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Archive > Volume 32 Issue 8 > 2025,32(8):862-869. DOI:10.3872/j.issn.1007-385X.2025.08.010 Prev Next
Technigue and Method
Establishment and verification of a detection method for the cytotoxic activity of human cascade-primed immune cell injection against non-small cell lung cancer
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Abstract:
[Abstract] Objective: To create a luciferase (Luc) / green fluorescent protein (GFP) dual-fluorescent labeled tumor cell line and establish a detection method for the cytotoxic activity of human cascade-primed immune cell injection against non-small cell lung cancer (NSCLC), and to conduct preliminary verification. Methods: NSCLC lines, which possessed homozygous HLA-A, HLA-B, and HLA-C genotypes and an allelic distribution frequency higher than 2.500% were screened through high-resolution HLA typing for use as cell models. The cell line stably expressing the green fluorescent protein (GFP) and luciferase (Luc) was obtained through infecting the original cell line with a recombinant lentivirus that carries the GFP gene and Luc gene. This cell line was then used as the target cell. Through co-culturing effector cells and target cells as well as optimizing parameters including the pretreatment steps of target cells, co-culture duration, and effector-to-target proportion, a detection method for the cytotoxic activity of human cascade-primed immune cell injection against NSCLC was established, Subsequently, both the specificity and precision of this method were thoroughly verified. Results: Through high-resolution HLA typing, the non-small cell lung cancer cell line HCC827 harboring high-frequency alleles HLA-A11:01:01 (20.893%), HLA-B52:01:01 (2.991%), and HLA-C*12:02:02 (3.139%) was successfully selected as the cellular model. The HCC827 cell line with GFP and Luc dual fluorescence labeling was successfully constructed using a lentiviral vector, with a 96% GFP-positive rate. The titer of the recombinant lentivirus was 1.83 × 10? TU/mL. Significant differences in the cytotoxic activity were observed among groups with effector-to-target (E∶T) ratios of 5∶1, 10∶1, 15∶1, and 20∶1 (P < 0.05), and the cytotoxic activity increased significantly with prolonged co-culture duration (P < 0.000 1). After comprehensive evaluation, the optimal parameters were determined as an effector-to-target (E∶T) ratio of 10:1 and a co-culture duration of 72 h. Methodological verification demonstrated that the established method exhibited strong specificity, with a coefficient of variation of 0.80% - 1.86% for repeatability and 1.00% - 1.58% for precision. Furthermore, no significant differences were observed via variance analysis (P > 0.05), confirming good repeatability of the method. Conclusion: A detection method for the cytotoxic activity of human cascade-primed immune cell injection against NSCLC has been successfully established and verified. This method might help human cascade-primed immune cell injection play an important role in the effectiveness evaluation of cellular immunotherapy.
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