空间转录组（ST）以空间层面提供肿瘤细胞的分子特征，为肿瘤和肿瘤微环境的研究带来了新的进展。本研究总结了我们机构对不同病理类型的鼻部和颅底肿瘤进行空间切片制备的经验和教训，旨在为研究人员提供指导，避免浪费珍贵样本，并为ST在临床实践中的应用提供依据。我们使用本机构诊断为鳞癌和腺癌的鼻部和颅底肿瘤患者的冰冻组织块进行制备。以RNA完整性指数（RIN）和HE评分为标准，探讨了不同程序和病理组织类型对切片质量的影响，并探索了不同RIN值对ST测序数据的影响。共从26名患者中获得43个样本，其中包括22个鳞癌和21个腺癌。对13个RNA质量控制良好、组织学形态良好的样本进行ST测序。样本的隔离时间<15分钟和放弃快速冻结异戊烷显著改善了RNA质量（p=0.004，p<0.0001）和组织形态完整性（p=0.02，p=0.02）。选择适当的组织RNA提取试剂盒对RNA质量至关重要（p<0.0001）。ST测序结果显示RIN≥6可以作为合格的RNA质量控制标准，6≤RIN<7和RIN>7之间没有差异。因此，用冷PBS尽快清洗新鲜组织，然后使用OCT干燥进行快速冻结，是目前制备不同病理类型的鼻和颅底肿瘤组织的空间切片的最佳方法。本研究是第一个研究在不同病理类型的鼻和颅底肿瘤中应用ST的可行性，并证明了ST在肿瘤中的广泛应用。合理优化空间切片制备程序和探索个体化的前测序协议是确保空间测序质量的第一阶段，并为随后的空间分析奠定基础。 © 2023作者

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.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.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.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.© 2023 The Author(s).