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 heterogeneity of disease that occurs in response to availability of niche derived cues in which tumor cell subpopulations with very specific interactions with niche cells derive enhanced protection. Standard 2D co-culture models that co-culture bone marrow derived stromal cells and leukemic cells have somewhat addressed the contribution of the marrow niche and have shown that interaction with bone marrow microenvironment cells increases their resistance to chemotherapy and alters their growth characteristics^9-14. These models however often fail to recapitulate long term survival of tumor cells and do not accurately inform the outcomes associated with the most resistant leukemic cell populations that contribute to MRD. In vivo models remain critical and define the "gold standard" for investigation of innovative therapies prior to clinical trials but they are often challenged by the time and cost required to test hypotheses related to resistant tumors and relapse of disease. As such, development of more informative 2D models would be of benefit for pilot investigations to better inform the design of subsequent murine based pre-clinical design.

The 2D in vitro model presented in this report lacks the complexity of the true in vivo microenvironment, but provides a cost effective and reproducible means to interrogate tumor interactions with the microenvironment that lends itself specifically to enrichment of the chemoresistant subpopulation of tumor cells. This distinction is valuable as evaluation of the entire population of tumor cells may mask the phenotype of a minor group of therapy resistant tumor cells that comprise the most important target. An additional advantage is the scalability of the model to fit the analysis of interest. Bulk cultures can be established for those analyses requiring significant recovery of tumor cells, while small scale co-cultures in multi-well plates can be utilized for PCR based analysis or microscopy based evaluations.

Based on this need we developed an in vitro model to address the heterogeneity of disease that is characteristic of B-lineage acute lymphoblastic leukemia (ALL). We demonstrate that ALL cells, which share many characteristics in common with their healthy counterparts, localize to distinct compartments of BMSC or OB co-culture. Three populations of tumor cells are generated that have distinct phenotypes that are valuable for investigation of therapeutic response. Specifically, we demonstrate that (ALL) cells recovered from the "phase dim" (PD) population of co-culture are consistently refractory to therapy with survival that approximates tumor cells that have not been exposed to cytotoxic agents. These ALL cells, from either established cell lines or primary patient samples, migrate beneath adherent stromal cells or osteoblast layers but can be captured following trypsinization of cultures and separation of cell types by utilization of gel type 10 cross-linked dextran (G10) particle columns¹⁵.

Here we present a setup of a 2D co-culture that can be employed to model interactions between bone marrow microenvironment stromal cells (BMSC/OB) and leukemic cells. Of particular importance is the observation that leukemic cells form three spatial subpopulations relative to the stromal cell monolayer and that the PD population represents a chemotherapy resistant tumor population due to its interaction with the BMSC or OB. Furthermore, we demonstrate how to effectively isolate the leukemic cell populations by G10 columns. Of note, we have found that isolation of these subpopulations allows for downstream analysis of the most resistant PD population to determine potential modes of resistance that are conferred to these cells due to their interaction with the bone marrow microenvironment stromal cells or osteoblasts. Techniques that we have utilized downstream of this co-culture and isolation model include flow cytometric evaluation, proteomic analysis and targeted protein expression evaluation as well as more recently developed laser ablation electrospray ionization (LAESI) and Seahorse analysis to evaluate metabolic profiles. Through use of this model in combination with the techniques above we have found that the PD population of leukemic cells has a chemotherapy resistant phenotype that is unique when compared to leukemic cells cultured in media alone or those recovered from the other subpopulations in the same co-culture. As such, this model lends itself to more rigorous evaluation to test strategies targeting the most chemotherapy resistant leukemic cells which derive their resistant phenotype through interaction with the bone marrow microenvironment.

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.
   2. Trypsinize BMSC or OB cells and split 1:2 onto new 10 cm plates. The cells are grown to these standards until needed for leukemic co-culturing.

2. Establishing and Maintaining Co-culture

1. Add 5-20 x 10⁶ leukemic cells in 10 ml of tumor specific culture media onto an 80%-90% confluent BMSC or OB plate.
   NOTE: Our lab maintains co-cultures at 37 °C in 5% O₂ to better recapitulate the bone marrow microenvironment which has been shown to range from 1% to 7%^16-18 . However, maintaining co-cultures at this oxygen tension is not critical for the establishment of the three leukemic subpopulations and is at the discretion of the lab.
2. Every 4^th day remove all but 1 ml of media (including leukemic cells in suspension) and replace with 9 ml fresh leukemic culture media. When removing 9 ml of media from plate, be careful not to disturb the BMSC or OB adherent layer.
   1. Remove media by tilting plate to the side and aspirate media in the corner of the plate. Additionally, when adding fresh media, be sure to add drop wise in the corner of the plate against the sidewall to ensure minimal disruption of the BMSC or OB adherent layer.
3. After the 12^th day of co-culture, rinse leukemic cells from BMSC or OB layer by pipetting culture media from dish up and down gently over the dish approximately 5 to 10 times and then collect in 15 ml conical tube. Reseed onto new 80%-90% confluent BMSC or OB plate as described in step 2.1.
   NOTE: The gentle rinsing of the co-culture as described in step 2.3 will remove S and PB leukemic cells without disrupting the BMSC or OB monolayer. This allows only tumor cells to be transferred to the next co-culture plate. This 12 day cycle can be repeated as many times as needed based on user needs.

3. Preparing G10 Bead Columns

NOTE: If sterile downstream analysis or culturing is required following G10 column separation the following steps should be carried out using sterile technique and G10 columns should be setup in a sterile biological hood.

1. Pre-warm cell culture media to 37°C in water bath (~30 ml per column). Using a 10 ml disposable syringe, remove and discard plunger. Add glass wool to syringe.
2. Using tweezers, pull apart glass wool into thin loose strands. Add multiple layers of lightly packed glass wool to the syringe until 2/3 of the syringe is filled with glass wool.
   NOTE: The glass wool is crucial to prevent loose G10 particles from contaminating the leukemic cell collection. Make sure glass wool is packed enough to support the G10 particles, but not too densely packed to block media flow through the column.
3. Attach 1-way stopcock to the tip of the syringe in the closed position.
4. Clamp syringe column to ring stand high enough so a 50 ml conical tube (collection tube) can be placed underneath stopcock. Place collection tube under syringe column.
5. Using a 10 ml pipette add, drop-wise, G10 particles resuspended in PBS to the column on top of the glass wool. Continue adding G10 particles until a ~2 ml pellet (as measured by graduations on syringe) of G10 particles forms on top of the glass wool.
6. Equilibrate the G10 column with pre-warmed media.
   1. Add 2 ml of pre-warmed media to column. Open stopcock valve slowly so that media flows out of the column drop-wise.
   2. Repeat step 3.6.1 until a total of 10 ml of pre-warmed media have been ran across the column.
      NOTE: If G10 particles are seen in the flow through in the collection tube, either 1) add more G10 particles to maintain ~2 ml pellet making sure no additional G10 particles escape from the column or 2) replace column with an unused one and repeat steps 3.4-3.6.1.
   3. After the pre-warmed media drains from the column, close the stopcock and discard collection tube with flow through. Add new collection tube under column. Column is ready to be loaded with media + cell mixture.
      NOTE: Columns should be used immediately and not allowed to dry.

4. Separating 3 Subpopulations within Co-culture

1. Collection of suspension (S) tumor subpopulation.
   1. Aspirate media from co-culture plate with pipette and gently re-apply the same media to rinse the plate and collect media containing leukemic cells in a 15 ml conical tube. The leukemic cells collected are the S subpopulation.
2. Collection of Phase Bright (PB) tumor subpopulation.
   1. Add 10 ml fresh media back onto co-culture plate. Rinse vigorously by pipetting added media up and down approximately 5 times to remove adherent leukemic cells but not hard enough to dislodge adherent BMSC/OB component.
   2. Aspirate with pipette and collect media in a 15 ml conical tube. The collected cells are the PB subpopulation.
3. Collection of Phase Dim (PD) tumor subpopulation.
   1. Rinse plate with 1 ml PBS to remove remaining media. Trypsinize co-culture plate with 3 ml trypsin and place into 37 °C incubator for 5 min.
   2. Remove plate out of incubator and gently tap sides of the plate to dislodge adherent BMSC/OB.
   3. Add 1 ml fetal bovine serum (FBS) and pipette up and down 3-5 times to break apart large cell aggregates.
   4. Collect media with cells in a 15 ml conical tube. These cells are the unpurified PD subpopulation with BMSC/OB as well.
4. Centrifuge 3 isolated subpopulations at 400 x g for 7 min. Aspirate and discard supernatant then individually resuspend pellets in 1 ml pre-warmed media. Cells are ready to be loaded onto a G10 column.

5. Loading Co-culture Cells onto G10 Column

NOTE: Make sure stopcock is completely closed before adding media containing cells to G10 column. Also, each subpopulation must be ran over a separate G10 column so not to introduce any bias between populations in downstream analysis.

1. Using a 1,000 µl pipette, add 1 ml of each cell subpopulation in pre-warmed media to a separate G10 column drop-wise. Ensure that the media containing the cells remains on top or within G10 pellet. Allow cells to incubate on G10 pellet for 20 min at RT.
   NOTE: Stopcock remains closed for duration of incubation.

6. Collecting Leukemic Cells from G10 Column

1. Add 1-3 ml pre-warmed media to each G10 column. Open stopcock valve and allow media to slowly exit the column drop-wise.
   NOTE: It is crucial to maintain a slow flow rate from the column or the G10 pellet containing BMSC/OB can wash out of the column and contaminate the isolated leukemic cells.
2. Continue to add pre-warmed media in small increments (1-2 ml) to G10 column until a total of 15 to 20 ml has run through column and has been collected. Close stopcock valve and cap collection tube.
   NOTE: If a G10 particle pellet is seen at the bottom of collection tube, gently remove media from the tube leaving G10 particle pellet undisturbed and transfer to new tube.
3. Centrifuge collected media at 400 x g for 7 min at RT. Remove supernatant and resuspend cell pellet in buffer appropriate for downstream application.
4. Cells are now a pure population of leukemic cells free of BMSC or OB contamination and are ready to be applied to downstream applications at user discretion.
   NOTE: Leukemic cell viability should remain unchanged when passing cells through G10 columns.

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

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.