(161n) Evaluating Parameters Associated with the Activation of Immune Response in Phosphatidylserine Targeted Photothermal Therapy of Triple Negative Breast Cancer Tumor Model in Mice

Although the available treatments for cancer have improved for the past years, they still promote debilitating side effects. Current therapies are not adequately effective for treating some types of tumors, especially for more advanced stages of the disease, when metastasis has occurred.

We have developed a cancer targeted photosensitizer for photothermal therapy (PTT) aiming to treat solid tumors based on the functionalization of single-walled carbon nanotubes (SWCNTs) to annexin A5 (ANXA5) to create the SWCNT-ANXA5 conjugate. PTT benefits from the peak absorbance of SWCNTs in the near-infrared (NIR) range to efficiently convert light into thermal energy, aiming to ablate the primary tumor. ANXA5 is a protein that has specificity for cancer due to the strong binding (Kd~ 1 nM) to externalized phosphatidylserine (PS) on the surface of tumoral cells and tumor vasculature. PS constitutes 15% of the phospholipid bilayer and is a biomarker of various types of cancers, including breast, bladder, lung and colorectal carcinoma and melanoma, but is absent on the surface of healthy cells.

ANXA5 is a relatively small protein that can be produced in bacterial cell culture. We have genetically engineered E. coli to produce human recombinant ANXA5 that contains six histidine residues (HisTag) enabling purification by affinity liquid chromatography in nickel column. The high purity protein (above 95%, confirmed by SDS-page electrophoresis) is then linked to SWCNTs with a linker DSPE-PEG-MAL (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)]), which interacts with the highly hydrophobic SWCNTs and bonds to ANXA5 through the cysteine reactive maleimide group.

Our research group have studied the combination of two strategies for cancer treatment to induce a systemic response against metastatic breast cancer: (1) SWCNT mediated PTT of breast cancer primary tumor model; (2) immunostimulation of the checkpoint inhibitor anti-CTLA-4 to downregulate the mechanisms that suppress immune system’s natural antitumoral response to cancer.

The irradiation of solid tumors with the SWCNT-ANXA5 mediated PTT efficiently ablates EMT6 tumors, a triple negative breast cancer tumor model in mice. The combination of PTT and anti-CTLA-4 has been able to improve the long-term survival of mice (55% survival at 100 days post tumor inoculation). The extended survival is correlated with evidence that only the combined therapy significantly increases the number of helper T cells and cytotoxic T cells, cell types that are associated to antitumoral effects. Additionally, the combined therapy increases the levels of pro-inflammatory cytokines in serum (IL-6, IFN-γ, and TNF-α) confirming the systematic activation of the immune system against metastatic tumors.

Further studies have shown that injection of the SWCNT-ANXA5 conjugate either intratumorally or intravenously is effective at increasing the levels of tumor necrosis factor alpha (TNF-α) cytokine in the serum 1 day after the PTT treatment compared to untreated control. Additionally, the final tumor surface temperatures of the PTT were measured by a thermal camera. Treatment with lower final temperature (45°C or 50°C) was able to promote an increase of the concentration of proinflammatory cytokines in serum compared to untreated control. This evidence suggests that even a reduced amount of energy applied during PTT induces the activation of immune response, increasing the safety of PTT to surrounding healthy tissue.