Guiding Ca2+ Channel-Based Cancer Treatment Using Mn2+-Enhanced MRI

描述(申请人提供)：某些钙离子(Ca~(2+))通道，如电压门控(VGCC)和瞬时受体电位(Trp)Ca~(2+)通道，在某些癌症中高度表达，提示它们可能成为治疗靶点。事实上，专门针对这些通道亚类的药物已经存在，目前有两种处于I期临床试验，但很难揭示在个别肿瘤中针对哪一种特定通道。我们的LNG长期目标是开发一种分析性的、非侵入性的体内肿瘤钙通道活性分析方法，最终可以用于临床，作为一种个性化的钙通道靶向治疗方法。为了实现这一目标，我们将测量肿瘤对锰离子(Mn2+)的摄取，Mn2+是一种MRI造影剂，主要通过钙通道进入细胞。我们的初步研究表明，在锰增强磁共振成像(MEMRI)检查中，不同类型的人类癌细胞以钙通道依赖的方式摄取外源性Mn2+，导致MRI纵向松弛速率增加，即R1值。此外，具体和系统地管理 VGCC通道的激动剂和拮抗剂可改变体内非肿瘤组织中Mn2+的积累。这一建议的总体假设是，在存在钙通道特异性激动剂或拮抗剂的情况下，使用锰增强磁共振成像(MEMRI)测量的肿瘤对Mn2+的摄取程度是肿瘤中特定钙通道活性的有用生物标志物，因此是预测基于钙通道的治疗的潜在肿瘤治疗反应的合理方法。为了充分检验这一假设，我们将解决两个具体目标。在目标1中，我们将定义MEMRI的敏感性和特异性的钙通道调节作为一种方法来确定在体外不同类型的人肿瘤细胞中表达的主要钙通道。我们将利用蛋白质印迹技术鉴定不同肿瘤细胞系中普遍存在的钙离子通道，用激动剂和拮抗剂处理不同的细胞，然后用MEMRI检测每种细胞系对Mn2+的摄取。预测是，特定的激动剂和/或拮抗剂将改变细胞的R1值，从而识别特定肿瘤细胞表达的关键通道(S)。在目标2中，我们将确定MEMRI和特定的钙通道修饰剂的组合是否可以在体内识别不同人类肿瘤移植瘤中的主要钙通道类型。在体外研究的基础上，将选定的激动剂和拮抗剂注射到带人皮下移植瘤的SCID小鼠体内，并在注射MnCl2后测定肿瘤的平均R1值。预测是注射特定的 激动剂和/或拮抗剂将改变肿瘤R1值，从而识别特定异种移植瘤表达的关键钙通道(S)。我们预见，结合使用MEMRI和调节特定的钙通道活性，最终将有助于无创性地确定靶向钙通道是否在患者的肿瘤中表达，从而指导后续的基于钙通道的治疗。

DESCRIPTION (provided by applicant): Certain calcium ion (Ca2+) channels, e.g., voltage-gated (VGCC) and transient receptor potential (TRP) Ca2+ channels, are highly expressed in some cancers, suggesting that they could be therapeutic targets. Indeed, drugs specifically targeting subclasses of these channels already exist and two are currently in Phase I clinical trials, but it is difficult to unravel which specific channel to target in individual tumors. Our lng-term goal is to develop an analytical, non-invasive assay of tumor calcium channel activity in vivo that could eventually be used clinically as a means to individualize Ca2+ channel-targeted therapies. To achieve this goal, we will measure tumor uptake of manganese ion (Mn2+), an MRI contrast agent that largely enters cells via Ca2+ channels. Our preliminary studies show that on manganese-enhanced MRI (MEMRI) examination, different types of human cancer cells take up exogenous Mn2+ in a calcium channel dependent fashion, resulting in an increase in MRI longitudinal relaxation rates, i.e., R1 values. In addition, specific and systemically administered agonists and antagonists of VGCC channels modify non-tumor tissue Mn2+ accumulation in vivo. The overall hypothesis of this proposal is that the extent of tumor uptake of Mn2+ in the presence of Ca2+ channel-specific agonists or antagonists, measured using manganese enhanced MRI (MEMRI), is a useful biomarker of specific calcium channel activity in the tumor and thus a rational approach for predicting potential tumor therapeutic response to Ca2+ channel-based therapies. To fully test this hypothesis, we will address two specific aims. In Aim 1, we will define the sensitivity of MEMRI and specific Ca2+ channel modulation as a method to identify the predominant Ca2+ channels expressed in different types of human tumor cells in vitro. We will characterize the prevalent Ca2+ channels in various human tumor cell lines using Western blots, treat the different cells with agonists and antagonists to specific Ca2+ channels, and then measure Mn2+ uptake in each cell line using MEMRI. The prediction is that specific agonists and/or antagonists will change the cellular R1 values, thereby identifying the key channel(s) expressed by the specific tumor cells. In Aim 2, we will determine if the combination of MEMRI and specific Ca2+ channel modifiers can identify dominant Ca2+ channel types in different human tumor xenografts in vivo. Based on the in vitro studies, selected agonists and antagonists will be injected into SCID mice bearing subcutaneous human xenografts, and average R1 values will be determined in the tumors following MnCl2 injection. The prediction is that injection of specific agonists and/or antagonists will change the tumor R1 values, thereby identifying the key Ca2+ channel(s) expressed by the specific xenografts. We envision that the combined use of MEMRI and modulation of specific Ca2+ channel activity will eventually be useful to noninvasively determine whether a targeted Ca2+ channel is expressed in a patient's tumor, thereby guiding subsequent Ca2+ channel-based treatment.