PKH⁺cells from primary xenografts have a higher tumorigenic ability upon secondary transplantation. (A) PKH⁺ MM cells from primary mice xenografts showed higher engraftment rates in secondary recipient mice than cells isolated from other niches. PKH⁺ cells and PKH^– (CD138⁺) cells were isolated from the spleen and the VS and OS niches of primary recipient mice 48 hours after transplantation. PKH^– cells do not have markers to distinguish human cells from mouse cells; therefore, we isolated and sorted the PKH^–CD138⁺ cells from the primary xenografts from the PKH^– cells. These cells (5 × 10⁴ cells/mouse) were then intravenously injected into irradiated secondary recipient mice and incubated for 3 months in vivo before sacrifice to evaluate engraftment. Tumor engraftment was analyzed using FACS against CD138 antibodies. (B) Detailed analysis of CD138⁺ engraftment levels analyzed in the OS niche, VS niche, and spleen. PKH⁺ or PKH^–CD138⁺ cells from different niches of primary NOD/SCID mice were sorted and subsequently injected into secondary NOD/SCID mice. After 3 months, the secondary xenograft mice were sacrificed, and the spleens and bones were isolated. The cells from each niche were isolated and analyzed for CD138⁺ cell engraftment using FACS. Between 15 and 17 mice were analyzed for each niche, and the standard deviation was calculated accordingly. (C) Immunohistochemistry analyses confirmed the FACS data. Confocal microscopy was performed using different sections of paraffin-embedded organs. Each indicated organ was isolated from secondary xenograft NOD/SCID mice. PKH67⁺ cells are shown in green, CD138⁺ cells are shown in red, and the nucleus is shown in blue. This confocal staining is in accordance with the observations from the FACS analysis, mainly that the CD138⁺ engraftment was higher in secondary xenograft mice when cells from the osteoblastic or VS niches were injected. The spleens showed lower levels of CD138⁺ engraftment. (D) Cytotoxicity was assessed using the fluorometric cell viability assay CellTiter-Blue (Promega, Madison, WI). Sorted PKH⁺ and PKH^–CD138⁺ cells (5 × 10⁴) were incubated for the indicated times with the indicated drug doses for 24 hours. Cell viability was assessed based on the value of the fluorescent signal from the live cells with no drug treatments. The viabilities of the drug-treated cells were calculated based on a ratio of the fluorescent signal as shown in previous studies.^7,8