Identification of therapeutic targets for treating fibrotic diseases and cancer remains challenging. Our study aimed to investigate the effects of TGF-β1 and TGF-β3 on myofibroblast differentiation and extracellular matrix deposition in different types of fibroblasts, including normal/dermal, cancer-associated, and scar-derived fibroblasts. When comparing the phenotype and signaling pathways activation we observed extreme heterogeneity of studied markers across different fibroblast populations, even within those isolated from the same tissue. Specifically, the presence of myofibroblast and deposition of extracellular matrix were dependent on the origin of the fibroblasts and the type of treatment they received (TGF-β1 vs. TGF-β3). In parallel, we detected activation of canonical signaling (pSMAD2/3) across all studied fibroblasts, albeit to various extents. Treatment with TGF-β1 and TGF-β3 resulted in the activation of canonical and several non-canonical pathways, including AKT, ERK, and ROCK. Among studied cells, cancer-associated fibroblasts displayed the most heterogenic response to TGF-β1/3 treatments. In general, TGF-β1 demonstrated a more potent activation of signaling pathways compared to TGF-β3, whereas TGF-β3 exhibited rather an inhibitory effect in keloid- and hypertrophic scar-derived fibroblasts suggesting its clinical potential for scar treatment. In summary, our study has implications for comprehending the role of TGF-β signaling in fibroblast biology, fibrotic diseases, and cancer. Future research should focus on unraveling the mechanisms beyond differential fibroblast responses to TGF-β isomers considering inherent fibroblast heterogeneity.

Agrimonia eupatoria L. (AE) has a rich tradition of use in wound healing improvement across various cultures worldwide. In previous studies, we revealed that Agrimonia eupatoria L. water extract (AE) possesses a rich polyphenolic composition, displaying remarkable antioxidant properties. Our investigations also demonstrated that lipophosphonoxin (LPPO) exhibited antibacterial efficacy in vitro while preserving the proliferation and differentiation of fibroblasts and keratinocytes. Building upon our prior findings, in this study, we intended to examine whether a combination of AE and LPPO could enhance skin wound healing while retaining antibacterial attributes. The antibacterial activity of AE/LPPO against Staphylococcus aureus was evaluated, alongside its effects on fibroblast-to-myofibroblast transition, the formation of extracellular matrix (ECM), and endothelial cells and keratinocyte proliferation/phenotype. We also investigated AE/LPPO’s impact on TGF-β1 and VEGF-A signaling in keratinocytes/fibroblasts and endothelial cells, respectively. Additionally, wound healing progression in rats was examined through macroscopic observation and histological analysis. Our results indicate that AE/LPPO promotes myofibroblast-like phenotypic changes and augments ECM deposition. Clinically relevant, the AE/LPPO did not disrupt TGF-β1 and VEGF-A signaling and accelerated wound closure in rats. Notably, while AE and LPPO individually exhibited antibacterial activity, their combination did not lead to synergism, rather decreasing antibacterial activity, warranting further examination. These findings underscore substantial wound healing improvement facilitated by AE/LPPO, requiring further exploration in animal models closer to human physiology.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense desmoplastic stroma that contributes to tumor progression, therapeutic resistance, and disease aggressiveness. Cancer-associated fibroblasts (CAFs) represent the predominant cellular component of this stroma and are a major source of extracellular matrix (ECM), playing a central role in shaping the tumor microenvironment. However, the reliable isolation of CAFs from human PDAC tissue remains technically challenging due to extensive matrix deposition, limited tissue availability, and an elevated risk of microbial contamination. In this study, we comparatively evaluated two isolation workflows using freshly resected human PDAC specimens: (i) mechanical explant-based outgrowth and (ii) combined mechanical-enzymatic dissociation with trypsin pre-digestion. In parallel, we evaluated graded antibiotic and antimycotic regimens to optimize contamination control during the initial culture period. The addition of amphotericin B to the culture medium effectively reduced contamination risk without compromising fibroblast outgrowth when withdrawn in a stepwise manner. Furthermore, gentle enzymatic pre-digestion enhanced overall isolation efficiency without affecting the timing of initial fibroblast outgrowth. Phenotypic characterization confirmed fibroblast identity and demonstrated comparable expression of activation-associated markers, although inter-sample variability was observed in signaling receptor expression, including PDGFR- α and PDGFR- β . Collectively, these findings establish a practical and reproducible protocol for isolating primary PDAC-derived CAFs suitable for downstream experimental studies of the tumor microenvironment.

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. The aim of this study was to evaluate the cytotoxic and chemosensitizing effects of tigecycline in combination with clinically relevant PDAC chemotherapy regimens, namely FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, and oxaliplatin) and Gem-Pac (gemcitabine plus nab-paclitaxel). The individual components of these regimens were applied at clinically relevant molar ratios reflecting patient treatment protocols. 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 assay, BrdU incorporation, and CellTiter-Glo ^® 3D assays, and spheroid growth dynamics were quantified by imaging-based area measurements after 3 days of treatment. Tigecycline enhanced the cytotoxic effects of both chemotherapy regimens in a concentration- and cell line-dependent manner, with a more pronounced effect observed in combination with Gem-Pac. In MTS assays, tigecycline converted otherwise chemoresistant cell lines (PaTu-8902, PANC-1, and MIAPaCa-2) into more responsive phenotypes, with the strongest sensitization observed in PaTu-8902 cells. Δ effect analysis further revealed that tigecycline-mediated sensitization to Gem-Pac occurred across a broad concentration range. Notably, this effect was evident even at lower, otherwise ineffective doses in Capan-2 cells. In contrast, the effect of tigecycline in combination with FOLFIRINOX was rather moderate and predominantly observed at higher concentrations. These trends were consistent with BrdU and 3D spheroid models, which supported the observed cell line-dependent differences in treatment response. Clinically relevant, tigecycline alone exhibited concentration-dependent cytotoxicity, with minimal effects at lower concentrations and more pronounced reductions in viability at higher doses. 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.