Abstract A42: Impact of liposomal drug delivery and alendronate co-encapsulation on the immune modulatory effects of doxorubicin in the tumor microenvironment
Authors: Islam Md Rakibul , Patel Jalpa , Back Patricia Ines , Shmeeda Hilary , Adamsky Konstantin , Yang Hui , Alvarez Carlos , Gabizon Alberto A , La-Beck Ninh M 
Published: December 2022

Abstract
Although Doxorubicin is the standard of care in advanced soft tissue sarcomas, therapeutic efficacy is minimal. A major contributing factor appears to be an immunosuppressive tumor microenvironment (TME). We have shown that liposomal alendronate, an amino-bisphosphonate, can remodel the immunosuppressive TME towards an immune-permissive milieu. We postulate that co-encapsulating alendronate with doxorubicin in pegylated liposomes (PLAD) will increase the efficacy of doxorubicin as alendronate abrogates the activity of suppressive myeloid cells and liposomes facilitate the accumulation of drugs in tumors. PLAD was formulated with hydrogenated soybean phosphatidyl-choline, methoxy-polyethylene glycol2000-distearoyl-phosphatidylethanolamine, and cholesterol at a molar ratio of 55:5:40, with 1 mg/ml doxorubicin and 0.43 mg/ml alendronate. WEHI-164 fibrosarcoma cells were implanted subcutaneously in male balb/c mice. For assessing the tumor immunologic milieu, mice were randomized when tumor size reached ~300 mm3 to receive PLAD, pegylated liposomal doxorubicin (PLD), or free doxorubicin (FDox) at 8 mg/kg of doxorubicin (n=9/group) or vehicle (n=5) IV. Tumors were collected 5 days post-treatment for FACS analysis. To assess antitumor efficacy, mice were randomized when tumor size reached ~150 mm3 to receive PLAD, PLD, or FDox at 5 mg/kg of doxorubicin (n=9/group) or vehicle (n=5) IV weekly and euthanized at humane endpoints. PLAD, and to a lesser extent PLD, shifted cellular drug uptake to tumor-associated macrophages (TAM) and to monocytic myeloid-derived suppressor cells (mMDSC) compared to free doxorubicin which had cellular drug uptake below detectable levels. PLAD and PLD were associated with fewer total TAMs and a decreased polarization to pro-tumoral M2-TAMs compared to free doxorubicin. Only PLAD significantly increased tumor infiltration of antigen cross-presenting dendritic cells, while both PLAD and PLD significantly increased the number and functionality of NKT and NK cells that are consistent with enhanced antitumor immune responses in the TME. All treatments significantly reduced regulatory T cell infiltration which could help promote antitumor responses in the TME. Interestingly, FDox treatment induced significant changes in the TME that could diminish antitumor immune responses by decreasing M1 TAMs and NK cells. These effects are reflected in the therapeutic study which demonstrates that PLAD and PLD inhibited tumor growth and significantly prolonged survival, while free doxorubicin showed little or no anticancer activity in this advanced fibrosarcoma model. We found that liposomal delivery is required for effective drug uptake by the TAMs and mMDSCs. Co-encapsulation of alendronate and doxorubicin led to a remodeling of the TME by abrogating pro-tumoral immune responses. This strongly suggests that the PLAD formulation is a promising liposome-based drug carrier to potentiate immunotherapy in synergy with its cytotoxic effects.
Citation Format: Md Rakibul Islam, Jalpa Patel, Patricia Ines Back, Hilary Shmeeda, Konstantin Adamsky, Hui Yang, Carlos Alvarez, Alberto A Gabizon, Ninh M La-Beck. Impact of liposomal drug delivery and alendronate co-encapsulation on the immune modulatory effects of doxorubicin in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A42.