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Abstract：
BACKGROUND: Although great progress has been made in anti-cancer therapy, the prognosis of laryngeal squamous cell carcinoma (LSCC) patients remains unsatisfied. Quantities of studies demonstrate that glycolytic reprograming is essential for the progression of cancers, where triosephosphate isomerase 1 (TPI1) serves as a catalytic enzyme. However, the clinicopathological significance and potential biological functions of TPI1 underlying LSCC remains obscure.
METHODS: We collected in-house 82 LSCC tissue specimens and 56 non-tumor tissue specimens. Tissue microarrays (TMA) and immunohistochemical (IHC) experiments were performed. External LSCC microarrays and bulk RNA sequencing data were integrated to evaluate the expression of TPI1. We used a log-rank test and the CIBERSORT algorithm to assess the prognostic value of TPI1 and its association with the LSCC microenvironment. Malignant laryngeal epithelial cells and immune-stromal cells were identified using inferCNV and CellTypist. We conducted a comprehensive analysis to elucidate the molecular functions of TPI1 in LSCC tissue and single cells using Pearson correlation analysis, high dimensional weighted gene co-expression analysis, gene set enrichment analysis, and clustered regularly interspaced short palindromic repeats (CRISPR) screen. We explored intercellular communication patterns between LSCC single cells and immune-stromal cells and predicted several therapeutic agents targeting TPI1.
RESULTS: Based on the in-house TMA and IHC analysis, TPI1 protein was found to have a strong positive expression in the nucleus of LSCC cells but only weakly positive activity in the cytoplasm of normal laryngeal cells (p < 0.0001). Further confirmation of elevated TPI1 mRNA expression was obtained from external datasets, comparing 251 LSCC tissue samples to 136 non-LSCC tissue samples (standardized mean difference = 1.06). The upregulated TPI1 mRNA demonstrated a high discriminative ability between LSCC and non-LSCC tissue (area under the curve = 0.91; sensitivity = 0.87; specificity = 0.79), suggesting its potential as a predictive marker for poor prognosis (p = 0.037). Lower infiltration abundance was found for plasma cells, naïve B cells, monocytes, and neutrophils in TPI-high expression LSCC tissue. Glycolysis and cell cycle were significantly enriched pathways for both LSCC tissue and single cells, where heat shock protein family B member 1, TPI1, and enolase 1 occupied a central position. Four outgoing communication patterns and two incoming communication patterns were identified from the intercellular communication networks. TPI1 was predicted as an oncogene in LSCC, with CRISPR scores less than -1 across 71.43% of the LSCC cell lines. TPI1 was positively correlated with the half maximal inhibitory concentration of gemcitabine and cladribine.
CONCLUSIONS: TPI1 is dramatically overexpressed in LSCC than in normal tissue, and the high expression of TPI1 may promote LSCC deterioration through its metabolic and non-metabolic functions. This study contributes to advancing our knowledge of LSCC pathogenesis and may have implications for the development of targeted therapies in the future.
METHODS: We collected in-house 82 LSCC tissue specimens and 56 non-tumor tissue specimens. Tissue microarrays (TMA) and immunohistochemical (IHC) experiments were performed. External LSCC microarrays and bulk RNA sequencing data were integrated to evaluate the expression of TPI1. We used a log-rank test and the CIBERSORT algorithm to assess the prognostic value of TPI1 and its association with the LSCC microenvironment. Malignant laryngeal epithelial cells and immune-stromal cells were identified using inferCNV and CellTypist. We conducted a comprehensive analysis to elucidate the molecular functions of TPI1 in LSCC tissue and single cells using Pearson correlation analysis, high dimensional weighted gene co-expression analysis, gene set enrichment analysis, and clustered regularly interspaced short palindromic repeats (CRISPR) screen. We explored intercellular communication patterns between LSCC single cells and immune-stromal cells and predicted several therapeutic agents targeting TPI1.
RESULTS: Based on the in-house TMA and IHC analysis, TPI1 protein was found to have a strong positive expression in the nucleus of LSCC cells but only weakly positive activity in the cytoplasm of normal laryngeal cells (p < 0.0001). Further confirmation of elevated TPI1 mRNA expression was obtained from external datasets, comparing 251 LSCC tissue samples to 136 non-LSCC tissue samples (standardized mean difference = 1.06). The upregulated TPI1 mRNA demonstrated a high discriminative ability between LSCC and non-LSCC tissue (area under the curve = 0.91; sensitivity = 0.87; specificity = 0.79), suggesting its potential as a predictive marker for poor prognosis (p = 0.037). Lower infiltration abundance was found for plasma cells, naïve B cells, monocytes, and neutrophils in TPI-high expression LSCC tissue. Glycolysis and cell cycle were significantly enriched pathways for both LSCC tissue and single cells, where heat shock protein family B member 1, TPI1, and enolase 1 occupied a central position. Four outgoing communication patterns and two incoming communication patterns were identified from the intercellular communication networks. TPI1 was predicted as an oncogene in LSCC, with CRISPR scores less than -1 across 71.43% of the LSCC cell lines. TPI1 was positively correlated with the half maximal inhibitory concentration of gemcitabine and cladribine.
CONCLUSIONS: TPI1 is dramatically overexpressed in LSCC than in normal tissue, and the high expression of TPI1 may promote LSCC deterioration through its metabolic and non-metabolic functions. This study contributes to advancing our knowledge of LSCC pathogenesis and may have implications for the development of targeted therapies in the future.
摘要：
背景：尽管在抗癌治疗方面取得了很大进展，喉鳞状细胞癌(LSCC)患者的预后仍不满意.大量的研究表明，糖酵解重编程对于癌症的进展至关重要，其中磷酸丙糖异构酶1（TPI1）充当催化酶。然而,作为LSCC基础的TPI1的临床病理意义和潜在生物学功能仍不清楚.
方法：我们内部收集了82个LSCC组织标本和56个非肿瘤组织标本。进行组织微阵列(TMA)和免疫组织化学(IHC)实验。整合外部LSCC微阵列和大量RNA测序数据以评估TPI1的表达。我们使用对数秩检验和CIBERSORT算法来评估TPI1的预后价值及其与LSCC微环境的关联。使用feerCNV和CellTypist鉴定恶性喉上皮细胞和免疫基质细胞。我们进行了全面分析，以阐明TPI1在LSCC组织和单个细胞中的分子功能，使用Pearson相关分析，高维加权基因共表达分析，基因集富集分析，并成簇规则间隔的短回文重复序列（CRISPR）筛选。我们探索了LSCC单细胞和免疫基质细胞之间的细胞间通讯模式，并预测了几种靶向TPI1的治疗剂。
结果：基于内部TMA和IHC分析，发现TPI1蛋白在LSCC细胞核中具有强阳性表达，但在正常喉细胞的胞浆中仅具有弱阳性活性（p<0.0001）。从外部数据集中获得了TPI1mRNA表达升高的进一步证实，比较251个LSCC组织样本和136个非LSCC组织样本（标准化平均差=1.06）。上调的TPI1mRNA在LSCC和非LSCC组织之间表现出很高的辨别能力（曲线下面积=0.91；灵敏度=0.87；特异性=0.79），提示其作为不良预后的预测标志物的潜力(p=0.037)。发现浆细胞的浸润丰度较低，幼稚B细胞，单核细胞,和中性粒细胞在TPI高表达的LSCC组织中。糖酵解和细胞周期是LSCC组织和单细胞的显着富集途径，其中热休克蛋白家族B成员1，TPI1和烯醇化酶1占据中心位置。从蜂窝间通信网络中识别出四种传出通信模式和两种传入通信模式。TPI1被预测为LSCC的癌基因，在71.43%的LSCC细胞系中，CRISPR得分低于-1。TPI1与吉西他滨和克拉屈滨的半数最大抑制浓度呈正相关。
结论：TPI1在LSCC中比在正常组织中显著过表达，TPI1的高表达可能通过其代谢和非代谢功能促进LSCC恶化。这项研究有助于提高我们对LSCC发病机制的认识，并可能对未来靶向治疗的发展产生影响。
方法：我们内部收集了82个LSCC组织标本和56个非肿瘤组织标本。进行组织微阵列(TMA)和免疫组织化学(IHC)实验。整合外部LSCC微阵列和大量RNA测序数据以评估TPI1的表达。我们使用对数秩检验和CIBERSORT算法来评估TPI1的预后价值及其与LSCC微环境的关联。使用feerCNV和CellTypist鉴定恶性喉上皮细胞和免疫基质细胞。我们进行了全面分析，以阐明TPI1在LSCC组织和单个细胞中的分子功能，使用Pearson相关分析，高维加权基因共表达分析，基因集富集分析，并成簇规则间隔的短回文重复序列（CRISPR）筛选。我们探索了LSCC单细胞和免疫基质细胞之间的细胞间通讯模式，并预测了几种靶向TPI1的治疗剂。
结果：基于内部TMA和IHC分析，发现TPI1蛋白在LSCC细胞核中具有强阳性表达，但在正常喉细胞的胞浆中仅具有弱阳性活性（p<0.0001）。从外部数据集中获得了TPI1mRNA表达升高的进一步证实，比较251个LSCC组织样本和136个非LSCC组织样本（标准化平均差=1.06）。上调的TPI1mRNA在LSCC和非LSCC组织之间表现出很高的辨别能力（曲线下面积=0.91；灵敏度=0.87；特异性=0.79），提示其作为不良预后的预测标志物的潜力(p=0.037)。发现浆细胞的浸润丰度较低，幼稚B细胞，单核细胞,和中性粒细胞在TPI高表达的LSCC组织中。糖酵解和细胞周期是LSCC组织和单细胞的显着富集途径，其中热休克蛋白家族B成员1，TPI1和烯醇化酶1占据中心位置。从蜂窝间通信网络中识别出四种传出通信模式和两种传入通信模式。TPI1被预测为LSCC的癌基因，在71.43%的LSCC细胞系中，CRISPR得分低于-1。TPI1与吉西他滨和克拉屈滨的半数最大抑制浓度呈正相关。
结论：TPI1在LSCC中比在正常组织中显著过表达，TPI1的高表达可能通过其代谢和非代谢功能促进LSCC恶化。这项研究有助于提高我们对LSCC发病机制的认识，并可能对未来靶向治疗的发展产生影响。
