Adhesion of chronic lymphocytic leukemia (CLL) cells to stromal cells in the marrow and secondary lymphoid tissues confers drug resistance and may account for survival and maintenance of residual CLL cells after conventional treatments, paving the way to relapses. Therefore, targeting the cross talk between CLL cells and stromal cells represents an attractive and necessary approach to overcome minimal residual disease in CLL. We previously reported that fractions of CLL cells and other neoplastic B cell spontaneously migrate beneath marrow stromal cells (MSC) in a CXCR4- and CD49d-(VLA-4) dependent fashion, an in vitro phenomenon termed pseudoemperipolesis (PEP). Also, we reported that cells that migrated beneath and underneath MSC were largely protected from cytotoxic drugs, in contrast to cells that remained in the supernatant (Blood 113:4604-13, 2009). The aim of this study was to identify correlations between CLL surface markers and their ability to migrate beneath MSC.
We tested samples from 116 different CLL patients in co-culture assays for their ability to migrate beneath MSC (KUSA H1 cells). After 6 hours of incubation, CLL cells were vigorously washed off the MSC layers, and PEP+ cases were identified by phase contrast microscopy as cases, in which CLL cells were visualized within the stromal layers, characterized by the dark appearance of CLL cells that had migrated into the same focal plane as the MSC. We characterized 45 PEP+ and 71 PEP- CLL cases. Next, we profiled the expression of adhesion molecules and chemokine receptors, including CD44, CD49d, CD62L, CXCR3, CXCR4 and CXCR5 by flow cytometry in the PEP+ and PEP- subgroups. Based on the mean fluorescence intensity ratios (MFIR), we found that both groups displayed comparable levels of CD44 (58.8±4.6 in PEP+ vs. 58.7±3.5 in PEP-), CD62L (7.8±1.3 vs. 9.3±1.3), CXCR3 (7.9±1.0 vs. 8.0±0.8), CXCR4 (87.6±9.3 vs. 81.4±4.6), and CXCR5 (47.4±2.9 vs. 49.3±2.7). In contrast, the levels of CD49d were significantly different between these two groups; the MFIR for CD49d was 14.0±2.4 in PEP+ cases and 4.0±0.7 in PEP- cases (p<0.0001, see Fig. 1). Interestingly, in PEP+ cases, higher levels of CD49d correlated with lower levels of CXCR4 (r=-0.43; p<0.003), which could be due to higher affinity to a CXCL12-rich microenvironment, with respective CXCR4 down regulation. Analysis of prognostic factors (CD38, ZAP-70 and mutational status) revealed that PEP+ cases displayed higher level of CD38 expression: 29.8±5.8% vs. 15.6±2.7% (MFIR, p<0.05), whereas there was no difference in ZAP-70 expression or mutational status between PEP+ or PEP- cases. When comparing cytogenetic profiles, favorable risk (del13q14.3 and normal karyotype) cases represented 43.6% in the PEP+ vs. 71.2% in PEP- groups; trisomy12 cases 23.1% vs. 4.5%, and poor risk cytogenetics (del11q, del17p, complex) represented 33.3% in the PEP+ vs. 24.3% in PEP- group.
Collectively, the distinct immunophenotype of CD49d+CD38+ identifies CLL cases with a higher affinity for migration beneath MSC. Consequently, CLL cells in such cases are expected to be more difficult to eradicate by conventional treatments because of higher levels of stroma-mediated drug resistance. Drugs, such as Natalizumab or Plerixafor, that target CLL-stroma interactions, could be of particular benefit for such patients by disrupting CLL-stroma crosstalk and mobilization of CLL cells from protective marrow niches.
No relevant conflicts of interest to declare.
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