Abstract:

Objective ·To analyze the association between T cell receptor repertoire diversity in colorectal cancer (CRC) patients and clinical characteristics and Fusobacterium nucleatum (Fn) abundance. Methods ·Sixty-three surgical CRC specimens and adjacent normal mucosa tissues were collected from the Department of Gastrointestinal Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine. Totally 53-paired qualified samples were subsequently sequenced on an Illumina HiSeq 4000 for paired-end 150 bp (PE150) sequencing. Quality control and genome annotation were carried out for the original sequencing data. Then a TRUST4 algorithm was further used to acquire TCR repertoire information from RNA sequencing results of tumor tissues and adjacent normal tissues. Meanwhile, the specific sequences of all samples' 16S rDNA were selected for high-throughput sequencing analysis. Clustered from the sequencing data after quality control, the operational taxonomic units (OTU) information was annotated based on rdp_16s_v16.fa taxonomic database to acquire bacterial abundance information, especially Fn. Immunarch R package was utilized to evaluate the characteristics of TCR repertoire clonotypes and diversity. Chao1 index, inverse Simpson index and Hill numerical curve were then conducted to assess the TCR repertoire diversity. Clinical features, such as gender, age, tumor location, and TNM stage, and biological factors like Fn load, were included to dichotomize patients into different groups, and repertoire diversity comparison between subgroups within different clinical features and Fn loads was conducted by Wilcoxon test subsequently. Results ·The number of TCR repertoire clonotypes in CRC tissues was lower compared to that in adjacent normal tissues (P=0.000). Chao1 index, inverse Simpson index and Hill number in tumor tissues were significantly lower than those in adjacent normal tissues (P=0.000). The Chao1 indexes, and inverse Simpson indexes between different gender, age (≥65 years vs <65 years) or TNM stage (Ⅰ/Ⅱ stage vs Ⅲ/Ⅳ stage) group showed no statistically significant difference (P>0.05). Tumors located in rectum had higher TCR clonotypes than those located in non-rectum (P=0.040). In addition, CRC patients with high Fn load demonstrated lower Chao1 indexes than those with low Fn load (P=0.030). Conclusion ·The TCR repertoire diversity is correlated with multiple clinical features as well as the abundance of Fn, indicating that the analysis based on TCR repertoire diversity of CRC patients can provide potential value for CRC tumorigenesis and development.

Key words: colorectal cancer (CRC), T cell receptor (TCR), RNA sequencing, 16S rDNA sequencing, Fusobacterium nucleatum (Fn)