Identification and Characterization of Adhesive and Non-Adhesive Sub-Populations of Multiple Myeloma.
Authors: Nadav Liat , Katz Ben-Zion , Naparstek Elizabeth , Geiger Benjamin 
Published: April 2020

Abstract
Multiple myeloma (MM) is an incurable malignancy of terminally differentiated B cells with heterogeonous clinical presentation. Although MM is viewed clinically as a disease restricted to the bone marrow, it is considered to be a systemic malignancy and peripheral blood MM cells can be detected in the majority of patients at diagnosis. In an attempt to clarify the diverse clinical presentation of this disease, we hypothesized that MM cells comprise a heterogeneous cell population which can be distinguished by their motile and adhesive phenotype: while most of the cells strongly adhere to the bone marrow environment, other non-adhesive sub-populations are able to invade into adjacent sites within the bone marrow, or circulate to distant locations. In order to study the motile and adhesive diversity within malignant plasma cell populations we used the ARH77 plasma cell leukemia cell line. These cells were fractionated into adherent and poorly-adherent sub-populations by continuous passages on fibronectin coated tissue culture plates. The enriched sub-populations were the basis for further experiments of adhesion, motility, cytoskeletal organization and flow cytometric profile. The adhesive sub-population readily attached and spread on fibronectin, forming paxillin-rich focal adhesions. In contrast, the poorly-adherent sub-population attached loosely to fibronectin, did not spread and failed to organize paxillin into discrete structures. The differences between these sub-populations of ARH77 cells were not observed when the cells were plated on hyaluronan or control, uncoated dish, indicating that the differences are in the integrin-mediated adhesion system. Although the flow cytomentry analysis indicated that the surface expression of integrins b1, a5 and aV is identical in the two sub-populations, specific inhibitory anti-integrin a5b1 antibodies inhibited the poorly-adherent sub-population, without significantly affecting the highly adhesive sub-population. These results suggest different mechanisms that regulate the adhesive interactions of the two sub-populations. To elucidate the motile phenotype of these cells, the two sub-populations were plated on fibronectin and hyaluronan. The migration velocity of the poorly-adherent cells on fibronectin was significantly higher compared to that of the adhesive cells. Moreover, the adhesive sub-population exhibited higher binding to physiologically-relevant substrates (cultured stromal cells and bone fragments). Furthermore, flow cytometry indicated that while both sub-populations expressed similar levels of the Ig kappa light chain, the adhesive sub-population expressed significantly higher levels of CD44 and CD45 and lower levels of CD138, compared to the poorly-adhesive population. In conclusion, we have shown that MM cells differ by their adhesive and motile properties and can be identified and separated according to their flow cytometric profile, thus providing useful tools for studying the differential properties of the various MM sub-populations in patients, and their clinical relevance. Moreover, this molecular diversity of MM cells can be further employed to design new drugs that can specifically inhibit the survival and dissemination of clinically important sub-populations.