Engineering Subcutaneous Ectopic Tertiary Lymphoid Structures as Personalized Immune-Activation Niches for Cancer Immunotherapy

Tumor-associated tertiary lymphoid structures (TLSs) can promote anti-tumor immune responses and survival. However, intratumor TLS formation and maturation are limited in tumors with a “cold” immune phenotype, including reduced infiltration of immune cells and a suppressive tumor microenvironment (TME). Here, we present a personalized immunotherapy strategy utilizing a subcutaneously injected, bioengineered hydrogel (scTLS) that acts as an ectopic immune-activation center, effectively decoupled from the suppressive TME. Native immune cells and antigen-presenting cells (APCs) with autologous antigens are recruited through the release of chemokines. An agonist for CD40 is modified on the gel, enabling an immune-activated environment. We demonstrate that scTLSs promote robust dendritic cell (DC) activation and antigen presentation, driving the formation of organized T and B cell zones as confirmed by 3D imaging and transcriptomic analysis. This personalized, autologous vaccine-based approach elicited potent anti-tumor immune responses and effectively inhibited tumor progression across multiple murine models, with enhanced efficacy upon combination with doxorubicin-induced immunogenic cell death and immune checkpoint blockade. By overcoming the spatial and immunosuppressive limitations of the native TME, our scTLS platform offers a versatile, "off-the-shelf" engineering strategy for inducing functional TLSs to enhance therapeutic outcomes in a broad spectrum of malignancies.