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

尽管进行了广泛的化疗、放射治疗和自体干细胞移植，但患有颅外实体瘤、出现转移疾病或在标准治疗后复发的儿童患者的长期无病存活率仍然很低。免疫治疗的出现提高了人们的期望，在黑色素瘤的免疫检查点(IC)阻断或血液系统恶性肿瘤的嵌合抗原受体T细胞(CAR-T)方面显示出巨大的临床反应。相比之下，这两种方法在儿童实体癌中只取得了微乎其微的临床疗效。各种机制可能导致疗效不足，即低突变负荷，导致IC阻断所需的肿瘤特异性T细胞的缺乏，以及肿瘤微环境(TME)的主动抑制和异种抗原的表达，从而导致CAR-T治疗中的抗原逃逸。合成生物学已经创造了一个不断扩大的修饰工具箱，以使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优化合理的治疗方法和应用新的T细胞工程工具是必不可少的。利用尖端的单细胞技术、单细胞/核RNA测序(sc/nRNASeq)和空间蛋白质组学，我们将综合分析75例确诊和复发的儿童肉瘤样本，包括横纹肌肉瘤、尤文肉瘤和骨肉瘤。Chromium平台(X10基因组学)将用于自动化的单核条形码和cDNA文库制备，随后将进行短读测序。自动超高含量成像的空间蛋白质组学将在MACSima成像平台(Miltenyi Biotec)上进行。数据分析的目的是a)确定适合于单抗(MAbs)或CAR-T治疗的靶抗原，b)评估靶抗原表达的异质性，c)深入表型渗透免疫和基质细胞并研究它们的轨迹，d)识别TME内可作为免疫治疗靶点的个体免疫逃避特征。CAR-T和肿瘤/TME之间的功能相互作用将在人源化患者来源的异种移植(PDX)模型中进行研究。将分析CAR-T诱导的转录反应以及肿瘤/TME对CAR-T的编辑，以揭示导致免疫逃避的多向细胞串扰。总而言之，我们希望产生一个疾病定义的里程碑式的数据集，这将对我们对儿童肉瘤免疫状况的理解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T细胞工程和儿童实体癌症循证免疫疗法设计的先决条件。

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.