Cell-Cell Communication Between Multiple Myeloma (MM) Cells and Cord Blood Derived NK Cells (CB-NK) Regulates Both Tumor Cell Death and Tumor Cell Survival
Authors: Martin-Antonio Beatriz , Serrano Erica , Suñe Guillermo , Perez Lorena , Calderón Marcos , Estanyol Jose María , Najjar Amer , Bueno Clara , Fernandez de Larrea Carlos , Urbano-Ispizua Alvaro 
Published: October 2019

Abstract
In previous work we have demonstrated that CB-NK exert a cytotoxicity towards MM cells which involves lipid-protein vesicle transfer, which it is secondarily transferred from recipient MM cells to neighboring MM cells. This transmissible cytotoxicity causes a 5-7% of neighboring MM cell death (CDD 2015;22(1):96-107). However, this transmission between MM cells could have a diluent effect of the CB-NK cytotoxicity which could be also a tumor cell survival mechanism. To further analyze this mechanism, we determined the proteins transferred between cells and the role of lipids in this transfer.
We performed TRANS-SILAC proteomics using two different approaches: 1) Labeling CB-NK cells with heavy amino-acids (hAA) to identify both CB-NK proteins transferred to MM (1ºMM) and secondary CB-NK proteins transferred from 1ºMM to neighboring MM cells (2ºMM). 2) Labeling MM cells, before CB-NK contact, with hAA to identify transferred proteins from 1ºMM to 2ºMM cells, and from 1ºMM to CB-NK.
We found that 1ºMM cells acquired 9.5% of CB-NK proteins, and that these proteins were transferred to 2ºMM cells. As a consequence, 1ºMM cells diluted the CB-NK proteins from 9.5% to 3.8%; which represented 7.2% of proteins in 2ºMM cells. In the second approach, we observed that, MM cells transferred to neighboring MM cells only 1.9% of MM proteins in resting conditions. However, in the presence of CB-NK, this transfer was increased up to 7.7%. Furthermore, CB-NK cells acquired 7.3% of 1ºMM proteins. These findings demonstrate a secondary CB-NK protein transfer between MM cells, which represents a CB-NK protein dilution content from 1ºMM to 2ºMM, and an increased transfer of 1ºMM proteins to 2ºMM (Fig.1A). Proteomic analysis showed that transferred proteins were involved in FAS signaling, apoptosis, inflammation, chromatin organization, glycolysis, spliceosome, and rRNA metabolic process. Among these proteins we focused on histones and the 14-3-3 family which are associated to both cell death and cell survival. Confocal microscopy confirmed transfer of histones and 14-3-3 proteins.
Protein transfer occurred within neutral lipid vesicles-structures. We also observed that MM cells were inter-connected with a much higher number of neutral lipid structures (nanotubes and bigger structures similar to bags) than in K562 cells (Fig. 1B). Thus, we next analyzed the role of these neutral lipids in MM cell-cell communication. The cholesterol synthesis and lipid transport inhibitor U18666A significantly decreased the number of these lipid connective structures between MM cells, and it was toxic for MM cells. Furthermore, breaking the lipid connection between MM cells with U18666A increased CB-NK cytotoxicity (p<0.05). These effects were not observed in K562 cells. This could be explained by a blockage of the diluent effect mediated by MM cells of the proteins and lipids from CB-NK cells.
Last, we analyzed apoptotic levels of 1ºMM cells after co-culture with 2ºMM cells, and the apoptotic levels of the remaining 2ºMM cells. Both populations became early apoptotic, however, after 3-5 days they recovered from early apoptosis, decreasing from 36% of early apoptotic cells to 3%, which was their apoptotic basal levels, and most importantly, without causing cell death, as no difference in the cell number in comparison to control MM cells was observed. Therefore, our data demonstrate that this diluent transmission effect between MM cells might be a new cell mechanism that contributes to tumor cell survival. Importantly, because lipid structures mediate this diluent transmission effect, this tumor cell survival mechanism might be eliminated by using lipid metabolism inhibitors.
In conclusion, cell-cell communication increases when MM cells are stressed because of CB-NK presence. Although this causes a transmissible cytotoxicity in a small % of MM cells, a diluent effect also occurs between cells helping MM cells to recover and promoting MM cell survival. Lipid structures play an important role in this cell-cell communication, which may represent a new therapeutic target for MM treatment.

Disclosures
No relevant conflicts of interest to declare.