Melanoma-Derived Extracellular Vesicles Induce CD36-Mediated Pre-Metastatic Niche

Abstract

CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.

Keywords: CD36; extracellular vesicles; macrophages; melanoma; premetastatic niche; sentinel lymph node.

Figure 1

Role of CD36 in the clinical prognosis and immune infiltration correlation in cutaneous melanoma: (A) Hallmark enrichment plot of CD36 generated with core cancer hallmark gene set (n = 1574) shows that CD36 is associated with invasion and metastasis of melanoma as well as reprogramming energy metabolism (p < 0.05). (B) Progression-free survival with CD36 expression in melanoma patients, using upper (red) and lower (blue) quartile data from the SKCM dataset. (C) Correlation of CD36 with CD163, CD14, CD209, and FAP in SKCM dataset (n = 471). (D) CD36 positively correlates with the infiltration level of M2 macrophages in the SKCM-metastasis dataset (n = 368). (E) Correlation between CD36 and infiltration level of endothelial cells in SKCM-metastasis dataset (n = 368).

Figure 2

Melanoma-derived EVs upregulate CD36 expression in the recipient cells. (A) Brief procedure for LEV and melanoma-cell-derived EV collection and characterization. Summarily, afflicted afferent lymph channels were surgically excised, and effluents of channels were utilized to collect LEVs through size-exclusion chromatography. Cell line culture-conditioned media was used to collect EVs by ultracentrifugation. All collected EV characterizations were carried out with NTA (see method in Section 2.3 for more details). (B) LEVs collected from patients possess a significantly higher percentage of CD36 + EVs in total LEVs than control LEVs. (C) SKMEL28 and C32TG melanoma-cell-derived EVs increase expression of CD36 in HLEC (endothelial) and THP1 (monocytic) cells after 24 h of exposures. (D) Microscopic observation of CFSE-labeled-SKMEL28-derived EVs in HLEC cells (scale bar: 100 µm). (E) ImageStream analysis of CFSE-labeled-SKMEL28-derived EVs in THP1 cells (scale bar: 7 µm). (F) The box plot indicates the real-time PCR data by Cq values of CD36 and GAPDH gene expression in EVs collected from melanoma cell lines. Original images of (A,C) can be found in Supplementary Materials.

Figure 3

Melanoma-derived EVs regulate M2-macrophage-like characteristics by upregulating CD36. (A) CD36 surface expression was increased upon EV exposure to cells compared to the control in M0 macrophages derived from THP1 cells. (B) Melanoma-derived EV exposure increases M2-macrophage-like characteristics by upregulating CD163 in PMA-pretreated THP1 cells. (C) EVs collected from CD36-silenced SKMEL28 cells decrease M2 macrophage characteristics in THP1-treated cells through CD36 downregulation.

Figure 4

Spatial MxIF image analysis of SLN (−), SLN (+) tissue from patients, and LN tissue from control subjects. (A) Box plot showing the percentage of cells expressing a specific marker of all cells in a field of view (FOV) (center panel) and proportion of CD36+ cells per FOV in the three groups of samples. (B) Estimated probability of CD36 cells colocalized with other immune cells (within a 40-micron radius of a CD36 cell) in an analyzed panel of markers using spatial regression models.

Figure 5

Primary-melanoma-secreted EVs in lymphatic fluid enter the SLN via afferent lymphatic vessels. The SLN components, including macrophages and endothelial cells, receive these EVs to upregulate CD36, possibly through CD36 cargoes (mRNA and protein). The upregulation of CD36 in the targeted cells can mediate the immunosuppression mechanism to promote the tumor-promoting niche in the SLN.