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In pancreatic ductal adenocarcinoma (PDAC), several clinical trials are underway to improve outcomes, yet a significant knowledge gap prevails involving the cell-extracellular matrix (ECM) crosstalk. Herein, we hypothesized that treatment with TTFields influence this crosstalk, which is reflected by the dynamic alteration in nanomechanical properties (NMPs) of cells and the ECM in a co-culture system. We employed an ECM gel comprising collagen, fibronectin, and laminin mixed in 100:1:1 stoichiometry to co-culture of Panc1 and AsPC1 individually. This ECM mixture mimics the in vivo tumor microenvironment closely when compared to the individual ECM components studied before. A comprehensive frequency-dependent study revealed the optimal TTFields frequency to be 150 kHz. We also observed that irrespective of the ECM\u2019s presence, TTFields increase cell membrane stiffness and decrease deformation several-folds in both Panc1 and AsPC1 cells at both 48 h and 72 h. Although adhesion for AsPC1 decreased at 48 h, at 72 h it was observed to increase irrespective of ECM\u2019s presence. Moreover, it significantly alters the NMPs of ECM gels when co-cultured with PDAC cell lines. However, AsPC1 cells were observed to be more detrimental to these changes. Lastly, we attribute the stiffness changes in Panc1 cells to the membrane F-actin reorganization in the presence of TTFields. This study paves a path to study complex PDAC TME as well as the effect of various chemotherapeutic agents on such TME with TTFields in the future.<\/jats:p>","DOI":"10.3390\/jfb16050160","type":"journal-article","created":{"date-parts":[[2025,5,4]],"date-time":"2025-05-04T20:09:14Z","timestamp":1746389354000},"page":"160","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Tumor-Treating Fields Alter Nanomechanical Properties of Pancreatic Ductal Adenocarcinoma Cells Co-Cultured with Extracellular Matrix"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9194-2460","authenticated-orcid":false,"given":"Tanmay","family":"Kulkarni","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sreya","family":"Banik","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1858-5054","authenticated-orcid":false,"given":"Debabrata","family":"Mukhopadhyay","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA"},{"name":"Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9368-6296","authenticated-orcid":false,"given":"Hani","family":"Babiker","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Hematology and Oncology, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3579-4317","authenticated-orcid":false,"given":"Santanu","family":"Bhattacharya","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA"},{"name":"Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine and Science, 4500 San Pablo Road South, Jacksonville, FL 32224, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Vergote, I., Macarulla, T., Hirsch, F.R., Hagemann, C., and Miller, D.S. 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