Development and validation of 3D in vitro dormant myeloma cell models to reduce and replace animal studies

科学意义：多发性骨髓瘤(MM)是一种B细胞癌，由骨髓中恶性浆细胞的生长引起。尽管针对骨髓瘤细胞(MC)固有通路的治疗方法已经开发出来，但它仍然是无法治愈的。疾病复发被认为源于潜伏的MC，它们位于专门的骨内壁龛中，抵抗化疗并重新填充肿瘤[1，2]。我们之前曾通过多光子成像技术在体内跟踪单个MC，当它们定植于骨内壁龛，进入休眠状态，然后被激活形成克隆[1]。MC休眠已被证明是一种可逆状态，通过与成骨细胞(在骨内壁龛)接触而开启，通过破骨细胞性骨吸收而关闭。我们已经确定，休眠的MC对以分裂细胞为靶点的化疗马法兰具有耐药性。骨内壁龛在控制MC休眠中起关键作用，这一证明突出了靶向细胞-外在机制以克服细胞内在耐药性和防止疾病复发的潜力。由于BM的细胞复杂性、血管系统和三维(3D)骨结构，在体外环境下重述BM微环境是非常具有挑战性的。然而，由于我们知道成骨细胞在MC休眠中起着关键作用，并且它们位于离细胞外基质(ECM)很近的骨表面，这将为在体外模拟这一方面提供一个平台。因此，这个以3Rs为中心的博士研究生的目标是建立几个MC休眠的体外3D模型，然后将它们与新的数据和我们已建立的临床相关的MC休眠和耐药小鼠模型的现有数据进行比较来验证它们。研究计划和目标：我们的目标是建立和验证几个MC休眠的3D体外模型，它概括了体内MC-成骨细胞在BM微环境中的相互作用，即诱导MC休眠。我们将通过评估和表征这些新建立的3D体外模型中的休眠MC来实现这一点，这样它们就可以作为小鼠骨髓瘤模型的有效替代品来评估新的和新的抗骨髓瘤疗法及其作用机制。我们假设，在体外，MC休眠的3D模型将模拟目前已建立的体内骨髓瘤临床前模型的BM中骨内壁龛中观察到的休眠MC。

Scientific importance: Multiple myeloma (MM) is a B cell cancer caused by the growth of malignant plasma cells in the bone marrow (BM). It remains incurable, despite the development of therapies that target myeloma cell (MC)-intrinsic pathways. Disease relapse is thought to originate from dormant MCs, localised in specialised endosteal niches, which resist chemotherapy and repopulate the tumour [1, 2]. We have previously tracked individual MC's in vivo by multi-photon imaging as they colonise the endosteal niche, enter a dormant state and subsequently become activated to form colonies [1]. MC dormancy has been demonstrated to be a reversible state that is switched 'on' by engagement with osteoblasts (in the endosteal niche) and 'off' by osteoclastic bone resorption. We have determined dormant MCs are resistant to the chemotherapy melphalan that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling MC dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse. To recapitulate the BM microenvironment in an in vitro setting is highly challenging because of the cellular complexity, vasculature and 3-dimensional (3D) bone structure. However, since we know osteoblasts play a key role in MC dormancy and they reside on bone surfaces in close proximity to the extracellular matrix (ECM), this will provide a platform to mimic this in vivo aspect in vitro. Therefore, the aim of this 3Rs-focussed PhD studentship is to develop several in vitro 3D models of MC dormancy and then validate them by comparing them to new data and our existing data from established clinically relevant murine models of MC dormancy and drug resistance.Research plan and objectives: We aim to develop and validate several 3D in vitro models of MC dormancy which recapitulates the interaction of MC-osteoblastic cells in the BM microenvironment in vivo i.e. induces MC dormancy. We will do this by assessing and characterising dormant MCs in these newly established 3D in vitro models so they can be used reliability as effective alternatives to murine models of myeloma to assess new and novel anti-myeloma therapies and their mechanisms of action. We hypothesise that in vitro 3D models of MC dormancy will mimic the dormant MC's observed in the endosteal niche in the BM of currently established in vivo pre-clinical models of myeloma.