Blocking CCR8 inhibits lymph node metastasis. (A and B) Expression of CCR8 inhibitor MC148 in tumor cell lines as indicated. (A) RT-PCR of cultured tumor cells for hCCL1, MC148, and hCCR8 mRNA. cDNA from LECs served as a positive control for CCL1. β-Actin served as a loading control. (B) Western blot for hCCR8 protein in lysates of cultured cells, MC148 protein in tumor cell CM in vitro, and MC148 in tumor lysates in vivo. β-Actin served as a loading control. C, pcDNA vector control; M, MC148. (C) Tumor growth of pcDNA control and MC148-expressing MDAcl.6 or MDAcl.13 cells. Each data point represents mean tumor volume ± SEM, n = 10 mice per group. (D) Quantification of total vessels (CD31⁺) in tumors as indicated. Bars represent the mean vessel area ± SEM per 10⁶ µm² total area; MDAcl.13, n = 4; SK-MEL-25, n = 3 tumors. (E and F) Efficiency of shRNA-mediated CCR8 knockdown in tumor cells. qPCR for human CCR8 (E) and Western blot analysis (F) in different tumor cell lines in vitro and in tumors in vivo, as indicated. (F) CCR8 knockdown in MDA-MB-435 cells was performed with shCCR8 sequence 1, shCCR8(1); in SK-MEL-25 with the sequence 2, shCCR8(2); and in MDA cl. 13 with shCCR8 sequences 1 and 2. Error bars indicate mean ± SD, n = 3. (G) Incidence of intranodal metastases upon CCR8 inhibition with the MC148 soluble antagonist or upon CCR8 knockdown with shRNA. Metastasis incidence is calculated as the number of LN with GFP⁺ tumor cells, divided by the total number of LN examined. ND, not determined; ns, not significant; ***, P < 0.001.