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Peritoneal carcinomatosis (PC) from ovarian cancer (OC) has poor prognosis and is in need for innovative therapies. Such clinical condition displays a composite structure with floating cell aggregates within ascites and solid-like masses invading the peritoneum. A deep understanding of the dynamics is thus crucial to propose more effective treatments. Chimeric antigen receptor (CAR)-based immunotherapies are emerging as promising approaches, and ex vivo expanded cytokine-induced killer lymphocytes (CIK), intrinsically endowed with HLA-independent antitumor activity, are a valid platform to be exploited against solid tumors. Appropriate preclinical models, recapitulating the clinical complexity, are needed in the perspective of clinical translation. Here we explored the activity of mesothelin (MSLN) CAR-redirected CIK (CAR.CIK) against advanced OC, by developing 3D models resembling PC structural complexity. CAR.CIK were generated from patients’ PBMC (n=11), engineered with 2nd generation MSLN-CAR with 4-1BB costimulation. As tumor targets we employed and characterized a panel of ascites-derived OC cell lines (aOC). Anti-aOC CAR.CIK killing ability was evaluated mostly by flow cytometry. CAR.CIK were successfully generated with a mean CAR expression of 42%. MSLN was highly expressed (>75%) in 5/6 aOC. CAR.CIK effectively killed tumor targets in standard 2D assays, even at very low effector/target ratios (E/T), with significant improvement compared to unmodified CIK (NTD.CIK): 78% vs 36%, 46% vs 15% respectively at 5:1 and 1:2 E/T (p In order to recapitulate the clinical complexity we generated 3D aOC spheroids, either floating in liquid medium or embedded in a 3D hydrogel and co-cultured with CAR.CIK. To measure CAR.CIK activity we employed live-imaging and image processing. CAR.CIK were found to localize faster than NTD.CIK in both liquid and solid settings. Recruitment kinetics of CAR.CIK on aOC aggregates in liquid was significantly faster in shaking cultures, reminiscent of spontaneous patient movement, than in absence of flow. Even in the 3D solid setting, CAR.CIK were functionally more efficient and localized faster on aOC spheroids than NTD.CIK. Our data indicate that MSLN-CAR.CIK are effective against aOC in both 2D and 3D settings. Kinetics measurements in 3D show that recruitment in liquid requires shorter times when coupled to fluid flow. The solid setting is instead characterized by slower kinetics but CAR.CIK anti-tumor activity remains comparable to the liquid condition. The kinetics of recruitment should therefore be considered when assessing CAR.CIK killing. Our findings provide reliable translational bases for clinical studies to explore intraperitoneal cellular immunotherapy with CAR.CIK against OC PC. Citation Format: Federica Galvagno, Valeria Leuci, Chiara Donini, Annamaria Massa, Ramona Rotolo, Sonia Capellero, Alessia Proment, Letizia Vitali, Valentina Tuninetti, Martina Olivero, Elisa Vigna, Giorgio Valabrega, Luca Primo, Alberto Puliafito, Dario Sangiolo. Anti-mesothelin CAR.CIK lymphocytes are effective against ovarian cancer in 3D models of peritoneal carcinomatosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3194.