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. 고급 준비 <ol><li> 덱스 트란 G10 입자를 준비합니다. <ol><li> 10g의 G10 입자로 1X PBS 50 ㎖를 첨가하여 G10 슬러리를 준비한다. 반전에 의해 믹스와 G10가 4 ° CO / N에서 인산염 완충 생리 식염수 (PBS)에서 정착 할 수 있습니다. </li><li> G10 컬럼 분리의 날, 정착 G10 입자에서 흡인 PBS 및 50 ml의 신선한 PBS를 추가합니다. 반전으로 섞는다. 사용할 준비가 될 때까지 4 ° C에서 정착 G10 입자와 저장소에 50 ml의 신선한 PBS를 추가, 두 번 반복합니다. </li></ol></li><li> BMSC와 OB를 배양. <ol><li> 90 % 컨 플루 언시에 도달 할 때까지 6 % CO 2 및 10cm 조직 배양 플레이트에서 성장하고 37 ° C에서 BMSC 또는 OB를 모두 유지한다. </li><li> 새로운 10cm 플레이트에 2를 Trypsinize BMSC 또는 OB 세포와 1 분할. 백혈병 공동 배양에 필요한 때까지 세포는 이러한 표준으로 성장하고 있습니다. </li></ol></li></ol> 2. 구축 및 공동 문화를 유지 <ol><li> 5 ~ 20 × 10 (6) 백혈병 세포를 추가하기 전n은 80 % -90 %의 합류 BMSC 또는 OB 판 상에 종양 특이 적 배양 배지 10 ㎖. 참고 : 우리의 실험실 더 나은 1 %에서 7 % 16 ~ 18의 범위 것으로 나타났다 골수 미세 환경을 요점을 되풀이하는 5 % O 2에서 37 ° C에서 공동 문화를 유지한다. 그러나,이 산소 장력 공동 배양을 유지하는 세 백혈병 개체군의 확립에 중요하지 않으며 실의 재량이다. </li><li> 매 4 번째 날 (정지에서 백혈병 세포를 포함) 미디어의 1ml를 제외하고 모두 제거하고 9 ml의 신선한 백혈병 배양 배지로 교체. 판에서 미디어의 9 ml의를 제거 할 때, BMSC 또는 OB 접착층을 방해하지 않도록주의. <ol><li> 플레이트의 모서리에있는 측과 대기음 미디어에 판을 기울여 용지를 제거합니다. 신선한 매체를 추가 할 때 또한, BMSC 또는 OB 접착 층의 중단을 최소화 할 수 있도록 측벽에 대한 플레이트의 모서리에 적가를 추가해야합니다. </li></ol></li><li> 공동 문화의 12 일 하루를 보낸 후, 약 5 ~ 10 배를 요리부터 문화 미디어를 피펫 팅 아래로 부드럽게 접시 위에 의해 BMSC 또는 OB 층에서 백혈병 세포를 씻어 다음 15 ML 원뿔 튜브에 수집합니다. 단계 2.1에 설명 된대로 새 80 % -90 %의 합류 BMSC 또는 OB 판에 시드. 참고 : BMSC 또는 OB 단층을 방해하지 않고 S 및 PB 백혈병 세포를 제거 단계 2.3에 설명 된대로 공동 문화의 부드러운 세정. 이것은 종양 세포는 다음 공동 배양 플레이트로 전달 될 수있다. 시간 12 일주기는 사용자의 필요에 따라 필요한만큼 여러 번 반복 될 수있다. </li></ol> 3. 준비 G10 구슬 열 주 : 멸균 하류 분석 또는 배양이 G10 컬럼 분리 한 다음 필요한 경우 다음 단계 멸균 기법을 이용하여 수행되어야하고, G10 컬럼 멸균 생물학적 후드에 설치되어야한다. <ol><li> 사전 WARM은 세포 배양 미디어 수조에서 37 ° C까지 (~ 30 ml의 컬럼 당). 10 ㎖의 일회용 주사기를 사용하여 제거하고 플런저를 폐기합니다. 주사기에 유리 섬유를 추가합니다. </li><li> 핀셋을 사용하여, 얇은 느슨한 가닥에 유리 섬유를 따로 빼냅니다. 주사기의 2/3이 유리 털실로 가득 채운 때까지 주사기로 가볍게 포장 유리 섬유의 여러 레이어를 추가합니다. 주 : 유리솜은 백혈병 세포 콜렉션을 오염시키는 이른바 G10 입자를 방지하는 것이 중요하다. 확실히 유리 섬유는 G10 입자를 지원하기에 충분 포장,하지만 너무 조밀하게 열을 통해 미디어의 흐름을 차단하는 포장되지되어 있는지 확인합니다. </li><li> 폐쇄 위치에서 주사기의 끝 부분에 1 방향 스톱 콕을 부착합니다. </li><li> 링 클램프 주사기 열 때문에 50 ML 원뿔 튜브 (포집 관)는 꼭지 아래에 배치 할 수 있습니다 충분 서있다. 주사기 열에서 수집 튜브를 놓습니다. </li><li> 10 ML의 피펫의 상단에 열을 PBS에 재현 탁, G10 입자를 추가 드롭 현명한 사용유리솜. 유리면 위에 G10 입자 형태 (주사기 눈금으로 측정) ~ 2 mL의 펠렛까지 G10 입자를 계속 추가. </li><li> 미리 예열 미디어와 G10의 열 평형. <ol><li> 열을 미리 예열 미디어 2 ML을 추가합니다. 그래서 오픈 스톱 콕 밸브는 천천히 미디어 드롭 현명한 열 밖으로 흐른다. </li><li> 미리 예열 미디어 10 ㎖의 총 때까지 단계를 반복 3.6.1은 열 가로 질러왔다. 참고 : G10 입자 포집 관에서를 통해 흐름에서 볼, 중 1)보다 G10 입자를 추가하는 경우 추가 G10 입자가 열에서 탈출하지 확인한 ~ 2 ml의 펠렛을 유지하거나 2)하지 않는 한 반복과 열을 교체 3.4-3.6.1 단계를 반복합니다. </li><li> 열에서 미리 예열 미디어 빼낸 후, 꼭지를 닫고 통해 흐름을 수집 튜브를 폐기합니다. 열에서 새 컬렉션 튜브를 추가합니다. 열 매체 + 세포 혼합액로드 될 준비가되어있다. 참고 : 열즉시 사용 및 건조 허용되지해야합니다. </li></ol></li></ol> 4. 공동 문화 내에서 3 부분 집단을 분리 <ol><li> 서스펜션 (S) 종양 모집단의 컬렉션입니다. <ol><li> 공동 배양 접시에서 대기음 미디어 피펫 부드럽게 플레이트를 씻어 15 ML 원뿔 튜브에 백혈병 세포를 포함하는 미디어를 수집하기 위해 같은 미디어를 신청 다시. 수집 백혈병 세포 S 아군이다. </li></ol></li><li> 단계 밝은 (PB) 종양 모집단의 컬렉션입니다. <ol><li> 공동 배양 접시에 다시 10 ml의에게 신선한 매체를 추가합니다. 부착 백혈병 세포 만 부착 BMSC / OB 성분을 제거하기 어려운 충분하지를 제거하기 위해 위 아래로 약 5 시간을 추가 한 미디어를 피펫 팅에 의해 적극적으로 씻어. </li><li> 피펫 기음과 15 ML 원뿔 튜브 미디어를 수집합니다. 수집 된 세포는 PB 아군이다. </li></ol></li><li> 단계 어둡게 (PD) 종양 모집단의 컬렉션입니다. <ol><li> 렘 1 ml의 PBS와 린스 판비켜 남은 용지. 5 분 동안 37 ° C 배양기에 3 ㎖ 트립신과 장소를 Trypsinize 공동 문화 판. </li><li> 부착 BMSC / OB을 제거하기 위해 플레이트의 측면을 누릅니다 부드럽게 인큐베이터에서 플레이트를 제거합니다. </li><li> 떨어져 큰 세포 집합체을 깰 1 ml의 소 태아 혈청 (FBS) 및 피펫 위아래로 3-5 시간을 추가합니다. </li><li> 15 ML 원뿔 튜브에 세포와 미디어를 수집합니다. 이러한 세포뿐만 BMSC / OB로 미정 PD 아군이다. </li></ol></li><li> 원심 분리기 3 7 분 동안 400 XG에서 소집단을입니다. 기음과 폐기 뜨는는 개별적으로 1 ml의 사전 예열 매체에 펠렛을 재현 탁. 세포는 G10 컬럼에로드 할 준비가 된 것입니다. </li></ol> G10 열 상 (5)로드 공동 배양 세포 참고 : 확인 꼭지를 확인 완전히 G10 컬럼 미디어 포함하는 셀을 추가하기 전에 폐쇄된다. 또한, 각각의 하위 집단은부터 소개하기 위해 그렇게하지 ​​별도의 G10 컬럼을 통해 실행해야합니다다운 스트림 분석 인구 사이의 바이어스를 전자. <ol><li> 1,000 μL 피펫을 사용하여, 드롭 현명한 별도의 G10 컬럼에 미리 예열 미디어에서 각 셀의 모집단 1 ㎖를 추가합니다. 세포를 포함하는 미디어가 상단이나 G10 펠릿 내에 남아 있는지 확인합니다. 세포가 실온에서 20 분 동안 G10 펠릿에 품어 할 수 있습니다. 참고 : 조절판은 배양 기간 동안 폐쇄 남아있다. </li></ol> 6. G10 열에서 백혈병 세포를 수집 <ol><li> 각 G10 컬럼에 1-3 ml의 사전 예열 매체를 추가합니다. 오픈 스톱 콕 밸브와 미디어 천천히 열을 적가 종료 할 수 있습니다. 참고 : 열 또는 열에서 씻고 고립 된 백혈병 세포를 오염시킬 수 BMSC / OB를 포함하는 G10 펠릿에서 느린 유속을 유지하는 것이 중요하다. </li><li> 15 ~ 20 ML의 총까지 G10 컬럼 작은 단위 (1-2 ml)에 미리 예열 미디어를 계속 추가 칼럼을 통해 실행하고 수집하고 있습니다. 닫기 스톱 콕 버지니아LVE와 모자 포집 관. 참고 : G10 입자 펠릿 수집 튜브의 하단에 볼 경우, 부드럽게 G10 입자 펠릿 그대로 새로운 튜브로 전송 떠나는 튜브에서 용지를 제거합니다. </li><li> 원심 분리기는 실온에서 7 분 동안 400 XG에 미디어를 모았다. 상층 액을 제거하고 다운 스트림 응용 프로그램에 대한 적절한 버퍼에 세포 펠렛을 재현 탁. </li><li> 셀은 BMSC 또는 OB 오염없는 백혈병 세포의 순수 인구이며 사용자 재량 하류 어플리케이션에 적용 할 준비가되어있다. 참고 : G10 컬럼을 통해 세포를 통과 할 때 백혈병 세포 생존 능력은 변경되지 않은 상태로 유지해야합니다. </li></ol>

<ol>
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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. 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.

질병의 재발에 기여 최소 잔존 질환 (MRD)는 모든 공격 내화물의 치료에서 주요 임상 과제이며, 또한, 다른 혈액 학적 악성 종양의 호스트로 계속된다. 골수 미세 환경 모두에서 재발 -3,8-의 일반적인 위치이다. 이와 같이, 골수 미세 환경을 모델링 모델 요법 노광 중에 백혈병 종양 세포 생존 및 MRD의 유지에 관한 가설을 테스트하기 위해 중요한 도구이다. 마우스 모델은 약물 효능에 관한 ?...

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.

이 공동 문화 모델의 성공적인 설치 및 문화. 부착 BMSC 또는 OB 단층에 비해 백혈병 세포의 3 개체군의 설립 결과 1 BMSC 단일 층으로 시드 ALL 세포가 초기에 중단의 같은 단 하나의 인구를 표시하는 방법을 보여줍니다 것이다 백혈병 세포. 백혈병 세포는 백혈병 세포의 개체군 3 공간을 형성하도록 상호 작용 BMSC 사일의 과정 ((현탁 S) 상 밝은 (PB) 및 ?...

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.

모델링 화학 요법 내성 백혈병<em&gt; 체외</em

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

Universidad de Cartagena UDEC

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Medicine

Modeling Chemotherapy Resistant Leukemia In Vitro

9.0K 조회수.  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. 

비디오: 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.

에 약물 저항 관련 소설 스플 라이스 변종을 감지하는 RNA 시퀀싱을 사용하여<em&gt; 체외</em&gt; 암 모델

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

연구

암 연구학

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.

반대로 mitotic 에이전트 Docetaxel에 전립선 암 세포 모델의 생성

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.

Oncogene 의존에 c Fos의 역할 및 Dusp1를 평가 하기 위한 방법

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.

급성 백혈병의 치료에 대한 티로신 키나제 억제제의 전임상 평가

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

의학

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).

약물 내성 표현형을 조절하는 인자를 식별하기 위한 풀링된 shRNA 라이브러리 스크리닝

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

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.

차 다발성 골수종 세포의 약물 민감도의 특성에 대한의 Organotypic 높은 처리량 시스템

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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://jove.unicartagenaproxy.elogim.com/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.

백혈병 세포와 BMSC의 2D 공동 배양: 백혈병 세포의 하위 집단을 얻는 방법

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 ...

JoVE Encyclopedia of Experiments

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://jove.unicartagenaproxy.elogim.com/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.

Leukemic Subpopulation Harvest: 2D 공동 배양에서 백혈병 세포 하위 집단의 공간적 분리 방법

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://jove.unicartagenaproxy.elogim.com/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.

모델링 화학 요법 내성 백혈병

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