Transcriptomic Profile of Bone Marrow-Derived Mesenchymal Stromal Cells of Pediatric Pre-B Acute Lymphoblastic Leukemia Patients and Healthy Donors after Interaction with Leukemic Cells

Background: Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by the abnormal expansion of clonal lymphoblasts in the bone marrow (BM). The behavior of malignant lymphoblasts is not only dependent on their genomic abnormalities but also, on their complex and reciprocal relationships with their local niche. BM microenvironment is a dynamic network of growth factors, cytokines, and stromal cells, providing a permissive environment for leukemogenesis and progression. Despite some evidence about the implication of the bidirectional interaction between mesenchymal stromal cells (MSCs) and leukemic cell on growth and survival, it is still largely unknown. Although an increasing number of studies are reporting on the expression of specific genes in leukemic cells-interacting MSCs, gene expression profiling in cocultured MSCs (with respect to mono culture conditions) has not been done on a transcriptome-wide basis. Taking all this into consideration, we have established cocultures between BM-derived MSCs and the leukemic mononuclear cells and performed gene expression profile (GEP) studies on the MSC population to determine those deregulated genes due to the coculture condition with respect to MSCs in mono culture.

Methods: Primary MSCs from BM samples of healthy donors (n=4) and pediatric pre-B ALL patients (n=4) were isolated and expanded and mononuclear cells were, also, isolated from patients peripheral blood. At passage 2, healthy/patient (h/p) MSCs were cocultured with the leukemic cells for 72 hours and subsequently subjected to GEP analyses. RNA isolated from h/pMSCs and biotinylated amplified RNA was synthesized. For the hybridization HG-U133 Plus, 2.0 GeneChip oligonucleotide arrays (Affymetrix) was used. Raw microarray data were processed into expression values through the Affy package in R. RMA (robust multi-array average) algorithm was used for preprocessing. The linear regression model package LIMMA (Linear Models for Microarray Data) was used to identify significant differential expression of genes (DEGs) compared to h/pMSCs after coculture with MSCs from the same origin in mono culture. Minimum 1.5 fold change and p-value < 0.001 were defined as the threshold. Functional enrichment, gene network and pathway analysis, were performed on selected sets of genes after differential expression analyses using two bioinformatics tools: DAVID Bioinformatics Resources 6.8 and WebGestalt 2019.

Results: We identified 293 DEGs when analyzing pMSC samples vs. hMSC in coculture condition and 853 DEGs of pMSC vs. hMSC analyze in mono culture condition and only 15 DEGs common in both group. According to the result of functional enrichment analysis of pMSC vs. hMSC in coculture condition, 29 gene ontology (GO) term biological process including (top five) apoptotic process, axon guidance, regulation of JNK cascade, cell-cell adhesion and protein phosphorylation and 4 KEGG pathways of DEGs including axon guidance, FoxO signalling pathway, Wnt signalling pathway and signalling pathways regulating pluripotency of stem cells were determined. Comparing pMSC vs. hMSC in mono culture condition, 292 GO term biological process including extracellular matrix organization, skeletal system development, angiogenesis and signal transduction and 43 KEGG pathways of DEGs including pathways in cancer, TGFß signalling pathway, ECM-receptor interaction, p53 signalling pathway and PI3-Akt signalling pathway were determined.Five genes with upregulated expression after coculture (COL18A1, EFNA5, MAP3K8, CASP1, IGF1R) and after mono culture (MMP1, CXCL1, IL1B, PCDH10, CXCL6) were identified.

Conclusions: Deregulated genes especially after coculture condition in axon guidance and ephrin receptor-mediated signalling pathway of which the importance is not yet understood in cancer microenvironment including EPHB1, ephrinA5, ephrinB3, p21 activated kinase 2 (PAK2), and ROBO3 showed highly differential expression pattern between pMSCs and hMSCs. This is the first report indicating the importance of axon guidance genes as a result of interaction between MSCs and leukemic cells. The elucidation of deregulated mechanisms and pathways provides new molecular insights to the contribution of these cells to ALL pathophysiology and understanding cross talk between these cells may lead to found new therapeutic targets.