Megakaryocytes Support Viability Proliferation and Protection of Primary Pre-B ALL Cells from Chemotherapy

BACKGROUND: The bone marrow is known to shelter leukemia cells from chemotherapy and contributes to the survival of chemotherapy resistant residual cells, termed minimal residual disease (MRD).  We have studied in situ the location of MRD+ ALL cells using a xenograft model of primary ALL cells and have found a novel co-localization of megakaryocytes (MK) with ALL cells. Mature hematopoietic cells have been implicated in modifying the local normal hematopoietic stem cell environment including MK. We hypothesize that MK are associated with the survival of MRD+ ALL cells. For this purpose, we tested the role of MK cells in maintenance and chemoprotection of ALL cells.

METHODS: Patient-derived (primary) pre-B ALL cells were engrafted into non-irradiated NOD/SCID IL2Rγ-/- (NSG) mice. Leukemia-bearing mice received 4 weeks of chemotherapy treatment (Vincristine, Dexamethasone, L-Asparaginase; VDL). MRD status of mice was confirmed by detection of human CD45 + CD19+ leukemia cells in the bone marrow by flow cytometry. In situ location of the MRD⁺ALL cells was determined by histological analysis and quantitation was performed by Fiji Image J. For in vitro studies, primary pre-B ALL cells were co-cultured for up to 2 days with murine calvaria-derived stromal OP9 cells or MK isolated from C57/BL6 BM primed for with murine thrombopoietin (mTPO) and sorted by flow cytometry for CD41+ MK. Annexin V/7-AAD staining was used for viability determination by flow cytometry. Boyden chamber system with either OP9 cells or MK cells seeded on the bottom and ALL cells on the top of the system was used for migration assays.

RESULTS: In situ analysis of MRD⁺ ALL recipient mice showed that cells MRD⁺ALL cells (huCD45⁺) are located in close proximity to MKs, with 11.57±2.94% of MRD+ ALL cells lying directly adjacent to MKs(0-5μm distance to MK). To further assess the role of MKs in ALL survival in vitro, we compared if MK cells can sustain proliferation and viability of primary leukemia cells like the OP stromal co-culture model that we have established previously. MKs were isolated from BM of C57BL/6 mice by FACS sorting for CD41⁺ cells from BM primed with mTPO. The sorted population showed a 90% purity of CD41⁺ cells. MKs were able to maintain ALL cell proliferation 1.90e⁶ cell count ± 0.48e⁶ cell count on  day 2) and provide chemoprotection from VDL treatment (77.24 ± 2.03% on  day 2), which  was  similar to the effect of OP9 cells on sustained proliferation and viability. Interestingly ALL cells cultured with MKs had a slight reduction in G2/M-phase (8.46±0.31%)  3 days after culture set up without treatment compared to cultures with OP9 stromal cells (13.59±0.14%; P-value < 0.0005). In a migration assay, MKs stimulated migration of ALL cells (5.42e⁵±0.72 migrated cells) significantly more than OP9 stromal cells (2.92e⁵±0.72 migrated cells) over a 24 hour period (P-value = 0.0132). Using the SDF1α inhibitor AMD3100 (100µM), migration of ALL cells was only partially inhibited (2.92e⁵±0.72), suggesting additional MK produced factors influence mobilization of human leukemia cells besides SDF1α. Using the recombinant forms of stromal cell-derived factor 1 (SDF1α) and von Willebrand factor (VWF), ALL cell migration was successfully stimulated over 24 hours (6.25e⁴±1.25 and 3.33e⁴±0.72 migrated cells, respectively), and this effect was inhibited using AMD3100, small molecule inhibitor of CXCR4, and anti-VWF antibody respectively.

CONCLUSION: Here we show for the first time that co-culture of MK and primary pre-B ALL cells supports   their proliferation, viability and protection from chemotherapy similar to murine OP9 stromal cells. Our data warrants further investigation of the underlying mechanism.