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Background Adoptive cell therapy (ACT) using tumour-infiltrating lymphocytes (TILs) has shown remarkable and durable anti-tumour responses in metastatic melanoma, even in patients unresponsive to immune checkpoint inhibitors. 1 2 Encouraged by these outcomes, TIL therapy is being explored in a variety of solid tumours, including breast, lung, ovarian, and endometrial cancers.3–5 However, the broader application of TIL therapy remains constrained by two major challenges: the laborious and time-intensive manufacturing process, and the limited availability of tumour tissue.In this study, we present a novel and robust method to generate and expand TILs from tumour samples of ovarian and endometrial cancer patients. Our approach utilizes mature dendritic cells derived from the leukemic cell line DCOne (DCOne mDC) to stimulate TIL proliferation.Methods Patient TILs were isolated from fresh tumour samples that were cut into pieces of <1 mm 3, digested overnight with collagenase and DNAse, centrifuged over a Ficoll-Plaque gradient. Isolated lymphocytes were subsequently cryopreserved until further use. TILs were stimulated with either high dose of IL-2 or with DCOne mDC in the presence of IL-2 up to maximum of 28 days to obtain young TILs. Young TILs were further expanded for 14 days rapid expansion protocol (REP) using irradiated allogeneic PBMC as feeder cells in the presence of anti-CD3 antibody and IL-2. Purity, viability, phenotype and functionality of expanded TILs was assessed pre- and post-REP expansion.Results TILs cultured in the presence of DCOne mDC and IL-2 resulted in at least average of 5-fold higher expansion up to day 28 compared to IL-2 cultures. The young TILs expanded in the presence of DCOne mDC contained mainly CD3 + T cells with a larger proportion of CD8+ T cells than CD4+ T cells compared to IL-2 cultures. Both CD4 and CD8 cells expanded in DCOne cultures were effector memory T cells. Furthermore, increased expansion of TILs from DCOne cultures was also observed during the REP phase compared to IL-2 TILs. The DCOne mDC expanded TILs showed increased CD107+IFN-g+effector T cells against autologous tumour.Conclusions The presented data indicate that DCOne-derived mature DC are equipped with the ability to promote strong ex vivo expansion of TILs compared to conventional IL-2-based protocols, yielding higher numbers of functional, cytotoxic T cells with a favourable effector memory phenotype. These findings offer a promising strategy to overcome current bottlenecks in TIL therapy and support the feasibility of extending ACT to treat patients with broader range of solid tumours successfully and timely manufactured TIL product.References Besser MJ, et al. Adoptive transfer of tumor-infiltrating lymphocytes in patients with metastatic melanoma: intent-to-treat analysis and efficacy after failure to prior immunotherapies. Clinical cancer research 2013;17:4792–800.Rohaan MW, et al. Tumor-infiltrating lymphocyte therapy or ipilimumab in advanced melanoma. N. Engl. J. Med. 2022;387:2113–2125.Zacharakis N, et al. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med. 2018; 24:724–30.Ben-Avi R, et al. Establishment of adoptive cell therapy with tumor infiltrating lymphocytes for non-small cell lung cancer patients. Cancer Immunol Immunother 67(8):1221–1230Pedersen M, et al. Adoptive cell therapy with tumor-infiltrating lymphocytes in patients with metastatic ovarian cancer: a pilot study. Oncoimmunology 2018;7(12)e1502905.Ethics Approval Tissue was collected through the OncoLifeS initiative. The OncoLifeS initiative has been approved by the medical ethics committee of the UMCG (no. 2010/109) and has been ISO certified (9001:2008 Healthcare). It was registered in the Dutch Trial Register under the number: NL7839. All patients signed informed consent.