Background The presence of tumor-localized tertiary lymphoid structures (TLSs) has been increasingly shown to correlate with improved survival in certain solid tumor types.^1-3 There is an unmet need to generate TLSs in tumors of patients where their absence is noted, with the intent to potentially improve outcomes. To address this, we employed murine models and hypothesized that encapsulating lymphoid chemokines and cytokines in controlled-release biomaterials would allow for their sustained delivery to the local tissue microenvironment, promote immune T cell and B cell accumulation, and lead to the in situ formation of TLS-like structures.

Methods We encapsulated mouse homeostatic chemokines CCL19, CCL21, CXCL13, and a tumor necrosis factor (TNF) superfamily cytokine lymphotoxin-alpha1/beta2 in lipid-enveloped mesoporous microparticles. In vitro characterization studies were performed to evaluate the encapsulation efficiency, release kinetics by ELISA, and chemotactic index of encapsulated chemokines by transwell assay. Chemokine/cytokine-releasing microparticles were next embedded in a thermosensitive chitosan-based hydrogel and injected subcutaneously into C57BL/6 mice. At weeks 1-4 post-injection, hydrogels, along with any newly formed surrounding tissues, were excised for histological analysis via hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining, and flow cytometry.

Results In vitro characterization studies indicated over 90% encapsulation efficiency for CCL19 in microparticles, with a sustained release profile where 78% of the chemokine was released over the first three days, as compared to levels measured on day seven. Additionally, chemotaxis experiments confirmed that all three chemokines—CCL19, CCL21, and CXCL13—retained their functional capacity to trigger lymphoid cell migration after being released from microparticles. Subcutaneous injections of chemokine/cytokine-releasing microparticles embedded in chitosan hydrogel led to the in vivo formation of structures enriched with immune cell aggregates, exhibiting more than a 10-fold increase in the number of CD45⁺ immune cells (p < 0.0001) within the newly formed structures surrounding the residual hydrogel deposit. This was accompanied by a marked increase in the number of CD19⁺ B cells (p < 0.0001) and CD3⁺ T cells (p < 0.0001), with 55% and 22% of CD45⁺ cells, respectively.

Conclusions We have developed an injectable biopolymer scaffold delivery system that can induce the formation of lymphoid aggregates resembling TLSs in mice. Given the importance of TLSs as reservoirs for tumor-infiltrating B and T lymphocytes and their established prognostic and predictive significance in certain human cancers, engineering such structures could serve as a vital platform for generating in situ anti-tumor immune responses, and thereby potentially enhancing the clinical efficacy of immunotherapies.

Acknowledgements We thank Dr. John Mullinax and Dr. Shari Pilon-Thomas for providing human TIL. This work has been supported by the Moffitt/USF Vivarium, Flow Cytometry, Analytical Microscopy, and Tissue Core Histology; all comprehensive cancer center facilities designated by the National Cancer Institute (P30-CA076292). This work was funded by the NCI-NIH (P30 CA076292, and P50 CA168536), CJG Fund, Chris Sullivan Fund, V Foundation, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, and the Melanoma Research Foundation Career Development Award.

References

1. Coppola D, Nebozhyn M, Khalil F, Dai H, Yeatman T, Loboda A, Mulé JJ. Unique ectopic lymph node-like structures present in human primary colorectal carcinoma are identified by immune gene array profiling. Am J Pathol 2011;179(1):37.

2. Messina JL, Fenstermacher DA, Eschrich S, Qu X, Berglund AE, Lloyd MC, Schell MJ, Sondak VK, Weber JS, Mulé JJ. 12-Chemokine gene signature identifies lymph node-like structures in melanoma: potential for patient selection for immunotherapy?. Scientific Reports 2012;2(1):765.

3. Schumacher TN, Thommen DS. Tertiary lymphoid structures in cancer. Science 2022;375(6576):eabf9419.