Close interaction with bone marrow mesenchymal stromal cells induces the development of cancer stem cell-like immunophenotype in B cell precursor acute lymphoblastic leukemia cells

概要

Introduction
Bone marrow (BM) mesenchymal stromal cells (MSCs) sustain the hematopoietic stem/progenitor cells. These interactions can also support the survival and proliferation of leukemic cells. In this study, we hypothesized that direct contact with B M・MSCs might contribute to the development of chemoresistance in B cell precursor、 (BCP)・acute lymphoblastic leukemia (ALL) cells and alter their im munophenotype.

Materials and methods
Five BCP-ALL cell lines established in our laboratory were used in this study. Immortalized human telomerase reverse transcriptase-and GFP-transduced MSC cell line (MSC-TERT) was gifted from St. Jude Children's Research Hospital. The effect of cell-to-cell contact on chemosensitivity and immunophenotype was determined by plating leukemic cells onto a confluent layer of (immortalized human telomerase reverse transcriptase- and GFP-transduced MSC cell line) MSC-TERT. Non-contact-dependent interactions between leukemic cells and MSC-TERT were studied using a double chamber co-culture system. Primary leukemic cells floating in BM blood were collected from a BM aspirate of a BCPALL patient. Leukemic cells within the BM niche were isolated from a BM-biopsy. Chemosensitivity of MSC-TERT-adhering leukemic cells was assayed after treating the leukemia cells for four weeks with cytarabine methotrexate, vincristine and prednisolone at the ICso for each cell line in suspension culture. Leukemic cells cultured in four different conditions - single-cell culture, cocultured with MSC-TERT in separate chambers of a double chamber system, suspended in the co-culture medium and adhering to MSC-TERT -were examined for the expression of cell differentiation, cell proliferation, chemoresistance and cell adhesion markers. Cell cycle and proliferation time were also analyzed.

Results
Adhering to the BM・MSCs protected the BCP・ALL cells from cytotoxicity of the drugs. The ALL cells that remained to be suspended in the culture medium expressed CD38 and were negative for CD34, CD133, P・glycoprotein, and BCRP/ABCG2 regardless of the pr噌 esenceor absence of MSC・TERT. In contrast, the MSC・TERT・adhering ALL cells expressed CD34, CD133, P・glycoprotein and BCRP/ABCG2, and turned CD38 negative. The MSC・TERT・adhering cells also expressed Tyrosine Kinase Non Receptor l(TNKl), a negative growth regulator. The primary samples showed the similar expression profiles. The MSC・TERT adhering ALL cells took a significantly longer time for cell division compared to that of the suspended ALL cells. The Go/G1 fraction was significantly higher when the cells were adhering to the MSC・TERT than that in suspension. MSC・TERT・ adhering ALL cells showed a strong expression of N・cadherin. Proteins in the Integrin signaling pathway were expressed in MSC・TERT・adhering ALL cells.

Discussion
Our study revealed that BCP・ALL cells start expressing hematopoietic stem/progenitor cell markers, CD34 and CD133, and down・regulated their expression of CD38 on attachment with MSC・TERT. CD133 is recognized as one of the cancer stem cell(CSC) markers, and CD34+ CD38・AML cells are considered as leukemia stem cells(LSCs). The MSC・TERT・adhering BCP・ALL cells showed an increase in the Go/G1 fraction. This might have contributed to the development of chemoresistance in these cells. These findings suggest that the contact with BM・MSCs induced a CSC・like phenotypic change in BCP-ALL cells. Our results suggest that detaching from MSC・TERT might reverse the CSC/LSC・ like phenotype. The cell adhesion of ALL cells with BM-MSCs could be a potential target to eradicate minimal residual disease (MRD) residing in the BM microenvironment. In conclusion, therapies targeting cell-to・cell contact in the BM microenvironment could be a promising strategy for the eradication of MRD in BCP-ALL.