(T1130-11-74) Multi-targeted Nanomedicine with Synergistic Doxorubicin and Oligo Hyaluronic Acid for Enhancing Anti-tumor Immunity in the Metastatic Triple-Negative Breast Cancer

Purpose: In the tumor microenvironment (TME), abundant hyaluronic acid (HA) and immunosuppressive cells negatively impact the treatment efficacy of metastatic triple-negative breast cancer (TNBC). The native HA interacts with the cluster of differentiation 44 (CD44) and receptors for hyaluronan mediated motility (RHAMM) that are overexpressed in TNBC activating the downstream oncogenic and pro-metastatic pathways. Immunosuppressive cells such as tumor associated macrophages (TAMs) produce anti-inflammatory cytokines and impair the function of cytotoxic T cells. To overcome the drug resistance and immunosuppression, our laboratory has devised αvβ3 and αvβ5-targeting polymer-lipid hybrid nanoparticles iRGD-DOX-oHA-PLNs to synergistically deliver the chemotherapy drug doxorubicin (DOX) and the CD44 regulator HA oligomers (oHA, < 10 kDa).¹ In this study, we aim at investigating the effect of the multi-targeted iRGD-DOX-oHA-PLNs on enhancing immune response in a syngeneic murine TNBC model.

Methods: The induction of immunogenic cell death (ICD) was evaluated in vitro by measuring the ATP release level and calreticulin (CRT) expression of 4T1 cells. The cytotoxicity of nanoparticles on mouse RAW 264.7 macrophages was evaluated by MTT assay. The secretion level of the anti-inflammatory cytokine interleukin-6 (IL-6) by 4T1-RAW 264.7 co-culture and in the 4T1 tumors was measured by the enzyme-linked immunosorbent assay (ELISA). The infiltration and activation of CD8⁺ T cells and depletion of TAMs in 4T1 tumors was examined by immunohistochemical (IHC) staining. The anti-tumor and anti-metastasis effect of iRGD-DOX-oHA-PLNs was evaluated by measuring tumor sizes and monitoring bioluminescence signals.

Results: The iRGD-DOX-oHA-PLNs promoted ATP generation and CRT expression by 4T1 cells, suggesting the stimulation of ICD. iRGD-DOX-oHA-PLNs efficiently killed RAW 264.7 macrophages with a half-maximal inhibitory concentration (IC50) of 1.49 μg/mL. DOX alone was found to promote IL-6 production in 4T1 tumors probably due to the elevated oxidative stress and nuclear factor kappa B (NF-κB) activation,² while oHA-containing formulations efficiently reduced the IL-6 level in a dose-dependent manner. The induction of anti-tumor immunity was evidenced by the IHC staining of CD8, granzyme B, and F4/80, which showed that iRGD-DOX-oHA-PLNs fostered cytotoxic CD8⁺ T cell infiltration and meanwhile depleted TAMs. iRGD-DOX-oHA-PLNs greatly repressed tumor growth and lung metastasis development with a significant decrease in the tumor volume and invisible bioluminescence signal in the lung area.

Conclusion: The co-delivery of oHA and DOX in the iRGD-DOX-oHA-PLNs efficiently boosted anti-tumor immunity and exhibited excellent anti-tumor and anti-metastasis efficacy. Conceivably, this nanoparticle system could be a promising option for metastatic TNBC treatment.

References: 1. Zhang, T. et al. Targeting Signaling Pathways of Hyaluronic Acid and Integrin Receptors by Synergistic Combination Nanocomposites Inhibits Systemic Metastases and Primary Triple Negative Breast Cancer. Advanced Therapeutics 4 (2021).
2. Bent, E.H., Gilbert, L.A. & Hemann, M.T. A senescence secretory switch mediated by PI3K/AKT/mTOR activation controls chemoprotective endothelial secretory responses. Genes Dev 30, 1811-1821 (2016).

Acknowledgements: This work is supported by the Canadian Institutes of Health Research and National Science and Engineering Research Council (NSERC) of Canada; King Abdulaziz City for Science and Technology (KACST) for the scholarship to I. Alradwan; the Chinese Scholarship Council for the scholarship to P. Zhi.