Fig 1: CCRL2 is the upregulated Chemerin receptor in cutaneous carcinoma cell lines and human squamous cell carcinoma(A) Heat map representing the gene expression profile of Chemerin receptors CCRL2, GPR1 and CMKLR1. Relative gene expression was calculated from all three independent experiments normalized to the level of Actin housekeeping gene expression, and relative to the mean expression in normal keratinocytes. Genes were rank-ordered by Pearson correlation and depicted by a pseudo color scale based on qRT-PCR delta-CT values with green and red demonstrating low and high mRNA abundance, respectively. CMKLR1 had very low to no mRNA transcripts; CCRL2 was highly upregulated in cSCC cells; and GPR1 was either downregulated or not changed in cSCC cells compared to keratinocytes. (B–C) Graphs depict the relative fold changes of CCRL2 and GPR1 mRNA expression in cSCC lines compared with normal keratinocytes as defined by qRT-PCR. Shown is one representative of three independent experiments, each with triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001 calculated by Bonferroni post hoc test after ANOVA. (Note that due to low standard deviations of some measurements, error bars are not visible for all data points.) (D–E) Cell surface expressions of CCRL2, GPR1 and CMKLR1 were assessed by flow cytometry analysis in SCL-1 cells and normal keratinocytes. Displayed are the representative fluorescence histograms depicting the relative fluorescence intensity of cells stained with (D) APC-conjugated anti-CCRL2 and (E) A488-conjugated anti-GPR1 antibodies (black histograms) compared with isotype control antibodies conjugated with corresponding fluorochromes (gray histograms). The expression of CMKLR1 was not detectable (not shown). Bar charts represent one of three independent experiments demonstrating the mean percentage ± S.D. for (d) CCRL2-positive and (E) GPR1-positive SCL-1 cells vs. keratinocytes. *p < 0.05, **p < 0.01 and ***p < 0.001 calculated by two-tailed student t-test (n = 8 biological replicates). (F) Representative Western bot analysis of cSCC and keratinocyte cell lysates, confirming the elevated protein level of CCRL2 in cSCC cells compared with normal keratinocytes. (G) Representative photomicrographs of skin biopsies derived from patients suffering from invasive cSCC vs. normal healthy controls stained for Hematoxylin and Eosin (H & E), CCRL2 in red and cytokeratin in green. Nuclear was stained with DAPI (blue). Arrows show the CCRL2-positive cells. Appropriate isotype controls were used to determine the background. H&E images were taken at ×200 magnification and immunofluorescence at ×400 magnification; Scale bars = 50 μm. Orange boxes depict the magnified area. (H) Graph representing the quantification of CCRL2-positive epidermal-derived cells in the skin specimens of patients suffering from cSCC (n = 8) vs. normal healthy individuals (n = 6). **p < 0.01 by two-tailed student t-test.