This study aims to elucidate the cellular heterogeneity of lung adenocarcinoma through single-cell transcriptomics, identify the key gene TK1 associated with tumor metastasis, and explore its underlying mechanisms and potential as a therapeutic target using spatial transcriptomics. The research focuses on validating the function of TK1 in tumor growth, metastasis, and the immune microenvironment, providing new targets and strategies for precision treatment of lung adenocarcinoma.

We employed a multidisciplinary approach, integrating single-cell transcriptomics, spatial transcriptomics, in vitro cell experiments, in vivo animal studies, clinical sample validation, and drug screening. We integrated datasets from GSE1239021, GSE1893572, and GSE1319073, processing 364,430 cells to identify eight epithelial cell subclusters with distinct biological functions. GeneNMF analysis highlighted the MP6 subcluster, which overexpresses TK1 and correlates with poor prognosis, further validated by spatial transcriptomics. In vitro, we used CRISPR/Cas9 to knock out TK1 in lung adenocarcinoma cell lines A549 and PC-9, and overexpressed TK1 to assess proliferation (CCK-8), migration, and invasion (Transwell) capabilities, as well as cell cycle and apoptosis (flow cytometry). In vivo, we established subcutaneous and pulmonary metastasis models in nude mice to observe tumor growth and metastasis. We also validated TK1 protein expression in clinical samples using tissue microarrays and immunohistochemistry. Finally, we screened for drugs sensitive to TK1-overexpressing cell lines and tested combination therapies in mouse models.

Single-cell transcriptomics identified the MP6 subcluster with high TK1 expression, which was significantly associated with metastasis and poor prognosis. In vitro experiments showed that TK1 knockout significantly inhibited cell proliferation, migration, and invasion, induced apoptosis, and arrested the cell cycle at G1 phase. In vivo studies confirmed that TK1 knockout suppressed tumor growth and pulmonary metastasis in nude mice, with immunohistochemistry revealing a positive correlation between TK1 and the proliferation marker Ki-67. Clinical sample validation indicated that TK1 protein was highly expressed in lung adenocarcinoma tissues and correlated with tumor stage, lymph node metastasis, and poor prognosis. Drug screening demonstrated that TK1-overexpressing cell lines were sensitive to chemotherapeutic agents such as paclitaxel and vincristine, with combination therapies showing significant antitumor effects in mouse models.

TK1 plays a crucial role in the proliferation, migration, and invasion of lung adenocarcinoma, with its high expression closely linked to tumor metastasis and poor prognosis. TK1 represents a promising therapeutic target for lung adenocarcinoma, with combination therapies showing potential for clinical application. This study provides new targets and strategies for precision treatment of lung adenocarcinoma, with future research focusing on elucidating the molecular mechanisms of TK1 and exploring its clinical translation.