Liposome-induced immunosuppression and tumor growth is mediated by macrophages and mitigated by liposome-encapsulated alendronate

Abstract

Liposomal nanoparticles are the most commonly used drug nano-delivery platforms. However, recent reports show that certain pegylated liposomal nanoparticles (PLNs) and polymeric nanoparticles have the potential to enhance tumor growth and inhibit antitumor immunity in murine cancer models. We sought herein to identify the mechanisms and determine whether PLN-associated immunosuppression and tumor growth can be reversed using alendronate, an immune modulatory drug. By conducting in vivo and ex vivo experiments with the immunocompetent TC-1 murine tumor model, we found that macrophages were the primary cells that internalized PLN in the tumor microenvironment and that PLN-induced tumor growth was dependent on macrophages. Treatment with PLN increased immunosuppression as evidenced by increased expression of arginase-1 in CD11b⁺Gr1⁺ cells, diminished M1 functionality in macrophages, and globally suppressed T-cell cytokine production. Encapsulating alendronate in PLN reversed these effects on myeloid cells and shifted the profile of multi-cytokine producing T-cells towards an IFNγ⁺ perforin⁺ response, suggesting increased cytotoxic functionality. Importantly, we also found that PLN-encapsulated alendronate (PLN-alen), but not free alendronate, abrogated PLN-induced tumor growth and increased progression-free survival. In summary, we have identified a novel mechanism of PLN-induced tumor growth through macrophage polarization and immunosuppression that can be targeted and inactivated to improve the anticancer efficacy of PLN-delivered drugs. Importantly, we also determined that PLN-alen not only reversed protumoral effects of the PLN carrier, but also had moderate antitumor activity. Our findings strongly support the inclusion of immune-responsive tumor models and in-depth immune functional studies in the preclinical drug development paradigm for cancer nanomedicines, and the further development of chemo-immunotherapy strategies to co-deliver alendronate and chemotherapy for the treatment of cancer.

Keywords: Alendronate; Cancer immunology; Immune modulation; Liposome; Nanoparticle.

Fig. 1

A proposed model of how interactions between the nanoparticle carrier, drug cargo, and immune system impact overall therapeutic efficacy of nanoparticle-delivered drugs. EPR, enhanced permeability and retention effect; MPS, mononuclear phagocyte system.

Fig. 2

PLN-induced tumor growth is mediated by macrophages. (A–C) PLNs are internalized by macrophages and CD11b⁺Gr1⁺ cells in the tumor microenvironment but not by other leukocytes. Mice (n = 8 total) bearing TC-1 tumors were treated intravenously with fluorescent NBD-labelled PLN (NBD-PLN) or vehicle control, tumors were harvested 24 h later and dissociated to obtain cells for FACS analysis. Representative images of macrophages (CD11b⁺F4/80⁺) (A) with and (B) without internalized NBD-PLN. (C) NBD fluorescence was determined in each cell population. (D) In vivo depletion of systemic macrophages abolished PLN-induced tumor growth. MaFIA transgenic mice (n = 24 total) bearing TC-1 tumors were treated with PLN or vehicle control, with macrophage depletion (AP20187) or mock depletion (vehicle). Data are mean + SEM, P-value based on (C) Wilcoxon two-sample rank-sum test, and (D) Holm-Tukey simultaneous multiple comparisons for two-factor analysis of variance with repeated measures; n.s., not significant.

Fig. 3

PLN-associated accumulation of splenic macrophages, monocytes, and granulocytes is mitigated by the alendronate cargo. Mice were treated with PLN, PLN-alen, or vehicle control and splenocytes were harvested 7 days post dose for immunophenotyping. (A–B) PLN increase splenic accumulation of CD11b + Gr1+ cells while PLN-alen did not. (C) The CD11b⁺Gr1⁺ subpopulations that increased were inflammatory monocytes and granulocytes, but an increase in resident monocytes was also observed. Total n = 11; data are mean + SEM (rank-transformed data in Supplemental Tables S4–S9); one-way analysis of variance based on ranks; n.s., not significant.

Fig. 4

PLN-associated polarization of splenic macrophages and immature myeloid cells towards an M2-like phenotype is differentially affected by the alendronate cargo. (A) PLN treatment increased arginase-1 in the non-macrophage CD11b⁺Gr1⁺ cells although this effect was reversed when alendronate was loaded into the carrier. (B) Intracellular iNOS and arginase-1 expression was used to define M1 (iNOS⁺ arg1⁻), M2 (iNOS⁻ arg1⁺), and mixed M1/M2 (iNOS⁺ arg1⁺) phenotypes in macrophages. Both PLN and PLN-alen increased M2 macrophages and decreased M1 macrophages. Total n = 11; data are mean + SEM (rank-transformed data in Supplemental Table S10); (A) one-way analysis of variance based on ranks and (B) multiple comparisons based on observations.

Fig. 5

PLN inhibit global cytokine production in CD8⁺ T-cells. Both PLN and PLN-alen inhibited global cytokine production in CD8⁺ T-cells. Cytokine production was determined by ex vivo stimulation with PMA/ionomycin in the presence of brefeldin A and analyzed by FACS. Total n = 10; data are group means.

Fig. 6

PLN-associated tumor progression is abolished by encapsulated alendronate but not free alendronate. Mice implanted with TC-1 tumorigenic cells were treated intravenously with PLN, PLN + free alendronate (PLN + alen), PLN-alen, or vehicle control. Data are from two independent experiments (total n = 28), (A) tumor volumes are expressed as mean + SEM; Holm-Tukey simultaneous multiple comparisons for two-factor analysis of variance with repeated measures, and (B) Kaplan-Meier survival curves of time to tumor growth to > 100 mm³ volume with Log-Rank test comparing the groups.

Fig. 7

PLN-alen mitigates carrier-associated effects on tumor-associated leukocytes. Mice bearing implanted TC-1 tumors were treated intravenously with PLN, PLN-alen, free alendronate (alen), or vehicle and sacrificed 48 h later to obtain tumor-derived single cell suspensions for immunophenotyping. Intracellular inducible nitric oxide synthase (iNOS) and arginase-1 (Arg1) expression was used to define M1 (iNOS⁺Arg1⁻), M2 (iNOS⁻Arg1⁺), and mixed M1/M2 (iNOS⁺Arg1⁺) TAMs; unactivated (iNOS⁻Arg1⁻) are not shown. (A) PLN polarized TAMs towards a mixed M1/M2 phenotype, whereas this was abolished by PLN-alen. (B) PLN, but not PLN-alen, increased Arg1 expression in myeloid-derived suppressor cells (MDSC). Total n = 24, expressed as mean + SEM (rank-transformed data in Supplemental Table S11); one-way analysis of variance based on ranks for vehicle, PLN, and PLN-alen.

Fig. 8

Proposed mechanisms of PLN-induced tumor growth and immunosuppression. A: Liposomes penetrate and deposit in the tumor via enhanced permeability and retention (EPR) effect; B: Tumor-associated macrophages phagocytose liposomes, and respond with increased arginase production, decrease iNOS, and shift towards an M2 phenotype; C: An autocrine/paracrine loop of TGFβ ensues and leads to secretion of CCL2 (MCP-1); D: CCL2 attracts peripheral migration of immature myeloid cells which increase arginase production and turn into suppressive cells; E: TAM and myeloid-derived suppressor cells (MDSC) impair T-cell immunity; F: Cytotoxic T lymphocyte (CTL) antitumor effects are inhibited.