Micro-environmental cross-talk in an organotypic human melanoma-in-skin model directs M2-like monocyte differentiation via IL-10

Abstract

Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response. Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks. Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane. This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells. These features resemble the initial stages of invasive melanoma. Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts. This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment. Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade. Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163⁺PD-L1⁺ M2-like phenotype upon IL-10 blockade. Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.

Keywords: IL-10; M2 macrophages; Melanoma; Reconstructed human skin; Tumor microenvironment; Tumor progression.

Fig. 1

The melanoma reconstructed human skin (Mel-RhS) model recapitulates the initial stages of invasive melanoma. Comparison of morphology and phenotype between human native skin, melanoma biopsies, and Mel-RhS cultured for 2, 4, and 6 weeks at the air–liquid interface. a Melan-A staining shows melanoma growth and nest formation in the melanoma biopsies and in Mel-RhS over time. b PCNA and c Ki-67 staining shows a decrease in mitotically active cells in the Mel-RhS over time. d Collagen type IV staining shows a heterogeneous and disorganized protein deposition in the Mel-RhS, with expression and sporadic interruptions around melanoma nests (indicated by black arrows). e MMP-9 staining highlights its expression in SK-MEL-28 within the Mel-RhS. Representative stainings (paraffin-embedded 5-μm-thick tissue sections) of at least four independent experiments (each with an intra-experiment replicate) are shown. Scale bar = 100 µm

Fig. 2

Cytokine release by the melanoma reconstructed human skin (Mel-RhS) compared to its control (RhS). After 4 weeks culture at the air–liquid interface, medium was refreshed and culture supernatant was collected over a period of 24 h. Cytokines were detected by means of ELISA. Results are shown as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001; paired t test; N = 7 independent experiments performed in duplicate for CCL2, CCL22, IL-8, and GM-CSF; N = 9 independent experiments performed in duplicate for CXCL10, IL-10, and VEGF; N = 10 independent experiments performed in duplicate for M-CSF and TGF-β1)

Fig. 3

Keratinocytes, fibroblasts, and melanoma cells produce IL-10 mRNA in the melanoma reconstructed human skin (Mel-RhS). a IL-10 mRNA was detected at single-cell level by fluorescent RNAish in the reconstructed human skin (RhS) and Mel-RhS model. Each dot (green) indicates one IL-10 mRNA molecule. b IL-10 fluorescent RNAish (cyan) was combined with Melan-A (red; melanocytes and melanoma cells), cytokeratin (green; keratinocytes), and DAPI (blue; nuclei of all cells, including fibroblasts) immunofluorescence staining. IL-10 mRNA spots in nuclei of keratinocytes (green) and melanoma cells (red) are clearly detectable, as well as in (typically elliptical) nuclei of unstained dermal fibroblasts, as indicated by white arrows. Scale bar = 20 µm

Fig. 4

moDC phenotype after exposure to melanoma reconstructed human skin (Mel-RhS) culture supernatants for 6 days compared to its control (RhS) and its relation to IL-10 levels. a Frequency percentage of CD45⁺ cells expressing the surface markers CD1a, CD14, BDCA3, CD163, or CD16 and with an M2-like phenotype (defined as CD14⁺BDCA3⁺CD163⁺CD16⁺PD-L1⁺PD-L2⁺), and geometric mean intensity (MFI) of PD-L1 and PD-L2 in the CD45⁺ cells (i.e. monocytes cultured in the presence of the DC differentiation-inducing cytokines GM-CSF and IL-4), exposed to culture supernatants from either RhS (+RhS SN; white circles) or Mel-RhS (+Mel-RhS SN; black circles). Results are shown as mean ± SEM (*p < 0.05 and **p < 0.01; paired t test; N = 10). b Geometric mean intensity (MFI) and positive percentages of CD163, CD16, PD-L1, and PD-L2 expression on the CD45⁺CD14⁺ cells upon exposure to either RhS- or Mel-RhS-derived supernatants (RhS SN and Mel-RhS SN, respectively). c Percentages of CD1a⁺ or M2-like cells (defined as CD14⁺BDCA3⁺CD163⁺CD16⁺PD-L1⁺PD-L2⁺) within the CD45⁺ monocytic cell population and their correlation with the IL-10 levels found to be secreted in the supernatants derived from the RhS (white circles) or Mel-RhS (black circles) models. Results are shown with the 95% confidence bands of the best-fit line. Both p-value and Pearson r value are shown

Fig. 5

High-dimensional analysis of the phenotype of monocytes conditioned by supernatants derived from the melanoma reconstructed human skin (Mel-RhS) model cultured in the presence or absence of IL-10 neutralizing antibodies. a Differences in the t-SNE analyses between IgG1 and anti-IL-10 conditions. Two gates with shifting subsets between conditions are shown with the percentage of total CD45⁺ monocytes in that particular gate. b Differences between IgG1 and anti-IL-10 in the intensity and the distribution of expression of CD14, BDCA3, PD-L1, CD163, and CD16 in the t-SNE analysis. The same gates as in a are depicted in b. c Percentage of M2-like cells (defined as CD14⁺BDCA3⁺CD163⁺CD16⁺PD-L1⁺PD-L2⁺) within the CD45⁺ cell population after incubation with Mel-RhS supernatant pre-treated with either IgG1 or anti-IL-10 (N = 3; mean ± SEM is shown)