Vienna, Austria

ESTRO 2023

Session Item

Tumour radiobiology
Poster (Digital)
Single cell sequencing is feasible in delineating chemoradiation resistance in rectal cancer
Michelle Or, Australia


Single cell sequencing is feasible in delineating chemoradiation resistance in rectal cancer

Michelle Or1, Purnima Sundaresan1,2, Joey Lai3, Brian Gloss4, Han Shen5,6, Ankur Sharma7, Hema Mahajan5,8,9, Nimalan Pathma-Nathan5,10,11, Toufic El-Khoury10,11,12, James Toh5,10,11, Jayasingham Jayamohan13,5, Mark Wong5,14, Adnan Nagrial5,14, Eric Hau13,5,15

1Westmead Hospital, Sydney, Australia, Radiation Oncology, Westmead, Australia; 2The University of Sydney, Sydney Medical School, Sydney, NSW, Australia; 3Westmead Institute of Medical Research, Genomics Facility, Westmead, Australia; 4Westmead Institute of Medical Research, Bioinformatics and Data Analysis, Westmead, Australia; 5The University of Sydney, Sydney Medical School, Sydney, Australia; 6Westmead Institute of Medical Research, Radiation Oncology, Westmead, Australia; 7Harry Perkins Institute of Medical Research, Single Cell Genomics, Perth, Australia; 8NSW health Pathology, Tissue pathology ICPMR Westmead hospital, Sydney, Australia; 9Western Sydney University, School of Medicine, Sydney, Australia; 10Westmead Institute of Medical Research, Surgery, Sydney, Australia; 11Westmead Hospital, Colorectal Surgery, Sydney, Australia; 12University of Notre Dame, School of Medicine, Sydney, Australia; 13Westmead Hospital, Radiation Oncology, Sydney, Australia; 14Westmead Hospital, Medical Oncology, Sydney, Australia; 15Westmead Institute of Medical Research, Radiation Oncology, Sydney, Australia

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Purpose or Objective

Curative management of locally advanced rectal cancer (LARC) involves neoadjuvant chemoradiation therapy (nCRT) followed by radical surgery. Pathological complete response (pCR) is seen in 15-27% of patients. The genomic basis of chemoradiation resistance in LARC patients remains poorly understood. Intra-tumoural genetic heterogeneity (ITH) is the proposed mechanism underlying disease progression and treatment failure. Single cell transcriptomics is a revolutionary approach for the in-depth exploration of ITH. We aim to ensure technical feasibility of single RNA sequencing in LARC patients before and after nCRT.

Material and Methods

LARC patients undergoing nCRT followed by surgery provided peripheral blood and tissue samples prior to chemoradiation and following surgery. We plan to recruit total of 12 patients. Tissue samples were collected and banked from examination under anaesthesia and prior to treatment and at time of surgery for single RNA sequencing. Clinicopathological information were obtained. The biopsies specimen underwent sample preparation and dissociation, single cell isolation (cell sorting and dividing cell suspension with CD45 positive and negative in 50:50 ratio) and library preparation and single cell RNA sequencing. The first 2 patients will be analysed for feasibility prior to completion of recruitment.


We successfully processed tumour tissue for single cell RNA sequencing in the 2 recruited patients using our methods with good sequencing and cell quality. The samples did not undergo preservation and all samples were processed fresh. There was high loss of tumour cells following cryopreservation due to epithelial cell type. Transcripts from approximately 4000 viable cells per sample were captured using the Chromium Gene Expression Assay (10x Genomics) and sequencing, read mapping and cell quality metrics passed acceptable thresholds as defined in Cell Ranger. We separately analysed each cell type by splitting into main subsets as determined by scPRED(1). Granulocytes and plasma cells were removed. The top 10 markers for each cell type were plotted in a heatmap. Our early analysis reveals distinct patterns of cell heterogeneity in patients with LARC. The rectal cell markers appeared to be driven by a subset of cells in one patient. There are clear differences between the gene expression signatures in the heat map for most cell types between pre- and post-treatment.

1 Alquicira-Hernandez J, Sathe A, Ji HP, Nguyen Q, Powell JE. scPred: accurate supervised method for cell-type classification from single-cell RNA-seq data. Genome Biology 2019;20:264.


Our study demonstrates feasibility of single cell RNA sequencing in tissue of patients with LARC.