Research paperA novel extracellular matrix-based leukemia model supports leukemia cells with stem cell-like characteristics
Graphical abstract
Introduction
Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy characterized by an aberrant clonal expansion of undifferentiated myeloid blasts. Studies have shown that leukemia stem cells (LSCs) contribute to relapse after chemotherapeutic treatment. [1] Like normal hematopoietic stem cells (HSCs), LSCs maintain their self-renewal ability while generating clonogenic leukemic progenitors capable of producing leukemic cells [2]. Anti-proliferative chemotherapeutic agents commonly target the rapidly cycling leukemic cells, but they generally are ineffective against the quiescent LSCs, partly because of enhanced drug efflux in LSCs [3]. Therefore, it is important to develop therapeutic strategies which eliminate the LSCs in the bone marrow, where they share the “hematopoietic niche” along with normal HSCs [4]. The LSC niche, similar to the hematopoietic niche, is a 3D microenvironment composed of bone marrow stromal cells and ECM components like collagen, fibronectin and tenascin [4]. These components create compartments that not only provide structural support to the cells in the bone marrow, but also provide chemokines and cytokines that are important in regulating LSC self-renewal, trafficking, proliferation and differentiation [5].
Currently, most leukemia in vitro studies are based on conventional two-dimensional (2D) cultures in tissue culture polystyrene (TCP) dishes/ flasks and stromal co-cultures. These models are useful in elucidating some of the molecular mechanisms of leukemia initiation and progression. However, 2D culture systems lack the leukemia-microenvironment interaction present in the 3D bone marrow microenvironment. Therefore, the LSCs in 2D culture frequently differentiate and lose their “stem-ness”. The development of a 3D model that replicates the in vivo mechanical and biochemical properties of bone marrow could allow maintenance of a true LSC-state.
Our laboratory has been focused on characterizing decellularized Wharton’s jelly matrix (DWJM) and examining its potential for regenerative medicine applications. [[6], [7], [8]], We hypothesized that DWJM would provide a similar environment to the bone marrow ECM, because its components, such as collagen, fibronectin, hyaluronic acid, and sulphated proteoglycan [8], also exist in the bone marrow hematopoietic niche. Moreover, because cell-ECM interactions play an important role in chemoresistance in leukemia cells [9], the environment provided by DWJM could support the maintenance of LSCs.
We therefore used DWJM as an ECM to examine leukemia cell-ECM interactions, hypothesizing that DWJM would support leukemia cells with LSC-like characteristics. In this model, we investigated the growth pattern of 3 human leukemia cell lines (HL60, Kasumi-1, and MV411), with a focus on proliferation, viability, morphology and myeloid differentiation. We also studied the drug resistance and stem cell characteristics of leukemia cells cultured in this model, compared to leukemia cells cultured in suspension. We found that leukemia cells cultured in our DWJM-based ECM model had LSC characteristics, suggesting that DWJM may prove useful in LSC characterization and in developing therapeutic interventions that target LSCs.
Section snippets
Cell culture
Human AML cell lines HL60, Kasumi I and MV 411 (ATCC, Manassas, VA) were maintained in T 75 tissue culture flasks with Advanced Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco), supplemented with 5% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (pen/strep) (Life Technologies). Cells were maintained at 37 °C in a fully humidified 5% CO2 incubator.
DWJM scaffold preparation
The preparation of DWJM was previously described. [8,10], Briefly, to prepare DWJM scaffolds, fresh human umbilical cords
Leukemia cells cultured in DWJM demonstrated reduced proliferation while maintaining cell viability and undifferentiated state
Since LSCs are quiescent, we first examined leukemia cell proliferation over time in our model, hypothesizing that leukemia-DWJM interactions result in decreased leukemia cell proliferation over time. Using AlamarBlue assay on days 2, 4, and 6 after leukemia cell seeding, we demonstrated decreased proliferation in all three leukemia cell lines (HL 60, Kasumi I and MV411) (Fig. 2A). Despite reduced proliferation, all three cell lines maintained nearly unchanged viability at the three time
Discussion
In this study, we examined DWJM as a 3D ECM-based model to learn about leukemia cell behavior. Our findings support the hypothesis that an in vitro matrix modeling the in vivo ECM maintains leukemia cell quiescence and LSC traits. In support of this interpretation, we found that leukemia cells maintained in DWJM showed an increase in ALDH+ population in both Kasumi 1 and MV411 cell lines. Since ALDH activity has been reported to be increased in LSCs in bone marrow samples of AML patients [14],
Acknowledgments
We thank Constance D. Baldwin, PhD for reviewing the manuscript for clarity. We thank Dr. Linheng Li for his advice in studying N-cadherin expression in our model. We also thank Dr. Fariba Behbod for her help with ALDH-related studies and Dr. Eduardo Rosa-Molinar for his help with confocal microscopy studies. This work was partly supported by the Robert K. Dempski Cord Blood Research Fund.
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