Metabolic Therapy of GBM guided by MRS of hyperpolarized 13C-pyruvate

描述(申请人提供)：除了异常高的增殖率，癌细胞普遍表现出异常代谢，其特征是过度利用糖酵解(GLY)而不是能量效率更高的线粒体氧化磷酸化(OXPHOS)。这种新陈代谢的改变最初是由Warburg在80多年前描述的，最近被称为新陈代谢重编程，现在被视为癌症的标志[1]，似乎与增殖状态密切相关。这些对肿瘤代谢的观察的新的临床含义是，他们提出了一个潜在的抗癌策略在于将这种代谢恢复到正常水平，即强制OXPHOS。虽然这本身不会导致细胞死亡，但它应该会以最小的毒性稳定生长。然而，尽管存在许多可以针对活性药物的靶点，我们认为将其推向临床的主要障碍是无法测量完整生物体中的癌症新陈代谢。超极化~(13)C磁共振波谱(MRS)的最新发展首次实现了对体内代谢的实时研究，信号比传统的~(13)C方法增加了10,000多倍。使用13C标记的丙酮酸(PYR)作为底物，使我们能够定量跟踪丙酮酸在体内的命运，丙酮酸在葡萄糖转化为乳酸(Lac；反映GLY)或乙酰辅酶A(在此过程中生成碳酸氢根[Bic]；反映OXPHOS)的代谢途径中占据关键节点。因此，使用这项技术，可以测量注射超极化[1-13C]-PYR后乳酸盐和碳酸氢盐的13C标记，从而允许计算Lac/Bic比率，我们建议将其作为治疗反应的标记进行研究。贝伐单抗(Bev；Avestin®)是一种能与血管内皮生长因子(VEGF)结合，从而抑制血管生成的单抗。它被广泛应用于许多肿瘤类型，包括恶性程度最高的原发脑肿瘤--多形性胶质母细胞瘤。虽然它可以有戏剧性的初始效果，但其持续时间往往相对较短，并与难治性肿瘤进展有关。虽然血流和新陈代谢之间的密切关系已经得到了很好的证明，但关于BEV对肿瘤代谢的影响的研究很少。我们提出BEV可以在组织水平上剧烈地干扰肿瘤的代谢，比如强迫OXPHOS，并且这种瞬时效应与肿瘤的稳定相关[2]。通过改进Bic检测以实现定量，我们观察到在抗VEGF治疗后移植的胶质母细胞瘤组织中Lac/Bic比率显著降低，这种效果可以在给药后三个小时内看到，这为这一反直觉的假设提供了初步支持。考虑到尽管对其抗血管生成作用进行了广泛的研究，但既没有阐明BEV效应的可靠早期临床标志物，也没有阐明其耐药性的发展，我们的结果为改善这种非常有价值的肿瘤治疗的影响提供了一个新的和令人兴奋的方向。这个项目代表了一个一直处于这项技术前沿的团队与P.I.之间的合作，P.I.是一名熟悉脑癌临床试验和实验室模型的临床科学家。多形性胶质母细胞瘤(GBM)是最常见的原发脑肿瘤，因其高GLY发生率和治疗难治性，是评价代谢治疗的良好典型肿瘤。然而，重要的是要注意到，得出的发现应该适用于所有癌症。我们的实验设计如下：到本资助期结束时，我们将改进这项技术，以便进行高分辨率成像以及评估谷氨酸等分子在OXPHOS途径(SA1)中的“更深层次”，确定BEV效应在移植性胶质母细胞瘤(SA2)中的时程和剂量关系，评估抗VEGF治疗对“代谢共生”(SA3)的影响，并评估这些发现是否推断为更临床相关的模型，在该模型中，脑瘤在子宫内暴露于神经致癌物质后“自发”发展(SA4)。因此，这些实验应该使我们更接近我们的最终目标，即将BEV的治疗影响与最佳的“乳酸盐/碳酸氢盐比例”联系起来，该比例不仅可以用作临床疗效的衡量标准，也可以作为治疗目标。最近授予斯坦福大学购买临床级偏振器的资金，我们也应该加快将研究结果从工作台转移到床边的速度。

DESCRIPTION (provided by applicant): In addition to their abnormally high proliferative rates, cancer cells universally demonstrate an abnormal metabolism that is characterized by an overutilization of glycolysis (GLY) relative to the more energy efficient mitochondrial oxidative phosphorylation (OXPHOS). Originally described over 80 years ago by Warburg, this altered metabolism, more recently termed metabolic reprogramming, is now viewed as a hallmark of cancer [1], and appears intimately tied to the proliferative state. The novel clinical implicationof these observations on tumor metabolism is that they suggest that a potential anticancer strategy lies in reverting this metabolism towards normal levels, i.e., forcing OXPHOS. Although this would not result in cell kill per se, it should result in stabilization of growth with minimal toxiity. However, although a number of targets exist for which active drugs can be directed, we contend that the major obstacle towards taking this to the clinic is the inability to measure cancer metabolism in the intact organism. The recent development of hyperpolarized 13C magnetic resonance spectroscopy (MRS) enables for the first time the real-time investigation of in vivo metabolism with more than a 10,000-fold signal increase over conventional 13C methods. Using 13C-labeled pyruvate (Pyr) as a substrate allows us to quantitatively follow the in vivo fate of pyruvate, which occupies a key nodal point in the metabolic pathway in which glucose is either converted to lactate (Lac; reflecting GLY) or acetyl CoA (generating bicarbonate [Bic] in the process; reflecting OXPHOS). With this technology, it is therefore possible to measure the 13C labeling of lactate and bicarbonate following the bolus injection of hyperpolarized [1-13C]-Pyr, thus permitting a Lac/Bic ratio to be calculated, which we propose to study as a marker of therapeutic response. Bevacizumab (BEV; Avastin®) is a monoclonal antibody (mab) that binds vascular endothelial growth factor (VEGF), thus inhibiting angiogenesis. It is widely used in a number of tumor types, including glioblastoma multiforme, the most malignant of the primary brain tumors. Although it can have dramatic initial effects, its duration tends to be relatively short-lived and associated with the development of refractory tumor progression. Although an intimate relationship between flow and metabolism is well documented, there has been little study of the impact of BEV on tumor metabolism. We have proposed that BEV acutely disrupts tumor metabolism at the tissue level, such as to force OXPHOS, and that this transient effect correlates with tumor stabilization [2]. By improving Bic detection to enable quantitation, we have observed a marked decrease in Lac/Bic ratio in transplanted glioblastoma tissue after anti-VEGF therapy, an effect that can be seen within three hours of administration, providing initial support for this counterintuitive hypothesis. Considering that despite extensive study into its antiangiogenic effects, neither a reliable early clinical marker of BEV effect nor o resistance development has been elucidated, our results offer a new and exciting direction for improving the impact of this very valuable oncotherapeutic. This project represents a collaboration between a group that has been on the cutting edge of this technology with the P.I., a clinician scientist who is familiar with clinical trials as well as laboratory models of brain cancer. Glioblastoma multiforme (GBM), the most commonly occurring primary brain tumor, is an excellent prototypical cancer with which to assess metabolic therapies because of its high rate of GLY and treatment refractoriness. However, it is important to note that the derived findings should apply to all cancer. Our experiments are designed in such a way that by the end of this funding period, we will have refined this technology so as to perform imaging with high resolution as well as to assess molecules such as glutamate which are "deeper" into the OXPHOS pathway (SA1), determine the time course and dose relationships of the BEV effect in transplanted glioblastoma (SA2), assess the impact of anti-VEGF therapy on "metabolic symbiosis" (SA3) and assess whether these findings extrapolate to a more clinically relevant model in which brain tumors develop "spontaneously" after exposure to a neurocarcinogen in utero (SA4). These experiments therefore should move us close to our ultimate goal of linking BEV's treatment impact with an optimal "lactate/bicarbonate ratio" that can be used clinically not only as a measure of therapeutic efficacy, but also as a therapeutic goal. The recent awarding of funds to purchase a clinical grade polarizer at Stanford should also us to accelerate the translation of findings from bench to bedside.