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. 2022 Apr 27;3(5):100621. doi: 10.1016/j.xcrm.2022.100621

Figure 1.

Figure 1

Cellular maps of metastatic melanoma tumors

(A) Example of melanoma whole section scan. From left to right: melanoma antigens (blue) and CD45 (green); or CD3 (red); or CD14 (green); or CD3 (red) and CD14 (green). Scale bar, 700 μm.

(B) Composition of CD45+ infiltrate in metastatic melanoma by histocytometry. Each square represents a different sample, bar indicates median value with 95% CI; red square represents tissues of non-lymphatic origin; n = 20.

(C) Top two panels depict iCD14+ cells (red) within melanoma clusters (green), left panel and iCD14 cells in close contact with CD3+ T cells (cyan), right panel. Lower two panels depict sCD14+ cells (red) in stromal area, left panel, which are also in close contact with CD3+ T cells (cyan), right panel. Scale bar, 20μm.

(D) Neighborhood probability analysis reveals proximity of CD3+ T cells with melanoma antigen-loaded CD14+ cells and other CD3+ T cells versus lower probability of proximity of CD3+ T cells with melanoma cells. CD3+ T cell proximity was determined with respect to melanoma cells (Mel+CD14−), melanoma antigen-loaded CD14+ cells (CD14+Mel+), melanoma antigen-lacking CD14+ cells (CD14+Mel−), other CD3+ T cells (CD3+), and other cell types (other). Log2FC greater than zero indicates increased likelihood of proximity; n = 20. Line at Median with 95% CI.

(E) Ratio of CD14+ cells loaded with unprocessed melanoma antigen. Tumor-infiltrating iCD14+ cells have a significantly higher ratio compared with stromal sCD14+ cells. Each square represents a different sample, bar indicates median value with 95% CI; n = 20.

(F) STED imaging of individual iCD14+ cells reveals intracytoplasmic localization of non-processed melanoma antigen. Surface rendering of DAPI (blue), CD14 (red) and melanoma antigen (green). Left panel shows overlay of all channels, top right panel shows CD14 vs DAPI; lower right panel shows DAPI versus melanoma antigen. Scale bar, 2 μm, n = 3.

(G) STED imaging reveals melanoma cells (white) interacting with iCD14+ cells (blue), which are also in contact with a CD3+ (green) CD8+ (red) T cells. Left panel shows opaque surface rendering for all channels together; scale bar, 5 μm. Right panel shows transparent surface rendering for CD14 channel to allow visualization of intracytoplasmic melanoma antigen in iCD14+ cells and close interaction with CD3+CD8+ T cells. Scale bar, 3 μm, n = 4.