Comprehensive Functional Immunological Profiling of Pediatric Solid Tumors by Single Cell RNA Sequencing and Spatial Proteomics

通过单细胞 RNA 测序和空间蛋白质组学对儿科实体瘤进行全面的功能免疫学分析

• 批准号: 458891500
• 负责人: Professor Dr. Thomas Grünewald, Ph.D.
• 金额: --
• 依托单位: Abteilung V: Kinderchirurgie und Kinderurologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/458891500?language=en
• 关键词: Comprehensive; Functional; Immunological; Profiling; Pediatric

Long-term, disease-free survival rates in pediatric patients with extracranial solid tumors, presenting with metastatic disease or relapse after standard therapy remain poor, despite extensive chemotherapy, radiation and autologous stem cell transplantation. The advent of immunotherapy has raised great expectations, demonstrating tremendous clinical responses for immune checkpoint (IC) blockade in melanoma or chimeric antigen receptor T cell (CAR-T) in hematological malignancies. In contrast, both approaches have achieved only marginal clinical efficacy in pediatric solid cancers. Various mechanisms have been identified that might contribute to insufficient efficacy, namely low mutational burden, resulting in a lack of tumor-specific T cells required for IC blockade as well as impaired trafficking and infiltration, active suppression in the tumor microenvironment (TME), and heterogeneric antigen expression, leading to antigen evasion in CAR-T therapy. Synthetic biology has created a constantly expanding tool box of modifications to equip T cells with functions to counteract these limitations. Thus, comprehensive understanding of the cellular composition and functional interaction between tumor, immune, and stromal cells in the TME is indispensable for optimal development of rational therapeutic approaches and to apply novel T cell engineering tools. Utilizing cutting-edge single cell technologies, single cell/nucleus RNA sequencing (sc/nRNASeq), and spatial proteomics, we will comprehensively analyze 75 pediatric sarcoma samples at diagnosis and relapse, comprising rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma. The Chromium platform (X10 Genomics) will be used for automated single nucleus barcoding and cDNA library preparation followed by short-read sequencing. Spatial proteomics by automated ultra-high content imaging will be performed on the MACSima imaging platform (Miltenyi Biotec). Data will be analyzed aiming to a) identify target antigens suitable for monoclonal antibody (mAbs) or CAR-T therapy, b) assess intratumoral heterogeneity of target antigen expression, c) deeply phenotype infiltrating immune and stromal cells and to study their trajectories and d) identify individual immune evasive signatures within the TME that could be targeted for immunotherapy. Functional interaction between CAR-T and tumor/TME will be studied in humanized patient-derived xenograft (PDX) models. Transcriptional response induced by CAR-T as well as CAR-T editing by the tumor/TME will be analyzed to unveil the multidirectional cellular crosstalk leading to immune evasion. In summary, we expect to generate a disease defining landmark data set which will have major implications on our understanding of the immune landscape in pediatric sarcomas as well as the future therapeutic interventions. Our data will serve as a prerequisite for informed T cell engineering and the design of evidence-based immunotherapies for pediatric solid cancers.

尽管进行了广泛的化疗、放疗和自体干细胞移植，但患有颅外实体瘤的儿科患者的长期无病生存率，在标准治疗后出现转移性疾病或复发仍然很差。免疫疗法的出现引起了极大的期望，证明了黑色素瘤中免疫检查点（IC）阻断或血液恶性肿瘤中嵌合抗原受体T细胞（CAR-T）的巨大临床反应。相比之下，这两种方法在儿科实体癌中仅取得了边缘临床疗效。已经确定了可能导致疗效不足的各种机制，即低突变负荷，导致缺乏IC阻断所需的肿瘤特异性T细胞以及受损的运输和浸润，肿瘤微环境（TME）中的主动抑制，以及异源性抗原表达，导致CAR-T治疗中的抗原逃避。合成生物学创造了一个不断扩大的修改工具箱，使T细胞具有抵消这些限制的功能。因此，全面了解TME中肿瘤细胞、免疫细胞和基质细胞之间的细胞组成和功能相互作用对于合理治疗方法的最佳开发和应用新型T细胞工程工具是必不可少的。利用最先进的单细胞技术，单细胞/核RNA测序（sc/nRNASeq）和空间蛋白质组学，我们将全面分析75例诊断和复发的儿科肉瘤样本，包括横纹肌肉瘤，尤文肉瘤和骨肉瘤。Chromium平台（X10 Genomics）将用于自动化单核条形码和cDNA文库制备，然后进行短读测序。将在MACSima成像平台（Miltenyi Biotec）上通过自动超高含量成像进行空间蛋白质组学。分析数据的目的是：a）鉴定适用于单克隆抗体（mAb）或CAR-T治疗的靶抗原，B）评估靶抗原表达的肿瘤内异质性，c）深入表型浸润免疫和基质细胞并研究其轨迹，d）鉴定TME内可靶向免疫治疗的个体免疫逃避特征。CAR-T和肿瘤/TME之间的功能性相互作用将在人源化患者来源的异种移植物（PDX）模型中进行研究。将分析CAR-T诱导的转录应答以及肿瘤/TME的CAR-T编辑，以揭示导致免疫逃避的多向细胞串扰。总之，我们期望产生一个疾病定义的里程碑数据集，这将对我们理解儿科肉瘤的免疫景观以及未来的治疗干预产生重大影响。我们的数据将作为知情的T细胞工程和儿科实体癌循证免疫疗法设计的先决条件。