Quiescent PKH67⁺MM cells prefer to reside within the osteoblastic niche in the bones. (A) MM cells (10⁶ cells/mouse) were stained with PKH67 and were injected into lightly irradiated (225 cGy) NOD/SCID mice via intravenous (IV) injection. Before injection, all cells were analyzed using FACS to ensure that >99% of the cells were PKH⁺. The mice were then sacrificed after 48 hours, and the spleens and bones were isolated. Cells were isolated from the osteoblastic (OS) and VS niches of the bones as indicated in the supplemental Methods. The number of PKH⁺ cells was calculated based on FACS analysis, which was gated on the PKH67⁺ cells (excitation wavelength 490 nm and emission wavelength 502 nm), and unstained samples were used as negative controls. (B) Immunohistochemistry analyses confirmed the FACS analyses. Each slide was examined for the presence of CD138, PKH, and Draq5. Image acquisition for the CD138, PKH, and Draq5 staining of each slide was performed using the same parameters of the confocal microscope in sequential mode. (C) PKH⁺CD138^– MM cells exhibited slightly higher engraftment rates in bones compared with CD138⁺ cells. The CD138⁺ and CD138^– cells were separated from MM cells (4 patients and 1 cell line) and were stained with PKH, followed by transplantation into irradiated NOD/SCID mice. (D) PKH⁺ MM cells isolated from the OS niches of the bones form more colonies in PHA-LCM medium than cells isolated from other niches. PKH⁺ MM cells were sorted from different organs of the mice 48 hours post-transplantation. PKH⁺ cells isolated from OS niches formed more colonies than PKH⁺ or PKH^– cells from other niches. In addition, the colonies formed from the PKH⁺ cells from VS niches (PKH⁺/VS) were composed of fewer cells compared with PKH⁺/OS cells. In addition, the shape of the colonies was not as distinguishable from the colonies formed by the PKH⁺/OS cells. Although some PKH⁺ cells reside in the spleen (PKH⁺/SP) 48 hours post-transplantation, those cells failed to form colonies. All samples were plated in triplicate. *P < .01.