Invited Speaker Multi-Omics Inaugural Conference 2022

Towards improved phenotyping of single-cell and spatial datasets through idem-cell integrated proteogenomics (#42)

Ghamdan Al-Eryani 1 2 , Holly Holliday 1 2 , Nenad Bartonicek 1 2 , Chia-Ling Chan 1 2 , Alma Anderson 3 , Taopeng Wang 1 2 , Ludvig Larsson 3 , Kate Harvey 1 2 , Sunny Wu 1 2 , Dan Roden 1 2 , Jessica Yang 1 2 , Mandeep Singh 1 2 , Clara Young 1 2 , Christopher Goodnow 1 2 , Cindy Ma 1 2 , Simon Junankar 1 2 , Camelia Quek 4 5 , James Wilmott 4 5 , Joakim Lundeberg 3 , Marlon Stoeckius 6 , Alex Swarbrick 1 2
  1. Garvan Institute of Medical Research, Sydney, NSW, Australia
  2. St Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia
  3. Royal Institute of Technology (KTH), Science For Life Laboratory, Stockholm, Sweden
  4. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  5. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia
  6. 10X Genomics, Stockholm, Sweden

High-throughput single-cell RNA sequencing (scRNA-Seq) has become a routine platform for the dissection of solid tumours into their cellular components. The development of methods to incorporate detection of cellular protein epitopes via barcoded antibodies (CITE-seq) has enabled advances in immunophenotyping but its application to investigating tissue immunology is limited. Here we present an optimised experimental and analytical framework for the phenotyping of normal and malignant human tissues by same-cell integrated transcriptome and barcoded antibody analysis of single cell suspensions and tissue sections. We describe SPatial Indexing of Transcriptomes and Epitopes (SPITE-Seq), a method for spatially resolved joint transcriptome and epitope analysis of snap frozen tissues using a modified 10X Visium protocol. We benchmark this method on immunofluorescent (IF) stained Jurkat/PBMC pseudo-tissue, four IF stained breast cancers and melanoma samples. We show the integration of RNA and protein resolves immune subsets at higher resolution than either modality alone. By estimating the contribution of individual antibodies to cell clustering, and their optimal concentration, we guide the development of antibody panels for the analysis of solid tissues. We then applied integrated proteogenomic analysis to a cohort of six breast cancer samples to reveal the improved resolution of the tumour microenvironment and novel markers for resting and activated tissue infiltrating lymphocytes (TILs). We reveal cell types previously not described in breast tissues, one associated with markers of tissue residency and exhaustion, and a previously undescribed phenotype marked by the expression of markers found to be associated with poor prognosis in several cancers, and response to checkpoint immunotherapy in breast cancer. We additionally show how SPITE-seq can enable the identification of phenotypes challenging to capture from single-cell datasets. This work highlights the importance of multi-omic methods for the phenotyping of cell states and the emergence of novel cellular states in the tumour microenvironment.