Fig 1: Total RNA was extracted and trimmed from formalin‐fixed, paraffin‐embedded (FFPE) sections of three cases of CRC and CRLM (A, dotted line) and subjected to RNA sequencing. The top five genes related to the decreased expression of TAMs in CRLM compared with that in CRC are listed as targeted genes. The data are normalized by TPM (B). RNA scope® assay displays CCL1 mRNA in the FFPE tissues from CRC and CRLM. The housekeeping gene PPIB stained positive in CRC and CRLM (C [a, b]). Little CCL1 mRNA expression is detected in the stroma of CRC (C [c], black arrowheads), while none is detected in that of CRLM (C [d]).
Fig 2: Immunohistochemical expression of CCL1 and CCR8 in 16 cases of CRC and CRLM (A) and quantification by the manual‐visual counting method (B). CCL1‐positive cells are accumulated mainly in the stroma of CRC (A [a,b]), while few low‐intensity CCL1‐positive cells are observed in the stroma of CRLM (A [c,d]). A significant difference is observed in the quantity of CCL‐1 positive cells between CRC and CRLM. A significant difference is observed in the distribution of CCR8‐positive cells in CRC and CRLM (A [e–h]). Double immunofluorescence staining of CCL1 (green) and CD68 (red) with DAPI (blue) in CRC and CRLM (C). CD68 and CCL1 immunoreactivity is observed more in CRC than in CRLM (C [a–d], D [a, b]). CCL1 immunoreactivity is colocalized mostly with CD68 in CRC (C [g]), but rarely in CRLM (C [h]). The percentage of CCL1 in CD68 positive cells (CCL1/CD68) was significantly higher in CRC than in CRLM (D [c]). *p < 0.05.
Fig 3: Cell-based assays for detection and functional characterization of anti-C-C motif chemokine receptor 8 autoantibodies.A Representative histograms from flow cytometry showing binding of anti-C-C motif chemokine receptor 8 (CCR8)-positive IgG from systemic sclerosis (SSc) patients or healthy controls (HC) to CCR8-overexpressing HEK293 cells. A fluorophore-conjugated anti-human IgG Fc antibody was used for detection. B Quantification of mean fluorescence intensity (MFI) in CCR8-overexpressing cells incubated with anti-CCR8-positive or control IgG, with or without preincubation with blocking anti-CCR8 monoclonal antibody (n = 4 technical replicates per group). C ERK phosphorylation levels in CCR8-overexpressing HEK293 cells after stimulation with CCL1 in the presence of anti-CCR8-positive or control IgG, as measured by ELISA (n = 8 biological replicates per group). D Treg migration assay in Transwell culture systems. The number of migrated Tregs in response to CCL1 with anti-CCR8-positive IgG or control IgG (n = 6 biological replicates per group) are presented. Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human CCL1/I-309 Protein