Journal: bioRxiv

Article Title: Ex vivo drug testing in metastatic biopsies reveals patient-specific vulnerabilities to cancer targeting and immune activating drugs

doi: 10.64898/2026.02.06.704037

Figure Lengend Snippet: ( A ) Schematic illustration of the drug response assay. Created with BioRender.com. TME, tumor microenvironment; IF, immunofluorescence. ( B ) Cellular composition of malignant pleural effusions (MPEs) from five NSCLC patients as determined by flow cytometry. ( C ) ATP-based viability of a MPE in different culture media. Each dot represents the mean ± standard deviation (shaded envelope) of n = 5 technical replicates from one representative donor (P179). ( D ) Flow cytometric analysis of MPE viability in HPLM medium. Each dot represents an individual donor (n = 5). P-values were calculated using Wilcoxon matched-pairs signed rank test. ( E ) Log2-fold change (Log2FC) in cellular composition of MPEs after 5 days in HPLM medium compared to baseline, analyzed by flow cytometry. Each dot represents an individual donor. Samples from n = 5 donors were measured. To illustrate fold changes, cell fractions below the 2%-detection limit were excluded from analysis. P-values were determined using a one-sample Wilcoxon test against zero. ( F ). As for (E), but for marker expression of cancer cells (PD-L1, Nectin-4, TROP2) and T cells (PD-1). ( G ) Cytokine secretion and immune checkpoint expression of MPEs (n = 5) after 5 days of ex vivo culture. Color scale represents Z-scores normalized across patients for each cytokine or marker. nMFI, mean fluorescence intensity normalized to fluorescence minus one (FMO) control. ( H ) Relative cell loss of two non-adherent cell lines following the optimized IHC liquid handling protocol. Dots represent outliers among technical replicate wells across n = 3 biological replicates (384-well plates). P-values were calculated using a one-sample Wilcoxon test. ( I ) Pseudocolor plot depicting MFIs of CD45 and EpCAM signal for all segmented masks of a spike-in of MCF-7 cells in lymph node cells. Cell populations were classified based on manual gating. ( J ) Number of cancer (left) and immune cells (right) detected using our image analysis pipeline. Box plots represent data of n = 10 technical replicate wells. ( K ) Cancer cell fractions in malignant pleural effusions (MPEs) from NSCLC patients (n = 5), determined by EpCAM-based immunocytochemistry. The dashed line indicates the detection limit (10 cells per well) of the drug response assay.

Article Snippet: Cells were resuspended in 100 μL of FACS buffer (1% (w/v) BSA, 2 mM EDTA in DPBS) per 1 x 10 6 cells and stained using the following antibodies: Alexa Fluor® 488 anti-human CD3, clone HIT3a (BioLegend, #300319, RRID:AB_493690), PerCP/Cyanine5.5 anti-human CD14, clone HCD14 (BioLegend, # 325621, RRID:AB_893252), PE anti-human CD279 (PD-1), clone EH12.2H7 (BioLegend, # 329905, RRID:AB_940481), PE/Cyanine7anti-human CD11c, clone Bu15 (BioLegend, # 337215, RRID:AB_2129791), APC-Vio770 anti-human CD19, clone LT19 (1:50; Miltenyi Biotec, # 130-098-073, RRID:AB_2661296), Brilliant VioletTM 421 anti-human CD45, clone 2D1 (BioLegend, # 368522, RRID:AB_2687375), Brilliant VioletTM 605 anti-human HLA-DR, clone L243 (BioLegend, # 307639, RRID:AB_11219187), Brilliant VioletTM 785 anti-human CD56 (NCAM), clone 167 (BioLegend, # 362549, RRID:AB_2566058), Alexa Fluor® 488 anti-human CD326 (EpCAM), clone Co17-1A (BioLegend, # 369808, RRID:AB_2650905), PE anti-human TROP2, REAfinityTM (1:50; Miltenyi Biotec, # 130-115-097, RRID:AB_2726914), APC anti-human Nectin-4, REAfinityTM (1:50; Miltenyi Biotec, # 130-116-103, RRID:AB_2727350), Brilliant VioletTM 421 anti-human CD274 (B7-H1, PD-L1), clone 29E.2A3 (BioLegend, # 329714, RRID:AB_2563852), Brilliant VioletTM 785 anti-human CD45, clone HI30 (BioLegend, # 304048, RRID:AB_2563129), Zombie AquaTM (1:1,000; BioLegend, # 423102), Zombie NIRTM (1:500; BioLegend, # 423106).

Techniques: Immunofluorescence, Flow Cytometry, Standard Deviation, Marker, Expressing, Ex Vivo, Fluorescence, Control, Immunocytochemistry

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: High PD-L1 expression is associated with poorer survival outcomes (A and B) Kaplan-Meier curve comparing progression-free survival (PFS) (A) and overall survival (OS) (B) with first-line osimertinib in patients with EGFR -mutated advanced NSCLC having high (tumor proportion score [TPS] ≥50%) or low (TPS <50%) PD-L1 TPS. (C and D) Outcomes of patients stratified by PD-L1 expression: PD-L1 negative (A, TPS <1%), PD-L1 low (B, TPS 1%–49%), and PD-L1 high (C, TPS ≥50%). Tick marks represent censored patients. Risk table below indicates the number of patient at risk at each time point. Hazard ratios and p values were calculated using the log rank test. (E and F) Univariate and multivariate Cox regression analyses identifying risk factors for PFS (E) and OS (F) with first-line osimertinib of patients with EGFR -mutated advanced NSCLC.

Article Snippet: APC anti-human PD-L1 antibody was purchased from Proteintech.

Techniques: Expressing

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: Upregulation of IFNG and IL-6/JAK/STAT3 signaling pathways in patients with high PD-L1 expression (A) Volcano plot showing differentially expressed genes between high and low PD-L1 expression groups. (B) Reactome pathway enrichment analysis of the differentially expressed genes in tumors with high PD-L1 expression compared to those with low expression. (C) Gene set enrichment analysis highlighting key pathways enriched in tumors with high PD-L1 expression compared to those with low expression. (D) Deconvolution analysis of immune cell infiltration between PD-L1 expression groups. Group 1: PD-L1 TPS <50%; group 2: PD-L1 TPS ≥50% (data are presented as mean ± SD; ∗ p < 0.05; ns, not statistically significant).

Article Snippet: APC anti-human PD-L1 antibody was purchased from Proteintech.

Techniques: Protein-Protein interactions, Expressing

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: Increased proportion of CD56 bright NK cells in patients with high PD-L1 expression (A and B) Flow cytometric analysis of CD56 + and CD16 + cells within CD3 − populations in patients with high PD-L1 expression (A) and low PD-L1 expression (B). (C) Bar graphs showing the ratio of CD56 dim NK cell (CD56 + CD16 + ) subsets to CD56 bright NK cell (CD56 + CD16 − ) subsets across all patients (data are presented as mean ± SD; ∗ p < 0.05).

Article Snippet: APC anti-human PD-L1 antibody was purchased from Proteintech.

Techniques: Expressing

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: STAT3 expression is elevated in tumor tissues from patients with high PD-L1 expression (A) Representative immunohistochemical images showing PD-L1 and STAT3 expression in high vs. low PD-L1 tumors. Scale bars: 100 and 25 μm for full and zoom images. (B) Boxplots comparing the percentage of STAT3-positive cells in high vs. low PD-L1 expression groups (data are presented as mean ± SD; ∗∗∗ p < 0.001).

Article Snippet: APC anti-human PD-L1 antibody was purchased from Proteintech.

Techniques: Expressing, Immunohistochemical staining

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: IFN-γ induces PD-L1 expression in EGFR -mutant NSCLC cell lines via STAT3 activation (A) Quantitative real-time PCR analysis of IFNG and PD-L1 gene expression in various lung cancer cell lines. (B–D) Western blot analysis demonstrating PD-L1 expression across cell lines (B), STAT3 phosphorylation following treatment with STAT3 inhibitor C188-9 in PC-9 (left) and HCC827 (right) cells (C), and PD-L1 expression and STAT3 phosphorylation after IFN-γ treatment for 30 min or 18 h in PC-9 (left) and HCC827 (right) cells. Ctrl denotes vehicle control. (E) Flow cytometry analysis of cell surface PD-L1 expression following IFN-γ stimulation (∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (F and G) Western blot analysis of PD-L1 expression in PC-9 (F) and HCC827 (G) cells after STAT3 knockdown, STAT3 overexpression, or C188-9 treatment. (H) Flow cytometry analysis of cell surface PD-L1 expression following C188-9 treatment (∗∗∗ p < 0.001). (I and J) Immunofluorescence analysis of STAT3 localization in HCC827 (I) and PC-9 (J) cells that overexpress STAT3 (STAT3-OE) or treated with IFN-γ (IFNG). NC indicates negative control. Nuclei were stained with DAPI. Red boxes in the merge images highlight areas shown at higher magnification in the fourth column. Scale bars: 10 and 2 μm for full and zoom images (data are presented as mean ± SD).

Article Snippet: APC anti-human PD-L1 antibody was purchased from Proteintech.

Techniques: Expressing, Mutagenesis, Activation Assay, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Phospho-proteomics, Control, Flow Cytometry, Knockdown, Over Expression, Immunofluorescence, Negative Control, Staining

Journal: iScience

Article Title: A translational study on the survival and molecular mechanism of PD-L1 expression in EGFR-mutant NSCLC treated with osimertinib

doi: 10.1016/j.isci.2025.114175

Figure Lengend Snippet: Schematic model of PD-L1 regulation in EGFR -mutated NSCLC cells EGFR -mutated NSCLC tumors with low PD-L1 expression harbor a higher proportion of CD56 dim natural killer (NK) cells, whereas tumors with high PD-L1 expression exhibit increased CD56 bright NK cells, which secrete IFN-γ, leading to STAT3 activation and upregulation of PD-L1 expression.

Article Snippet: APC anti-human PD-L1 antibody was purchased from Proteintech.

Techniques: Expressing, Activation Assay