Lactate Is a Crucial Energy Source For Multiple Myeloma (MM) Cells In Bone Marrow Microenvironment
Authors: Fujiwara Shiho , Wada Naoko , Kawano Yawara , Kikukawa Yoshitaka , Mitsuya Hiroaki , Hata Hiroyuki 
Published: October 2019

Abstract

Introduction
It has been reported that the growth of certain cancer cells depends on the Warburg effect (aerobic glycolysis) for such cells to adapt to hypoxic environment and obtain ATP efficiently via glycolysis. We have previously reported that genes related to aerobic glycolysis are up-regulated in MM cells, and MM cells produce large amounts of lactate (Br J Cancer 2013, 108, 170-8). In the last ASH meeting, we reported that the expressions of lactate transporters were critical for the survival of MM cells. In this study, we further investigated the roles of lactate transporters in MM cells, focusing on the role of stromal cells as the supplier of lactate in microenvironment.

Methods
Six MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, and KMM1 were utilized. Bone marrow samples were obtained from newly diagnosed MM patients. Bone marrow mononuclear cells were isolated by Ficoll-Hypaque density sedimentation. To obtain stromal cells, bone marrow mononuclear cells were cultured in RPMI1640 medium supplemented with 10% FCS and allowed to attach to the bottom of plastic wells. After 3-4 weeks incubation, adherent cells were harvested and utilized as stromal cells. The expression of three components of the lactate transporter complex [monocarboxyl transporter 1 (MCT1), MCT4, and CD147] were analyzed with western blotting or flow cytometry. Lactate uptake was quantified using [14C]-Lactate. Cellular ATP production was quanatified with the ATP Determination Kit (Molecular Probes). An inhibitor of MCT1, a-cyano-4 hydroxycinnamic acid (CHC), was utilized to analyze cytotoxic effects on MM cells under both normoxia ( O2 20% ) and hypoxia (O2 1% ). AnnexinV/PI staining was performed to quantify cytotoxicity. Knockdown of MCT1 was achieved using siRNA.

Results
Knockdown of MCT1, a molecule responsible for lactate incorporation into cytoplasm, induced apoptosis in MM cells, while lactate uptake and ATP production of MM cells were reduced, suggesting that MCT1 inhibition might induce apoptosis in MM cells by decreasing the lactate-derived ATP production. On the other hand, we found significant lactate production by stromal cells. We also found an abundant amount of MCT4, a molecule responsible for lactate excretion, in bone marrow-derived stromal cells. By contrast, lactate amount in the culture supernatant of stromal cells was significantly decreased when they were co-cultured with MM cells. Therefore, it appears that lactate produced by stromal cells is incorporated into MM cells as an energy source. Interestingly, we found that the expression of MCT1 is up-regulated in MM cells under an aerobic condition, while that of MCT4 is up-regulated under a hypoxic condition. The treatment of MM cells with a-cyano-4 hydroxycinnamic acid (CHC), a MCT1 inhibitor, induced apoptosis in MM cells, suggesting that lactate may be more preferentially incorporated into MM cells under a normoxic than hypoxic condition (Figure A).

Conclusion
Our results suggest that the growth of MM cells, at least in part, depends on lactate under a normoxic condition. The data also suggest that lactate may play a role as an energy interplay shuttle between stromal cells and MM cells (Figure B). Therefore, regulating lactate incorporation by MM cells may provide a new avenue for a therapeutic strategy for multiple myeloma.

Disclosures:
No relevant conflicts of interest to declare.