Abstract 1372: Development of a multi-omic spatial phenotyping assay for affirmative detection of CAR-T cells in the tumor immune microenvironment of solid pediatric tumors

Abstract Certain pediatric cancers, like blood leukemia, have shown high cure rates, but effective treatments of solid tumors remain elusive and survival rates for affected children remain low. While CAR-T therapy has demonstrated success in treating acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), its effectiveness against solid tumors has been limited. Yet, pediatric patients with solid tumors may still receive CAR-T therapy with minimal prior evidence of treatment efficacy. It is imperative to understand why CAR-T treatments succeed or fail in solid pediatric tumors, as such knowledge can guide the development of future treatment options. This study focuses on a pediatric patient enrolled in a Phase 1/2 clinical trial exploring the use of CAR-T therapy for solid tumors. The patient underwent tumor resection before receiving CAR-T therapy (PRE). Following treatment, the tumor did not respond, leading to recurrence and a second resection (POST).Using advanced spatial transcriptomic profiling we compared the tumor immune microenvironment (TiME) between the PRE and POST treatment samples. Our findings revealed significant differences, including changes in immune cell populations and the spatial distribution of T cells in the TiME. However, while we observed alterations in T cell distribution, the precise localization of CAR-T cells relative to other cell types remained unclear. To address this, we developed a novel targeted spatial multi-omics assay that combines next-generation RNA fluorescence in situ hybridization (HCR™ Gold RNA-FISH) with multiplex immunofluorescence (mIF). HCR™ Gold RNA-FISH is broadly compatible with protein imaging methods, and therefore enables RNA and protein co-detection on the same tissue sample, which is needed to affirmatively detect and map the putative infiltration of CAR-T cells in the TiME. CAR-T cell detection was enabled by custom-designed HCR™ HiFi probes, targeted against B3-H7. Affirmative identification, phenotyping, and spatial profiling of these cells were enabled via VistaPlex™ mIF assay panels deployed serially on the same formalin-fixed paraffin-embedded (FFPE) tissue samples. All experiments were automated on the CellScape™ spatial biology platform and image data were processed and integrated with a custom image analysis pipeline. Our method enables conclusive follow-up investigations into the efficacy of CAR-T treatments in solid pediatric tumors. With this toolkit in hand, it is possible to (a) confirm the status of the CAR-T cell infiltration, (b) define the phenotype and activation states of these cells, and (c) map the spatial biology and cell-to-cell interactions of the CAR-T cells within the TiME. Citation Format: Jasmine T. Plummer, Felipe Segato-Dezem, Yutian Liu, Arjumand Wani, Hannah Chasteen, Arne Christians, Jannik Boog, Randy Chen, Harry Choi, Aneesh Acharya, Oliver Braubach, Giedre Krenciute, Jason Chiang, Christopher De Renzo, Kelsey Bertrand. Development of a multi-omic spatial phenotyping assay for affirmative detection of CAR-T cells in the tumor immune microenvironment of solid pediatric tumors [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 1372.