Cellular backpacks for macrophage immunotherapy

Abstract

Adoptive cell transfers have emerged as a disruptive approach to treat disease in a manner that is more specific than using small-molecule drugs; however, unlike traditional drugs, cells are living entities that can alter their function in response to environmental cues. In the present study, we report an engineered particle referred to as a "backpack" that can robustly adhere to macrophage surfaces and regulate cellular phenotypes in vivo. Backpacks evade phagocytosis for several days and release cytokines to continuously guide the polarization of macrophages toward antitumor phenotypes. We demonstrate that these antitumor phenotypes are durable, even in the strongly immunosuppressive environment of a murine breast cancer model. Conserved phenotypes led to reduced metastatic burdens and slowed tumor growths compared with those of mice treated with an equal dose of macrophages with free cytokine. Overall, these studies highlight a new pathway to control and maintain phenotypes of adoptive cellular immunotherapies.

Fig. 1. Schematic illustration of cellular backpacks for maintaining proinflammatory phenotypes of adoptive MΦ therapies.

(A) MΦs polarized with IFN-γ ex vivo quickly shift from proinflammatory to anti-inflammatory phenotypes after penetrating a solid tumor. (B) MΦs carrying IFN-γ–loaded backpacks maintain their proinflammatory phenotypes deep within the tumor microenvironment, altering the phenotypes of endogenous TAMs.

Fig. 2. Backpack preparation, characterization, and monocyte interactions.

(A) Schematic illustrations of a backpack (i) and its method of printing (ii); graphs of average backpack stiffness, thickness, and width (n ≥ 4) (iii). (B) Amount of active IFN-γ per backpack, determined by ELISA (n = 5). ***P < 0.001. (C) Cumulative release of IFN-γ from backpacks over 60 hours (n = 3). (D) Association of backpacks with primary murine macrophages over time in vitro (n = 3). (E) Proportion of backpacks that evaded phagocytosis over time compared with spheres of similar volume (n = 5). (F) Confocal micrographs of leukocytes (nucleus, blue; membrane, green) displaying PLGA discs (red).

Fig. 3. Phenotypic evaluation of macrophages (MΦs) carrying IFN-γ backpacks in vitro.

BMDMs were cultured for 5 days with free IFN-γ (16 ng/ml; black lines), blank backpacks (0 ng/ml IFN-γ; green lines), and IFN-γ backpacks (16 ng/ml equivalent) in normoxia (dark blue lines) and tumor-mimicking conditions (1% O₂ and 10 volume % tumor-conditioned media; light blue lines). Cellular expression of representative (A) M1 markers (iNOS, MHCII, and CD80) and (B) M2 markers [vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), and CD206], relative to that of unpolarized macrophages (without IFN-γ or backpacks). Graphs are logarithmic (n = 10,000 events per data point).

Fig. 4. IFN-γ backpacks promote proinflammatory phenotypes in solid tumors.

(A) Polarization of adoptively transferred macrophages (MΦs) 48 hours after injection. BMDMs were polarized ex vivo for 24 hours with IFN-γ (16 ng/ml) (i), left unpolarized and injected with 50 ng of free IFN-γ (ii) or left unpolarized, bound to IFN-γ backpacks at a dose of 50 ng equivalent IFN-γ and injected (iii). Bar graphs indicate the fold change in the median expression of representative M1 biomarkers (iNOS, MHCII, and CD80; top row) and M2 biomarkers (HIF-1α, CD206, and Arg-1; bottom row), relative to their native expression in endogenous TAMs. (B) Polarization of endogenous TAMs 48 hours after injection of groups described in (A). Bar graphs indicate the fold change in the median expression of representative M1 biomarkers (top row) and M2 biomarkers (bottom row) relative to the native expression of endogenous TAMs [leftmost bars in (B)]. For all bar graphs, n = 5. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. Efficacy of IFN-γ backpacks for reducing metastasis and tumor burden of 4T1 mammary carcinomas.

(A) In vivo bioluminescence imaging of metastatic colony formation in the chest cavities of mice burdened with 4T1-Luc cells 32 days after inoculation (primary tumor outside of view). Five representative images per treatment group are shown. (B) Average radiance from bioluminescence in the chest cavities of the mice in (A) (n = 9). (C) Representative histological section of a 4T1 tumor treated with macrophages carrying IFN-γ backpacks. Dotted line separates regions of cleared (top) and intact tumorous tissue (bottom). (D) Relative proportion of tumor-infiltrating dendritic cells (TIDCs) in solid 4T1 tumors revealed through tumor-associated immune cell phenotyping (determined by CD45⁺, SYTOX⁻, and CD11c⁺; n = 5). (E) Weight changes of mice burdened with 4T1-Luc tumors in different groups (n = 9). (F) Growth kinetics of tumors in the groups shown in (E). Black arrows indicate days of therapeutic injections. (G) Survival of mice in (E). Statistical significance was determined via a log-rank test. *P < 0.05; **P < 0.01; ***p < 0.001.