Investigating and Targeting TAMs in the Glioma Microenvironment

描述（由申请人提供）：高级别胶质瘤，也称为多形性胶质母细胞瘤（GBM），是最常见和最具侵袭性的成人原发性脑肿瘤。GBM患者对目前的治疗（包括手术、放疗和替莫唑胺化疗）的反应最小。 大多数患者在诊断后14个月内死亡，强调迫切需要新的治疗方法来对抗这种疾病。基于不同的基因表达特征，GBM可以分为几种分子亚型，包括前神经型、神经型、间充质型和经典型。 在这些亚型中，前神经GBM在年轻患者中特别具有侵袭性，并且旨在直接靶向该亚型中的肿瘤细胞的大多数治疗方法都失败了。 与高度突变的肿瘤细胞相反，在肿瘤微环境（TME）中支持肿瘤发生的非癌性基质细胞代表遗传稳定的治疗靶点。 这意味着靶向TME的治疗不太可能由于基质细胞的遗传变化而导致获得性耐药性的发展。肿瘤相关巨噬细胞（TAM）是TME中的一种重要细胞类型，与许多癌症（包括神经胶质瘤）的肿瘤分级增加和患者预后不良相关，表明具有重要的癌症促进功能。为了检查TAM对胶质瘤进展的贡献，我们使用了前神经GBM的基因工程小鼠模型。我们发现TAM在小鼠模型中随着肿瘤级别的升高而逐渐增加，这与在人类胶质瘤发展过程中观察到的增加平行。 为了研究TAM积累的功能意义，我们设计了前神经GBM模型中的临床前试验，以治疗性靶向集落刺激因子-1受体（CSF-1R），巨噬细胞依赖于该受体进行存活和分化。 CSF-1R抑制作为单一疗法显著增加了这些小鼠的存活率，并在短短7天后使已建立的肿瘤消退。正如我们预期的那样，正常大脑中的巨噬细胞被耗尽，但在治疗小鼠的胶质瘤中却没有。相反，胶质瘤分泌的因子促进TAM生存的CSF-1R抑制剂的存在。有趣的是，这些存活的TAM的基因表达分析揭示了交替激活/M2极化巨噬细胞标志物的显著减少，并且一致地，功能分析揭示了抗肿瘤发生表型。 因此，TAM消耗对于有效的巨噬细胞靶向治疗不是严格必要的。相反，我们提出，胶质瘤TME中巨噬细胞存活因子的存在不仅使TAM能够在暴露于CSF-1R抑制剂的情况下存活，而且通过这个过程进行“再教育”，从而产生显著的抗肿瘤反应。 我们的初步数据确定TAM作为前神经胶质瘤的一个有前途的治疗靶点，并建立了GBM中CSF-1R抑制的强大翻译潜力。在这项提议中，我们将扩展这些结果，以阐明TAM介导胶质瘤细胞表型的机制，并确定CSF-1R抑制剂如何干扰这种相互通信，以延迟和阻断胶质瘤进展。我们的目标是阐明TAM最初是如何被胶质瘤细胞训练的，然后在胶质瘤微环境中通过CSF-1R抑制再训练。 接下来，我们将研究哪些神经胶质瘤细胞信号通路被TAM增强，并确定对CSF-1R体内抑制功效至关重要的下游效应物。 最后，我们将确定胶质瘤是否对CSF-1R抑制产生抗性，并确定潜在的机制。 为了实现这些目标，我们将采用多种不同的方法，包括小鼠胶质瘤模型和广泛的共培养试验组，以研究胶质瘤TME中胶质瘤细胞、TAM和其他细胞之间的通讯。我们将结合联合收割机分析已知的信号通路与识别新的目标，使用蛋白质组学和表达谱的方法。除了剖析TAM促进胶质瘤进展的潜在生物学机制外，拟议的实验还将导致开发其特异性抑制的治疗策略，如果成功，最终可以在临床上进行测试。总的来说，这些研究对CSF-1R抑制剂的未来临床考虑以及针对癌症中TME的其他疗法具有重要意义。

DESCRIPTION (provided by applicant): High-grade gliomas, also known as glioblastoma multiforme (GBM), are the most common and aggressive adult primary brain tumors. GBM patients have a minimal response to current therapies, including surgery, radiation and temozolomide chemotherapy. Most patients die within 14 months following diagnosis, emphasizing the urgent need for new therapies to combat this disease. GBM can be grouped into several molecular subtypes, including proneural, neural, mesenchymal and classical, based on distinct gene expression signatures. Of these subtypes, proneural GBM is particularly aggressive in younger patients, and most therapeutic approaches aimed at directly targeting tumor cells in this subtype have failed. In contrast to highly mutable tumor cells, non-cancerous stromal cells that support tumorigenesis in the tumor microenvironment (TME) represent genetically stable therapeutic targets. This means therapies targeted against the TME are less likely to result in the development of acquired resistance as a result of genetic changes in the stromal cells. Tumor-associated macrophages (TAMs) are an important cell type in the TME that correlate with increased tumor grade and poor patient prognosis in many cancers, including gliomas, suggesting important cancer-promoting functions. To examine the contribution of TAMs to glioma progression, we have used a genetically engineered mouse model of proneural GBM. We found that TAMs progressively increase with higher tumor grade in the mouse model, which parallels the increase observed during human glioma development. To investigate the functional significance of TAM accumulation, we designed preclinical trials in the proneural GBM model to therapeutically target colony stimulating factor-1 receptor (CSF-1R), which macrophages depend upon for survival and differentiation. CSF-1R inhibition as a monotherapy dramatically increased survival in these mice, and regressed established tumors after just 7 days. Macrophages were depleted in the normal brain, as we had expected, but not in gliomas of treated mice. Instead, glioma-secreted factors facilitated TAM survival in the presence of CSF-1R inhibitors. Interestingly, gene expression analysis of these surviving TAMs revealed a significant decrease in alternatively activated/ M2 polarization macrophage markers, and consistently, functional analyses revealed anti-tumorigenic phenotypes. Thus, TAM depletion is not strictly necessary for effective macrophage-targeted therapy. Rather, we propose that the presence of macrophage survival factors in the glioma TME not only enables TAMs to survive exposure to a CSF-1R inhibitor, but to be 're-educated' through this process, resulting in a striking anti-tumor response. Our preliminary data identifies TAMs as a promising therapeutic target for proneural gliomas, and establishes strong translational potential of CSF-1R inhibition in GBM. In this proposal, we will expand on these results to elucidate the mechanisms by which TAMs mediate glioma cell phenotypes, and determine how CSF-1R inhibitors interfere with this reciprocal communication to delay and block glioma progression. Our objectives are to elucidate how TAMs are initially educated by glioma cells, and then re-educated by CSF-1R inhibition in the glioma microenvironment. Next, we will investigate which glioma cell signaling pathways are enhanced by TAMs, and determine the downstream effectors that are critical to CSF-1R inhibition efficacy in vivo. Finally, we will determine whether gliomas develop resistance to CSF-1R inhibition, and identify the underlying mechanisms. To address these goals, we will employ multiple different methods including mouse glioma models and an extensive panel of co-culture assays to investigate communication between glioma cells, TAMs, and other cells in the glioma TME. We will combine analyses of known signaling pathways with the identification of novel targets using proteomics and expression profiling approaches. In addition to dissecting the underlying biological mechanisms by which TAMs promote glioma progression, the proposed experiments will also result in the development of therapeutic strategies for their specific inhibition, which if successful could ultimately be tested in the clinic. Collectively, these studies have important implications for future clinical consideration of CSF-1R inhibitors, and for other therapies that target the TME in cancer.