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.先进的准备<ol><li>准备葡聚糖G10颗粒。 <ol><li>通过加入50ml 1×PBS中至10克G10颗粒制备G10浆料。通过颠倒混合并允许G10到在4℃CO / N沉淀出来的磷酸盐缓冲盐水（PBS）的。 </li><li> G10柱分离当天，吸出PBS尘埃落定G10颗粒和加入50毫升新鲜PBS。颠倒混合。重复两次，在4℃加入50毫升新鲜PBS入驻G10颗粒和储存待用。 </li></ol></li><li> BMSC的培养和OB。 <ol><li>在37℃的CO 2和生长在10厘米组织培养板6％维持两者的BMSC或OB直至达到90％汇合。 </li><li> Trypsinize BMSC或OB细胞分裂和1:2到全新的10 9cm培养皿中。直到所需的白血病共培养的细胞生长至这些标准。 </li></ol></li></ol> （二）建立和维护共同的文化<ol><li>添加5-20×10 6个白血病细胞我Ñ​​10毫升肿瘤特异性培养基到80％-90％汇合的BMSC或OB板。 注意:我们的实验室保持共培养物，在37℃，在5％ 的 O 2，以更好地概括这已显示的范围从1％至7％16-18骨髓微环境。然而，维护共同培养这个氧分压不是建立三个白血病亚群的关键，是在实验室的自由裁量权。 </li><li>每个第 4天去除所有除1毫升的媒体（包括悬浮白血病细胞）和9毫升的新鲜白血病培养基更换。当从板卸下9mL经媒体，要小心，不要打扰BMSC或OB胶粘层。 <ol><li>通过在板的拐角处的倾斜板的侧面和抽吸媒体取出介质。此外，加入新鲜介质时，一定要加一滴反对侧壁板的角落明智的，以确保BMSC或OB胶粘层的最小的中断。 </li></ol></li><li>联合培养的第 12天之后，从冲洗BMSC或OB层白血病细胞通过菜上来吹打培养基和在菜轻轻地放下约5至10次，然后在15毫升锥形管收集。补种到新的80％-90％汇合的BMSC或OB板作为在步骤2.1中描述。 注:在步骤2.3将删除S和PB白血病细胞，而不破坏BMSC或OB单层描述的共培养的温和冲洗。这仅允许肿瘤细胞被转移到下一个共培养板。这12个天的周期可被重复多次，根据需要根据用户的需求。 </li></ol> 3.准备G10珠列 注:如果需要以下G10柱分离无菌下游分析或培养以下 ​​步骤应该使用无菌技术进行，G10列应是无菌生物罩设置。 <ol><li>预WARM细胞培养基至37℃在水浴（〜每塔30毫升）中。用10ml的一次性注射器，取出并丢弃活塞。玻璃棉加入注射器。 </li><li>使用镊子，拉开玻璃棉成薄松股。 轻轻填充玻璃棉多层添加到注射器直到注射器的2/3填充有玻璃棉。 注:玻璃棉是防止松动G10颗粒污染的白血病细胞采集的关键。确保玻璃棉被打包足以支持G10粒子，但不能太密集阻止通过色谱柱媒体流。 </li><li>附加1路活塞，以在关闭位置的注射器的尖端。 </li><li>钳注射器列环站得高足以使50毫升的锥形管（收集管）可以放在水龙头下面。将收集管注射器下列。 </li><li>用10毫升吸管添加，逐滴重悬于PBS到列在上面，G10颗粒玻璃棉。继续添加G10颗粒直至约2ml粒料G10颗粒形式的玻璃棉顶部（由刻度上注射器作为测量）。 </li><li>平衡与预热媒体的G10列。 <ol><li> 2毫升预热媒体的添加到柱。开放式旋塞阀慢一点，让媒体流出列逐滴。 </li><li>重复步骤3.6.1至总共为10ml预热培养基的已穿过柱跑去。 注意:如果 G10颗粒通过在收集管见于的流动，是1）增加更多G10颗粒保持约2ml粒料确保没有额外G10颗粒从柱逃逸或2）与未使用的和重复更换柱步骤3.4-3.6.1。 </li><li>从柱中预热介质排水沟后，关闭旋塞并丢弃收集管与流过。柱下添加新的收集管。列准备就绪，可以加载介质+细胞混合物。 注:列应立即使用，而不是让其干燥。 </li></ol></li></ol> 4.分离共培养在3亚群<ol><li>悬浮液（S）的肿瘤亚群的集合。 <ol><li>从共培养板吸管轻轻吸媒体再次申请同一介质冲洗板并收集含白血病细胞在一个15毫升的锥形管媒体。收集的白血病细胞是在S亚群。 </li></ol></li><li>相亮（PB）的肿瘤细胞亚群的集合。 <ol><li>加10毫升的新鲜介质倒流到共培养板上。吹打添加的媒体上下约5倍，除去附着的白血病细胞，但不够硬打跑贴壁BMSC / OB分量大力冲洗。 </li><li>用吸吸管和收集在一个15毫升的锥形管媒体。收集的细胞是在PB亚群。 </li></ol></li><li>昏暗的阶段（PD）的肿瘤细胞亚群的集合。 <ol><li>冲洗板用1毫升PBS到REM奥雅纳剩余的介质。 Trypsinize共培养板用3ml胰蛋白酶和地点到37℃培养箱中培养5分钟。 </li><li>删除盘开出孵化器，轻轻敲击板面打跑贴壁BMSC / OB。 </li><li>加入1 ml胎牛血清（FBS）和移液器上下3-5倍掰开大细胞聚集体。 </li><li>收集介质在15ml锥形管中的细胞。这些细胞是未纯化的PD亚群与BMSC / OB为好。 </li></ol></li><li>离心3分离的亚群，在400×g离心7分钟。吸弃上清然后分别在1毫升预热的媒体悬浮颗粒。细胞准备被装载到一个G10柱。 </li></ol> 5.装载共培养细胞到G10柱 注:确保活塞加入含介质细胞G10柱之前完全关闭。此外，每个亚群必须在一个单独的G10柱跑，这样不会introducE在下游分析人群之间的任何偏差。 <ol><li>用1000微升吸管，在预热的媒体加入1毫升各种细胞亚群的一个单独的G10柱滴。确保含有细胞的媒体仍然在顶部或G10颗粒内。允许细胞在G10沉淀在室温下孵育20分钟。 注:旋塞阀仍然是孵化的时间关闭。 </li></ol> 6.从G10柱收集白血病细胞<ol><li>加入1-3毫升预热的媒体到每个G10列。开放式旋塞阀，并允许媒体要慢慢退出列逐滴。 注:这是保持从列或含有BMSC / OB可以洗出列，污染隔离白血病细胞的G10颗粒缓慢的流速是至关重要的。 </li><li>继续以小的增量（1-2毫升）G10柱添加预热的介质，直到总共15至20毫升已通过柱运行，并且已收集。关闭活塞VALVE和帽收集管。 注:如果 G10颗粒沉淀在收集管的底部可见，轻轻地从管离开G10粒子颗粒不受干扰，并转移到新的管取出介质。 </li><li>离心收集介质，在400×g离心在RT 7分钟。除去上清液，并在适当的下游应用程序缓冲区悬浮细胞沉淀。 </li><li>细胞现在可以自由的BMSC或OB污染的白血病细胞的纯群体，并准备在用户自行决定要施加到下游应用。 注:通过细胞通过G10列时，白 ​​血病细胞存活率应保持不变。 </li></ol>

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The authors have no competing financial interests.

微小残留病变（MRD），其有助于疾病的复发仍然是治疗侵袭性耐火ALL的一个主要的临床挑战，以及，其它血液恶性肿瘤的宿主。骨髓微环境是复发的所有3,8最常见的部位。这样，模型建模骨髓微环境是重要的工具来测试化疗曝光期间与白血病肿瘤细胞的存活和MRD的维护假设。虽然小鼠模型定义了相关的药物疗效测试题的黄金标准，2D共培养仍然是关系到明天骨微环境的支持白血病细胞存活的假设检验和药物的战略具有成本效益的方法。许多团体已经表明白血病细胞的共培养BMSC或当与化疗剂9,10,12-14,19-21挑战OB提供生存优势。建模工作正常不成熟CD34 + HEMA在共培养的间充质干细胞（MSC）topoietic细胞表明造血细胞将与​​MSCs的贴壁单层以形成造血细胞22,23的三个不同的空间群相互作用。扩散和CD34 +细胞分化相对于共培养22内的位置进行。我们在这一观察扩大到测试相关的二维共培养于骨髓微基质细胞支持肿瘤细胞的抗性群体及其下游分析隔离的问题。 不像标准的2D共培养模型通常采样除去悬浮肿瘤的白血病细胞，我们的模型表明，共培养表示在共培养BMSC或转播形式的白血病细胞相对于BMSC 3亚群更加动态的相互作用或OB单层（ 图1）。形成肿瘤亚群被暂停（S）的肿瘤，WHICh是自由浮动的媒体;相亮（PB），该附着于BMSC或OB的表面上;和相昏暗已被埋在BMSC或OB单层（ 图1）下方（PD）。在这个模型中，我们发现，以实现在一个共培养模型一致的结果，因此，我们喂共培养物在4天的间隔和转移肿瘤到新的BMSC或OB单层每12天严格馈送和补种时间表是非常重要的。根据需要，这可能需要修改替代肿瘤类型。将迁移的BMSC或OB以下肿瘤细胞的数目，以形成对PD群体可以不同白血病细胞之间变化。这可以是该模型的限制，当PD肿瘤细胞的数量是低使得难以收集足够的细胞用于下游分析。在某些情况下，这个问题可以通过建立复制共培养，以允许三个单独的亚群的池来克服。 由于这种模式重新位于肿瘤细胞相互作用与BMSC或OB具有以除去间质细胞污染处理白血病细胞的特异性生物学一种有效的方法是重要的。为了实现这种分离从基质我们使用G10列的肿瘤细胞。正确安装和使用这些列是为下游分析纯肿瘤人群的隔离至关重要。所强调在图2B中，在大于99％的纯度的G10柱分离结果中的肿瘤细胞的恢复的正确执行。这允许不将导致从基质细胞污染结果的解释复杂化的白血病细胞的下游分析。要注意的是所有白血病细胞类型是否细胞系或原患者样品将略微在其穿过G10柱的能力而变化是很重要的。在大规模的实验使用前用户应确定他们必须输入到回收所需的量需要˚F白血病细胞的数量或他们的特定下游需求。此外，洗涤介质的量轧过G10柱后培养可增加到尝试和增加回收的细胞的数量。应注意，以确保该附加洗涤不引起其可以通过细​​胞计数来确定交洗涤或通过流式细胞流分析基质细胞污染。 在建立这种模型中，我们观察到，从PD人口回收白血病细胞暴露于细胞毒性化疗时增加存活率相比，在培养基中生长单独，以及对那些在同一共培养的S或PB种群恢复白血病细胞（图3 AB）。这是显著，因为它代表白血病细胞在体外该派生化疗显保护的群体。这提供了测试旨在针对最抗性肿瘤人口，其通过支持治疗策略的有用工具骨髓微环境。此外，由于这些白血病细胞是由BMSC或OB共培养，以便很好地保护它是适合于在体外结合治疗策略，即在媒体单独或标准共培养模型似乎或不是将彻底杀死肿瘤始终代表耐微环境支持白血病人群看到效果。 最后，我们认为，使用这种模式可以提供有价值的洞察白血病细胞是负责对化疗治疗的抗性的BMSC / OB之间的相互作用，并且，导致MRD和随后复发。该模型提供了一种体外平台设计实验，这将更好地了解下游临床前模型。虽然我们展示使用这种模式来测试骨髓基质细胞和所有派生的白血病细胞之间的相互作用，我们希望，这种模式的未来发展方向和应用将是我们eful在各种恶性肿瘤，其中骨髓微环境的化学治疗干预过程中提供避难所的肿瘤的位点。

The overall goal of the method described is to provide an efficient, cost-effective in vitro approach that supports investigation of the mechanisms that underlie bone marrow supported survival of leukemic cells during chemotherapy exposure. It is well documented that surviving residual tumor cells that persist after treatment contribute to relapse of disease that is often more aggressive than that at diagnosis and is often less effectively treated1-8. Models that include leukemic cells in isolation, such as those limited to culture of cells in media alone, for testing of therapeutic approaches do not factor in these critical signals, or the heterogeneity of disease that occurs in response to availability of niche derived cues in which tumor cell subpopulations with very specific interactions with niche cells derive enhanced protection. Standard 2D co-culture models that co-culture bone marrow derived stromal cells and leukemic cells have somewhat addressed the contribution of the marrow niche and have shown that interaction with bone marrow microenvironment cells increases their resistance to chemotherapy and alters their growth characteristics9-14. These models however often fail to recapitulate long term survival of tumor cells and do not accurately inform the outcomes associated with the most resistant leukemic cell populations that contribute to MRD. In vivo models remain critical and define the "gold standard" for investigation of innovative therapies prior to clinical trials but they are often challenged by the time and cost required to test hypotheses related to resistant tumors and relapse of disease. As such, development of more informative 2D models would be of benefit for pilot investigations to better inform the design of subsequent murine based pre-clinical design. 
The 2D in vitro model presented in this report lacks the complexity of the true in vivo microenvironment, but provides a cost effective and reproducible means to interrogate tumor interactions with the microenvironment that lends itself specifically to enrichment of the chemoresistant subpopulation of tumor cells. This distinction is valuable as evaluation of the entire population of tumor cells may mask the phenotype of a minor group of therapy resistant tumor cells that comprise the most important target. An additional advantage is the scalability of the model to fit the analysis of interest. Bulk cultures can be established for those analyses requiring significant recovery of tumor cells, while small scale co-cultures in multi-well plates can be utilized for PCR based analysis or microscopy based evaluations. 
Based on this need we developed an in vitro model to address the heterogeneity of disease that is characteristic of B-lineage acute lymphoblastic leukemia (ALL). We demonstrate that ALL cells, which share many characteristics in common with their healthy counterparts, localize to distinct compartments of BMSC or OB co-culture. Three populations of tumor cells are generated that have distinct phenotypes that are valuable for investigation of therapeutic response. Specifically, we demonstrate that (ALL) cells recovered from the "phase dim" (PD) population of co-culture are consistently refractory to therapy with survival that approximates tumor cells that have not been exposed to cytotoxic agents. These ALL cells, from either established cell lines or primary patient samples, migrate beneath adherent stromal cells or osteoblast layers but can be captured following trypsinization of cultures and separation of cell types by utilization of gel type 10 cross-linked dextran (G10) particle columns15. 
Here we present a setup of a 2D co-culture that can be employed to model interactions between bone marrow microenvironment stromal cells (BMSC/OB) and leukemic cells. Of particular importance is the observation that leukemic cells form three spatial subpopulations relative to the stromal cell monolayer and that the PD population represents a chemotherapy resistant tumor population due to its interaction with the BMSC or OB. Furthermore, we demonstrate how to effectively isolate the leukemic cell populations by G10 columns. Of note, we have found that isolation of these subpopulations allows for downstream analysis of the most resistant PD population to determine potential modes of resistance that are conferred to these cells due to their interaction with the bone marrow microenvironment stromal cells or osteoblasts. Techniques that we have utilized downstream of this co-culture and isolation model include flow cytometric evaluation, proteomic analysis and targeted protein expression evaluation as well as more recently developed laser ablation electrospray ionization (LAESI) and Seahorse analysis to evaluate metabolic profiles. Through use of this model in combination with the techniques above we have found that the PD population of leukemic cells has a chemotherapy resistant phenotype that is unique when compared to leukemic cells cultured in media alone or those recovered from the other subpopulations in the same co-culture. As such, this model lends itself to more rigorous evaluation to test strategies targeting the most chemotherapy resistant leukemic cells which derive their resistant phenotype through interaction with the bone marrow microenvironment.

<table border="0" cellpadding="0" cellspacing="0" width="734">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:224px;">G10 sephadex beads</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">G10120</td>
			<td style="width:243px;">Referred to in manuscript as gel type 10 cross-linked dextran particles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">10 ml sterile syringe</td>
			<td style="width:149px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Glass wool</td>
			<td style="width:149px;">Pyrex</td>
			<td style="width:119px;">3950</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">1-way stopcocks</td>
			<td style="width:149px;">World Precision Instruments, Inc.</td>
			<td style="width:119px;">14054-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">50 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021039</td>
			<td>Used as collection tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">15 ml conical centrifuge tubes</td>
			<td style="width:149px;">World Wide Medical Products</td>
			<td style="width:119px;">41021037</td>
			<td>Used for cell collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">Fetal Bovine Serum</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:119px;">F6178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:224px;">0.05% Trypsin&#160;</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">100X20mm Cell Culture Dishes</td>
			<td style="width:149px;">Greiner Bio-One</td>
			<td style="width:119px;">664160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Culture media</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Osteoblast culture media&#160;</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-27001</td>
			<td>For human osteoblast media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">RPMI 1640 media</td>
			<td style="width:149px;">Mediatech, Inc.</td>
			<td style="width:119px;">15-040</td>
			<td>For tumor media prepation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Name</td>
			<td style="width:149px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Cell lines</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Adherent Cells</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">Human Osteoblasts</td>
			<td style="width:149px;">PromoCell</td>
			<td style="width:119px;">C-12720</td>
			<td style="width:243px;">Human osteoblast were cultured according to the supplier&#8217;s recommendations.&#160;</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:224px;">Human Bone Morrow Stromal Cells</td>
			<td style="width:149px;">WVU Biospecimen Core</td>
			<td style="width:119px;"></td>
			<td style="width:243px;">De-identified primary human leukemia and bone marrow stromal cells (BMSC) were provided by the Mary Babb Randolph Cancer Center (MBRCC) Biospecimen Processing Core and the West Virginia University Department of Pathology Tissue Bank. BMSC cultures were established as previously described (*)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Leukemic Cells:</td>
			<td style="width:149px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">REH</td>
			<td style="width:149px;">ATCC</td>
			<td style="width:119px;">ATCC-CRL-8286</td>
			<td style="width:243px;">REH cells were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">SD-1</td>
			<td style="width:149px;">DSMZ</td>
			<td style="width:119px;">ACC 366</td>
			<td style="width:243px;">SD-1 were cultured according to the supplier&#8217;s recommendations and recommended media.&#160;</td>
		</tr>
		<tr height="48">
			<td colspan="4" height="48" style="height:48px;width:735px;">(*) Gibson LF, Fortney J, Landreth KS, Piktel D, Ericson SG, Lynch JP. Disruption of bone marrow stromal cell function by etoposide. Biol Blood Marrow Transplant J Am Soc Blood Marrow Transplant. 1997 Aug;3(3):122&#8211;32.&#160;</td>
		</tr>
	</tbody>
</table>

modeling chemotherapy resistant leukemia in vitro

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

此共培养模型的成功建立和培养将导致建立相对于粘附BMSC或OB单层白血病细胞的3亚群。 图1示出了接种到BMSC单层ALL细胞如何最初显示为仅悬浮单个群体白血病细胞。超过4天的白血病细胞与BMSC相互作用形成白血病细胞的3空间亚群的过程（悬浮（S）相亮（PB）和相暗淡（PD））。而肿瘤细胞的3亚群可以通常后的BMSC或OB共培养24小时中可以看出，我们的共同培养​​细胞4天，以允许白血病细胞和BMSC / OB小区之间的完整动态和交互发生任何操纵或实验之前发生（图1）。另外请注意，我们保持在5％的氧张力的共培养来概括骨髓生理学，其具有蜂N报从1％-7％的范围16-18。 绝大多数下游分析的需要来自BMSC或OB白血病细胞的分离。为了实现这一目标，我们使用G10列收获白血病细胞（图2A）的纯人口。以下的BMSC和PD REH细胞胰蛋白酶混合群由通过流式细胞术两个不同正向/侧群体（图2B，上图）看出。以下G10分离仅REH ALL细胞的纯群体被回收，这是由前向/侧向散射流式细胞术（ 图2B，下图）确认。 此共培养模型和来自3亚群分离白血病细胞时在共培养BMSC或OB允许白血病细胞表型的询问与相对于它的空间位置相对于附着的BMSC或OB的能力的使用单层。特别感兴趣的是，从一个BMSC或OB共培养的PD人口回收ALL细胞暴露于细胞毒性化疗有很少或没有细胞死亡（图3A，B）。 <img alt="图1" src="/files/ftp_upload/53645/53645fig1.jpg" /> 图1. ALL细胞在共培养BMSC或转播形式三个空间群。我们的实验室使用的体外共培养体系建模与骨髓微环境衍生的基质细胞（BMSC或OB）白血病细胞的相互作用。建立共培养，白血病细胞（红色）接种到BMSC或OB（蓝），其被表示为"时间0"的80％-90％汇合单层。共培养物被保持在37℃，5％的氧来近似骨髓微环境的条件。白血病细胞会开始，早在24小时形成3亚群，但允许完全的互动，FORM我们允许联合培养4天利用白血病细胞进行实验前建立。由天4（右图），白血病细胞三个亚群将形成在相对于贴壁单层。示意图（右上）和相差显微镜（右下）示出悬浮（S）白血病细胞中的媒体自由浮动;相亮（PB），它附着在BMSC或OB单层的表面白血病细胞;和相暗淡（PD）已迁移的BMSC或OB单层之下的白血病细胞。比例尺代表10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53645/53645fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53645/53645fig2.jpg" /> 图2. G10柱的使用允许对从BMSC / OB ALL细胞的分离。采用G10山坳的过程中（A）示范UMN到ALL细胞从BMSC / OB共培养分离，以实现肿瘤细胞用于下游分析纯群体。从左至右，ALL细胞和BMSC / OB细胞的混合物被添加到G10柱的顶部; （中心）细胞混合物将在G10浆料沉降并应在RT孵育20分钟（注:旋塞处于关闭位置在整个前两步）; （右）白血病细胞通过打开旋塞，用预热介质漂洗柱回收。（B）的顶板G10分离有含BMSC（蓝色门）和REH ALL细胞的细胞的混合群（红色栅极之前显示）通过评估向前（FSC）和侧向散射（SSC）的分析。底板，下面G10仅分离REH的纯群体中所有细胞（红色门）留在不到1％的基质细胞污染（蓝色大门）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53645/53645fig2large.jpg" target="_blank">请点击此处查看THI一个更大的版本</a><html>的身影。 <img alt="图3" src="/files/ftp_upload/53645/53645fig3.jpg" /> 图3的PD白血病细胞增加抵抗化疗曝光。从BMSC共培养（A）的 PD人口回收不显示以下4天暴露于阿糖胞苷降低存活率SD-1白血病细胞[1μM] ，MTX [50μM]或VCR [25μM]，类似于未处理的对照组（请注意，阿糖胞苷第二剂量的增加在48小时以考虑由于稳定性的任何药物损失）。单从媒体的白血病细胞，经台盼蓝exclusion.SD-1白血病细胞与细胞OB共培养确定了活力（B）显示了类似的趋势S和PB数量已经减少了显著生存能力。结果表示为平均值±SEM表示。 （*）表示P &lt;，相对于未处理的对照组0.05非配对t检验。5fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

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.

建模化疗耐药白血病<em&gt;体外</em

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

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Medicine

Modeling Chemotherapy Resistant Leukemia In Vitro

9.0K 次观看.  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. 

视频: Modeling Chemotherapy Resistant Leukemia In Vitro

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

科研

医学

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

在小鼠的脑出血模型:注射自体血液或细菌胶原酶

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

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.

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

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

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

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

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

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

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

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

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

造型中风小鼠:在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

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

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

在C2C12肌细胞造型强直性肌营养不良1

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

建模化疗耐药白血病

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