Novel Combination Treatment for Melanoma: FLASH Radiotherapy and Immunotherapy Delivered by a Radiopaque and Radiation Responsive Hydrogel

Abstract

Immunotherapies have become the standard treatment for melanoma. To further improve patient responses, combinations of immunotherapies and radiotherapy (RT) are being studied, since radiotherapies can potentially provide additional immune stimulation, in addition to direct antitumor effects. FLASH-RT is a novel, ultrahigh dose rate, radiation delivery approach, with the potential of at least equivalent tumor control efficacy and reduced damage to healthy tissue. However, the effects of combining FLASH-RT and immunotherapy have not been extensively studied in melanoma. Toll-like receptor (TLR) agonists, such as imiquimod (IMQ), are potent immunostimulatory agents, although their utility is limited due to poor solubility and systemic side effects. We therefore developed a novel combination therapy for melanoma consisting of IMQ delivered to the tumor via a radiopaque and radiation responsive hydrogel combined with FLASH-RT. We found that FLASH was able to effectively stimulate IMQ release from the hydrogel. In addition, we found that the combination of FLASH and released IMQ resulted in synergistic melanoma cell killing in vitro. The combination therapy reduced tumor growth compared to controls, enhanced survival, and resulted in remarkable enhancements in certain tumor cytokine levels. CT imaging allowed the hydrogel to be monitored in vivo. In addition, no adverse effects of the treatment were observed. Overall, this IMQ-gel and FLASH-RT combination may have potential as an improved treatment for melanoma and indicates that the interactions of FLASH-RT and TLR agonists merit further study.

Figure 1.

(A) Schematic representation of the FLASH-RT triggered release of IMQ drug and AuNP from the AuNP-IMQ hydrogel. Influence of radiation on the release of payloads from hydrogels. Quantification of the released IMQ from the hydrogel irradiated with (B) FLASH-RT of 60 Gy, CONV-RT of 60 Gy, photon radiation of 60 Gy, or mock irradiation of 0 Gy. (C) FLASH-RT and CONV-RT with radiation dose from 0 to 60 Gy. Quantification of the released gold from the hydrogel irradiated with (D) FLASH-RT (E) CONV-RT with radiation dose from 0 to 60 Gy.

Figure 2.

Phantom imaging of hydrogels with a micro-CT. (A) CT phantom scans of hydrogels before and 7 days after FLASH-RT. (B) Quantification of CT attenuation values of the images in panel (A).

Figure 3.

(A) Effect of AuNP-IMQ-gel on B16–F10 cell viability after 8 h incubation. (B) Effect of 10 Gy FLASH-RT and 10 Gy CONV-RT on B16–F10 cell viability after 48 h incubation. (C) Viability of B16–F10 cells after receiving treatment of eluted drug from the nonirradiated AuNP-IMQ-gel, 10 Gy FLASH-RT, FLASH-triggered released drug from AuNP-IMQ-gel, or the combination of FLASH-RT and FLASH-triggered released drug from AuNP-IMQ-gel.

Figure 4.

In vivo evaluation of the tumor growth inhibition effect of the IMQ-loaded hydrogel and FLASH-RT over 18 days. The mice are divided into the following groups: DPBS (vehicle control group), FLASH (FLASH-RT only), Gel + FLASH (AuNP-hydrogel with FLASH-RT), IMQ-gel (AuNP-IMQ-gel), and IMQ-gel + FLASH (AuNP-IMQ-gel with FLASH-RT). (A) Experimental design of the study. (B) Kaplan–Meier curve for the experiment. Statistical comparisons were done with log-rank analysis using Datatab.

Figure 5.

Luminex multiplex analysis of cytokine levels in B16–F10 tumors 4 days after treatment. A combination of FLASH-RT and IMQ immunotherapy induces cytokine production in the B16–F10 tumors. (A) Cytokine profiles in B16–F10 tumors. (B) Concentrations of G-CSF, IL-9, IP-10, MCP-1, MIP-2, and VEGF in B16–F10 tumors 4 days after treatment.

Figure 6.

In vivo images and hydrogel volume analysis. (A) Representative CT images of AuNP-IMQ hydrogel injected in the right flank, at different time points: preinjection, 3 days postinjection, and 14 days postinjection. Insets are enlarged images of injected hydrogels highlighted in blue. (B) Representative 3D CT images of a mouse with the hydrogel highlighted in blue. (C) 3D reconstructions of the hydrogels based on CT images in panel (B). (D) Quantification of the hydrogel degradation by comparing the hydrogel volumes.