigecycline on cell proliferation determined by BrdU assay\u003c/h2\u003e \u003cp\u003eTo further validate the antiproliferative effect of tigecycline in combination with standard chemotherapeutic regimens, BrdU incorporation assays were performed under identical treatment conditions as the MTS assay. PDAC cell lines were exposed to FOLFIRINOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) or Gem-Pac (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), either alone or in combination with tigecycline, for a total of 72 hours, with BrdU labeling added during the final 12 hours of incubation.\u003c/p\u003e \u003cp\u003eConsistent with viability measurements, treatment with FOLFIRINOX resulted in measurable, dose-dependent reduction in proliferation in MIAPaCa-2 and Capan-2 cells, particularly at higher concentrations. In contrast, PaTu-8902 and PANC-1 cells demonstrated relative resistance, showing minimal proliferation decrease even at elevated doses. Tigecycline alone had minimal effect on DNA synthesis across all cell lines; however, when combined with FOLFIRINOX, a further reduction in proliferation was observed\u0026mdash;most noticeably in PaTu-8902 and MIAPaCa-2 cells. In PANC-1 and Capan-2 cells, this enhancement remained modest.\u003c/p\u003e \u003cp\u003eUnder Gem-Pac treatment, PaTu-8902, PANC-1, and MIAPaCa-2 cells displayed substantial sensitivity, with reduced proliferation observed even at the lowest tested concentrations, while Capan-2 cells followed a more gradual dose-dependent response. The addition of tigecycline further reduced BrdU incorporation across all Gem-Pac\u0026ndash;treated groups; however, similar to the FOLFIRINOX condition, this effect represented a visible trend rather than a consistently statistically significant enhancement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBrdU incorporation was measured after 72 hours in pancreatic cancer cell lines (PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2) treated with FOLFIRINOX (A) or Gem-Pac (B), either alone (yellow) or in combination with tigecycline (blue). Data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Viability of PDAC spheroids assessed by CellTiter-Glo assay\u003c/h2\u003e \u003cp\u003eTo further assess treatment efficacy under conditions that better approximate the \u003cem\u003ein vivo\u003c/em\u003e tumor architecture, 3D spheroid models were generated from PDAC cell lines. Compact and stable spheroids were successfully generated from PANC-1 and PaTu-8902 cells. In contrast, Capan-2 and MIAPaCa-2 cells produced only loose aggregates that disintegrated upon manipulation, preventing their use in subsequent analysis (data not shown). Cell viability within the spheroids was assessed using the CellTiter-Glo\u0026reg; 3D assay following 72 h of exposure to FOLFIRINOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) or Gem-Pac (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) with or without tigecycline.\u003c/p\u003e \u003cp\u003eFOLFIRINOX treatment induced a reduction in viability only in PANC-1 spheroids, and this effect became apparent only at higher concentrations, whereas PaTu-8902 spheroids remained largely resistant across the tested range. Tigecycline alone resulted in a modest decrease in viability in both cell models; however, this reduction was observed as a trend and did not reach statistical significance. When combined with FOLFIRINOX, tigecycline produced a significant viability decline in PaTu-8902 spheroids, while the effect in PANC-1 remained limited and did not differ substantially from FOLFIRINOX alone.\u003c/p\u003e \u003cp\u003eIn contrast, Gem-Pac treatment reduced viability in PaTu-8902 spheroids in dose-dependent manner, PANC-1 spheroids demonstrated a more resistant response profile. Importantly, co-treatment with tigecycline markedly potentiated the effects of Gem-Pac, resulting in a substantial decrease in viability across all tested doses. These findings indicate that tigecycline exerts a synergistic effect with Gem-Pac, particularly in metabolically active 3D PDAC models, while its impact in combination with FOLFIRINOX remains comparatively modest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpheroids derived from PaTu-8902 and PANC-1 cells were treated with FOLFIRINOX (A) or Gem-Pac (B), either alone (yellow) or in combination with tigecycline (green). Viability was measured after 72 hours using the CellTiter-Glo luminescence assay. Data represent mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent experiments; statistical significance was determined by two-way ANOVA with comparisons in each row (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of FOLFIRINOX and Gem-Pac treatments on spheroid growth dynamics\u003c/h2\u003e \u003cp\u003eTo assess growth dynamics in 3D models, spheroids derived from PaTu-8902 and PANC-1 were exposed to FOLFIRINOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) or Gem-Pac (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) treatment, either alone or in combination with tigecycline. Changes in spheroid size were monitored over time, and the fold change in spheroid area between day 3 and day 6 was calculated to quantify treatment-induced alterations in spheroid size. Representative images of PaTu-8902 and PANC-1 spheroids treated with increasing concentrations of FOLFIRINOX or Gem-Pac, in the presence or absence of tigecycline, are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, illustrating treatment-induced changes in spheroid morphology and growth dynamics.\u003c/p\u003e \u003cp\u003eUnder FOLFIRINOX treatment, PaTu-8902 spheroids showed a dose-dependent reduction in expansion, while PANC-1 spheroids remained largely unresponsive, indicating resistance across most tested concentrations. Tigecycline alone resulted in spheroid growth reduction in both cell lines reaching statistical significance in PaTu-8902 spheroids. Notably, combining tigecycline with FOLFIRINOX further suppressed spheroid growth, with a significant effect observed only in PaTu-8902 spheroids, whereas PANC-1 spheroids again showed limited sensitivity.\u003c/p\u003e \u003cp\u003eSimilarly, Gem-Pac treatment displayed a dose-dependent inhibition of spheroid growth in PaTu-8902 cells, while PANC-1 spheroids maintained high growth rates, indicating chemoresistance. Remarkably, co-treatment with tigecycline profoundly impaired PaTu-8902 spheroid expansion, effectively halting growth even at the lowest concentrations of Gem-Pac. Together, these results confirm that tigecycline exerts a measurable chemosensitizing effect in 3D PDAC models, most prominently in PaTu-8902 spheroids and particularly under Gem-Pac treatment.\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn the present study, we provide the first systematic evaluation of tigecycline in combination with the two most widely used chemotherapy regimens for PDAC, FOLFIRINOX and Gem-Pac. Using complementary 2D monolayer and 3D spheroid PDAC models, we demonstrate that tigecycline consistently increases chemosensitivity and enhances the efficacy of both standard treatments. Our results also indicate that PDAC cell lines that were otherwise refractory to conventional chemotherapy displayed a marked improvement in treatment response upon co-treatment with tigecycline, highlighting its potential role as a chemosensitizing adjunct in resistant disease settings.\u003c/p\u003e \u003cp\u003eWhen comparing responses across tested PDAC cell lines, distinct sensitivity profiles to standard chemotherapies were evident. Consistent with previous reports [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], Capan-2 and MIAPaCa-2 cells displayed greater sensitivity to Gem-Pac treatment, whereas PaTu-8902 and PANC-1 cells exhibited pronounced chemoresistance to both regimens. Importantly, tigecycline enhanced the efficacy of both regimens, with the most substantial improvement observed in the Gem-Pac\u0026ndash;treated PaTu-8902 cells, even at lower concentrations of chemotherapy.\u003c/p\u003e \u003cp\u003eThese response patterns closely reflect differences in epithelial\u0026ndash;mesenchymal transition (EMT) status and metabolic phenotype among the cell lines [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Capan-2 cells retain a predominantly epithelial-like state, characterized by higher E-cadherin expression and lower levels of mesenchymal markers such as vimentin, ZEB1, and SNAIL, which has been associated with increased susceptibility to cytotoxic therapies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, PaTu-8902 and PANC-1 exhibit a mesenchymal, EMT-enriched phenotype, with elevated expression of vimentin, N-cadherin, and EMT transcription factors, accompanied by enhanced migratory capacity, resistance to apoptosis, and increased metabolic flexibility [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEMT has been linked to mitochondrial reprogramming and a shift toward oxidative phosphorylation\u0026ndash;dependent energy production, enabling tumor cells to adapt to nutrient deprivation and chemotherapeutic stress [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These mesenchymal, metabolically adaptable cells also display stronger interactions with cancer-associated fibroblasts within the desmoplastic tumor microenvironment, further promoting therapy resistance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The ability of tigecycline to inhibit mitochondrial translation may therefore preferentially disrupt this EMT-associated metabolic dependency, rendering otherwise chemoresistant PDAC cells more vulnerable to standard chemotherapy. Thus, the observed chemosensitizing effect of tigecycline is mechanistically consistent with the EMT-driven metabolic state of resistant PDAC cell lines, providing a functional link between tumor cell plasticity, mitochondrial metabolism, and treatment response.\u003c/p\u003e \u003cp\u003eAcross assays, combination treatment generally reduced cell viability/proliferation, minor discrepancies were noted between metabolic and proliferation-based readouts. These differences likely reflect the distinct cellular processes each assay measures: MTS primarily captures mitochondrial activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], while BrdU incorporation directly indicates DNA synthesis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and CellTiter-Glo quantifies ATP levels as a measure of metabolic viability in 3D systems [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The observed variations may therefore arise from metabolic reprogramming induced by tigecycline [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which can reduce mitochondrial function [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] prior to detectable decreases in proliferation or viability.\u003c/p\u003e \u003cp\u003eThe results obtained from 3D spheroid models provide additional insight into the potential of tigecycline as a chemosensitizing agent under more physiologically relevant conditions. Compared to conventional 2D cultures, spheroids better mimic the tumor microenvironment, including nutrient gradients, hypoxia, and cellular heterogeneity\u0026mdash;factors known to drive drug resistance in PDAC [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In these 3D systems, FOLFIRINOX and Gem-Pac alone exhibited only modest efficacy on PANC-1 cells, while PaTu-8902 spheroids showed a stronger cytotoxic response. Importantly, co-treatment with tigecycline substantially enhanced the cytotoxic effects of FOLFIRNOX and Gem-Pac, leading to a pronounced decrease in PaTu-8902 viability and practically complete suppression of spheroid growth even at lower concentrations of chemotherapy.\u003c/p\u003e \u003cp\u003eThe observed chemosensitizing effects of tigecycline may stem from its interference with mitochondrial metabolism, potentially limiting oxidative phosphorylation, reducing energy production, and thereby weakening the metabolic adaptability of PDAC cells\u0026mdash;an effect more evident in 3D spheroid models that more closely mimic \u003cem\u003ein vivo\u003c/em\u003e tumor conditions. Although preliminary, this metabolic vulnerability represents a plausible mechanistic basis for the observed chemosensitization and warrants further detailed investigation.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. The present study focused on \u003cem\u003ein vitro\u003c/em\u003e systems, which do not fully capture the complexity of tumor\u0026ndash;stroma interactions, drug pharmacokinetics, or immune modulation \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Future work should therefore aim to validate these findings in animal models of PDAC, incorporating pharmacodynamic and metabolic profiling to clarify how tigecycline modulates tumor energy metabolism in the context of combination therapy. Such preclinical studies will be essential to to determine an appropriate therapeutic window \u003cem\u003ein vivo\u003c/em\u003e that maximizes chemosensitization while minimizing systemic toxicity arising from combination therapy, potentially improving therapeutic outcomes in this highly aggressive and chemoresistant malignancy.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study demonstrates that tigecycline markedly enhances the efficacy of Gem-Pac in PDAC, with pronounced chemosensitization observed particularly in chemoresistant PaTu-8902 models. Enhancement in combination with FOLFIRINOX was cell-line dependent and less pronounced. In 3D spheroid systems that more closely recapitulate the \u003cem\u003ein vivo\u003c/em\u003e tumor microenvironment, co-treatment with tigecycline led to marked reductions in viability and spheroid growth, reinforcing its potential as a chemosensitizing agent. Although preliminary, these results pave the way for future preclinical studies aimed at validating tigecycline usage as a strategy to enhance chemosensitivity in PDAC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Fellowships for Excellent Researchers R2, funded by the European Union NextGenerationEU through the Recovery and Resilience Plan for Slovakia under project no. 09I03-03-V04-00074.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by J.S., L.V., L.U. and M.Č. The first draft of the manuscript was written by P.G. and M.Č. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Martina H\u0026aacute;jikov\u0026aacute; for her excellent technical assistance in the cell culture laboratory.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A. Cancer Statistics, 2021. CA Cancer J Clin. 2021;71(1):7\u0026ndash;33. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21654\u003c/span\u003e\u003cspan address=\"10.3322/caac.21654\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurris HA, 3rd, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, et al. 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From in vitro Experiments to in vivo and Clinical Studies; Pros and Cons. Curr Drug Discov Technol. 2015;12(4):218\u0026ndash;24. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1570163813666160114093140\u003c/span\u003e\u003cspan address=\"10.2174/1570163813666160114093140\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PDAC, gemcitabine, nab-paclitaxel, chemoresistance","lastPublishedDoi":"10.21203/rs.3.rs-9084399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9084399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies and remains highly resistant to systemic chemotherapy. Tigecycline, a glycylcycline antibiotic that inhibits mitochondrial translation, has recently attracted attention as a potential candidate for drug repurposing in oncology because of its proposed chemosensitizing effects. In this study, we investigated the cytotoxic and chemosensitizing effects of tigecycline in combination with the clinically used PDAC chemotherapy regimens FOLFIRINOX and gemcitabine plus nab-paclitaxel (Gem-Pac). Experiments were performed using both 2D monolayer cultures and 3D spheroid models of four PDAC cell lines (PaTu-8902, PANC-1, MIAPaCa-2, and Capan-2). Cell viability and proliferation were assessed using MTS, BrdU incorporation, and CellTiter-Glo\u0026reg; 3D assays, and spheroid growth dynamics were quantified by imaging-based area measurements after 3 days of treatment. Tigecycline showed limited cytotoxicity as a single agent but moderately enhanced the antiproliferative effects of FOLFIRINOX in a cell-line-dependent manner, while a more pronounced and consistent chemosensitizing effect was observed in combination with Gem-Pac, particularly in PaTu-8902 and PANC-1 models. These effects were reproducible across independent assays and were most evident in 3D spheroids, where tigecycline markedly suppressed chemotherapy-induced spheroid growth. Overall, these findings demonstrate that tigecycline can enhance the efficacy of clinically relevant PDAC chemotherapy regimens in vitro and support its further preclinical evaluation as a potential adjunct to existing PDAC treatment strategies.\u003c/p\u003e","manuscriptTitle":"Tigecycline Enhances Sensitivity to FOLFIRINOX and Gemcitabine/Nab-Paclitaxel in In Vitro Models of Pancreatic Ductal Adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:41:34","doi":"10.21203/rs.3.rs-9084399/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-14T16:35:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-14T04:47:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-27T19:07:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"340085890035398657425069861938233852539","date":"2026-03-26T08:55:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306610117651996746032637699254952616800","date":"2026-03-25T20:56:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132381581821442556210890312300189520352","date":"2026-03-23T12:02:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200139396752869017182933172917864785344","date":"2026-03-16T08:30:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-13T13:04:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-11T11:01:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T11:00:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bratislava Medical Journal","date":"2026-03-10T13:02:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bratislava-medical-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Bratislava Medical Journal](https://link.springer.com/journal/44411)","snPcode":"44411","submissionUrl":"https://submission.springernature.com/new-submission/44411/3","title":"Bratislava Medical Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4d1cc9e2-ad90-4d1b-8ee0-cd656f47df6b","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T07:24:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:41:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9084399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9084399","identity":"rs-9084399","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}