Direct contact of MM cells with ECs leads to a dynamic and reversible increase of CD138^low/neg JAM-C⁺ cells in mice and humans. (A) Representative fluorescence-activated cell sorting (FACS) plots of surface CD138 and JAM-C expression on living MM cells at depicted time points of coculture without ECs (MM, upper panel) or with ECs (MM + ECs, lower panel) and (B) corresponding graphical display of increase in cell frequencies. (C) Representative FACS plots of surface CD138 and JAM-C expression on living MM cells grown without ECs for 48 hours in complete growth medium after the end of co-culture without ECs (upper panel) or with ECs (lower panel) and (D) corresponding graphical display of cell frequencies. Normalized relative transcription levels of (E) CD138 and (F) JAM-C messenger RNA (mRNA) within the MM cells cultured with ECs (MM + ECs) or without ECs (MM) determined by quantitative real-time polymerase chain reaction (qRT-PCR). (G) Normalized relative transcription levels of CD138 and JAM-C mRNA within MM cells cultured without ECS for 48 hours in complete growth medium after the end of EC co-culture compared with the expression level at 72 hours depicted in panels E and F (open columns). (H) Representative FACS plots of surface CD138 and JAM-C expression on living RPMI 8226 (MM) cells at depicted time points of co-culture without HUVECs (huMM, upper panel) or with HUVECs (huMM + ECs, lower panel) and (I) corresponding graphical display of increase in cell frequencies. (J) Representative FACS plots of surface CD138 and JAM-C expression on living huMM cells grown without ECs for 48 hours in complete growth medium after the end of co-culture without ECs (upper panel) or with ECs (lower panel) and (K) corresponding graphical display of cell frequencies. All experiments were independently repeated 3 times. Unpaired, two-tailed Student t tests were used. Error bars indicate SD for FACS analyses and standard error of the mean for qRT-PCR results.