Targeted intra-tumoral hyperthermia using uniquely biocompatible gold nanorods induces strong immunogenic cell death in two immunogenically 'cold' tumor models.

Introduction: Hyperthermia is an established adjunct in multimodal cancer treatments, with mechanisms including cell death, immune modulation, and vascular changes. Traditional hyperthermia applications are resource-intensive and often associated with patient morbidity, limiting their clinical accessibility. Gold nanorods (GNRs) offer a precise, minimally invasive alternative by leveraging near-infrared (NIR) light to deliver targeted hyperthermia therapy (THT). THT induces controlled tumor heating, promoting immunogenic cell death (ICD) and modulating the tumor microenvironment (TME) to enhance immune engagement. This study explores the synergistic potential of GNR-mediated THT with immunotherapies in immunogenically 'cold' tumors to achieve durable anti-tumor immunity. Methods: GNRs from Sona Nanotech Inc.™ were intratumorally injected and activated using NIR light to induce mild hyperthermia (42-48°C) for 5 minutes. Tumor responses were analyzed for cell death pathways and immune modulation. The immunogenic effects of THT were assessed alone and in combination with intratumoral interleukin-2 (i.t. IL-2) or systemic PD-1 immune checkpoint blockade. Immune cell infiltration, gene expression changes, and tumor growth kinetics were evaluated. Results: THT reduced tumor burden through cell death mechanisms, including upregulated ICD marked by calreticulin exposure within 48 hours. By 48 hours, CD45+ immune cell levels were increased, including increased levels of immunosuppressive M2 macrophages. While THT led to innate immune cell stimulations highlighted by gene expression upregulation in the STING cGAS pathway and enhanced M1 and dendritic cell levels, tumor regrowth was observed within six days post-treatment. To enhance THT's immunogenic effects, the therapy was combined with intratumoral interleukin-2 (i.t. IL-2) or systemic PD-1 immune checkpoint blockade. Sequential administration of i.t. IL-2 post-THT induced robust CD8+ T-cell infiltration and led to sustained tumor regression in both treated and distant tumors, accompanied by the emergence of memory T cells. However, IL-2-induced immunosuppressive T-reg populations were also sustained to tumor endpoint suggesting that therapy could be further enhanced. Additionally, PD-1 expression, which was upregulated in CD8+ T cells by THT, was targeted with systemic PD-1 inhibition, further augmenting immune engagement within the TME. Discussion: These combinatory treatments demonstrated synergistic effects, promoting durable anti-tumor responses and immune memory. Collectively, GNR-mediated THT effectively reduces tumor burden and remodels the TME, potentiating systemic immunity and enhancing the impact of complementary immunotherapies.