Myeloid gene signature originates from donor myeloma cells and not host myeloid cells. (A) Experimental workflow to examine the proliferative potential of dormant myeloma cells. eGFP⁺DiD⁺ 5TGM1 cells were injected IV into BKAL mice. Hind limbs were isolated, and eGFP⁺DiD^hi and eGFP⁺DiD^neg cells were index sorted into individual wells of 96-well plates. Plates were cultured and imaged over 14 days. (B) Representative images of individual eGFP⁺ cells isolated from eGFP⁺DiD^hi (top) and GFP⁺DiD^neg (bottom) cell populations at day 0 through day 14. Inset images show eGFP and DiD channels separately. Scale bar = 50 µm; inset scale bar = 20 µm. (C) Histogram showing the proportion of wells containing eGFP⁺ colonies after 14 days from 3 independent experiments. Red bars represent dormant MM cells; green bars represent reactivated MM cells. Numbers are individual cells evaluated from each experiment. (D) Histograms of the aggregated expression of myeloma cells and myeloid markers from index-sorted dormant (gold) and reactivated (gray) cells that formed colonies or those that did not. (E) Schematic workflow for the analysis of expression of 1571 Y-chromosome (chrY) genes from RM1 (positive control) and 5TGM1 scRNAseq data from cells isolated from male and female mice. (F) Heatmap of the top 41 chrY genes detected from raw unfiltered counts from RM1 cells (blue) and 5TGM1 cells isolated from male (green) and female (purple) mice. (G) Aggregated raw unfiltered counts of 41 chrY genes in RM1 single cells isolated from male mice and 5TGM1 single cells isolated from male and female mice. (H) Transcript profile of the top 5 chrY genes from raw unfiltered counts in RM1 single cells and 5TGM1 single cells isolated from male and female mice.