Flow cytometric measurement of surface receptors associated with BM homing and infiltration of myeloma cells.

<p>BALB/c wild type mice were injected with 1×10⁵ MOPC-315.BM luc⁺ cells via the tail vein. (<b>A</b>) 42 days after MM injection mice showed high BLI signals from hematopoietic compartments such as femur/tibia and spleen. Shown are two representative mice from ventral and dorsal view immediately before cells from BM and spleens were harvested for flow cytometry. (<b>B–D</b>) Besides BM and spleen derived MM cells, we also analysed MOPC-315.BM luc+ cells from culture. Dead cells were excluded by propidium iodide staining and MOPC cells identified as CD138⁺CD4⁺ double positive cells. (<b>B</b>) α4β1 integrin positive MOPC-315.BM luc⁺ cells were identified by flow cytometry as α4⁺ (CD49d⁺) and α4β7⁻. Representative FACS plots and the corresponding graph are shown, stating the frequency within CD138⁺CD4⁺ MOPC-315.BM luc⁺ cells expressing α4β1. For CXCR4 (<b>C</b>) and CD44 (<b>D</b>) representative histograms for each organ and cell line, including unstained fluorescence minus one (FMO) sample are displayed. Corresponding graphs state the fold difference in mean fluorescence intensity (MFI) related to the unstained FMO sample. BM and spleen: two independent experiments, n = 10, cells from cell culture: n = 4 for CXCR4 and CD44, n = 3 for α4β1. * indicates p<0.05 and ** indicates p<0.01 as determined by Kruskal-Wallis test with Dunn post test. (<b>E</b>) MOPC-315.BM luc⁺ cells were sorted for CXCR4^low and CXCR4^high expression. After 2 days in cell culture sorted cells regained the original CXCR4 expression level of the cell line. (<b>F</b>) 5×10⁵ sorted cells were i.v. injected into 4 female BALB/c mice each and BLI from ventral, lateral and dorsal was performed 10 days later. Sorted CXCR4^low as well as CXCR4^high CXCR4 cells readily homed to the BM compartment as well as to the spleen. (<b>G</b>) After BLI the mice were sacrificed, cells from left and right femur/tibia (separately) and the spleen extracted, and percentage as well as absolute numbers for CD138⁺CD4⁺ MM cells determined. From these values a ratio of spleen/BM was calculated to determine the homing capacity of the sorted populations. (<b>H</b>) Comparison of CXCR4 expression levels of sorted CXCR4^low and CXCR4^high cells immediately before injection and MM cells from BM and spleen after 10 days <i>in vivo</i> revealed a dynamic CXCR4 regulation.