Improvements and challenges of tissue preparation for spatial transcriptome analysis of skull base tumors.

Background: Spatial transcriptome (ST) provides molecular profiles of tumor cells at the spatial level, which brings new progress to the research of tumors and the tumor microenvironment. This study summarizes the experiences and lessons learned in the spatial section preparation of two different pathological types of nose and skull base tumors at our institution, with the aim of offering guidelines to researchers to avoid wasting precious samples and provide a basis for the application of ST in clinical practice. Methods: Frozen tissue blocks from patients with squamous cell carcinoma and adenocarcinoma of the nose and skull base diagnosed at our institution were prepared. The effects of different procedures and pathological tissue types on slide quality were explored and evaluated using RNA integrity number (RIN) and HE scores as criteria. The effects of different RIN values on ST sequencing data were explored. Results: A total of 43 samples were obtained from 26 patients, including 22 with squamous carcinomas and 21 with adenocarcinomas. Thirteen samples with satisfactory RNA quality control and good histological morphology were sequenced for ST. Sample isolation time <15 min and abandonment of snap-frozen isopentane significantly improved RNA quality (p = 0.004, p < 0.0001) and histomorphological integrity (p = 0.02, p = 0.02). Selection of a suitable tissue RNA extraction kit was critical for RNA quality (p < 0.0001). No difference between 6 ≤ RIN <7 and RIN >7 in ST sequencing results was found, indicating that RIN ≥6 can be used as a criterion for qualified RNA quality control. Therefore, fresh tissues washed as soon as possible with cold PBS and then dried using OCT for snap freezing are currently the best method for preparing spatial sections of nose and skull base tumor tissues of different pathological types. Conclusion: This study is the first to investigate the feasibility of applying ST to different pathological types of nose and skull base tumors and to demonstrate the widespread application of ST in tumors. Rational optimization of spatial slide preparation procedures and exploration of individualized pre-sequencing protocols are used as the first stage to ensure the quality of spatial sequencing and lay the foundation for subsequent spatial analysis.