Tumor-associated macrophages in the cutaneous SCC microenvironment are heterogeneously activated

Abstract

Tumor-associated macrophages (TAMs) may have an important role in tumor immunity. We studied the activation state of TAMs in cutaneous SCC, the second most common human cancer. CD163 was identified as a more abundant, sensitive, and accurate marker of TAMs when compared with CD68. CD163(+) TAMs produced protumoral factors, matrix metalloproteinases 9 and 11 (MMP9 and MMP11), at the gene and protein levels. Gene set enrichment analysis (GSEA) was used to evaluate M1 and M2 macrophage gene sets in the SCC genes and to identify candidate genes in order to phenotypically characterize TAMs. There was coexpression of CD163 and alternatively activated "M2" markers, CD209 and CCL18 (chemokine (C-C motif) ligand 18). There was enrichment for classically activated "M1" genes in SCC, which was confirmed in situ by colocalization of CD163 and phosphorylated STAT1 (signal transducer and activator of transcription 1), IL-23p19, IL-12/IL-23p40, and CD127. Also, a subset of TAMs in SCC was bi-activated as CD163(+) cells expressed markers for both M1 and M2, shown by triple-label immunofluorescence. These data support heterogeneous activation states of TAMs in SCC, and suggest that a dynamic model of macrophage activation would be more useful to characterize TAMs.

Figure 1. Macrophages were more abundant in SCC compared to normal skin

Representative immunohistochemistry (10x) and cell counts of the macrophage markers (a) CD163 (with an inset of CD163⁺ cells at 20x) and (b) CD68, showing a significantly increased number of macrophages surrounding SCC tumor nests compared to normal skin. Each dot represents one patient. ***P <0.001. (c) CD163 (green) co-localized with CD68 (red) shown as yellow, but there were CD163⁺ cells that did not co-express CD68. (d) CD163 (green) did not co-express CD11c (red), while (e) some CD68⁺ cells (green) did co-express CD11c (red) shown as yellow. Bar=100μm.

Figure 2. SCC TAMs expressed pro-tumoral products in the tumor microenvironment

Mean mRNA expression of (a) MMP9 and (b) MMP11 relative to HARP after adjustment for batch effect in normal skin (white bars), non-tumoral skin (NT, gray bars), and SCC (black bars) with standard error of the mean. *P <0.05, **P <0.01. CD163⁺ cells (green) demonstrated abundant co-localization with (c) MMP9 (red) and (d) MMP11 (red) in SCC compared to normal skin. Double positive cells appear yellow. Bar=100μm.

Figure 3. SCC TAMs expressed products of alternatively activated macrophages

Many CD163⁺ cells (green) co-expressed (a) phosphorylated STAT6 (STAT6p) (red), (b) CD209/DC-SIGN (red), and (c) CCL18 (red) compared to normal skin. Double positive cells appear yellow. Bar=100μm

Figure 4. SCC TAMs expressed characteristics of classically activated macrophages in a microenvironment with Type-1 activation

Compared to normal skin, CD163⁺ cells co-expressed (a) phosphorylated STAT1 (STAT1p) (red), (b) CD127/IL7R (red), (c) IL-23p19 (red), and (d) IL-12/IL-23p40 (green). Double positive cells appear yellow. Bar=100μm

Figure 5. A subset of SCC TAMs simultaneously expressed characteristics of both classical and alternative activation

Triple-labeled confocal immunofluorescence revealed the presence of (a) CD163⁺ cells (blue) that simultaneously co-expressed STAT6p (green) and STAT1p (red) and (b) CD163⁺ cells (green) that simultaneously co-expressed the markers CD127 (red) and CD209 (blue) in SCC. Triple-positive cells (white) are indicated by arrows. Bar=100μm. (c) The proposed model of SCC macrophage polarization. Th1 and Th2 cells produce cytokines, IFNγ and IL-4, respectively, and act on resident CD163⁺ macrophages to polarize these cells in several directions. IFNγ stimulates the M1 phenotype (CD127 and IL-23), and IL-4 stimulates towards the M2 phenotype (CD209 and CCL18). There is also production of mediators that are not driven by known polarizing cytokines, such as MMP9, MMP11, and VEGF-C. The overall outcome is a poly-activated TAM.