Histopathological, Immunohistochemical, and Flow Cytometry Data from Lung Cancer Mouse Models

Histopathological and Immunohistochemical AnalysisTumor and lung tissues were fixed in 4% paraformaldehyde for 48 hours, paraffin-embedded, and sectioned at 5 μm thickness. Sections were stained H&E using standard protocols for morphological assessment. Apoptosis was evaluated by Tunel assay using the Fluorescein (FITC) Tunel Cell Apoptosis Detection Kit (Servicebio, #G1501) according to the manufacturer’s protocol. Immunohistochemical staining (IHC) was performed to evaluate the expression of CD4, CD8, IFN-γ and Ki67 using the IHC Select HRP/DAB Detection Kit (Millipore, #DAB150) according to manufacturer specifications. All slides were scanned with a pannoramic SCAN panoramic scanner (3Dhistech, Hungary). The images were processed using caseviewer (v2.2) software, and the quantitative analysis of positively stained areas across 10 random fields per sample was conducted using ImageJ v1.53a with IHC Profiler plugin.Flow cytometry analysis of immune cellsAt the endpoint of the experiments, the blood, tumors, lungs, and CD8⁺ T cells were harvested for flow cytometry analysis. Single-cell suspensions were stained with antibodies targeting mouse CD markers: APC/Cy7 CD45 (clone 30-F11), PE CD3 (clone 17A2), PerCP CD8 (clone 53–6.7), FITC CD4 (clone RM4-5), and APC CD25 (clone PC61), all from Biolegend, USA. Cells underwent intracellular staining after a 4 h stimulation at 37℃ using PMA (50 ng/mL), ionomycin (1 μg/mL), and BD Golgi STOPT. Following fixation and permeabilization, cells were stained with APC/Cy7 anti-mouse IFNγ (clone XMG1.2) and PE anti-mouse FoxP3 (clone MF-14; both from Biolegend, USA). Flow cytometry was conducted with a FACSAria III flow cytometer (BD, USA). FlowJo software (version 10.4, USA) was used for data analysis.