Targeting a scavenger receptor on tumor-associated macrophages activates tumor cell killing by natural killer cells

Abstract

Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.

Keywords: immunotherapy; melanoma; tumor associated macrophages.

Fig. 1.

MARCO⁺ TAMs have a perivascular phenotype, and targeting results in less vessel tortuosity inside the tumor. (A) Phenotyping of MARCO⁺ TAMs compared with other TAMs in untreated B16 melanoma tumors on day 10. Pregating on CD45⁺ CD11b⁺ F4/80⁺. Mean fluorescence intensity (MFI) is shown. n = 9. (B) MARCO⁺ cells are mostly not located in hypoxic areas. Mice were injected i.p. with pimo 2 h before tumor collection on day 10. Representative staining of untreated B16 tumor. n = 3. (Scale bar: 50 µm.) (C) MARCO⁺ cells are closely located to CD4⁺ cells. Representative image. n = 3. (Scale bar: 50 µm.) (D) MARCO⁺ cells are in proximity to CD8⁺ cells. Representative image. n = 3. (Scale bar: 50 µm.) (E) MARCO⁺ cells are negative for proliferation marker Ki-67. Representative image. n = 3. (Scale bar: 50 µm.) (F) MARCO+ macrophages are closely located to blood vessels defined by CD31 expression. A representative image is shown from an untreated B16 tumor on day 10 (Scale bar: 50 µm.) (G) Representative images from whole tumor imaging following the iDISCO method. Tumors were stained for CD31 on day 10. n = 3 per group. (H) Decreased tortuosity of vessels after anti-MARCO Ab treatment analyzed by whole tissue imaging. Tortuosity reflects the ratio of chord length to curved length. Data are expressed as mean ±SD; P values calculated by Mann-Whitney U test.

Fig. 2.

Targeting of MARCO by monoclonal antibodies leads to antibody internalization and metabolic changes. (A) Representative image of internalized MARCO on peritoneal macrophages after 3 h of stimulation. Cells were pretreated with Fc block for 30 min before labeled anti-MARCO Ab was added to the cultures. On the right side is the quantification of all time points. MFI is shown. A representative experiment of three independent experiments is shown. (B) Internalization of MARCO by WT and FcγR/IIb KO peritoneal macrophages. Cells were incubated with labeled anti-MARCO Ab or rat IgG1 isotype control for the stated time before being analyzed on the image stream. MFI is shown. One experiment with two replicates was performed. (C) Tumor size of B16 melanoma tumors on day 10 in untreated and anti-MARCO Ab-treated mice from WT or P2X7R KO mice. n = 4 or 5 per group. (D) Tumor size of B16 melanoma tumors on day 10 in PBS-treated and anti-MARCO Ab-treated mice from WT or NLRP3 KO mice. n = 4 or 5 per group. (E) MARCO⁺ peritoneal macrophages were stimulated for 24 h and analyzed for glycolysis and glycolytic rate. The extracellular acidification rate (ECAR) was measured with a Seahorse XFe96 analyzer. One representative experiment out of five is shown. Data are expressed as mean ± SD, except in C, where data are mean ± SEM. P values were calculated using the Mann–Whitney U test, except in A and B, where one-way ANOVA was used.

Fig. 3.

Anti-MARCO Ab effect on B16 melanoma tumors is dependent on NK cells. (A) Tumor size of B16 tumors in WT or CD8⁺ T cell-depleted mice treated with either PBS or anti-MARCO Ab on day 10. n = 4 or 5 per group. (B) Tumor size of B16 tumors in WT or CD4⁺ T cell-depleted mice treated with PBS or anti-MARCO Ab on d 10. n = 4 or 5 per group. (C) Tumor size of B16 tumors in WT or TCR-β–deficient mice treated with either PBS or anti-MARCO Ab. n = 5 per group. (D) Tumor size of B16 tumors in WT or NK cell depleted mice treated with PBS or anti-MARCO Ab on d 10. n = 13 or 14 per group. Data are pooled from three independent experiments. (E) B16 tumor growth curves measured manually by caliper in mice treated with PBS or anti-MARCO Ab. Mice were either WT or depleted for NK cells. #P = 0.0004, PBS + PBS vs. PBS + anti-MARCO Ab; &P = 0.0555, PBS + PBS vs. anti-NK1.1 Ab + PBS; ¤P < 0.0004, PBS + anti-MARCO Ab vs. anti-NK1.1 Ab + anti-MARCO Ab. Data are expressed as mean ± SD (B and C) or mean ± SEM (D). P values were calculated using the Mann–Whitney U test.

Fig. 4.

The anti-MARCO–mediated anticancer effect attracts NK cells and is dependent on their TRAIL-dependent killing mechanism. (A) Frequency of B16 intratumoral NK cells in PBS or anti-MARCO Ab-treated mice on day 10 defined as CD45⁺ CD3⁻ NKp46⁺ cells. Histogram shows frequency of CD45⁺ immune cells inside the tumor. n = 22-24/group. Data pooled from four independent experiments. (B) Histogram showing IL-15 levels detected in the sera of PBS or anti-MARCO Ab treated mice. Detection by Mesoscale. n = 3. (C) Expression of effector molecule Perforin on B16 intratumoral NK cells in PBS or anti-MARCO Ab treated mice on d10. NK cells were gated as CD45⁺ CD3⁻ NKp46⁺ cells. (D) TRAIL expression on B16 intratumoral NK cells in PBS or anti-MARCO Ab treated mice on d10. NK cells were gated as CD45⁺ CD3⁻ NKp46⁺ cells. (E) NK1.1⁺ cells are near MARCO⁺ cells in untreated B16 tumors. Representative image shown from three tumors. (Scale bar = 50 μm.) (F) Size of B16 tumors in WT or FcγRIII-deficient mice treated with PBS or anti-MARCO Ab on day 10. n = 8–10/group. Data are pooled from two independent experiments. Datapoints represent female and male mice. (G) Size of B16 tumors in WT or perforin-deficient mice treated with PBS or anti-MARCO Ab treated on day 10. n = 4–5/group. (H) Histogram showing B16 tumor size on day 10 in WT mice and mice treated with TRAIL-blocking antibodies before tumor injection. Mice were then either PBS or anti-MARCO Ab treated. n = 9–10/group. Data are pooled from two independent experiments. Data are expressed as mean ± SEM except in B and D, where data are expressed as mean ± SD. P values were calculated by Mann-Whitney U test.

Fig. 5.

Motility of NK cells in metastatic lymph nodes increases after anti-MARCO Ab treatment. (A) Representative snapshots of NK cell tracks in the tumor region of popliteal lymph nodes injected with metastatic B16 tumor cells. Mice were treated with PBS or anti-MARCO Ab. (B) Quantification of mean speed by NK cells before and after anti-MARCO Ab treatment. Each dot represents one cell. (C) Quantification of total distance covered (i.e., track length) by NK cells before and after anti-MARCO Ab treatment. Each dot represents one cell. (D) Quantification of straightness in directionality of NK cells before and after anti-MARCO Ab treatment. Each dot represents one cell. Data are expressed as mean ± SEM. P values were calculated with the Mann–Whitney U test.

Fig. 6.

Combination of anti-MARCO Ab treatment with other immune checkpoint inhibitors enhances their efficacy and attracts NK cells. (A) Schematic of the B16 model for combinatorial treatments and Ab administration. (B) Size of B16 tumors in PBS, anti-MARCO Ab, anti–PD-L1 Ab and anti–PD-L1 + anti-MARCO Ab-treated mice on day 12. n = 13 to 15 per group. Data are pooled from three independent experiments. (C) Size of B16 tumors in PBS, anti-MARCO Ab, anti–PD-1 Ab, and anti–PD-1 + anti-MARCO Ab-treated mice on day 12. n = 13 to 15 per group. Data pooled from three independent experiments. (D) Histogram showing the frequency of NK cells in B16 tumors on day 12 after PBS, anti-MARCO Ab, anti–PD-L1 Ab, and anti–PD-L1 + anti-MARCO Ab treatment. n = 8 to10 per group. Data are pooled from two independent experiments. NK cells are defined as CD45⁺CD3⁻ NKp46⁺. (E) Histogram showing the expression of transcription factors Eomes and T-bet. Double-positive cells are defined as NK cells. n = 8 to 10 per group. Data are pooled from two independent experiments. Data are expressed as mean ± SEM. P values are calculated with the Mann–Whitney U test.

Fig. 7.

MARCO is also expressed by human immune suppressive macrophages, and targeting by anti-hMARCO antibodies can activate NK cells to increase their tumor killing. (A) MARCO expression on human macrophages shown by MFI. Tumor-conditioned macrophages with different human melanoma cell lines also up-regulate MARCO. n = 2 to 7. (B–D) Activation of NK cells after coculture with control or anti-MARCO Ab-treated macrophages measured by degranulation (CD107a), IFN-γ expression, and proliferation (Ki-67). NK cells were then stimulated with human melanoma cell line ANRU. Two independent experiments were pooled. Each dot represents one donor. n = 9 or 10. (E–G) Activation of NK cells after coculture with control or anti-MARCO Ab-treated macrophages measured by degranulation (CD107a), IFN-γ expression, and proliferation (Ki-67). NK cells were then stimulated with human melanoma cell line KADA. Two independent experiments were pooled. Each dot represents one donor. n = 9. (H) Human NK cells were cocultured with control or anti-MARCO Ab-treated macrophages, NK cells were transferred to the human melanoma cancer cell line ANRU, and tumor cell killing was measured. Two independent experiments were pooled. Each dot represents one donor. n = 9. (I) Human NK cells were cocultured with control or anti-MARCO Ab-treated macrophages, NK cells were transferred to the human melanoma cell line KADA, tumor cell killing was measured. Two independent experiments were pooled. Each dot represents one donor. n = 9. Data are expressed as mean ± SEM P values were calculated using the Wilcoxon test.