Name: modeling chemotherapy resistant leukemia in vitro
Uploaded: 2016-02-09T14:00:00
Description: Watch this Scientific Journal Video about Modeling Chemotherapy Resistant Leukemia In Vitro at JoVE.com

Supported by National Institutes of Health (NHLBI) R01 HL056888 (LFG), National Cancer Institute (NCI) RO1 CA134573NIH (LFG), P30 GM103488 (LFG), WV CTR-IDEA NIH 1U54 GM104942, the Alexander B. Osborn Hematopoietic Malignancy and Transplantation Program, and the WV Research Trust Fund. We are grateful for the support of Dr. Kathy Brundage and the West Virginia University Flow Cytometry Core Facility, supported by NIH S10-OD016165 and the Institutional Development Award (IDeA) from the NIH Institute of General Medical Sciences of the National Institutes of Health (CoBRE P30GM103488 and INBRE P20GM103434).

1. Advanced Preparation
<ol>
	<li>Preparing dextran G10 particles.
	<ol>
		<li>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 &#176;C O/N.</li>
		<li>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&#160;&#176;C until ready to use.</li>
	</ol>
	</li>
	<li>Culturing BMSC and O...

<ol>
	<li>Ayala, F., Dewar, R., Kieran, M., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+bone+microenvironment+to+leukemogenesis+and+leukemia+progression.">Contribution of bone microenvironment to leukemogenesis and leukemia progression.</a> Leukemia. 23 (12), 2233-2241 (2009).</li><li>Coustan-Smith, E., Sancho, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+importance+of+minimal+residual+disease+in+childhood+acute+lymphoblastic+leukemia.">Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia.</a> Blood. 96 (8), 2691-2696 (2000).</li><li>Gaynon, P. S., Qu, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+after+relapse+in+childhood+acute+lymphoblastic+leukemia.">Survival after relapse in childhood acute lymphoblastic leukemia.</a> Cancer. 82 (7), 1387-1395 (1998).</li><li>Kikuchi, M., Tanaka, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+of+minimal+residual+disease+in+adult+acute+lymphoblastic+leukemia.">Clinical significance of minimal residual disease in adult acute lymphoblastic leukemia.</a> Int. J. Hematol. 92 (3), 481-489 (2010).</li><li>Krause, D. S., Scadden, D. T., Preffer, F. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+Accessory+Cells+by+Adherence+to+Sephadex+G-10.">Depletion of Accessory Cells by Adherence to Sephadex G-10.</a> Curr. Protoc. , (2001).</li><li>Berniakovich, I., Giorgio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+oxygen+tension+maintains+multipotency,+whereas+normoxia+increases+differentiation+of+mouse+bone+marrow+stromal+cells.">Low oxygen tension maintains multipotency, whereas normoxia increases differentiation of mouse bone marrow stromal cells.</a> Int. J. Mol. Sci. 14 (1), 2119-2134 (2013).</li><li>Chow, D. C., Wenning, L. A., Miller, W. M., Papoutsakis, E. 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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 ALL3,8. 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...

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 treated1-8. 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 characteristics9-14. 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.

<table border="0" cellpadding="0" cellspacing="0" width="734">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:224px;">G10 sephadex beads</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">G10120</td>
			<td style="width:243px;">Referred to in manuscript as gel type 10 cross-linked dextran particles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">10 ml sterile syringe</td>
			<td style="width:149px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Glass wool</td>
			<td style="width:149px;">Pyrex</td>
			<td style="width:119px;">3950</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">1-way stopcocks</td>
			<td style="width:149px;">World Precision Instruments, Inc.</td>
			<td style="width:119px;">14054-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">50 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021039</td>
			<td>Used as collection tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">15 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021037</td>
			<td>Used for cell collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Fetal Bovine Serum</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">F6178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">0.05% Trypsin&#160;</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">100X20mm Cell Culture Dishes</td>
			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:119px;">664160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Culture media</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Osteoblast culture media&#160;</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-27001</td>
			<td>For human osteoblast media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">RPMI 1640 media</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">15-040</td>
			<td>For tumor media prepation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Cell lines</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Adherent Cells</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">Human Osteoblasts</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-12720</td>
			<td style="width:243px;">Human osteoblast were cultured according to the supplier&#8217;s recommendations.&#160;</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:224px;">Human Bone Morrow Stromal Cells</td>
			<td style="width:149px;">WVU Biospecimen Core</td>
			<td style="width:119px;"></td>
			<td style="width:243px;">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 (*)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Leukemic Cells:</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">REH</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:119px;">ATCC-CRL-8286</td>
			<td style="width:243px;">REH cells were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">SD-1</td>
			<td style="width:149px;">DSMZ</td>
			<td style="width:119px;">ACC 366</td>
			<td style="width:243px;">SD-1 were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="48">
			<td colspan="4" height="48" style="height:48px;width:735px;">(*) 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&#8211;32.&#160;</td>
		</tr>
	</tbody>
</table>

modeling chemotherapy resistant leukemia in vitro

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.

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...

Watch this Scientific Journal Video about Modeling Chemotherapy Resistant Leukemia In Vitro at JoVE.com

Modeling Chemotherapy Resistant Leukemia In Vitro

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.

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

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Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...