Abstract 647: Intratumoral activation of anti-tumor CD4Th1 cells prior to neoadjuvant chemotherapy eradicate metastasis initiating cancer cells in breast cancer patients

Abstract Clinical overt metastases emerge from metastasis initiating cancer cells (MICs) disseminated during early tumor progression prior to clinical diagnosis of apparent primary breast cancer (BC). Targeting and inhibiting dissemination of MICs from early tumor lesions can be a potential therapeutic strategy to prevent late recurrence and metastasis in patients. Unfortunately, no such therapy currently exists that inhibits MIC dissemination and metastatic seeding in distant organs. Our recent findings support the critical contribution of intratumoral delivery of type 1 conventional dendritic cells (cDC1) in complete eradication of primary BC in patients. We demonstrated that intratumoral activation of anti-tumor CD4T helper 1 (CD4Th1) cells by tumor antigen targeting cDC1 limits MIC dissemination mechanisms independent of CD8 T cells in BC mouse models. Anti-tumor CD4Th1 cells also restored the loss of nuclear receptor subfamily 2 group F member 1 (NR2F1) in human epidermal growth factor receptor 2 (HER2)+ early tumor lesions, reducing dissemination of MICs. Moreover, CD4Th1 cytokine Interferon gamma inhibited dissemination of MICs from early lesions and primary tumors via the modulation of HER2/progesterone receptor/Wnt4/receptor activator of nuclear factor-κB ligand (RANKL) dissemination mechanisms. Most strikingly, we provide clinical evidence that intratumoral activation of anti-tumor CD4Th1 cells inhibited disseminating MIC phenotypes in the tumor lesion of BC patients. This was further corroborated by the decreased seeding of disseminated MICs in the bone marrow of BC patients treated with intratumoral cDC1 prior to any standard neoadjuvant chemotherapy (NACT) compared to patients treated with standard NACT or patients with no prior treatment. Single cell RNA sequencing (scRNA-seq) of isolated MICs (disseminated cancer cells; DCCs) from bone marrow of BC patients with no prior treatment revealed that these patient MICs were highly enriched for cytokeratin 10/13, heterogeneity, stemness and loss of MHC class II and displayed a unique transcriptome profile. Our ongoing clinical study is investigating isolated MICs from patients treated with intratumoral cDC1 and standard NACT at a single cell level by scRNA-seq. Collectively, these results provide a new strategy to selectively target and inhibit dissemination of MICs from the early tumor lesion and prevent overt metastasis in patients. This is also the first study to delineate the transcriptome profile of MICs (DCCs) from BC patients using scRNA-seq. Citation Format: Ganesan Ramamoorthi, Marie Catherine Lee, Carly M. Farrell, Hyo S. Han, Caitlyn N. Balsay-Patel, Grace Spiegelhoff, Brian J. Czerniecki. Intratumoral activation of anti-tumor CD4Th1 cells prior to neoadjuvant chemotherapy eradicate metastasis initiating cancer cells in breast cancer patients [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 647.