CD200 Upregulation in Vascular Endothelium Surrounding Cutaneous Squamous Cell Carcinoma

Figure 1. Messenger RNA (mRNA) and protein expression of CD200 are significantly increased in the stromal regions of squamous cell carcinoma (SCC). A, Cells in the interstitium of SCC tumor nests (SCC stroma) had significantly greater mRNA expression of CD200 (adjusted to human acidic ribosomal protein [hARP], in log₂ scale) compared with cells in normal reticular dermis (RD); the average in normal RD was adjusted to 1.0. B, There was very low expression of CD200 in normal skin. C, CD200 was expressed in the dermis adjacent to SCC but was not highly expressed in tumor nests. D, The numbers of CD200⁺ cells were quantified. There was a significant increase in CD200⁺ cells in SCC stroma compared with normal RD. Error bars indicate mean (SEM). AK indicates actinic keratosis; EPI, epidermis.

Figure 2. The number of PAL-E⁺ structures that coexpress CD200 increases with increasing invasiveness of squamous cell carcinoma (SCC). A, Immunofluorescence showed little coexpression of CD200 (green, left upper panel) and PAL-E (red, right upper panel) in normal dermis (n = 5). (The bottom panels show the merged image where double positive cells are identified in yellow.) B, Some PAL-E⁺ cells coexpress CD200 in the stroma of SCC in situ (n = 5). C, Nearly all PAL-E⁺ cells coexpress CD200 in invasive SCC (n = 7). D, There was little coexpression of CD200 (green, left upper panel) and LYVE-1 (red, right upper panel) in normal dermis (n = 5). E, There was little coexpression of CD200 and LYVE-1 in the stroma of SCC in situ (n = 5). F, There was little coexpression of CD200 (green, left upper panel) and LYVE-1 (red, right upper panel) in invasive SCC (n = 5). Scale bars = 100 μm.

Figure 3. CD200 expression is inducible on human dermal blood endothelial cells (HDBECs) in culture using tumor supernatants. A, When cultured for 12 hours in media containing tumor supernatant, HDBECs had significantly greater messenger RNA (mRNA) expression (adjusted to human acidic ribosomal protein [hARP], in log₂ scale) than when cultured in media alone; media with 0.01–μg/mL lipopolysaccharide (LPS) was used as a positive control. Error bars indicate mean (SEM). B, When cultured for 36 hours in media containing tumor supernatant, HDBECs had greater CD200 surface expression than when cultured with media alone, as measured by mean fluorescence intensity. Error bars indicate mean (SEM). C, Superimposed histograms of fluorescence intensity of HDBECs from selected samples of media alone and tumor supernatant illustrate a shift toward greater surface expression.

Figure 4. CD200 expression is induced to a greater degree on CD34^high human dermal blood endothelial cells (HDBECs) treated with tumor supernatant compared with CD34^high HDBECs treated with normal skin supernatant. A, CD31⁺ HDBECs were further gated on CD34^high populations and analyzed for the expression of CD200. The value 25.3 reflects 25.3% of all cells. B, Superimposed histograms of fluorescence intensity of CD34^high HDBECs from selected samples of media plus lipopolysaccharide (LPS) (red), normal skin supernatant (blue), and squamous cell carcinoma (SCC) tumor supernatant (green) illustrate a stepwise shift toward greater surface expression of CD200. The orange line indicates background fluorescence intensity (FMO). C, When cultured for 36 hours in media containing tumor supernatant, HDBECs were found to have greater CD200 surface expression than when cultured with normal skin supernatant, as measured by mean fluorescence intensity (MFI). Error bars indicate mean (SEM).

Figure 5. CD200 receptor (CD200R) is located on most CD163⁺ macrophages and occasional CD11c⁺ dendritic cells in the squamous cell carcinoma (SCC) microenvironment. A, Immunofluorescence in invasive SCC shows that the majority of CD163⁺ cells (green, left upper panel) coexpress CD200R (red, right upper panel). (The bottom panels show the merged image where double positive cells are identified in yellow.) B, Some CD11c⁺ cells (red, left upper panel) coexpress CD200R (green, right upper panel). C, There was little coexpression of CD3 and CD200R in invasive SCC. D, There was little coexpression of CD207/Langerin and CD200R in invasive SCC. E, There was little coexpression of NKp46 and CD200R in invasive SCC. Scale bars = 100 μm.