primary uman lung fibroblasts hlf  (ATCC)

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) 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

Journal: Cell Death Discovery

Article Title: Trifluoperazine causes mast cell apoptosis through a secretory granule-mediated pathway

doi: 10.1038/s41420-026-03122-x

Figure Lengend Snippet: A Human lung smooth muscle cells (HLSMCs), lung fibroblasts (HLFs), and small airway epithelial cells (HSAECs) were treated with TFP at the indicated concentrations for 24 h. Human peripheral blood eosinophils and neutrophils were treated with TFP for 2 h. Cell viability was assessed by staining the cells with Annexin V (AnnV) and DRAQ7. Viable cells, AnnV − DRAQ7 − ; apoptotic cells, AnnV + DRAQ7 − ; necrotic/late apoptotic cells, AnnV + DRAQ7 + . HSAECs, n = 6 from three independent experiments; HLSMCs, eosinophils, neutrophils, n = 4 from four independent experiments; HLFs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA for HLSMCs, HLFs; Kruskal-Wallis for HSAECs; Friedman test for eosinophils, neutrophils). B – C Bone marrow-derived MCs (BMMCs) and peritoneal cell-derived MCs (PCMCs) treated under the same conditions as in ( A ) for 24 h. BMMCs, n = 5 from two independent experiments; PCMCs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA). Untreated (control) cells were used for statistical comparisons to all other groups in all figures. The bar charts show mean values + SEM or median + interquartile range. * P < 0.05; ** P < 0 .01; **** P < 0.0001. D Effect of TFP on DNA degradation. MCs were preincubated with bafilomycin A1 (Baf A1) (20 nM) for 2 h followed by treatment with TFP (10 μΜ) for 2 h. DNA was extracted from MCs and fragmentation was assessed by agarose gel electrophoresis. St standard marker.

Article Snippet: Primary human lung fibroblasts (HLFs) (PCS-201-013) and primary human lung smooth muscle cells (HLSMCs)(PCS-130-010) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Control, Agarose Gel Electrophoresis, Marker

Journal: Cell Death Discovery

Article Title: Trifluoperazine causes mast cell apoptosis through a secretory granule-mediated pathway

doi: 10.1038/s41420-026-03122-x

Figure Lengend Snippet: A Human lung smooth muscle cells (HLSMCs), lung fibroblasts (HLFs), and small airway epithelial cells (HSAECs) were treated with TFP at the indicated concentrations for 24 h. Human peripheral blood eosinophils and neutrophils were treated with TFP for 2 h. Cell viability was assessed by staining the cells with Annexin V (AnnV) and DRAQ7. Viable cells, AnnV − DRAQ7 − ; apoptotic cells, AnnV + DRAQ7 − ; necrotic/late apoptotic cells, AnnV + DRAQ7 + . HSAECs, n = 6 from three independent experiments; HLSMCs, eosinophils, neutrophils, n = 4 from four independent experiments; HLFs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA for HLSMCs, HLFs; Kruskal-Wallis for HSAECs; Friedman test for eosinophils, neutrophils). B – C Bone marrow-derived MCs (BMMCs) and peritoneal cell-derived MCs (PCMCs) treated under the same conditions as in ( A ) for 24 h. BMMCs, n = 5 from two independent experiments; PCMCs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA). Untreated (control) cells were used for statistical comparisons to all other groups in all figures. The bar charts show mean values + SEM or median + interquartile range. * P < 0.05; ** P < 0 .01; **** P < 0.0001. D Effect of TFP on DNA degradation. MCs were preincubated with bafilomycin A1 (Baf A1) (20 nM) for 2 h followed by treatment with TFP (10 μΜ) for 2 h. DNA was extracted from MCs and fragmentation was assessed by agarose gel electrophoresis. St standard marker.

Article Snippet: Primary human lung fibroblasts (HLFs) (PCS-201-013) and primary human lung smooth muscle cells (HLSMCs)(PCS-130-010) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Control, Agarose Gel Electrophoresis, Marker