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

• Applicants: Professor Dr. Thomas Grünewald, Ph.D.; Privatdozent Dr. Kristian Pajtler; Dr. Christian Seitz
• Subject Area: Pediatric and Adolescent Medicine ; Hematology, Oncology ; Immunology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 458891500

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.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Jörg Fuchs