建模化疗耐药白血病<em&gt;体外</em

摘要

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.

摘要

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.

引言

The overall goal of the method described is to provide an efficient, cost-effective in vitro approach that supports investigation of the mechanisms that underlie bone marrow supported survival of leukemic cells during chemotherapy exposure. It is well documented that surviving residual tumor cells that persist after treatment contribute to relapse of disease that is often more aggressive than that at diagnosis and is often less effectively treated^1-8. Models that include leukemic cells in isolation, such as those limited to culture of cells in media alone, for testing of therapeutic approaches do not factor in these critical signals, or the heterogeneity of disease that occurs in response to availability of niche derived cues in which tumor cell subpopulations with very specific interactions with niche cells derive enhanced protection. Standard 2D co-culture models that co-culture bone marrow derived stromal cells and leukemic cells have somewhat addressed the contribution of the marrow niche and have shown that interaction with bone marrow microenvironment cells increases their resistance to chemotherapy and alters their growth characteristics^9-14. These models however often fail to recapitulate long term survival of tumor cells and do not accurately inform the outcomes associated with the most resistant leukemic cell populations that contribute to MRD. In vivo models remain critical and define the "gold standard" for investigation of innovative therapies prior to clinical trials but they are often challenged by the time and cost required to test hypotheses related to resistant tumors and relapse of disease. As such, development of more informative 2D models would be of benefit for pilot investigations to better inform the design of subsequent murine based pre-clinical design.

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

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

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

研究方案

1.先进的准备

1. 准备葡聚糖G10颗粒。
   1. 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。
   2. 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。
2. BMSC的培养和OB。
   1. 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。
   2. 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。

（二）建立和维护共同的文化

1. 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。
2. 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。
   1. 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。
3. 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。

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。

1. 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。
2. 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。
3. 附加1路活塞，以在关闭位置的注射器的尖端。
4. 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。
5. 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）。
6. 平衡与预热媒体的G10列。
   1. 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。
   2. 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。
   3. 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。

4.分离共培养在3亚群

1. 悬浮液（S）的肿瘤亚群的集合。
   1. 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。
2. 相亮（PB）的肿瘤细胞亚群的集合。
   1. 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。
   2. 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。
3. 昏暗的阶段（PD）的肿瘤细胞亚群的集合。
   1. 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。
   2. 删除盘开出孵化器，轻轻敲击板面打跑贴壁BMSC / OB。
   3. 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。
   4. 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。
4. 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。

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。

1. 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。

6.从G10柱收集白血病细胞

1. 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。
2. 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。
3. 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。
4. 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。

结果

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?...

讨论

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...

材料

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