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: Materials Today Bio

Article Title: Metformin glycyrrhetinic acid binary injectable hydrogel for synergistic tumor immunotherapy via spatiotemporal microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102749

Figure Lengend Snippet: Systemic antitumor immune modulation and combined immune checkpoint blockade (ICB) therapy mediated by Fe 3 O 4 NPs@Met-GA-H. ( A ) Schematic illustration of the experimental timeline for treating B16F10 tumor-bearing C57BL/6 mice. ( B ) Representative images of excised tumors from each treatment group (n = 5). ( C ) Tumor growth curves across different treatments (n = 5). ( D ) Tumor inhibition rates following various treatments (n = 5). ( E ). Proposed mechanism of Fe 3 O 4 NPs@Met-GA-H-induced immune activation and tumor suppression. ( F ) Spleens collected from each group (n = 5). ( G ) Spleen weights under different treatment conditions (n = 5). ( H, I ) Flow cytometry analysis of CD80 + CD86 + expression (H) and MHC-II (I) expression in splenic DCs (n = 5). ( J ) Flow cytometry assay of CD4 + and CD8 + expression (gated on CD3 + ) from splenocytes (n = 5). ( K ) Flow cytometry analysis of CD86 + CD206 − and CD86 − CD206 + macrophages (gated on F4/80 + ) in tumors (n = 5). ( L, M ) Quantitative analysis of CD80 + CD86 DCs (L) and MHC-II DCs (M) in spleens (n = 5). ( N, O ) Quantitative analysis of CD4 + (N) and CD8 + (O) T-cell populations in spleen (n = 5). ( P ) Ratio of CD86 + CD206 − to CD86 − CD206 + (gated on F4/80 + ) tumor-associated macrophages (n = 5). Data are presented as mean ± SD. Statistical significance was determined using Student's t-test for pairwise comparisons and one-way ANOVA for multiple groups. Ns: not significant ( P > 0.05); ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus the Ctrl group. # P < 0.05, ## P < 0.01, ### P < 0.001 versus the anti-PD-1 group.

Article Snippet: Anti-mouse PD-1 antibodies (Cat. No. S-5001) were obtained from MedChemExpress.

Techniques: Inhibition, Activation Assay, Flow Cytometry, Expressing