Pegylated Liposomal Alendronate Biodistribution, Immune Modulation, and Tumor Growth Inhibition in a Murine Melanoma Model

Abstract

While tumor-associated macrophages (TAM) have pro-tumoral activity, the ablation of macrophages in cancer may be undesirable since they also have anti-tumoral functions, including T cell priming and activation against tumor antigens. Alendronate is a potent amino-bisphosphonate that modulates the function of macrophages in vitro, with potential as an immunotherapy if its low systemic bioavailability can be addressed. We repurposed alendronate in a non-leaky and long-circulating liposomal carrier similar to that of the clinically approved pegylated liposomal doxorubicin to facilitate rapid clinical translation. Here, we tested liposomal alendronate (PLA) as an immunotherapeutic agent for cancer in comparison with a standard of care immunotherapy, a PD-1 immune checkpoint inhibitor. We showed that the PLA induced bone marrow-derived murine non-activated macrophages and M2-macrophages to polarize towards an M1-functionality, as evidenced by gene expression, cytokine secretion, and lipidomic profiles. Free alendronate had negligible effects, indicating that liposome encapsulation is necessary for the modulation of macrophage activity. In vivo, the PLA showed significant accumulation in tumor and tumor-draining lymph nodes, sites of tumor immunosuppression that are targets of immunotherapy. The PLA remodeled the tumor microenvironment towards a less immunosuppressive milieu, as indicated by a decrease in TAM and helper T cells, and inhibited the growth of established tumors in the B16-OVA melanoma model. The improved bioavailability and the beneficial effects of PLA on macrophages suggest its potential application as immunotherapy that could synergize with T-cell-targeted therapies and chemotherapies to induce immunogenic cell death. PLA warrants further clinical development, and these clinical trials should incorporate tumor and blood biomarkers or immunophenotyping studies to verify the anti-immunosuppressive effect of PLA in humans.

Keywords: PD-1; cancer; immune checkpoint; immunotherapy; liposomal alendronate; melanoma; tumor.

Figure 1

In vitro drug cytotoxicity. (A) B16-OVA melanoma cells, (B) bone marrow-derived macrophages were treated for 48 h with liposomal alendronate (PLA), alendronate, or doxorubicin (positive control) at various concentrations. Cytotoxicity was assessed using the MTT assay. Each treatment condition was performed in duplicate or triplicate; coefficients of variation were <10% for all replicates in all experiments. Data shown are mean ± SEM from one representative experiment; at least two experimental replicates were conducted.

Figure 2

Liposomal alendronate induced M1 polarization and inflammatory markers in M0 and M2 bone marrow-derived macrophages (BMDMs). (A) Expression of iNOS as a marker of M1 polarization, (B) expression of Arg1 as a marker of M2 polarization, (C) expression of cytokines in unpolarized M0 macrophages, (D) expression of cytokines in M2-polarized macrophages, and (E) expression of cytokines in M1-polarized macrophages were determined by RT-qPCR. Fold changes were normalized to vehicle-treated unpolarized M0 cells. Results are expressed as mean ± SEM (n = 6/group from two experimental replicates); one-way ANOVA followed by Tukey’s multiple comparisons analyses; all pair-wise comparisons were performed, and only significant results are shown (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). PLA = liposomal alendronate.

Figure 3

Levels of (A) IL-6 and (B) CXCL-10 production in unpolarized M0, M1-polarized, and M2-polarized bone marrow-derived macrophages (BMDMs) were confirmed by ELISA analyses of the cell culture supernatants from these experiments. Results are expressed as mean ± SEM (n = 6/group from two experimental replicates); one-way ANOVA followed by Tukey’s multiple comparisons analyses; all pair-wise comparisons were performed and only significant results are shown (*** p < 0.001, **** p < 0.0001). PLA = liposomal alendronate.

Figure 4

Biodistribution of liposomal alendronate (PLA) in tumor-bearing mice. LC-MS/MS quantitation of alendronate concentrations in different tissues and serum at (A) 24 h for both treatments, and (B) at 1 h (free alendronate) or 48 h (PLA) after intravenous dosing (n = 2–4/per group). Data represent mean ± SEM; unpaired two tailed t-tests; *p < 0.05, **p < 0.01. (C) Representative fluorescence microscopy images of DiI-labeled PLA in different tissues at 24 h post-dose. (D) Mean DiI fluorescence intensity and (E) quantification of the proportion of tissue area that was DiI-positive for PLA-Dil and Dil only groups; each point represents one image. TLDN, tumor-draining lymph node; LN, lymph node.

Figure 5

Antitumor efficacy of liposomal alendronate (PLA) in B16-OVA melanoma. Mice were implanted with B16-OVA cells, and then randomized (n = 6–7 mice/group) to treatments that were initiated when tumors were ~50 mm³. Tumor volume (A), Kaplan–Meier curves (B) and tumor weight (C) data show that PLA significantly reduced tumor growth in B16-OVA melanoma model. Images of whole tumor at endpoint from each treatment group (D). Data represent mean ± SEM; Two-way ANOVA with Dunnett’s test for tumor volume analyses; Logrank test for the Kaplan–Meier curve; unpaired two tailed T-test for tumor weights.

Figure 6

B16-OVA tumor immunological milieu. (A,B) In a B16-OVA melanoma tumor model, PLA treatment reduced the total tumor-associated macrophages (TAM) in the tumor microenvironment (TME). Analyses of the subpopulation of TAM revealed that PLA increased non-polarized TAM, but the effects on M1- or M2-polarized TAM were not significantly different than the vehicle group. (C,D) Treatment with PLA increased the total myeloid-derived suppressor cells (MDSC) and the subset of neutrophilic myeloid-derived suppressor cells (nMDSC) in the TME. (E,F) The total T-cell population was decreased with PLA treatment. Further analyses of the subpopulation revealed that the helper T cells were decreased without any effect on the cytotoxic T cells. n = 6 for PLA + isotype; n = 7 for vehicle + anti-PD1; n = 6 for vehicle + isotype. Mean ± SEM shown; one-way ANOVA with Dunnett’s test. * p < 0.05, ** p < 0.01, *** p < 0.001.