biostudies-literatureUnknown29(1)Chen LThe evaluation of predictive biomarkers in advanced lung cancer requires methods that can comprehensively profile rare cell populations. We developed a dual-labeling assay integrating tyramide signal amplification immunofluorescence (TSA-IF) with fluorescence <i>in situ</i> hybridization (FISH) to concurrently detect protein expression and chromosomal aberrations. This approach was used to analyze PD-L1-positive circulating tumor cells (CTCs), circulating tumor endothelial cells (CTECs), and white blood cells (WBCs) from multiple biofluid types. Our assay improved signal integrity and revealed distinct clinical associations: specific PD-L1⁺ CTC phenotypes were linked to metastasis and correlated with improved immunotherapy response, whereas PD-L1⁺ CTECs were associated with treatment resistance and serum tumor markers. Furthermore, PD-L1⁺ WBC levels were strongly correlated with C-reactive protein, connecting them to systemic inflammation. This integrated liquid biopsy strategy enables a multifaceted view of the tumor microenvironment and host immune status, presenting a conceptual advance for monitoring treatment efficacy and inflammatory activity in advanced lung cancer.iScience114357https://www.ebi.ac.uk/biostudies/studies/S-EPMC12799786biostudies-literatureAnalyzed PD-L1-positive subpopulations by dual-labeling TSA-IF-FISH predicts immunotherapy efficacy in advanced lung cancer.PMC12799786Yang ZLi SLu YChen LTang DZhang LfalseAnalyzed PD-L1-positive subpopulations by dual-labeling TSA-IF-FISH predicts immunotherapy efficacy in advanced lung cancer.The evaluation of predictive biomarkers in advanced lung cancer requires methods that can comprehensively profile rare cell populations. We developed a dual-labeling assay integrating tyramide signal amplification immunofluorescence (TSA-IF) with fluorescence <i>in situ</i> hybridization (FISH) to concurrently detect protein expression and chromosomal aberrations. This approach was used to analyze PD-L1-positive circulating tumor cells (CTCs), circulating tumor endothelial cells (CTECs), and white blood cells (WBCs) from multiple biofluid types. Our assay improved signal integrity and revealed distinct clinical associations: specific PD-L1⁺ CTC phenotypes were linked to metastasis and correlated with improved immunotherapy response, whereas PD-L1⁺ CTECs were associated with treatment resistance and serum tumor markers. Furthermore, PD-L1⁺ WBC levels were strongly correlated with C-reactive protein, connecting them to systemic inflammation. This integrated liquid biopsy strategy enables a multifaceted view of the tumor microenvironment and host immune status, presenting a conceptual advance for monitoring treatment efficacy and inflammatory activity in advanced lung cancer.2026-01-01T00:00:00Z2026 Jan2026-06-06T13:18:31.638Z2026-05-31T03:07:26.622ZS-EPMC127997864154168510.1016/j.isci.2025.114357