An Ex Vivo 3D Tumor Microenvironment-Mimicry Culture to Study TAM Modulation of Cancer Immunotherapy

Abstract

Tumor-associated macrophages (TAMs) accumulate in the solid tumor microenvironment (TME) and have been shown to promote tumor growth and dampen antitumor immune responses. TAM-mediated suppression of T-cell antitumor reactivity is considered to be a major obstacle for many immunotherapies, including immune checkpoint blockade and adoptive T/CAR-T-cell therapies. An ex vivo culture system closely mimicking the TME can greatly facilitate the study of cancer immunotherapies. Here, we report the development of a 3D TME-mimicry culture that is comprised of the three major components of a human TME, including human tumor cells, TAMs, and tumor antigen-specific T cells. This TME-mimicry culture can readout the TAM-mediated suppression of T-cell antitumor reactivity, and therefore can be used to study TAM modulation of T-cell-based cancer immunotherapy. As a proof-of-principle, the studies of a PD-1/PD-L1 blockade therapy and a MAO-A blockade therapy were performed and validated.

Keywords: CAR-engineered T (CAR-T) cell; cancer immunotherapy; checkpoint inhibitor blockade; chimeric antigen receptor (CAR); ex vivo 3D TME-mimicry culture; monoamine oxidase A (MAO-A) blockade; tumor microenvironment (TME); tumor-associated macrophage (TAM).

Figure 1

Generation and validation of human monocyte-derived M2-polarized immunosuppressive macrophages. (A) Diagram showing the human monocyte-derived M2 macrophage culture and polarization. M-CSF, macrophage colony-stimulating factor; MDM, monocyte-derived macrophage; Mφ, macrophage. (B) Fluorescence-activated cell sorting (FACS) detection of CD11b and CD14 on M2-polarized macrophages. Healthy donor peripheral blood mononuclear cells (PBMCs) were included as a staining control. (C) FACS detection of surface markers on M2-polarized macrophages. Monocytes were included as a control. (D–J) In vitro mixed Mφ/T-cell reaction assay to study M2 macrophage-mediated T-cell suppression. (D) Experimental design. 1 × 10⁵ PBMCs were cultured in the assay. (E) PBMC-T-cell growth curve (n = 3). (F) FACS detection of surface marker (CD25) and intracellular cytotoxic molecules (Perforin and Granzyme B) of CD4⁺ and CD8⁺ T cells. (G) Quantification of F (n = 3). (H) ELISA analyses of cytokine (IFN-γ and TNF-α) production in the mixed reaction assay at day 3 (n = 3). (I) FACS analyses of PD-1 on CD4⁺ and CD8⁺ T cells (n = 3). (J) FACS analyses of PD-L1 on M2 macrophages (n = 3). Representative of 3 (D–J) and > 5 (A–C) experiments. Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by Student’s t test.

Figure 2

Validation of immune modulatory reagents on antagonizing M2 macrophage-mediated immunosuppression. (A–E) Study the effect of anti-PD-L1 antibody that blocks macrophage-T-cell inhibitory interaction. (A) Experimental design. 1 × 10⁵ PBMCs were cultured in the assay. (B) CD4⁺ PBMC-T-cell growth curve (n = 3). (C) CD8⁺ PBMC-T-cell growth curve (n = 3). (D,E) ELISA analyses of IFN-γ (D) and TNF-α (E) production in the mixed reaction assay at day 3 (n = 3). (F–K) Study the effect of MAO-A inhibitor phenelzine that reprograms M2 macrophage polarization. (F) Experimental design. About 1 × 10⁵ PBMCs were cultured in the assay. (G) FACS detection of CD206, PD-L1, and PD-L2 on phenelzine-treated or non-treated M2-polarized macrophages. Phe, phenelzine. (H) CD4⁺ PBMC-T-cell growth curve (n = 3). (I) CD8⁺ PBMC-T-cell growth curve (n = 3). (J,K) ELISA analyses of IFN-γ (J) and TNF-α (K) production in the mixed reaction assay at day 3 (n = 3). Representative of three experiments. Data are presented as the mean ± SEM. ns, not significant, *** p < 0.001, **** p < 0.0001, by one-way ANOVA.

Figure 3

Development of an ex vivo 3D TME-mimicry culture to study TAM modulation of T-cell antitumor reactivity. (A) Diagram of an ex vivo 3D TME-mimicry culture. Three human tumor cell lines were studied: MM.1S (multiple myeloma), OVCAR3 (ovarian), and A375 (melanoma). (B) Schematics showing the engineered MM.1S-FG, OVCAR3-FG, and A375-A2-ESO-FG cell lines. Fluc, firefly luciferase; EGFP, enhanced green fluorescent protein; FG, Fluc-EGFP; F2A, foot-and-mouth disease virus 2A; RFP, red fluorescent protein; NY-ESO-1, New York esophageal squamous cell carcinoma-1; ESOp, ESO peptide; IRES, internal ribosome entry site; HLA, human leukocyte antigen. (C–F) Generation of BCMA CAR-engineered T (BCAR-T), mesothelin CAR-engineered T (MCAR-T), and HLA-A2-restricted, NY-ESO-1 tumor antigen-specific human CD8 TCR-engineered T (ESO-T) cells. (C) Experimental design. (D–F) FACS detection of BCAR on BCAR-T cells (D), MCAR on MCAR-T cells (E), and ESO-TCR on ESO-T cells (F). Human T cells that received mock transduction were included as a staining control (denoted as Mock-T). (G–O) TAM modulation of T-cell antitumor reactivity. (G) Tumor killing data of MM.1S-FG by BCAR-T cells at 48 h (n = 3). (H) FACS detection of surface markers (CD25 and CD62L) and intracellular cytotoxic molecule (Granzyme B) of BCAR-T cells (I) Quantification of H (n = 3). (J) Tumor killing data of OVCAR3-FG by MCAR-T cells at 48 h (n = 3). (H) FACS detection of surface markers and intracellular cytotoxic molecule by MCAR-T cells (L) Quantification of K (n = 3). (M) Tumor killing data of A375-A2-ESO-FG by ESO-T cells at 48 h (n = 3). (N) FACS detection of surface markers and intracellular cytotoxic molecule by ESO-T cells. (O) Quantification of N (n = 3). Representative of three experiments. Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by Student’s t test (I,L,O), or by one-way ANOVA (G,J,M).

Figure 4

Application of the ex vivo 3D TME-mimicry culture: studying the PD-1/PD-L1 blockade therapy. (A) Experimental design. OVCAR-3-FG and MCAR-T cells were studied. (B) Tumor killing data at 48 h (n = 3). (C–E) FACS analyses of intracellular cytotoxicity molecule Granzyme B (C), and surface marker CD25 (D) and CD62L (E) of MCAR-T cells (n = 3). Representative of three experiments. Data are presented as the mean ± SEM. ns, not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by one-way ANOVA.

Figure 5

Application of the ex vivo 3D TME-mimicry culture: studying the MAO-A blockade therapy. (A) Experimental design. OVCAR-3-FG and MCAR-T cells were studied. (B) Tumor killing data at 48 h (n = 3). (C–E) FACS analyses of intracellular cytotoxicity molecule Granzyme B (C), and surface marker CD25 (D) and CD62L (E) of MCAR-T cells (n = 3). Representative of three experiments. Data are presented as the mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by one-way ANOVA.