Abstract 5250: Re-activating suppressed tumor-infiltrating lymphocytes from melanoma brain metastases
Authors: Mark W. Sweep, Imke van Wandeloo, Mark A. Gorris, Guido van Mierlo, Benno Küsters, Kiek Verrijp, Alfred C. Koetsveld, Mark ter Laan, I Jolanda de Vries, Carla M. van Herpen, Kalijn F. Bol
Journal: Cancer Research (2025)

Abstract Brain metastases (BrMs) occur in up to half of patients with metastatic melanoma and are a major cause of death. The response of BrMs to immunotherapy can be different from extracerebral metastases (ECMs). Hence, in this study the immune microenvironment of BrMs is compared with that of ECMs. Bulk RNA sequencing was performed on a retrospective cohort of 38 melanoma samples from 14 patients, consisting of 15 matched BrMs and 23 ECMs. This cohort was expanded to perform multiplex immunohistochemistry on a total of 107 matched BrM and ECM samples from 44 patients. Fresh BrM material was prospectively obtained from a total of 12 patients with melanoma BrM, who underwent surgical resection of their BrM. From this material, tumor-infiltrating lymphocytes (TILs) and corresponding tumor cells were cultured. These cells were characterized by flow cytometry. TIL cultures were expanded and tested for reactivity against autologous tumor cell lines using flow cytometry and ELISA. In a patient-matched comparison, gene set enrichment analysis showed that brain-specific pathways are enriched in BrMs, whereas immune activation pathways, especially pathways related to TILs, are suppressed in BrMs compared to ECMs. However, the computationally inferred abundance of TILs did not differ between BrMs and ECMs, but rather showed an increase of anti-inflammatory M2 macrophages in BrMs. This indicates that TILs are equally present in BrMs and ECMs, but that they are less activated in BrMs. Interestingly, M2 macrophages seemed reduced in patients who received prior treatment with BRAF/MEK inhibitors. When taken out of their native environment, ex vivo cultures of BrM-derived TILs and tumor cells are successful in 7/10 (70%) and 7/12 (58%) patients, respectively. In 3/6 (50%) patients with matched TIL and tumor cell cultures, tumor reactivity was indicated, without external stimulation, by production of TNFα, IFNγ, perforin, and expression of CD107a and CD137. Our data suggest that TILs are suppressed in melanoma BrMs as opposed to ECMs, possibly due to increased presence of anti-inflammatory M2 macrophages. Nevertheless, these TILs can efficiently be expanded ex vivo and show tumor reactive potential, providing opportunities for TIL-based and other immunotherapeutic treatments in BrM. Combined with macrophage targeting, TILs could synergize to provide effective targeting of the tumor. Citation Format: Mark W. Sweep, Imke van Wandeloo, Mark A. Gorris, Guido van Mierlo, Benno Küsters, Kiek Verrijp, Alfred C. Koetsveld, Mark ter Laan, I Jolanda de Vries, Carla M. van Herpen, Kalijn F. Bol. Re-activating suppressed tumor-infiltrating lymphocytes from melanoma brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5250.