The greatest contributors to cancer mortality are metastasis and the consequences of its treatment. Here, we present a novel treatment of metastatic breast cancer that combines photothermal therapy with targeted single-walled carbon nanotubes (SWCNTs) and immunostimulation with a checkpoint inhibitor. We find that the selective near-infrared photothermal ablation of primary orthotopic EMT6 breast tumors in syngeneic BALB/cJ mice using an annexin A5 (ANXA5) functionalized SWCNT bioconjugate synergistically enhances an anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4)-dependent abscopal response, resulting in an increased survival (55%) at 100 days after tumor inoculation. In comparison, there was no survival at 100 days for either photothermal therapy by itself or immunostimulation by itself. Prior to photothermal therapy, the SWCNT-ANXA5 bioconjugate was administered systemically at a relatively low dose of 1.2 mg/kg, where it then accumulated in tumor vasculature via ANXA5-dependent binding. During photothermal therapy, the average maximum temperature in the tumor reached 54 °C (duration 175 s). The mechanism of prolonged survival resulting from combinatorial photothermal ablation and immune stimulation was evaluated by flow cytometric quantification of splenic antitumoral immune effector cells and serum cytokine quantification.

The high prevalence of breast cancer is a global health concern, but there are no safe or effective treatments for it at its advanced stages. These facts urge the development of novel treatment strategies. Annexin A5 (ANXA5) is a natural human protein that binds with high specificity to phosphatidylserine, a phospholipid tightly maintained in the inner leaflet of the cell membrane on most healthy cells but externalized in tumor cells and the tumor vasculature. Here, we have developed a targeted photosensitizer for photothermal therapy (PTT) of solid tumors through the functionalization of single walled carbon nanotubes (SWCNTs) to ANXA5—the SWCNT-ANXA5 conjugate. The ablation of tumors through the SWCNT-ANXA5-mediated PTT synergizes with checkpoint inhibition, creating a systemic anti-cancer immune response. In vitro ablation of cells incubated with the conjugate promoted cell death in a dose-dependent and targeted manner. This treatment strategy was tested in vivo with the orthotopic EMT6 breast tumor model in female balb/cJ mice. Enhanced therapeutic effects were achieved by using intratumoral injection of the conjugate and treating tumors at a lower PTT temperature (45oC). When combined with checkpoint inhibition of anti-PD-1, SWCNT-ANXA5-mediated PTT increased survival and 80% of the mice survived for 100 days. Evidence of immune system activation by flow cytometry of splenic cells strengthens the hypothesis of an abscopal effect as a mechanism of prolonged survival.