Abstract 1375: Active inhibition of chemokine-mediated migration impairs tertiary lymphoid structure formation

Abstract The presence of tumor-infiltrating B cells (TIL-B) and tertiary lymphoid structures (TLS) in the primary tumor microenvironment carries prognostic value across cancer types. We have shown that triple-negative breast cancer (TNBC) patients with robust germinal center (GC) responses in their lymph nodes (LN) exhibit higher levels of TILs and more TLS. In this study, we investigated human and mouse immune-cold TNBCs to uncover mechanisms by which tumor-LN crosstalk may be impaired. In orthotopic TNBC mouse models, we observed robust GC responses in tumor-draining LNs. However, minimal TIL-Bs and no TLS were detected in the primary tumors. The tumor-draining LNs were confirmed as the source of the few TIL-B by using FTY720, a drug that blocks LN exit. Transcriptomic analysis of 124 untreated TNBC patients confirmed a strong association between TIL-B/TLS presence and chemokine-enriched pathways. Notably, in orthotopic TNBC mouse models, multiplex proteomic analyses of the tumor secretome revealed that most TIL-B- and TLS-associated chemokines were absent. Next, we conducted ex vivo migration assays and found unexpectedly that supplementing mouse tumor-conditioned medium with chemokines induced significantly less migration compared to chemokine-supplemented culture medium. In vivo, co-injection of genetically engineered chemokine-expressing cancer-associated fibroblasts (CAFs) did not elicit TLS formation nor increased TIL-B infiltration in the primary tumors. These findings suggest the presence of specific inhibitory factors within the tumor secretome that impair chemokine-mediated migration and immune cell infiltration. Using molecular weight-based fractionation and high-throughput analysis, we are narrowing down the identity of these components and conducting functional studies to validate their effects. Preliminary experiments show that removing these factors can restore chemokine activity, permitting B cell migration. In vivo, we are evaluating the combination of genetically engineered chemokine-expressing CAFs and systemic inhibition of these factors to promote TIL-B infiltration and TLS formation. Preliminary data indicate this dual treatment promotes the formation of large peritumoral B cell aggregates, providing critical insights into the factors enabling tumor immune infiltration and organization. Ongoing human transcriptomic analyses on internal and publicly available datasets support a strong association between chemokine expression, reduced inhibitory factor secretion, and TIL-B presence across human cancer types, including TNBC, colon and lung cancer. This work defines that the absence of TIL-B and TLS in cancer is the consequence of a combination of mechanisms: insufficient chemokine production by the tumor microenvironment to drive immune infiltration from the LN, coupled with the active secretion of factors that impair chemokine functionality. Citation Format: Victoire Boulat, Elena Alberts, Carin Andrea Brundin, Dinis P. Calado, Anita Grigoriadis. Active inhibition of chemokine-mediated migration impairs tertiary lymphoid structure formation [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 1375.