MicroRNA-200c Attenuates the Tumor-Infiltrating Capacity of Macrophages

Abstract

Macrophages constitute a major part of the tumor-infiltrating immune cells. Within the tumor microenvironment, they acquire an alternatively activated, tumor-supporting phenotype. Factors released by tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of hsa-miR-200c-3p (miR-200c) in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed the transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and identified numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. miR-200c-mediated reduction in infiltration further correlated with a miR-200c migration signature comprised of the four miR-200c-repressed, predicted targets PPM1F, RAB11FIB2, RDX, and MSN.

Keywords: breast tumor; macrophage; miR; tumor microenvironment.

Figure 1

Interaction with apoptotic MCF-7 cells leads to increased miR-200c levels in primary human MΦ. miR-200c levels in (A) primary human macrophages (MΦ) and MCF7 cells, (B) MΦ after 48 h coculture (co) with MCF7 cells, (C) apoptotic or viable MCF7 cell-conditioned medium (ACM/VCM), and (D) MΦ 24 h after treatment with ACM/VCM (1 h). miR-200c levels were determined using qPCR and normalized to either SNORD44 (cellular levels) or spiked-in cel-miR-39a (CM). Data are depicted as mean ± SEM (n ≥ 4; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

miR-200c is transferred from MCF7 cells to primary human MΦ. Primary human macrophages (MΦ) were cocultured with MCF7 cells before (pre-) miR-200c levels in MΦ were determined. (A) MΦ pre-miR-200c levels after coculture (48 h) with MCF7 cells. MΦ miR-200c levels upon (B) DICER knockdown, (C) cytochalasin D (CytD) treatment (10 µM), (D) carbenoxolone (CBX) treatment (100 µM), (E) neuropilin receptor (NRP) 1/2 knockdown, (F) CD36 receptor knockdown, (G) CD36 inhibitor sulfo-N-succinimidyl oleate (SSO) treatment (5 µM), or (H) neutralizing CD36 antibody treatment (2 µg/mL). (Pre-) miRNA levels were determined using qPCR and normalized to either SNORD44 or TBP (pre-miRNA). (B,E,F) Knockdown was achieved by transfection with specific siRNAs 24 h prior to coculture. Data are depicted as mean ± SEM (n ≥ 5; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

RNA-sequencing of miR-200c overexpressing MΦ identifies downregulation of migration-associated mRNAs. Primary human macrophages (MΦ) were transfected with miR-200c mimic (mimic) or mimic control (ctr) 72 h before mRNA was isolated for mRNA sequencing. (A) Heatmap of 649 differentially expressed genes in miR-200c-transfected MΦ (|log2FC| ≥ 0.66). (B) GO term analysis was performed with the 250 downregulated targets (log2FC ≤ −0.66) using GOrilla [39] and the top 10 processes (according to their p-value) are shown. (C) Venn diagrams intersecting predicted miR-200c targets (green) with targets upregulated (red; left panel) or downregulated (blue; middle panel) in mimic-transfected MΦ, and with targets downregulated in mimic-transfected MΦ associated with migration-related GO terms (yellow; right panel). (D) Heatmap depicting targets downregulated in mimic-transfected MΦ associated with migration-related GO terms (green: predicted miR-200c targets). (E) Expression of mimic downregulated, migration-associated, predicted miR-200c targets MMD (monocyte to macrophage differentiation-associated 1), PPM1F (protein phosphatase 1F), ARL2BP (ADP-ribosylation factor-like protein 2 binding protein), FRMD4B (FERM domain-containing 4B), RAB11FIP2 (RAB11 family-interacting protein 2), RDX (radixin), and MSN (moesin) was validated in MΦ 72 h after mimic transfection by qPCR. Data were normalized to ctr MΦ and are depicted as mean ± SEM (n ≥ 4; * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

miR-200c overexpression in MΦ alters spheroid infiltration. Primary human macrophages (MΦ) were transfected with miR-200c mimic (mimic) or mimic control (ctr). (A) Twenty-four hours after transfection, a scratch was generated and scratch closure was evaluated after 24 and 48 h by microscopy (representative pictures shown in the left panel). Quantitative assessment of the remaining scratch area was carried out using ImageJ (n = 6; right panel). (B) Forty-eight hours after transfection, MΦ were seeded on fibronectin-coated ibiTreat µ-slides. MΦ migration was determined by live cell tracking for 16 h and quantified using the tracking application of the AxioVisionSoftware. Fifteen representative tracks are depicted (left panel) and a total of 50 cells per condition were analyzed per replicate regarding distance and velocity (n = 4, right panel). (C) Forty-eight hours after transfection, MΦ were transferred onto 4-day-old MCF7 tumor spheroids and allowed to infiltrate for 24 h prior to flow cytometric analysis. Infiltration of mimic-transfected MΦ into spheroids was normalized to ctr-transfected MΦ. Infiltration was determined as number of CD45+ cells in single cell suspensions of infiltrated tumor spheroids. Data are presented as mean ± SEM (n = 13; *** p < 0.001). (D) Correlation of tumor spheroid infiltration with the expression of potential miR-200c targets of the migration-associated miR-200c signature including MMD (monocyte to macrophage differentiation-associated 1), PPM1F (protein phosphatase 1F), ARL2BP (ADP-ribosylation factor-like protein 2 binding protein), FRMD4B (FERM domain-containing 4B), RAB11FIP2 (RAB11 family-interacting protein 2), RDX (radixin), and MSN (moesin). mRNA expression in MΦ was determined at the beginning of the infiltration.

Figure 5

Model of the role of miR-200c in the interaction between tumor cells and MΦ. miR-200c is released from apoptotic, miR-200c-expressing tumor cells and taken up by MΦ via the CD36 receptor. In MΦ, miR-200c reduces the expression of a migration-associated gene signature, consequently limiting the tumor-infiltrating capacity of the MΦ.