Glioblastoma tumor microenvironmental influence on acquired and inherent cancer therapy resistance.

项目总结/摘要： 目前的潜在疗法的临床前测试方法在很大程度上没有给人留下深刻印象， 他们产生临床影响的能力。此外，癌症治疗抗性的临床前研究 机制面临着类似的挑战，至少部分是由于临床前模型系统的局限性， 缺乏可靠的生物标志物。一个主要的例子是胶质母细胞瘤（GBM），几乎没有治疗选择， 尽管进行了近80年的研究，但结果非常差（5年生存率<4%）。事实上，当前 护理标准（SOC）疗法包括最大安全手术切除，随后分次照射， 替莫唑胺（TMZ）化疗。这些患者往往分为两类：那些谁有固有的 对放射和TMZ的抵抗以及对这些疗法获得抵抗的那些（通常在6个月内）。 虽然许多研究小组试图开发更有效的治疗方法或克服GBM耐药机制， 实际上所有的尝试都依赖于在主要生长促进条件下的高度人工模型， 高度增殖的肿瘤不再像病人的肿瘤。为了解决这些问题，研究人员 越来越多地利用患者来源的癌症模型（PDMC）， 分析，以建立更可靠的模型，用于检查治疗耐药性和开发新的疗法。 在一系列合作和资助项目的基础上，我们的调查团队开发了一个大型小组， GBM异种移植物（PDX），可以在没有血清的情况下培养，作为衍生的PDMC模型， （神经球）和基质包埋的微肿瘤。我们的亲本U 01（U 01-CA 223976）试图研究肿瘤 微环境（TME）应激源在分子生物学方面对这3种PDMC模型的影响（例如，转录组 和激酶组相似性）和表型（例如，辐射和TMZ响应）保真度。在本U 01修订版中 应用，我们寻求通过开发新的体外和体内模型来扩展我们的工作，以研究固有的 以及获得性对SOC GBM疗法的抗性。我们已经表征了基线体内辐射和TMZ 在20个GBM PDX中筛选出了8个抗辐射和5个抗TMZ的等基因系 从最初敏感的肿瘤。对这些对的转录组学和激酶组学测试已经确定了几个基因 和与抗性相关的激酶。我们将利用这一非常独特的资源来研究SOC治疗 阻力Aim 1利用高通量地理空间控制的3D生物打印系统在体内复制 通过使用PDX细胞和血管内皮细胞在各种基质中的共培养， 其他TME应激源（例如，患者中观察到的缺氧和营养缺乏）。这些结构将在 高达384孔格式，以促进抑制SOC抗性的高优先级靶标，如从我们的 转录组学和激酶组学分析。目标2将探索肠道微生物组的作用，这是一种未充分研究的宿主TME 我们的初步数据表明，肠道微生物组改变了TMZ， 灵敏度选定的PDX将在具有人粪便移植物的Rag 1-/-小鼠的脑中生长，用于SOC测试。

PROJECT SUMMARY/ABSTRACT: Current methods of preclinical testing of potential therapeutics have been, for the most part, underwhelming in terms of their ability to yield a clinical impact. Moreover, preclinical investigation of cancer therapy resistance mechanisms faces similar challenges due, at least in part, to both limitations of the preclinical model systems and lack of reliable biomarkers. A prime example is glioblastoma (GBM) with few therapeutic options yielding incredibly poor outcomes (5-year survival <4%) despite nearly eight decades of research. Indeed, current standard of care (SOC) therapy includes maximal safe surgical resection followed by fractionated irradiation and temozolomide (TMZ) chemotherapy. These patients tend to fall into two categories: those who have inherent resistance to radiation and TMZ and those who acquire resistance to those therapies (typically within 6 months). While many groups have tried to develop more effective therapeutics or overcome GBM resistance mechanisms, virtually all attempts have relied on highly artificial models under major growth promoting conditions that select for highly proliferative tumors that no longer resemble the patient’s tumor. To address these issues, investigators are increasingly utilizing patient-derived models of cancer (PDMC) coupled with comprehensive molecular profiling to build more reliable models for examining therapeutic resistance and for developing novel therapies. Building on a series of collaborations and funded projects, our investigative team has developed a large panel of GBM xenografts (PDX) that can be cultured without serum as derivative PDMC models including spheroids (neurospheres) and matrix-embedded microtumors. Our parent U01 (U01-CA223976) seeks to investigate tumor microenvironmental (TME) stressors on these 3 PDMC models in terms of molecular biology (e.g., transcriptome and kinome similarity) and phenotype (e.g., radiation and TMZ response) fidelity. In this U01 Revision Application, we seek to extend our work by developing new in vitro and in vivo models to investigate inherent and acquired resistance to SOC GBM therapy. We have characterized baseline in vivo radiation and TMZ sensitivity in 20 of our GBM PDX and have developed 8 radiation resistant and 5 TMZ resistant isogenic lines from initially sensitive tumors. Transcriptomic and kinomic testing of these pairs have identified several genes and kinases associated with resistance. We will leverage this very unique resource to examine SOC therapeutic resistance. Aim 1 utilizes a high-throughput geospatially controlled 3D bioprinting system to replicate in vivo conditions by using co-culture of PDX cells and vascular endothelial cells in a variety of matrices with and without other TME stressors (e.g. hypoxia and nutrient deprivation seen in patients). These constructs will be tested in up to 384-well format to facilitate inhibition of high-priority targets of SOC resistance as identified from our transcriptomic and kinomic profiling. Aim 2 will explore the role of the gut microbiome, an understudied host TME factor, in SOC therapeutic resistance as our preliminary data indicate that the gut microbiome alters TMZ sensitivity. Selected PDX will be grown in the brains of Rag1-/- mice with human fecal transplants for SOC testing.