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

Abstract

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.

Keywords: breast cancer; gold nanorods; hyperthermia; immunotherapy; interleukin-2; melanoma; photothermal therapy.

Figure 1

Biochemical properties of Sona Nanotech^TM’s GNRs. Representative TEM images of GNRs samples (A), measurement bar is 200 nm. Images were taken at 100,000× magnification. (B) Representative DLS for a GNR sample diluted in different media, showing size distribution by intensity.

Figure 2

GNR-Enhanced Laser Irradiation Induces Tumor THT, Absent in Laser-Only Controls. (A) Photo of the experimental setup, showing a BALB/c mouse bearing a 4T1 tumor, with two internal temperature probes used to monitor the effects of THT versus controls. (B) Representative temperature profiles of 4T1 tumors in BALB/c mice generated from internal probes during NIR light, comparing the temperature changes in GNR-injected tumors to those in laser-only controls. (C, D) Quantitative analysis of surface temperatures in BALB/c mice with 4T1 tumors (n=18 control, 19 THT) and C57BL/6 mice with B16-F10 tumors (n=11 control, 14 THT). **** P ≤ 0.0001.

Figure 3

GNR-Mediated THT Induces Tumor Shrinkage, Cell Death, and Immune Activation in 4T1 Models and Growth Suppression in B16-F10 Models Within 48 Hours Post-Treatment. (A) Tumor volumes in 4T1 tumor models following GNR-mediated THT treatment (n=24 control, n=28 THT), error bars represent SEM. (B) Representative images of a control mouse and a THT-treated mouse 48 hours post-irradiation, alongside a comparison of tumor volumes between GNR-treated and control groups. (C) Flow cytometry analysis of cell viability (as determined by BD Horizon™ Fixable Viability Stain 510) in all cells of a 4T1 tumors 24 and 48 hours post-THT (n=5). (D) Levels of extracellular calreticulin in 4T1 tumors (CD45-) 24 and 48 hours post-THT (n=5). (E) Flow cytometry data showing the percentage of CD45⁺ immune cells in 4T1 tumors 24 and 48 hours post-treatment (n=5). (F) Analysis of M2 macrophage levels (gated on CD45+/CD11b+F480+CD206+) in 4T1 tumors 24 and 48 hours post-THT (n=5). (G) Tumor volume measurements in the B16-F10 model within 48 hours post-laser treatment (n=19 control, n=23 THT), error bars represent SEM. *p < 0.05, **p <0.01.

Figure 4

GNR-Induced THT Results in Tumor Regrowth After Initial Reduction, Despite Upregulation of STING Pathway Genes and Increased Innate Immune Cell Levels. (A) Tumor volume measurements in 4T1 models following THT treatment compared to control (n=16 control, n=15 THT), error bars represent SEM. (B) Tumor volume analysis in B16-F10 models (n= 9 control, n=11 THT), error bars represent SEM. (C, D) Gene expression analysis 8 days post-laser treatment showing upregulation of STING pathway genes in 4T1 (n= 8 control, 7 THT) and B16-F10 tumors (n = 4 control, 7 THT) subjected to THT or control. (E-G) Flow cytometry analysis of dendritic cells (CD45+CD11b+F4/80-CD11c+MHCII+), M1 macrophages (CD45+CD11b+F4/80+CD80+CD86+), and macrophages (CD45+CD11b+F4/80+) in 4T1 tumors post-THT, (n=6 control, n= 7 THT). (H-K) Flow cytometry analysis of dendritic cells, M1 macrophages, macrophages and NK cells (CD3- NK1.1+) in B16-F10 tumors post-THT, (n=3 control, n= 5 THT). *p < 0.05, **p <0.01.

Figure 5

I.t. IL-2 Treatments Prevent Tumor Regrowth Following GNR-Induced THT. (A) Tumor volume measurements over a 14-day period post-laser treatment for each group: THT alone, i.t. IL-2 alone, THT plus i.t. IL-2, and control in the 4T1 model (n=16 control, 14 IL2, 15 THT, 12 THT+IL2), error bars represent SEM. (B) Representative images of extracted 4T1 tumor (C) Tumor volume measurements up to day 8 post-laser treatment in the B16-F10 model, showing the comparison between the same groups (n=9 control, 7 IL2, 10 THT, 10 THT+IL2), error bars represent SEM. (D) Representative images of extracted B16-F10 tumor. *p < 0.05 vs. control, ⁺p < 0.05 vs. IL-2, ^#p < 0.05 vs. THT (ANOVA), ^ap < 0.05 (t-test).

Figure 6

IL-2 Treatment Enhances CD8+ T Cell Infiltration, Central Memory Differentiation, and PD-1 Expression in Tumors Following GNR-Induced THT. Panel (A) shows the quantification of CD8+ T cells (gated on CD3+CD8+) in 4T1 tumors across different treatment groups (n= 6 Control, 3 IL2, 7 THT, 6 THT+IL2). Panel (B) illustrates the expression of PD-1+ on CD8+ T cells (gated on PD-1+CD8+CD3+) in 4T1 tumors. Panel (C) displays the frequency of CD8+ central memory (CM) T cells (gated on CD62L+CD44+CD8+CD3+) in 4T1 tumors. Panel (D) presents the quantification of M2 macrophages (gated on CD45+/CD11b+F480+CD206+) in 4T1 tumors. In the B16-F10 model, panel (E) quantifies CD8+ T cells (gated on CD3+CD8+) across different treatment groups (n= 4 Control, 4 IL2, 5 THT, 7 THT+IL2). Panel (F) shows the expression of PD-1+ on CD8+ T cells (gated on PD-1+CD8+CD3+) in B16-F10 tumors. Panel (G) illustrates the frequency of CD8+ central memory (CM) T cells (gated on CD62L+CD44+CD8+CD3+) in B16-F10 tumors, while panel (H) quantifies M2 macrophages (gated on CD45+/CD11b+F480+CD206+) in B16-F10 tumors. (I) Tumor volume measurements over a 14-day period post-laser treatment for each group: THT alone, i.t. IL-2 alone, THT plus i.t. IL-2, and control in the 4T1 model (n=16 control, 14 IL2, 15 THT, 12 THT+IL2, 2 PD1, 4 PD1+THT), error bars represent SEM. *p<0.05, **p <0.01.

Figure 7

GNR-Induced THT Combined with IL-2 Reduces Contralateral 4T1 Tumor Size and Enhances CD8+ T Cell Infiltration. (A) shows the tumor volume of contralateral (untreated) 4T1 tumors in mice with bilateral tumors (n= 8 Control, 6 IL2, 8 THT, 7 THT+IL2). (B) quantifies CD8+ T cells (gated on CD3+CD8+) in contralateral tumors. (C) CD4+ T cells in contralateral tumors. (D) Treg (CD3+CD4+FoxP3+CD25+) levels in contralateral tumors, (for all flow analysis: n= 6 Control, 3 IL2, 6 THT, 6 THT+IL2). *p<0.05 vs. control, ^#p<0.05 vs. THT. **p<0.01, ***p<0.001.