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. 2021 Feb;70(2):289-298.
doi: 10.1007/s00262-020-02676-8. Epub 2020 Jul 23.

Therapeutic potential of anti-VEGF receptor 2 therapy targeting for M2-tumor-associated macrophages in colorectal cancer

Aung Kyi Thar Min  1 , Kosaku Mimura  2   3 , Shotaro Nakajima  1 , Hirokazu Okayama  1 , Katsuharu Saito  1 , Wataru Sakamoto  1 , Shotaro Fujita  1 , Hisahito Endo  1 , Motonobu Saito  1 , Zenichiro Saze  1 , Tomoyuki Momma  1 , Shinji Ohki  1 , Koji Kono  1
Affiliations

Therapeutic potential of anti-VEGF receptor 2 therapy targeting for M2-tumor-associated macrophages in colorectal cancer

Aung Kyi Thar Min et al. Cancer Immunol Immunother. 2021 Feb.

Abstract

Background: Although immunotherapy with immune checkpoint inhibitors (ICIs) has become a standard therapeutic strategy in colorectal cancer (CRC) exhibiting microsatellite instability-high, limited patients benefit from this new approach. To increase the efficacy of ICIs in CRC patients, it is crucial to control the function of immunosuppressive cells in the tumor microenvironment. M2-tumor-associated macrophages (TAMs) are key immunosuppressive cells and promote tumor growth, angiogenesis, and epithelial-mesenchymal transition. In the present study, we focused on the VEGF signaling pathway in M2-TAMs to control their inhibitory function.

Methods: We evaluated the population of M2-TAMs, the VEGF receptor 2 (VEGFR2) expression on M2-TAMs, and the correlation between HIF-1α-positive cells and VEGFR2 expression levels on M2-TAMs in CRC using the analysis of The Cancer Genome Atlas colorectal adenocarcinoma dataset (n = 592), the flow cytometry of freshly resected surgical specimens of CRC (n = 20), and the immunofluorescence staining of formalin-fixed paraffin-embedded whole tissue samples of CRC (n = 20). Furthermore, we performed a functional assay of M2 macrophages through the VEGF/VEGFR2 signaling pathway in vitro.

Results: The population of M2-TAMs and their VEGFR2 expression significantly increased in the tumor compared to the normal mucosa in the CRC patients. HIF1-α-positive cells significantly correlated with the VEGFR2 expression level of M2-TAMs. M2 macrophages induced by cytokines in vitro produced TGF-β1 through the VEGF/VEGFR2 signaling pathway.

Conclusions: Our results suggest that anti-VEGFR2 therapy may have therapeutic potential to control the immune inhibitory functions of M2-TAMs in CRC, resulting in enhanced efficacy of immunotherapy with ICIs.

Keywords: Colorectal cancer (CRC); M2-tumor-associated macrophages (M2-TAMs); TGF-β; VEGF receptor 2 (VEGFR2).

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
The number of M2-TAMs increased in the tumor. a The gating method of flow cytometry for freshly resected surgical specimens to detect M1-TAMs (CD14+CD11c+CD163−) and M2-TAMs (CD14+CD11c−CD163+). b The summary of population of M1- or M2-TAMs in CD14-positive cells in the normal mucosa (normal) and the tumor (tumor) by flow cytometric analysis (M1-TAMs; CD14+CD11c+CD163−, M2-TAMs; CD14+CD11c−CD163+). c Representative images showing the immunofluorescence staining of M2-TAMs in the normal mucosa (normal) and the tumor (tumor) samples. Green staining; CD163, red staining; HIF-1α, blue staining; DAPI. d The number of M2-TAMs in the normal mucosa (normal) and the tumor (tumor). *p < 0.05, ****p < 0.0001
Fig. 2
Fig. 2
VEGFR2 expression on M2-TAMs. a Summarized data for VEGFR2 expression on M1- and M2-TAMs in the normal mucosa (normal) and the tumor (tumor) by flow cytometric analysis (n = 20) (M1-TAMs; CD14+CD11c+CD163−, M2-TAMs; CD14+CD11c−CD163+). Representative histogram of VEGFR2 expression on M1- and M2-TAMs (upper). b mRNA expression z-scores in 592 samples with colorectal adenocarcinoma were obtained from TCGA (PanCancer Atlas) dataset. We utilized multi-gene expression signatures for M2-TAMs signature and the signature score was calculated by averaging the expression levels of included genes. Correlation of mRNA levels of KDR (VEGFR2) with M2-TAMs signature. *p < 0.05, ***p < 0.001, ****p < 0.0001
Fig. 3
Fig. 3
HIF1-α+ M2-TAMs increased in the tumor. a mRNA expression z-scores in 592 samples with colorectal adenocarcinoma were obtained from TCGA (PanCancer Atlas) dataset. Correlation of KDR (VEGFR2) with HIF1A. b The number of HIF-1α+ cells (left) and HIF-1α+ M2-TAMs (right) in the normal mucosa (normal) and the tumor (tumor). c The summary of correlation of VEGFR2 expression on M1-TAMs (CD14+ CD11c+ CD163−) and M2-TAMs (CD14+CD11c−CD163+) from flow cytometric analysis with the number of HIF-1α+ cells from immunofluorescence staining analysis in each patient. ***p < 0.001
Fig. 4
Fig. 4
M2 macrophages expressing VEGFR2 produced TGF-β1 through the VEGF/VEGFR2 signaling pathway. a VEGFR2 expression on induced M1 and M2 macrophages by flow cytometric analysis. b Western blot analysis showing the VEGF signaling molecules in induced M1 and M2 macrophages after stimulation by VEGF with/without selective VEGFR2 inhibitor (small molecule). c ELISA analysis showing the TGF-β1 secretion from induced M2 macrophages after stimulation by VEGF with/without selective VEGFR2 inhibitor (small molecule). *p < 0.05
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