Modeling Chemotherapy Resistant Leukemia In Vitro

Summary

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

Abstract

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Introduction

The overall goal of the method described is to provide an efficient, cost-effective in vitro approach that supports investigation of the mechanisms that underlie bone marrow supported survival of leukemic cells during chemotherapy exposure. It is well documented that surviving residual tumor cells that persist after treatment contribute to relapse of disease that is often more aggressive than that at diagnosis and is often less effectively treated^1-8. Models that include leukemic cells in isolation, such as those limited to culture of cells in media alone, for testing of therapeutic approaches do not factor in these critical signals, or the heterog....

Protocol

1. Advanced Preparation

1. Preparing dextran G10 particles.
   1. Prepare G10 slurry by adding 50 ml 1x PBS to 10 g G10 particles. Mix by inversion and allow G10 to settle out of phosphate buffered saline (PBS) at 4 °C O/N.
   2. The day of G10 column separation, aspirate PBS from settled G10 particles and add 50 ml fresh PBS. Mix by inversion. Repeat twice, adding 50 ml fresh PBS to settled G10 particles and store at 4 °C until ready to use.
2. Culturing BMSC and OB.
   1. Maintain both BMSC or OB at 37 °C in 6% CO₂ and grown on 10 cm tissue culture plates until 90% confluency is reached.

Results

Successful setup and culture of this co-culture model will result in the establishment of 3 subpopulations of leukemic cells relative to the adherent BMSC or OB monolayer. Figure 1 shows how ALL cells seeded into a BMSC monolayer initially appear as only a single population of suspended leukemic cells. Over the course of 4 days leukemic cells interact with the BMSC to form 3 spatial subpopulations of leukemic cells (suspended (S), phase bright (PB), and phase dim (PD)). W.......

Discussion

Minimal residual disease (MRD) which contributes to relapse of disease continues to be a major clinical challenge in the treatment of aggressive refractory ALL, as well as, a host of other hematological malignancies. The bone marrow microenvironment is the most common site of relapse in ALL^3,8. As such, models that model the bone marrow microenvironment are vital tools to test hypotheses related to leukemic tumor cell survival and maintenance of MRD during chemotherapy exposure. While mouse models define the g.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
G10 sephadex beads	Sigma	G10120	Referred to in manuscript as gel type 10 cross-linked dextran particles
10 ml sterile syringe	BD	309604
Glass wool	Pyrex	3950
1-way stopcocks	World Precision Instruments, Inc.	14054-10
50 ml conical centrifuge tubes	World Wide Medical Products	41021039	Used as collection tubes
15 ml conical centrifuge tubes	World Wide Medical Products	41021037	Used for cell collection
Fetal Bovine Serum	Sigma	F6178
0.05% Trypsin	Mediatech, Inc.	25-053-CI
100 x 20 mm Cell Culture Dishes	Greiner Bio-One	664160
Culture media
Osteoblast culture media	PromoCell	C-27001	For human osteoblast media
RPMI 1640 media	Mediatech, Inc.	15-040	For tumor media prepation
Cell lines
Adherent Cells:
Human Osteoblasts	PromoCell	C-12720	Human osteoblast were cultured according to the supplier’s recommendations.
Human Bone Morrow Stromal Cells	WVU Biospecimen Core		De-identified primary human leukemia and bone marrow stromal cells (BMSC) were provided by the Mary Babb Randolph Cancer Center (MBRCC) Biospecimen Processing Core and the West Virginia University Department of Pathology Tissue Bank. BMSC cultures were established as previously described (*)
Leukemic Cells:
REH	ATCC	ATCC-CRL-8286	REH cells were cultured according to the supplier’s recommendations and recommended media.
SD-1	DSMZ	ACC 366	SD-1 were cultured according to the supplier’s recommendations and recommended media.
(*) Gibson LF, Fortney J, Landreth KS, Piktel D, Ericson SG, Lynch JP. Disruption of bone marrow stromal cell function by etoposide. Biol Blood Marrow Transplant J Am Soc Blood Marrow Transplant. 1997 Aug;3(3):122–32.