Name: modeling chemotherapy resistant leukemia in vitro
Uploaded: 2016-02-09T14:00:00.000+00:00
Duration: 8 min 41 s
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 OB.
	<ol>
		<li>Maintain both BMSC or OB at 37 &#176;C in 6% CO2 and grown on 10 cm tissue culture plates until 90% confluency is reached.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Establishing and Maintaining Co-culture
<ol>
	<li>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 &#176;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.</li>
	<li>Every 4th 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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>After the 12th 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.</li>
</ol>
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.
<ol>
	<li>Pre-warm cell culture media to 37&#176;C in water bath (~30 ml per column). Using a 10 ml disposable syringe, remove and discard plunger. Add glass wool to syringe.</li>
	<li>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.</li>
	<li>Attach 1-way stopcock to the tip of the syringe in the closed position.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Equilibrate the G10 column with pre-warmed media.
	<ol>
		<li>Add 2 ml of pre-warmed media to column. Open stopcock valve slowly so that media flows out of the column drop-wise.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Separating 3 Subpopulations within Co-culture
<ol>
	<li>Collection of suspension (S) tumor subpopulation.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Collection of Phase Bright (PB) tumor subpopulation.
	<ol>
		<li>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.</li>
		<li>Aspirate with pipette and collect media in a 15 ml conical tube. The collected cells are the PB subpopulation.</li>
	</ol>
	</li>
	<li>Collection of Phase Dim (PD) tumor subpopulation.
	<ol>
		<li>Rinse plate with 1 ml PBS to remove remaining media. Trypsinize co-culture plate with 3 ml trypsin and place into 37&#160;&#176;C incubator for 5 min.</li>
		<li>Remove plate out of incubator and gently tap sides of the plate to dislodge adherent BMSC/OB.</li>
		<li>Add 1 ml fetal bovine serum (FBS) and pipette up and down 3-5 times to break apart large cell aggregates.</li>
		<li>Collect media with cells in a 15 ml conical tube. These cells are the unpurified PD subpopulation with BMSC/OB as well.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Using a 1,000 &#181;l 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.</li>
</ol>
6. Collecting Leukemic Cells from G10 Column
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge collected media at 400 x g for 7 min at RT. Remove supernatant and resuspend cell pellet in buffer appropriate for downstream application.</li>
	<li>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.</li>
</ol>

<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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hematopoietic+stem+cell+niche-home+for+friend+and+foe?.">The hematopoietic stem cell niche-home for friend and foe?.</a> Cytometry B Clin. Cytom. 84 (1), 7-20 (2013).</li><li>Meads, M. B., Hazlehurst, L. A., Dalton, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bone+Marrow+Microenvironment+as+a+Tumor+Sanctuary+and+Contributor+to+Drug+Resistance.">The Bone Marrow Microenvironment as a Tumor Sanctuary and Contributor to Drug Resistance.</a> Clin. Cancer Res. 14 (9), 2519-2526 (2008).</li><li>Nguyen, K., Devidas, M., 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=Factors+Influencing+Survival+After+Relapse+From+Acute+Lymphoblastic+Leukemia:+A+Children's+Oncology+Group+Study.">Factors Influencing Survival After Relapse From Acute Lymphoblastic Leukemia: A Children's Oncology Group Study.</a> Leuk. Off. J. Leuk. Soc. Am. Leuk. Res. Fund UK. 22 (12), 2142-2150 (2008).</li><li>Szczepanek, J., Styczy&#324;ski, J., Haus, O., Tretyn, A., Wysocki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapse+of+Acute+Lymphoblastic+Leukemia+in+Children+in+the+Context+of+Microarray+Analyses.">Relapse of Acute Lymphoblastic Leukemia in Children in the Context of Microarray Analyses.</a> Arch. Immunol. Ther. Exp. (Warsz.). 59 (1), 61-68 (2011).</li><li>Boyerinas, B., Zafrir, M., Yesilkanal, A. E., Price, T. T., Hyjek, E. M., Sipkins, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+osteopontin+in+the+bone+marrow+niche+regulates+lymphoblastic+leukemia+cell+dormancy.">Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.</a> Blood. 121 (24), 4821-4831 (2013).</li><li>Bradstock, K., Bianchi, A., Makrynikola, V., Filshie, R., Gottlieb, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+and+proliferation+of+precursor-B+acute+lymphoblastic+leukemia+cells+on+human+bone+marrow+stroma.">Long-term survival and proliferation of precursor-B acute lymphoblastic leukemia cells on human bone marrow stroma.</a> Leukemia. 10 (5), 813-820 (1996).</li><li>Clutter, S. D., Fortney, J., Gibson, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP-2+is+required+for+bone+marrow+stromal+cell+support+of+pro-B-cell+chemotaxis.">MMP-2 is required for bone marrow stromal cell support of pro-B-cell chemotaxis.</a> Exp. Hematol. 33 (10), 1192-1200 (2005).</li><li>Manabe, A., Murti, K. G., 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=Adhesion-dependent+survival+of+normal+and+leukemic+human+B+lymphoblasts+on+bone+marrow+stromal+cells.">Adhesion-dependent survival of normal and leukemic human B lymphoblasts on bone marrow stromal cells.</a> Blood. 83 (3), 758-766 (1994).</li><li>Mudry, R. E., Fortney, J. E., York, T., Hall, B. M., Gibson, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+regulate+survival+of+B-lineage+leukemic+cells+during+chemotherapy.">Stromal cells regulate survival of B-lineage leukemic cells during chemotherapy.</a> Blood. 96 (5), 1926-1932 (2000).</li><li>Tesfai, Y., Ford, 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=Interactions+between+acute+lymphoblastic+leukemia+and+bone+marrow+stromal+cells+influence+response+to+therapy.">Interactions between acute lymphoblastic leukemia and bone marrow stromal cells influence response to therapy.</a> Leuk. Res. 36 (3), 299-306 (2012).</li><li>Hathcock, K. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+pO(2)+distributions+in+the+bone+marrow+hematopoietic+compartment.+II.+Modified+Kroghian+models.">Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models.</a> Biophys. J. 81 (2), 685-696 (2001).</li><li>Holzwarth, C., Vaegler, M., 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=Low+physiologic+oxygen+tensions+reduce+proliferation+and+differentiation+of+human+multipotent+mesenchymal+stromal+cells.">Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells.</a> BMC Cell Biol. 11, (2010).</li><li>O'Leary, H., Akers, S. M., 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=VE-cadherin+Regulates+Philadelphia+Chromosome+Positive+Acute+Lymphoblastic+Leukemia+Sensitivity+to+Apoptosis.">VE-cadherin Regulates Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Sensitivity to Apoptosis.</a> Cancer Microenviron. Off. J. Int. Cancer Microenviron. Soc. 3 (1), 67-81 (2010).</li><li>Wang, L., Chen, L., Benincosa, J., Fortney, J., Gibson, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-induced+phosphorylation+of+Bcl-2+influences+B+lineage+leukemic+cell+response+to+apoptotic+stimuli.">VEGF-induced phosphorylation of Bcl-2 influences B lineage leukemic cell response to apoptotic stimuli.</a> Leukemia. 19 (3), 344-353 (2005).</li><li>Wang, L., Coad, J. E., Fortney, J. M., Gibson, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-induced+survival+of+chronic+lymphocytic+leukemia+is+independent+of+Bcl-2+phosphorylation.">VEGF-induced survival of chronic lymphocytic leukemia is independent of Bcl-2 phosphorylation.</a> Leukemia. 19 (8), 1486-1487 (2005).</li><li>Jing, D., Fonseca, A. V., 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=Hematopoietic+stem+cells+in+co-culture+with+mesenchymal+stromal+cells--modeling+the+niche+compartments+in+vitro.">Hematopoietic stem cells in co-culture with mesenchymal stromal cells--modeling the niche compartments in vitro.</a> Haematologica. 95 (4), 542-550 (2010).</li><li>Jing, D., Wobus, M., Poitz, D. M., Bornh&#228;user, M., Ehninger, G., Ordemann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+plays+a+critical+role+in+the+hematopoietic+microenvironment+in+vitro.">Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro.</a> Haematologica. 97 (3), 331-339 (2012).</li></ol>

The authors have no competing financial interests.

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 gold standard for testing questions related to drug efficacy, 2D co-culture continues to be a cost effective methodology for testing hypotheses and drug strategies related to bone morrow microenvironment support of leukemic cell survival. Many groups have shown that co-culture of leukemic cells with BMSC or OB provides a survival advantage when challenged with chemotherapy agents9,10,12-14,19-21. Work modeling normal immature CD34+ hematopoietic cells in co-culture with mesenchymal stem cells (MSC) revealed that hematopoietic cells will interact with the adherent monolayer of MSCs to form three distinct spatial populations of hematopoietic cells22,23. Proliferation and differentiation of the CD34+ cells was effected relative to their location within the co-culture22. We have expanded on this observation to test questions related to bone marrow microenvironment stromal cell support of a resistant population of tumor cells within a 2D co-culture and its isolation for downstream analysis.
Unlike standard 2D co-culture models which typically sample leukemic cells by removal of the suspended tumor, our model shows that co-culture represents a more dynamic interaction in which leukemic cells in co-culture with BMSC or OB form three subpopulations relative to the BMSC or OB monolayer (Figure 1). The tumor subpopulations that form are suspended (S) tumor, which is freely floating in the media; phase bright (PB) that is adhered to the surface of the BMSC or OB; and the phase dim (PD) which have buried beneath the BMSC or OB monolayer (Figure 1). In this model, we found that a strict feeding and reseeding schedule is important to achieve consistent results in a co-culture model and therefore we feed the co-cultures at 4 day intervals and transfer tumor to new BMSC or OB monolayers every 12 days. This may require modification for alternative tumor types as needed. The number of tumor cells that will migrate below the BMSC or OB to form the PD population may vary between different leukemic cells. This can be a limitation of the model, when the number of PD tumor cells are low making it difficult to collect enough cells for downstream analysis. In some cases this problem can be overcome by establishing replicate co-cultures to allow for pooling of the three individual subpopulations.
As this model relies on tumor cell interaction with the BMSC or OB it is important to have an effective method to remove stromal cell contamination to address specific biology of the leukemic cells. To accomplish this separation of tumor cells from stroma we use G10 columns. Proper setup and use of these columns is crucial for isolation of pure tumor populations for downstream analysis. As highlighted in Figure 2B, proper execution of the G10 column separation results in recovery of tumor cells at greater than 99% purity. This allows for downstream analysis of the leukemic cells without complication of interpretation of results that would result from stromal cell contamination. It is important to note that all leukemic cell types whether cell lines or primary patient samples will vary slightly in their ability to pass through the G10 columns. Before use in large scale experiments users should determine the number of leukemic cells they must input to recover the desired amount needed for their specific downstream needs. In addition, the amount of wash media ran over the G10 column post incubation can be increased to try and increase the number of cells recovered. Care should be taken to insure that the additional washes do not cause stromal cell contamination which can be determined post wash by cell counts or flow cytometry analysis of flow through.
In establishing this model, we observed that leukemic cells recovered from the PD population have increased viability compared to leukemic cells grown in media alone, as well as to those recovered from the S or PB populations in the same co-culture when exposed to cytotoxic chemotherapy (Figure 3 A-B). This is significant because it represents a population of leukemic cells in vitro that derive pronounced protection from chemotherapy. This provides a useful tool to test treatment strategies aimed at targeting the most resistant tumor population, which is supported by the bone marrow microenvironment. Furthermore, because these leukemic cells are so well protected by the BMSC or OB co-culture it is amenable to in vitro combination treatment strategies, that in media alone or standard co-culture models would appear to or would completely kill the tumor which is not always representative of effects seen in resistant microenvironment supported leukemic populations.
Finally, we believe that use of this model can provide valuable insight into the interactions between BMSC/OB, and leukemic cells that are responsible for resistance to chemotherapy treatment, lead to MRD, and subsequently relapse. This model provides an in vitro platform to design experiments which will better inform downstream pre-clinical models. Though we show use of this model to test interactions between bone marrow stromal cells and ALL derived leukemic cells, we are hopeful that future directions and applications of this model will be useful in a variety of malignancies in which the bone marrow microenvironment provides a site of sanctuary for tumors during chemotherapeutic intervention.

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><tbody><tr><td>G10 sephadex beads</td><td>Sigma</td><td>G10120</td><td>Referred to in manuscript as gel type 10 cross-linked dextran particles</td></tr><tr><td>10 ml sterile syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Glass wool</td><td>Pyrex</td><td>3950</td><td></td></tr><tr><td>1-way stopcocks</td><td>World Precision Instruments, Inc.</td><td>14054-10</td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td>World Wide Medical Products</td><td>41021039</td><td>Used as collection tubes</td></tr><tr><td>15 ml conical centrifuge tubes</td><td>World Wide Medical Products</td><td>41021037</td><td>Used for cell collection</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F6178</td><td></td></tr><tr><td>0.05% Trypsin&amp;nbsp;</td><td>Mediatech, Inc.</td><td>25-053-CI</td><td></td></tr><tr><td>100 x 20 mm Cell Culture Dishes</td><td>Greiner Bio-One</td><td>664160</td><td></td></tr><tr><td>&lt;strong&gt;Culture media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Osteoblast culture media&amp;nbsp;</td><td>PromoCell</td><td>C-27001</td><td>For human osteoblast media&amp;nbsp;</td></tr><tr><td>RPMI 1640 media</td><td>Mediatech, Inc.</td><td>15-040</td><td>For tumor media prepation&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Adherent Cells:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human Osteoblasts</td><td>PromoCell</td><td>C-12720</td><td>Human osteoblast were cultured according to the supplier&amp;rsquo;s recommendations.&amp;nbsp;</td></tr><tr><td>Human Bone Morrow Stromal Cells</td><td>WVU Biospecimen Core</td><td></td><td>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><td>&lt;strong&gt;Leukemic Cells:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>REH</td><td>ATCC</td><td>ATCC-CRL-8286</td><td>REH cells were cultured according to the supplier&amp;rsquo;s recommendations and recommended media.&amp;nbsp;</td></tr><tr><td>SD-1</td><td>DSMZ</td><td>ACC 366</td><td>SD-1 were cultured according to the supplier&amp;rsquo;s recommendations and recommended media.&amp;nbsp;</td></tr><tr><td colspan="4">(*) 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&amp;ndash;32.</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)). While the 3 subpopulations of tumor cells can commonly be seen after 24 hr of co-culture with BMSC or OB we co-culture the cells for 4 days to allow the full dynamics and interactions between the leukemic cells and BMSC/OB cells to take place before any manipulation or experimentation takes place (Figure 1). Also, note that we maintain the co-cultures at an oxygen tension of 5% to recapitulate bone marrow physiology, which has been reported to range from 1%-7%16-18.
A vast majority of downstream analysis requires the separation of the leukemic cells from the BMSC or OB. To achieve this we use G10 columns to harvest a pure population of leukemic cells (Figure 2A). Following trypsinization of BMSC and PD REH cells a mixed population is seen by two distinct forward/side populations by flow cytometry (Figure 2B, top panel). Following G10 separation a pure population of only REH ALL cells is recovered, which was confirmed by forward/side scatter flow cytometry (Figure 2B, bottom panel).
Use of this co-culture model and the ability to isolate leukemic cells from 3 subpopulations when in co-culture with BMSC or OB allows for interrogation of leukemic cell phenotype with relation to its spatial location relative to the adherent BMSC or OB monolayer. Of particular interest, is that ALL cells recovered from the PD population of a BMSC or OB co-culture have little to no cell death following exposure to cytotoxic chemotherapy (Figure 3A,B).
<img alt="Figure 1" src="/files/ftp_upload/53645/53645fig1.jpg" /> 
Figure 1. ALL cells in co-culture with BMSC or OB form three spatial populations. Our lab uses an in vitro co-culture system to model leukemic cell interactions with bone marrow microenvironment derived stromal cells (BMSC or OB). To establish co-culture, leukemic cells (Red) are seeded onto an 80%-90% confluent monolayer of BMSC or OB (Blue), which is denoted as &#39;Time 0&#39;. Co-cultures are maintained at 37&#160;&#176;C at 5% oxygen to approximate conditions of the bone marrow microenvironment. Leukemic cells will begin to form 3 subpopulations as early as 24 hr, but to allow for complete interactions to form we allow co-cultures 4 days to establish before utilizing leukemic cells for experiments. By day 4 (right panels), three subpopulations of leukemic cells will form in relation to the adherent monolayer. The schematic (top right) and the phase contrast microscopy (bottom right) show the suspended (S) leukemic cells freely floating in the media; phase bright (PB) leukemic cells which are adhered to the surface of the BMSC or OB monolayer; and the phase dim (PD) leukemic cells that have migrated beneath the BMSC or OB monolayer. Scale bar represents 10 microns. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53645/53645fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53645/53645fig2.jpg" /> 
Figure 2. Use of G10 columns allows for separation of ALL cells from BMSC/OB. (A) Demonstration of the process of using a G10 column to separate ALL cells from BMSC/OB co-culture to achieve a pure population of tumor cells for downstream analysis. From left to right, a mixture of ALL cells and BMSC/OB cells is added to the top of the G10 column; (Center) cell mixture will settle in the G10 slurry and should be incubated at RT for 20 min (Note: Stopcock is in the closed position throughout first two steps); (right) leukemic cells are recovered by opening the stopcock and rinsing column with pre-warmed media. (B) Top panel shows before G10 separation that there is a mixed population of cells containing BMSC (blue gate) and REH ALL cells (red gate) by evaluating forward (FSC) and side scatter (SSC) analysis. Bottom panel, following G10 separation only the pure population of REH ALL cells (red gate) remain with less than 1% stromal cell contamination (blue gate). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53645/53645fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53645/53645fig3.jpg" /> 
Figure 3. PD leukemic cells have increased resistance to chemotherapy exposure. SD-1 leukemic cells recovered from the PD population of a BMSC co-culture (A) do not display reduced viability following a 4 day exposure to Ara-C [1 &#181;M], MTX [50 &#181;M], or VCR [25 &#181;M], similar to untreated controls (note that a second dose of Ara-C added at 48 hr to account for any drug loss due to stability). Leukemic cells from the media alone, S, and PB populations have significantly reduced viability as determined by trypan blue exclusion.SD-1 leukemic cells co-cultured with OB cells display similar trends in viability (B). Results are expressed as mean &#177; SEM. (*) denotes p &#60; 0.05, unpaired t-test relative to untreated controls. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53645/53645fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

modeling-chemotherapy-resistant-leukemia-in-vitro

Nanyang Technological University

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9.0K Views.  West Virginia University School of Medicine. 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. 

Video: Modeling Chemotherapy Resistant Leukemia In Vitro

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Cancer Research

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

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Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

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Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

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2D Co-culture of Leukemic Cells with BMSCs: A Method to Obtain Subpopulations of Leukemic Cells

Source: Slone, W. L. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes the technique for long term 2D co-culture of leukemia cells with bone marrow stromal cells to study their interaction with the bone marrow microenvironment. The 2D coculture model utilizes the acute lymphoblastic leukemia cells and the bone marrow stromal or osteoblasts that influence tumor cell phenotype. This study will support investigation of the mechanisms that underlie bone marrow supported survival of leukemic cells during chemotherapy exposure.

Source: Slone, W. L. et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes the technique for long term 2D ...

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Leukemia

In vitro techniques & assays

Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

Source: Slone, W. L., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes a method for spatial separation of three leukemic cell subpopulations: the suspended leukemic cells freely floating in the media, phase bright leukemic cells adhered to the surface of the mesenchymal stromal cell monolayer, and the phase dim leukemic cells that have migrated beneath the mesenchymal stromal cell monolayer from 2D co-culture.

Source: Slone, W. L., et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes a method for spatial separation ...

Cell isolation & characterization

G-10 Column Based Leukemia Cell Sorting: A Method to Purify Acute Lymphoblastic Leukemia Cells from Bone Marrow Stromal Cells

Source: Slone, W. L.,et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes the separation of acute lymphoblastic leukemia cells from bone marrow stromal cells using gel-type ten cross-linked dextran G-10 columns. This method helps recover the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells generating a &#34;phase dim&#34; tumor cell population.

Modeling Chemotherapy Resistant Leukemia In Vitro

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