P-233: Comparative analysis of CXCL12-abundant Reticular (CAR) Cell subpopulations in the Bone Marrow of Healthy Individuals versus MGUS and Multiple Myeloma Patients

Introduction:

Studies demonstrate that MM cells present constitutive activation of the canonical Wnt pathway, inducing cell proliferation, disease progression, dissemination, and drug resistance. Mechanistically, the oncogenic activity of canonical Wnt in MM is due to the abnormal secretion of R-spondins (RSPOs) within the BM, which promotes the stabilization of the Wnt receptor at the plasma membrane of MM cells and increases their responsiveness to Wnts. Nevertheless, the BM microenvironment is heterogeneous, and it is currently unknown what the specific stromal cell type responsible for RSPO secretion is. Considering that CAR cells are the main constituents of the hematopoietic niche and that murine CAR cells express RSPOs, we hypothesized that these cells would be the primary source of RSPOs in human BM, contributing to MM progression. Therefore, this study aimed to map the distribution of the distinct CAR cell subpopulations, identified by the differential expression of leptin receptor (LEPR) and CD56, in BM samples of healthy individuals (sustaining orthopedic conditions) versus MGUS and MM patients.





Methods:

Healthy BM samples were collected from bone discards of patients undergoing primary hip arthroplasty. MM and MGUS samples were collected from patients undergoing BM aspiration for diagnostic examination. Nucleated cells were isolated and incubated for 30 minutes with fluorochrome-conjugated primary antibodies against CD3, CD31, CD45, CD235a, CD71, LEPR, and CD56. Immunophenotyping was performed using a FACS Canto II flow cytometer. Analysis was performed with the DIVA software.



Results: Flow cytometry analysis of healthy BM samples (n=6) revealed the presence of two CAR cell subpopulations - LEPR+/CD56- and LEPR+/CD56+ - at similar frequencies. In MGUS (n=3), an expansion of the LEPR+/CD56- population was verified, accompanied by a decrease in the LEPR+/CD56+ pool. As for the MM samples (n=6), we observed two patient profiles: one group with an increase in the LEPR+/CD56+ fraction and another in which this population was decreased.

Conclusions:
Considering the literature, evidence points out that the expression of CD56 in cells of the BM is a distinguishing criterion between MM and MGUS, and CD56 expression is related to the presence of lytic bone lesions in MM. Our results suggest that (i) the CAR cell compartment is altered in MGUS and MM; (ii) the level of CD56 expression in CAR cells might serve as a criterion to stratify MM stages; (iii) the LEPR+/CD56+ CAR pool might be the primary source of RPSOs that promote MM progression. Additional flow cytometry and qRT-PCR analysis are ongoing to evaluate the correlation of CD56 expression in CAR cells to the MM stage and the expression of RSPOs in each CAR cell subpopulation.