Osteoblast Differentiation of Stromal Cells Induced By Leukemic Cells
Authors: Ganesan Saravanan , Palani Hamenth Kumar , Lakshmanan Vairavan , Balasundaram Nithya , Alex Ansu Abu , David Sachin , George Biju , Balasubramanian Poonkuzhali , Vyas Neha , Palakodeti Dasaradhi , Mathews Vikram 
Published: October 2019

Abstract
There are numerous reports on bone marrow micro-environment mediated drug resistance (EM-DR) in cancer. We had previously reported, that there was significant EM-DR to ATO in APL. We had also shown that the effect of cross talk between leukemic cells and stromal cells leads to increased activation of NF-kB pathway in the leukemic cells. There are very limited data available on the effect of leukemic cell interaction on the stromal cells. We undertook a study to evaluate the changes induced by leukemic cells (NB4 cell line) on the HS-5 stromal cell line.
After 2 days of co-culture with NB4 cells, the RNA from HS-5 stromal cells were subjected to gene expression profiling and small RNA sequencing. In a gene expression profiling, we observed an enrichment of Wnt signaling and BMP signaling pathway. When we subjected the array to functional analysis, a significant enrichment of osteoblast differentiation genes was observed in the stromal cells co-cultured with leukemic cells in comparison to HS-5 cells alone. A stringent miRNA analysis using DESeq (miRNAs whose FDR corrected p values < 0.05 are considered significant) revealed 21 miRNA were differentially regulated in the HS-5 cells co-cultured with leukemic cells in comparison to HS-5 cells alone. Consistent with the gene expression profile, we observed that most of the miRNA differentially regulated were involved in the regulation of osteoblast differentiation (such as hsa-miR-33b-5p, hsa-miR-210-3p and hsa-miR-222-5p).
As previously reported (PNAS 2013;110 (23):9469) we also observed upon co-culture with leukemic cells a down-regulation of NF-kB pathway in HS-5 cells. This was demonstrated by documenting decreased translocation of p65 subunit in the nuclear compartment of HS-5 cells on day 2 and day 7 of co-culture. This was further validated by documenting downregulation of NF-kB pathway genes by real time PCR on day 2. Along with this we also observed an increased translocation of B-catenin in the nuclear compartment of HS-5 cells on day 2 and day 7 of co-culture. This observation was validated by a real time PCR assay where an upregulation of its target gene was seen (Fig 1a). We observed that there is an increased expression of osteoblast differentiation transcription factor (RUNX2) in HS-5 cells upon co-culture on day 2 by real time PCR and western blot (Fig 1a, 1b). We also observed an increase in alkaline phosphatase activity in HS-5 cells upon co-culture with NB4 cells at different time points detected by BCIP-NBT staining. Further validation of differentiation of HS-5 cells into osteoblast was done using alizarin red S stain on day 7.
Similar differentiation effects were seen in HS-5 cells when it is co-cultured with other malignant cells (U937, U266 and Jurkat) where an increased expression of RUNX2 on day 2 of co-culture was observed, suggesting that this effect is common with different subtypes of leukemia. We did not observe any effect of PBMNC / normal cord blood CD34+cells on HS-5 cells in inducing osteoblast differentiation (no expression of RUNX2; Fig 1b).
This study demonstrates a possible role of leukemic cells in establishing leukemic niche in the bone marrow by inducing osteoblast differentiation of stromal cells. The role of this leukemic cell induced osteoblast differentiation of stroma in drug resistance against various chemotherapeutic agents needs further evaluation.

Disclosures
No relevant conflicts of interest to declare.