Abstract A041: Therapeutic induction of tertiary lymphoid structures by STING and lymphotoxin-β receptor agonists sensitizes poorly immunogenic rhabdomyosarcoma to PD-1 blockade

Metastatic rhabdomyosarcoma (RMS) remains a clinically challenging pediatric malignancy with limited response to immunotherapy and poor prognosis. The 76-9 murine RMS exhibits low MHC class I expression and poor immunogenicity, making it a model to evaluate immune-activating treatment strategies. Tertiary lymphoid structures (TLS), ectopic lymphoid aggregates resembling secondary lymphoid organs, are increasingly recognized as critical sites for local lymphocyte priming and have been associated with improved survival and responsiveness to immune checkpoint inhibitors in several adult cancers. However, their role and therapeutic potential in RMS remain largely unexplored. We investigated whether TLS could be therapeutically induced in RMS to potentiate anti-tumor immunity and sensitize tumors to immune checkpoint blockade. Here we show that a combination therapy with stimulator of interferon genes (STING) and lymphotoxin-β receptor (LTβR) agonists induces the formation of intratumoral TLS exhibiting germinal center B cell responses in 76-9 RMS tumors, leading to the inhibition of tumor growth and metastasis and prolonged survival. Increased levels of tumor-specific IgG were detected in blood serum following this treatment. Histological examination showed no significant differences in the number or size of germinal centers in draining lymph nodes between the treated and untreated groups, suggesting that intratumoral TLS were the main source of the anti-tumor IgG. Functional assays showed that the tumor-specific IgG bound to RMS cells and triggered NK cell–mediated antibody-dependent cellular cytotoxicity (ADCC), resulting in tumor cell killing in vitro. Flow cytometry demonstrated increased accumulation of TCF1+CD8+ progenitor exhausted/stem-like T cells in the treated tumors, and immunofluorescence revealed that the stem-like T cells were localized in the close proximity to TLS that created unique niches for these T cells. Finally, the administration of anti–PD-1 antibody following agonist-induced TLS formation significantly prolonged survival while PD-1 blockade alone did not improve survival, indicating that TLS reprogrammed the tumor microenvironment to heighten the responsiveness to immune checkpoint blockade therapy. In conclusion, our study demonstrates that STING/LTβR agonist therapy induces functional TLS in “immune cold” RMS tumors, overcoming the inherent resistance to immunotherapy through the development of humoral and cellular immunity against RMS. TLS induction by the agonist combination represents a promising strategy for potentiating immunotherapy in sarcomas with low immunogenicity. Citation Format: Yasuhiro Kikuchi, Maxwell Duah, Tomoko Stansel, Fumiaki Kanamori, Masanobu Komatsu. Therapeutic induction of tertiary lymphoid structures by STING and lymphotoxin-β receptor agonists sensitizes poorly immunogenic rhabdomyosarcoma to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2): nr A041.