Table_2_Asparagine Synthetase-Mediated l-Asparagine Metabolism Disorder Promotes the Perineural Invasion of Oral Squamous Cell Carcinoma.xlsx

Dysregulated amino acids metabolism reciprocally interplays with evolutionary phenotypic characteristics of cancer cells to enhance metastasis. The high metastasis potential of oral squamous cell carcinoma (OSCC) can manifest with perineural invasion (PNI). We here aimed to determine the role of amino acids metabolism in OSCCs with different PNI statuses. Targeted metabolomics was used to quantify 48 amino acids in 20 fresh OSCC samples and 25 amino acids were successfully detected, within which 9 were significantly up-regulated in PNI positive (PNI+) samples. As its highest area under the curve value (0.9063), l-asparagine was selected as the biomarker to distinguish PNI+ from PNI negative (PNI−). Then, the key enzyme of l-asparagine, asparagine synthetase (ASNS), was investigated using immunohistochemistry with 86 OSCC patients. The results showed that ASNS mainly expressed in tumor epitheliums and positively correlated with lymph node metastasis and PNI. Moreover, subgroup survival analysis revealed that ASNS expression combined with PNI status significantly improved their prognostic value, which was confirmed by the TCGA OSCC cohort (n = 279). To validate whether ASNS promotes PNI, we determined ASNS expression levels in five OSCC cell lines and one normal oral keratinocyte, and HSC3 showed the lowest ASNS level but CAL33 had the highest. Therefore, HSC3 and CAL33 (or PBS as control) were selected and injected separately into sciatic nerves to construct the in vivo PNI mouse models. Although both models eventually developed the hind-limb paralysis, nerve dysfunction in the CAL33 model progressed significantly earlier than HSC3 (Day 9 vs. Day 24). Besides, CAL33 migrated significantly farther than HSC3 in the nerve microenvironment (P = 0.0003), indicating high ASNS expression is indispensable for OSCC progression, especially PNI formation, through l-asparagine metabolism alteration. This study provides novel insights into how amino acids metabolism disorders alter tumor neurotropism which helps cancer metastasis.

氨基酸代谢紊乱与癌细胞进化获得的表型特征相互调控，进而促进肿瘤转移。口腔鳞状细胞癌（Oral Squamous Cell Carcinoma, OSCC）具有高转移潜能，其临床表现可包括神经周围侵犯（Perineural Invasion, PNI）。本研究旨在探究氨基酸代谢在不同神经周围侵犯状态的OSCC中的作用。研究采用靶向代谢组学技术定量20例新鲜OSCC样本中的48种氨基酸，最终成功检出25种，其中9种在神经周围侵犯阳性（PNI+）样本中显著上调。鉴于L-天冬酰胺拥有最高的曲线下面积值（0.9063），故将其选为区分PNI+与PNI阴性（PNI−）样本的生物标志物。随后，针对L-天冬酰胺的关键酶——天冬酰胺合成酶（Asparagine Synthetase, ASNS），本研究纳入86例OSCC患者样本开展免疫组化检测。结果显示，ASNS主要表达于肿瘤上皮细胞中，且与淋巴结转移及PNI呈正相关。此外，亚组生存分析结果显示，将ASNS表达水平与PNI状态相结合可显著提升预后评估价值，该结论在癌症基因组图谱（TCGA）的OSCC队列（n=279）中得到验证。为验证ASNS是否可促进PNI发生，本研究检测了5株OSCC细胞系及1株正常口腔角质形成细胞中的ASNS表达水平，结果显示HSC3细胞的ASNS表达水平最低，而CAL33细胞则最高。因此，本研究选取HSC3与CAL33细胞（以磷酸盐缓冲液（Phosphate Buffered Saline, PBS）作为空白对照），分别注射至小鼠坐骨神经中，以此构建体内PNI小鼠模型。尽管两组模型最终均出现后肢麻痹症状，但CAL33模型的神经功能障碍出现时间显著早于HSC3模型（第9天 vs 第24天）。此外，在神经微环境中，CAL33细胞的迁移距离显著远于HSC3细胞（P=0.0003），这表明通过改变L-天冬酰胺代谢，高表达ASNS对于OSCC进展，尤其是PNI的形成不可或缺。本研究为氨基酸代谢紊乱如何改变肿瘤嗜神经性以促进癌症转移提供了全新的研究视角。