Journal: Cell Death & Disease

Article Title: Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K

doi: 10.1038/s41419-020-2349-8

Figure Lengend Snippet: a Effects of 5-FU on proliferation of HCT116 cells, alone (IC 25 : 2.2 µM; IC 50 : 13.7 µM) or in combination with Cla (IC 25 : 40 µM; IC 50 : 80 µM). Experiments were carried out as described for Fig. . Data points give the number of trypan blue-negative cells ( n = 3, each carried out in triplicate). Full statistics are reported in Table . b Percentages of apoptotic (early apoptotic plus late apoptotic from Annexin V/PI staining) HCT116 cells treated as in ( a ) for 24 and 48 h ( n = 3). c Time course of spheroids’ volume relative to their initial volume (time 0) for the indicated conditions. Drugs were added at their IC 50 and IC 25 values, obtained in 2D cultures ( n = 3; 8 samples for each condition). Spheroids’ photographs, taken with an inverted microscope (Nikon Eclipse TE300, with a ×10 objective), were used to calculate spheroid volumes as in ref. by SpheroidSizer1_0, a MATLAB-based and open-source software (MATLAB 2015a, MathWorks Inc.). The reported significance values refer to the comparison between each condition and control cells. d , e Volumes ( d ) and cell viability ( e ) of spheroids treated as in ( c ) for 120 h. Spheroid volumes are reported as the percentage of control ( n = 3; 8 samples for each condition). Cell viability was assessed by the live/dead imaging (see Materials and Methods) ( n = 2; 6 samples for each condition). f Representative 3D reconstruction of tumor masses, performed in B-Mode imaging with VevoLAZR-X (scale bar, 5 mm) (left panel). Time course of tumor growth relative to the volume determined at the beginning of treatment (day 7 after the inoculum) (right panel). One week after subcutaneous (s.c) inoculation (3 × 10 6 HCT116 cells/flank), mice were treated for two weeks with saline (control group), Cla (40 mg kg −1 , twice daily by oral gavage (o.g.), 5-FU (30 mg kg −1 , twice a week intraperitoneally (i.p.)), or the combination of Cla plus 5-FU ( n = 2 mice; n = 4 tumor masses, for each for each experimental group), as reported in the schematic representation of treatment regime. Statistical analysis was performed with one-way ANOVA: Cla + 5-FU vs. control mice, P = 0.019; Cla + 5-FU vs. Cla mice, P = 0.033; Cla + 5-FU vs. 5-FU mice, P = 0.046. g Expression of phospho-ERK1/2 Thr202/Tyr204 and pro-caspase 3 in tumor masses obtained from mice treated as in ( f ). Membranes were re-probed with anti-ERK1/2 or antitubulin antibodies, to normalize data. The corresponding densitometric analyses are given in the bar graphs ( n = 4 for each experimental group). h IHC analysis of hERG1 and p53 in tumor masses of HCT116 tumor xenografts of mice treated as in ( f ). Representative images are reported (original magnification, ×400; scale bar, 100 µm). The percentages of cells positive for either hERG1 or p53 are reported in the bar graph ( n = 2 mice, n = 4 tumor masses for each experimental group). Statistical significance was assessed with one-way ANOVA for ( a – h ); * P < 0.05; ** P < 0.01, and *** P < 0.001.

Article Snippet: Spheroids’ photographs, taken with an inverted microscope (Nikon Eclipse TE300, with a ×10 objective), were used to calculate spheroid volumes as in ref. by SpheroidSizer1_0, a MATLAB-based and open-source software (MATLAB 2015a, MathWorks Inc.).

Techniques: Staining, Inverted Microscopy, Software, Comparison, Control, Imaging, Saline, Expressing

Journal: Frontiers in Molecular Biosciences

Article Title: Morphological Response in Cancer Spheroids for Screening Photodynamic Therapy Parameters

doi: 10.3389/fmolb.2021.784962

Figure Lengend Snippet: Spheroids show variability in all morphometric parameters at all time points during growth. Parameters were obtained with widefield microscopy images and AnaSP with spheroids at day 4 after seeding ( N = 3, n = 6). Error bars represent standard deviation from the mean value of all samples (shown on the right). Each point represents a single spheroid before selection for PDT. Sphericity, area, and equivalent diameter were chosen to pre-select samples before continuing to in vitro PDT evaluation. Although solidity had low variation, it was not possible to distinguish between healthy and damaged samples. Spheroids continued to grow and maintain their morphology past day 7, reaching around 600 µm in diameter.

Article Snippet: Spheroid morphology after PDT was analyzed using AnaSP and ReViSP, MATLAB-based open-source software, obtaining nine different parameters.

Techniques: Microscopy, Standard Deviation, Selection, In Vitro

Journal: Frontiers in Molecular Biosciences

Article Title: Morphological Response in Cancer Spheroids for Screening Photodynamic Therapy Parameters

doi: 10.3389/fmolb.2021.784962

Figure Lengend Snippet: Parameter extraction improves as debris is cleared from the well. Automatic segmentation depends on initial widefield light microscope image quality. Conversion to binary causes accuracy to decrease as cellular debris accumulating in the well bottom is mistaken for spheroid mass. Debris removal significantly changes extracted parameters. Area and volume are highly affected as they depend on 3D projections from the binary mask obtained with AnaSP and ReViSP. ( N = 3, n = 6).

Article Snippet: Spheroid morphology after PDT was analyzed using AnaSP and ReViSP, MATLAB-based open-source software, obtaining nine different parameters.

Techniques: Extraction, Light Microscopy

Journal: Frontiers in Molecular Biosciences

Article Title: Morphological Response in Cancer Spheroids for Screening Photodynamic Therapy Parameters

doi: 10.3389/fmolb.2021.784962

Figure Lengend Snippet: Parameter extraction improves as debris is cleared from the well. Automatic segmentation depends on initial widefield light microscope image quality. Conversion to binary causes accuracy to decrease as cellular debris accumulating in the well bottom is mistaken for spheroid mass. Debris removal significantly changes extracted parameters. Area and volume are highly affected as they depend on 3D projections from the binary mask obtained with AnaSP and ReViSP. ( N = 3, n = 6).

Article Snippet: The development of open-source alternatives is a key step in the introduction of reliable computer-assisted image analysis to researchers working with MCTS. developed AnaSP (ANAlyse SPheroids) and ReViSP (Reconstruction and Visualization from a Single Projection), MATLAB-based tools for analyzing various spheroid parameters using widefield microscopy images ( ; ).

Techniques: Extraction, Light Microscopy