循环肿瘤细胞（CTC）的纵向监测揭示了在ALK阳性NSCLC中与疾病进展和生存相关的动态CTC行为

Introduction: Heterogeneous mutation profiles are common in anaplastic lymphoma kinase-positive non-small cell lung cancer (ALK+ NSCLC), especially upon progression, supporting that robust longitudinal monitoring to detect these changes early can play a role in disease management. Circulating tumor cells (CTCs) provide a minimally invasive modality for real-time monitoring. We longitudinally analyze CTC counts and sc-transcriptomes to assess their reflection of clinical responses and correlation with progression risk in ALK+ patients. Methods: Following informed consent, we collected 61 samples from 12 ALK patients (2-10 timepoints/patient; 11 Stage IV, 1 Stage III). Serial peripheral blood samples were processed using the in house developed microfluidic Labyrinth device for CTC enrichment. CTC subgroups were identified by immunofluorescence staining for Cytokeratin (CK), EpCAM, and Vimentin. At each sampling point, clinical response was assessed by a treating physician. Sc-RNA sequencing was performed on enriched CTCs, characterizing differentially expressed gene (DEG), survival, trajectory states, immune interactions, and inferred CNV profiles. Results: Patients receiving a single TKI treatment (Alectinib) with clinically stable disease showed decreasing CTC burden. Patients with prior exposure to multiple TKIs and progressive disease showed fluctuating dynamics. Kaplan-Meier analysis showed a decrease in total CTCs (77%, p=0.016) and a decrease in CK+ CTCs (6.05%, p=0.049) significantly correlated with lower progression risk. scRNA-seq on three Stage IV patient's CTC collected at multiple time points revealed dynamic immune populations changed between the visits. DEGs in CTC compared to the rest of immune cells in all 3 visits displayed EMT-associated upregulation (TUBB1, PPBP, ITGA2B); and downregulation of GATA2, consistent with its reported reduction in lung cancer. A composite of 45 consistently downregulated genes was correlated with reduced survival, using a TCGA cohort (n=500; p=0.039). Notably, downregulated genes obtained after the patient started progressing showed increasingly more significant associations with poor survival (p=0.21, p=0.019, p=0.0067). Trajectory analysis identified two CTC clusters, with later visits mapping to higher pseudotime states. CNV analysis revealed a CDK4 copy-gain specifically at progression. Intercellular communication analysis showed decreased CTC-B-cell and increased CTC-monocyte interactions at progression, which may suggest reduced anti-tumor surveillance and enhanced myeloid support. Conclusion: Longitudinal monitoring integrating CTC burden and scRNA analysis provides insights into tumor evolution and immune interactions in ALK+ NSCLC, potentially signaling imminent progression and may help guide personalized treatment strategies.