BM dissemination model with color-coded MM cells. (A) A schematic diagram illustrating the study procedures. MM cell lines were transduced with a lentiviral (LV) mixture carrying 4 fluorescent proteins (FPs; BFP [B], GFP [G], RFP [R], and iRFP [I]), generating 15 subpopulations with different fluorescence markers. Each cell population was sorted by flow cytometry, expanded in vitro, and then mixed together at an even proportion. Two million color-coded MM cells were injected into the BM cavity of femoral bones freshly resected from syngeneic donor mice. Then, myeloma-bearing bone chips were subcutaneously transplanted under the dorsal skin of recipient mice. (B) The proportion of each of the 15 subpopulations of cells passaged in vitro throughout the animal experiment did not change. (C) To assess in vivo clonal dynamics in animals during disease course, cells from the implanted bone chip (primary site), left and right femur BM (distant BM sites), and CTCs were analyzed upon symptoms of hindlimb paralysis. Each uniquely colored circle represents a single colored clone in an animal. The area of the circle is proportional to the size of each clone. The proportion of the 15 subpopulations of distant BM sites (left and right femurs) showed biased color distribution, compared with primary implanted sites. Color distributions of left and right femurs were similar to that of matched CTCs. (D-E) Confocal imaging of color-coded MM cells in the primary implanted bone (D) and femur BM (E). Scale bar, 100 μm.