Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Cytotoxicity of GBM cell lines A172 and U251 following prolonged arginine deprivation (A). Average ASS-1 levels, expressed as mean fluorescence intensity (MFI), for each condition (isotype, untreated and treated cells) at 24, 48, 72, 96, and 120 hours post-treatment. Stars indicate a significant increase in ASS-1 expression compared to both isotype control and untreated cells (B) ASS-1 expression of A172 and U251 cell lines at 72 hours post-treatment. MFI is on the X-axis (FL1-H) and forward scatter is on the Y-axis (C).

Article Snippet: Human Glioblastoma cell lines A172 and U251 MG were obtained from the American Type Culture Collection (A172 ATCC, CRL-1620) and from the European Collection of Authenticated Cell Cultures (U251 MG, ECACC, catalog # 09063001).

Techniques: Fluorescence, Expressing, Control

Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Autophagosome accumulation in GBM cells, expressed as mean fluorescence intensity (MFI), for control untreated cells and cells treated with [HuArgI (Co)-PEG5000] at 24, 48, 72, 96, and 120 hours post-treatment. Stars indicate a significant increase in autophagosome accumulation compared to untreated cells (A). Autophagosome accumulation in A172 and U251 cell lines at 72 hours post-treatment in untreated cells and cells treated with CQ, [HuArgI (Co)-PEG5000] and a combination of both. MFI is on the X-axis (FL1-H) and forward scatter is on the Y-axis (B). Immunohistochemistry staining of autophagosomes in A172 cells untreated and treated with CQ, [HuArgI (Co)-PEG5000], or a combination of both, at 24, 48, and 72 hours, post-treatment (C).

Article Snippet: Human Glioblastoma cell lines A172 and U251 MG were obtained from the American Type Culture Collection (A172 ATCC, CRL-1620) and from the European Collection of Authenticated Cell Cultures (U251 MG, ECACC, catalog # 09063001).

Techniques: Fluorescence, Control, Immunohistochemistry, Staining

Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Western blot of Atg13 and phosphor-Atg13 on protein extracts of A172 and U251 untreated and treated with [HuArgI (Co)-PEG5000] for up to 96 hours (Atg13) (A). Histogram of the ratio of p-Atg13 over Atg13 showing a significant decrease in the phosphorylation of Atg13 at 72- and 96-hours post-treatment. Stars indicate statistical significance (A). ULK phosphorylation levels expressed as mean fluorescence intensity (MFI), for untreated cells and cells treated with [HuArgI (Co)-PEG5000] at 24, 48, 72, 96, and 120 hours post-treatment. Double stars indicate significantly lower ULK phosphorylation in treated compared to untreated cells (B). ULK phosphorylation is also shown in A172 and U251 cell lines at 24- and 48-hours post-treatment in isotype controls, untreated cells and cells treated with [HuArgI (Co)-PEG5000]. MFI is represented on the X-axis (FL1-H) and forward scatter is represented on the Y-axis. Last panel on the right is a histogram representing the overlayed phospho-ULK MFI of the isotype control (red), untreated cells (black) and cells treated with [HuArgI (Co)-PEG5000] (blue) (B). Cytotoxicity of [HuArgI (Co)-PEG5000] alone and in combination with chloroquine (CQ) (50 µM) to A172 and U251 cells at 48, 72, 96, and 120 hours (C).

Article Snippet: Human Glioblastoma cell lines A172 and U251 MG were obtained from the American Type Culture Collection (A172 ATCC, CRL-1620) and from the European Collection of Authenticated Cell Cultures (U251 MG, ECACC, catalog # 09063001).

Techniques: Western Blot, Phospho-proteomics, Fluorescence, Control

Journal: CNS Oncology

Article Title: Arginine deprivation induces prolonged autophagy activation and ROS-dependent cell death in glioblastoma multiforme

doi: 10.1080/20450907.2026.2666026

Figure Lengend Snippet: Intracellular ROS accumulation in GBM cells following treatment with [HuArgI (Co)|-PEG5000], expressed as both percent positive cells (left panel) and mean fluorescence intensity (MFI) (right panel). Stars indicate a significant increase in ROS accumulation. The bottom histograms show ROS accumulation in treated cells (red) compared to controls (black) in A172 and U251 cells. (A) Autophagosome accumulation in GBM cells, expressed as MFI, for control untreated cells and cells treated with [HuArgI (Co)-PEG5000] alone or a combination of [HuArgI (Co)-PEG5000] and N-acetylcysteine (NAC) at 24, 48, 72, 96, and 120 hours post-treatment (B). Autophagosome formation in A172 and U251 cell lines. Control untreated cells (left panel) and cells treated with [HuArgI (Co)-PEG5000] (10 −7 M) (middle panel) or treated with [HuArgI (Co)-PEG5000] and NAC (0.82 g/L) (right panel) at 24, 48, 72, 96, and 120-hours post-treatment. In the histogram (right panel), control cells are in black, cells treated with [HuArgI (Co)-PEG5000] are in red and cells treated with the combination with NAC are in blue (C). Cytotoxicity of A172 and U251 cells treated with [HuArgI (Co)-PEG5000] alone or in combination with N-acetylcysteine (NAC) (0.82 g/L) (D).

Article Snippet: Human Glioblastoma cell lines A172 and U251 MG were obtained from the American Type Culture Collection (A172 ATCC, CRL-1620) and from the European Collection of Authenticated Cell Cultures (U251 MG, ECACC, catalog # 09063001).

Techniques: Fluorescence, Control

Journal: bioRxiv

Article Title: Intraventricular infusion to circumvent the blood-brain barrier to gemcitabine

doi: 10.64898/2026.05.01.722145

Figure Lengend Snippet: GB cells were exposed to GEM for 1h or 24 h at the indicated concentrations. After GEM exposure, cells were maintained in drug-free fresh culture medium for 72 h before a viability assay with Crystal Violet. IC50 of GEM on the A172, U87-MG and U118-MG human GB cell lines were indicated in the legend of . Phase contrast microscopy images of GB cells treated for 24 h at IC 90. Cells were maintained in culture in drug free medium and observed 24 h or 5 days after GEM treatment. Note the presence of enlarged / flattened cells.

Article Snippet: Human GB cell lines A172, U87-MG and U118-MG were purchased from American Type Culture Collection (ATCC) (LGC Standards, Strasbourg, France) and cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (Jacques Boy, Reims, France), 2 mM glutamine and penicillin / streptomycin.

Techniques: Viability Assay, Microscopy

Journal: bioRxiv

Article Title: Direct interaction between cancer cells and fibroblasts promotes early chemoresistance to standard-of-care drug therapy in small cell lung cancer (SCLC)

doi: 10.64898/2026.04.27.721078

Figure Lengend Snippet: (A) Representative images of Control and Etoposide-treated H209 cells acquired using the IncuCyte S3 live cell imaging system. Annexin V red dye was used to identify apoptotic cells. The pink mask represents the annexin V-positive apoptotic cells. Bar = 200 µm. (B-C) Etoposide dose response analysis in (B) H209 cancer cells and (C) CCD8 fibroblasts. Left panels show the time course increase in apoptotic cells number identified as annexin V-positive cells using IncuCyte S3 live microscopy. Graphs are presented as fold increase from T0 using the “Red Area / Phase Area Normalized to T0” software analysis. Right panels show “Area under the curve” (AUC) analysis using the Prism software. AUC was computed from “Fold increase annexin V-positive cells” vs time using the trapezoidal rule across 72 hours. Statistical significance between Control and treatment groups was assessed using one-way ANOVA with Dunnett’s post hoc correction. Data are shown as mean ± SEM from one representative experiment (6 replicates/plate). p-values are indicated in the graphs: ns, not significant; **** , p< 0.0001. (D) Immunoblot analysis of cleaved and uncleaved caspase-3 and caspase-7 in H209 cancer cells and CCD8 fibroblasts after etoposide treatment for 3 days.

Article Snippet: Small cell lung carcinoma cell lines H209 (HTB-172 ) and H69 (HTB-119), and normal lung fibroblast CCD8 (CCL-201), were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS.

Techniques: Control, Live Cell Imaging, Microscopy, Software, Western Blot

Journal: bioRxiv

Article Title: Direct interaction between cancer cells and fibroblasts promotes early chemoresistance to standard-of-care drug therapy in small cell lung cancer (SCLC)

doi: 10.64898/2026.04.27.721078

Figure Lengend Snippet: (A) Representative images acquired using the IncuCyte S3 live cell imaging system at different time points after treatment with etoposide in cancer cells-fibroblasts co-culture. Cancer cells only were labeled with a green fluorescent dye prior to co-culture. Annexin V red dye was used to identify apoptotic cells. The purple mask identifies live green cancer cells, while the orange mask identifies Annexin V-positive apoptotic cells. Cells positive for both green and red signals represent apoptotic cancer cells. Bar = 50 µm. (B) Etoposide dose response analysis in H209 cancer cells alone or in co-culture with fibroblasts was assessed using the IncuCyte S3 software. Left panel shows the time course increase in apoptotic cancer cells number identified as Annexin V-positive cells using IncuCyte S3 live microscopy. Graphs are presented as fold increase from T0 using the “Red Area / Green Area Normalized to T0” software analysis. Right panel shows AUC computed from “Fold increase Annexin V-positive cells” vs time using the trapezoidal rule across 72 hours. Statistical significance between H209 alone (SCLC alone) and H209 in co-culture (Direct co-culture) groups was assessed using one-way ANOVA with Šidák’s post hoc correction. Data are shown as mean ± SEM from one representative experiment (6 replicates/plate). p-values are indicated in the graphs: ***, p< 000.5; ****, p< 0.0001. (C) H209 cancer cells alone or in co-culture with fibroblasts were treated with the indicated doses of etoposide doses for 3 days. The cancer cells in mixed population were separated using FACS sorting after 3 days of treatment. Protein expression of cleaved and uncleaved caspase -3 and caspase-7 in both conditions was determined by immunoblotting. (D) H209 cancer cells in single and co-culture conditions were treated with a YAP1-inhibitor, verteporfin, for 24 hours prior to drug etoposide treatment. The cancer cells in mixed population were separated using FACS sorting after 3 days of treatment. Protein expression of cleaved and uncleaved caspase -3 and caspase-7 in verteporfin-treated and untreated conditions was determined by immunoblotting. (E) H209 cancer cells were grown in different conditioned media and left untreated or treated with the indicated doses of etoposide doses for 3 days. Protein expression of cleaved and uncleaved caspase -3 was determined by immunoblotting.

Article Snippet: Small cell lung carcinoma cell lines H209 (HTB-172 ) and H69 (HTB-119), and normal lung fibroblast CCD8 (CCL-201), were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS.

Techniques: Live Cell Imaging, Co-Culture Assay, Labeling, Software, Microscopy, Expressing, Western Blot

Journal: bioRxiv

Article Title: Direct interaction between cancer cells and fibroblasts promotes early chemoresistance to standard-of-care drug therapy in small cell lung cancer (SCLC)

doi: 10.64898/2026.04.27.721078

Figure Lengend Snippet: (A) The two models of SCLC-fibroblasts co-culture are represented: 2D model-cancer cells and fibroblasts are grown in direct contact with fibroblasts in a 75cm 2 flask; 3D model: spheroids are created in U-shaped bottom plates by mixing different numbers of cancer cells and fibroblasts to simulate in vivo conditions. (B) Mix spheroids with different CAFs to cancer cells ratios were treated with the indicated etoposide dose. Annexin V red dye was added at the time of treatment. IncuCyte S3 live microscopy captured the “Brightfield” and “Red fluorescent” spheroid images every 4 hours for 3 days. Microscopy graphs show Day 0 and Day 3 post-treatment in spheroids with different ratios of fibroblasts (CCD8) to cancer cells (H209). Bar = 400 µm. (C) Etoposide treatment analysis in H209 cancer cells in mixed spheroids with fibroblasts was assessed using the IncuCyte S3 software. Left panel show the time course increase in the intensity of Annexin V-positive spheroids using IncuCyte S3 live microscopy. Graphs are presented as a time course of “Total Red Object Integrated Intensity (RCU x µm 2 /image)” for 72 hours. Right panel shows AUC computed from “Total Red Object Integrated Intensity” curves vs time using the trapezoidal rule across 72 hours. Statistical significance between different spheroid mixed groups was assessed using one-way ANOVA with Šidák’s post hoc correction. Data are shown as mean ± SEM from one representative experiment (6 replicates/plate). p-values are indicated in the graphs: ns, not significant; ****, p< 0.0001.

Article Snippet: Small cell lung carcinoma cell lines H209 (HTB-172 ) and H69 (HTB-119), and normal lung fibroblast CCD8 (CCL-201), were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS.

Techniques: Co-Culture Assay, In Vivo, Microscopy, Software

Journal: bioRxiv

Article Title: Direct interaction between cancer cells and fibroblasts promotes early chemoresistance to standard-of-care drug therapy in small cell lung cancer (SCLC)

doi: 10.64898/2026.04.27.721078

Figure Lengend Snippet: (A) Representative images of “Control” and “Drug candidate” spheroids identified in a high-throughput screening of NIH Clinical Collection. Mixed spheroids of 1:1 ratio of H209 cancer cells and CCD8 lung fibroblasts were established for 3 days before treatment. Spheroid images were captured from Day 0 to Day 6 every 4 hours using IncuCyte S3 live microscopy. At the end of acquisition, a mask (in yellow, Spheroid mask) was designed using the IncuCyte S3 software to determine the size of the spheroid. Bar = 800 µm. (B) “Spheroid growth (size fold increase from Day 0)” graph shows all the drugs identified in the first screening as potential growth inhibitors of mixed H209:CCD8 spheroids. A <1.5-fold increase threshold was applied across all the drugs tested from the NIH Clinical Collection. (C) Mixed spheroid growth over 10 days for the six drugs with the strongest effect on growth in secondary screenings are presented as a time-course for “Brightfield Object Total Area” as determined by the IncuCyte S3 software (left panel) and “Fold increase from D0” (right panel). Statistical significance between Control and drug treated groups was assessed using one-way ANOVA with Dunnett’s post hoc correction. p-values are indicated in the graphs: ****, p< 0.0001.

Article Snippet: Small cell lung carcinoma cell lines H209 (HTB-172 ) and H69 (HTB-119), and normal lung fibroblast CCD8 (CCL-201), were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS.

Techniques: Control, High Throughput Screening Assay, Microscopy, Software

Journal: bioRxiv

Article Title: Direct interaction between cancer cells and fibroblasts promotes early chemoresistance to standard-of-care drug therapy in small cell lung cancer (SCLC)

doi: 10.64898/2026.04.27.721078

Figure Lengend Snippet: (A-B) Immunoblot detection of cleaved and uncleaved caspase-3 or caspase-7 in mono-culture of H209 cancer cells (A) or CCD8 fibroblasts (B) treated with 10µM of drugs identified from drug screening for 36 hours. (C) H209 cancer cells alone or in co-culture with fibroblast were treated with idarubicin at two different concentrations and apoptosis in different conditions was determined by immunoblotting detection of cleaved and uncleaved caspase-3 or caspase-7 as described. (D) H209 cancer cells alone or in co-culture with fibroblasts were treated with two different doses of etoposide or idarubicin and apoptosis was determined by immunoblotting detection of cleaved and uncleaved caspase-3 or caspase-7.

Article Snippet: Small cell lung carcinoma cell lines H209 (HTB-172 ) and H69 (HTB-119), and normal lung fibroblast CCD8 (CCL-201), were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS.

Techniques: Western Blot, Drug discovery, Co-Culture Assay

Journal: bioRxiv

Article Title: Direct interaction between cancer cells and fibroblasts promotes early chemoresistance to standard-of-care drug therapy in small cell lung cancer (SCLC)

doi: 10.64898/2026.04.27.721078

Figure Lengend Snippet: Cancer cells cultured alone or in co-culture with fibroblasts were treated with different doses of etoposide and idarubicin as indicated. Apoptosis was assessed using the FITC Annexin V Apoptosis Detection Kit with 7-AAD in H209 cancer cells alone (A) , H209 cancer cells from co-culture (B) , and CCD8 fibroblasts from co-culture (C) . Quadrant gating distinguishes cell populations as live (Annexin V − /7-AAD − ), early apoptotic (Annexin V + /7-AAD − ), late apoptotic (Annexin V + /7-AAD + ), or dead (Annexin V − /7-AAD + ).

Article Snippet: Small cell lung carcinoma cell lines H209 (HTB-172 ) and H69 (HTB-119), and normal lung fibroblast CCD8 (CCL-201), were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS.

Techniques: Cell Culture, Co-Culture Assay