Assistant professor Cleveland Clinic, Ohio, United States
Introduction:: Breast cancer is one of the leading causes of female mortality in the United States. Particularly, triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancers, which is notorious for its low response to therapeutics and highly invasive nature. The five-year survival rate is 65 % for regional TNBC, but only 11% for metastatic stage. Conventional cancer treatments, such as chemotherapy and radiation therapy are still the first-line treatments for metastatic breast cancer patients. Photonic therapy is a promising non-invasive method for treating cancer, utilizing light energy to efficiently destroy cancer cells. Conventional cancer treatments principally induce apoptosis, an anti-inflammatory programmed cell death. In contrast, photonic therapy generates high temperatures, and intense acoustic vibrations leading to proinflammatory necrotic cell death. Photonic therapy effectively destroys tumor cells and reprograms the tumor microenvironment by disrupting the physical barriers in solid tumors and removing the immunosuppressive milieu. Polyhydroxy fullerene (PHF), which has photothermal and photoacoustic properties, was used in the current study for photonic therapy.
Materials and Methods:: PHF purchased from Suzhou Dade Carbon Nanotechnologies (Suzhou, China) was purified by methanol wash and size-exclusion chromatography in our lab. Chitosan-PHF (CPHF) hydrogel was prepared by electrostatic crosslinking. The photothermal efficiency of nanomaterials was determined similar to our previous publication1 (Figure 1 A). The cytotoxicity and the cell death modality after photonic treatment in 4T1 TNBC cells and 3T3 fibroblasts were measured with XTT, live/dead, and apoptosis/necrosis assay kits. In vivo experiments were carried out with the 4T1 TNBC murine model. Mice were injected with 50% of the tumor volume of the photonic agent intratumorally. Photonic treatment was carried out with a near-infrared laser (785 nm, 600 mW, Ø6mm), and the temperature during treatment was recorded with a thermal camera. To achieve effective treatment on the 4T1 BALB/c mouse model, a set of in vivo studies was designed to identify the optimum setting for photonic therapy. Tumor recurrence and growth after treatment were recorded over 30 days. We found 46°C as the effective treatment temperature threshold with a better tumor-free rate. Linear correlation was found between treatment temperature and recurrence day. In separate experiments, tumors were harvested 6 and 24 hours after photonic therapy, and flow cytometry was used to characterize the innate immune cell populations using the untreated tumor as control.
Results, Conclusions, and Discussions:: The CPHF hydrogel possesses lower photothermal efficiency (η) compared to PHF nanoparticles. PHF nanoparticles affected cell viability when its concentration is higher than 1 mg/mL for 4T1 cells, and 2 mg/mL for 3T3 cells. No cytotoxicity was observed with CPHF hydrogel, which could be due to the crosslinking of PHF with chitosan that would impede the capability of PHF nanoparticles to enter cells. In cellular photonic therapy experiments, both PHF nanoparticle and CPHF hydrogel induced necrotic cell death. Furthermore, disassembly of cell nuclei, and extracellular DNA release were observed in 4T1 cells after PHF nanoparticle-mediated photonic therapy (Figure 1 B), which suggests additional damage occurred at the subcellular level. In vivo photonic therapy experiments with the 4T1 TNBC model also showed extracellular diffusion of DNA (staining with toluidine blue) from the treatment site (Figure 1 C). Flow cytometry revealed innate immune cell infiltration into the treated tumor, characterized by an increased population of neutrophils at the 6 and 24 hours timepoint and monocyte at the 24 hours timepoint (Figure 1 D).
In conclusion, photonic therapy has the potential to induce necrotic cell death and alter the tumor immune environment by promoting innate cell infiltration. A better understanding of the immunological milieu after photonic therapy can help develop strategies to enhance the patient’s immune system to recognize tumor-associated antigens, resulting in improved anti-cancer immunity and therapeutic outcomes.
Acknowledgements (Optional): : The authors acknowledge the financial support from the Lerner Research Institute.
References (Optional): : [1] Chen, A., Grobmyer, S. R., & Krishna, V. B. (2020). Photothermal response of polyhydroxy fullerenes. ACS omega, 5(24), 14444-14450.
