Temporal analysis of the GBM tumor microenvironment during myeloid cell activating therapy

摘要 多形性胶质母细胞瘤(GBM)的肿瘤微环境(TME)往往是复杂的、全面的 免疫抑制，可根据不同的治疗干预措施迅速改变。其原因是 我们对高度动态的网络的有限理解在很大程度上是因为i)当前的转录分析 几乎总是代表手术切除标本的单个时间点数据，ii)TME可以改变 在治疗期间迅速和iii)外周免疫细胞组成通常不能反映什么 发生在肿瘤内部。为了设计更有效的GBM疗法，我们将i)需要新的疗法 方法(药物、载体和组合)；ii)连续询问TME随时间的变化的工具，以便 可以确定新出现的抵抗力和免疫抑制的代偿机制。的目标 这个项目是为了测试他们已经证明的以Candi IL-12为靶向的新型髓系细胞激活疗法 在临床前GBM[Lugani等人，2022，ADV Mat，综述中]和其他癌症模型[Koch 等，2020，Cell Chem Biol，27，94-104.e5；Rodell等，2018，NAT Bimed Eng，2,578-588]和ii)改进 通过执行序列多路复用分析，我们对GBM TME的时间理解。这项建议建立了 关于以新方式解决上述问题的三种最近的新技术：i)FAST-FNA[Ko等人，2020， Angew Chem Weinheim Bergstr Ger，132,6906-6913；oh等人，2021，Clin Cancer Res]执行连续剧， GBM的深度多路分析，II)多路安全活体显微镜(IVM)[Ko等，2022，ADV Sci (Weinh)，e2200064；Ko等人，2022，NAT Biotechnol]，以分析GBM药物分布和细胞效应。 体内单细胞分辨率和III)Candi[Lugani等人，2022，ADV Mat，正在审查；Rodell等人，2018，NAT BioMed Eng，2,578-588]，一种新的髓系细胞靶向双重免疫刺激方法 启动GBM免疫反应。我们提出了三个目标：i)对两只小鼠的TME进行系列分析 使用新的生物正交法的模型(CT2a和005)(“基线研究”)；ii)确定疗效 和III)进行机制研究，以进一步了解 Candi在GBM中的作用及如何加强这一治疗。这些机械论研究的结果将是 重要的是，它们可以在机械水平上揭示治疗的作用机制，并从 从临床角度，明确哪些TME成分应该进行临床研究。最终，我们希望 提高我们对GBM TME的时间理解，并将所获得的知识应用于未来的设计 审判。

ABSTRACT The tumor microenvironment (TME) in glioblastome multiforme (GBM) is often complex, overall immunosuppressive, and can change quickly in response to different therapeutic interventions. The reason for our limited understanding of the highly dynamic network is largely because i) current transcriptomic analyses nearly always represent single time point data from surgical resection specimen, ii) the TME can change rapidly during treatment and iii) the fact that peripheral immune cell composition generally does not reflect what occurs inside tumors. In order to design more effective GBM therapies, we will i) require new therapeutic approaches (drugs, carriers and combinations); ii) tools to serially interrogate TME changes over time so that emerging compensatory mechanisms of resistance and immunosuppression can be identified. The goals of this project are to i) test the novel myeloid cell targeted CANDI IL-12 activating therapies as they have shown remarkable efficacy in preclinical GBM[Lugani et al., 2022, Adv Mat, in review] and other cancer models[Koch et al., 2020, Cell Chem Biol, 27, 94-104.e5; Rodell et al., 2018, Nat Biomed Eng, 2, 578-588] and ii) improve our temporal understanding of the GBM TME by performing serial multiplexed analyses. This proposal builds on three recent novel technologies to address the above problems in new ways: i) FAST-FNA[Ko et al., 2020, Angew Chem Weinheim Bergstr Ger, 132, 6906-6913; Oh et al., 2021, Clin Cancer Res] to perform serial, deep multiplexed analyses of GBM, ii) multiplexed SAFE-intravital microscopy (IVM)[Ko et al., 2022, Adv Sci (Weinh), e2200064; Ko et al., 2022, Nat Biotechnol] to analyze GBM drug distribution and cellular effects at single cell resolution in vivo and iii) CANDI[Lugani et al., 2022, Adv Mat, in review; Rodell et al., 2018, Nat Biomed Eng, 2, 578-588], a novel myeloid cell targeted dual immunostimulatory approach to efficiently jumpstart a GBM immune response. We propose three aims: i) serial analysis of the TME in two murine models (CT2A and 005) using the new bioorthogonal approaches (“baseline study”); ii) determine the efficacy of CANDI myeloid activating therapies in GBM and iii) perform mechanistic studies to gain further insight into the effects of CANDI and how to enhance this therapy in GBM. Findings from these mechanistic studies will be important because they could reveal at a mechanistic level the treatment's mechanisms of action, and from a clinical perspective, define which TME components that should be studied clinically. Ultimately, we hope to improve our temporal understanding of the GBM TME and apply the gained knowledge to the design of future trials.