Computational protocol: Anti-Influenza Neuraminidase Inhibitor Oseltamivir Phosphate Induces Canine Mammary Cancer Cell Aggressiveness

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Protocol publication

[…] CMA07 and CMT-U27 cell lines were cultured in 6-well plates and then treated with different concentrations of oseltamivir phosphate (0.305 μM, 3.05 μM, 30.5 μM and 305 μM, and PBS was used as control). After 24 hours of treatment, culture medium and tripsinized cells were collected and centrifuged for 10 minutes at 2000 rpm. Cells were washed in PBS and fixed in cold methanol for 20 minutes. After fixation, cells were ressuspended in 1 mL of PBS for cytospin procedure. Briefly, 100 μL of cell suspension were centrifuged in a cytospin3 centrifuge using polilysine coated slides. Slides were then used for in situ cell death detection using a commercially available kit (In situ cell death detection kit, fluorescein from Roche) based on labeling DNA double strand breaks (TUNEL technology), according to manufacturer's instructions. Slides were observed under a fluorescence microscope using a 488nm excitation wavelength and percentage of dead cells was calculated by recording positive TUNEL cells in relation to total cells using the ImageJ software. This assay was performed twice. [...] Whenever appropriate, the results are presented as mean ± standard deviation. Statistical analysis was performed using One Way ANOVA (Analysis of variance) test. For MTS, cell proliferation, and cell growth assays multiple comparisons Dunnett method were used. Student t test was performed to evaluate the wound healing assay results. Multiple comparisons Tukey’s test was used to study cell matrigel invasion assay and multiple comparisons Bonferroni test was used for Tunel assay analysis. GraphPad Prism 5.02 version was used to perform these statistical analysis.Imunohistochemical images of Ki-67 and caspase-3 expression were analyzed using the ImmunoRatio online analysis tools, under the high-power magnification in each area []. Tumor cases were divided into 3 categories according to the Ki67 score: less than 10% Ki67 positive cells (low), 10 to 50% Ki67-positive cells (moderate) and more than 50% Ki67-positive cells (high). To evaluate caspase 3 and SLex expression, tumors were grouped according to the percentage of immunoreactive cells throughout the whole lesion, into 3 classes: 0% (Negative); less than 5% (rare cells) or 6–10% (low). Regarding SNA and MAA I expression, a semi-quantitative analysis in which samples were scored according to the percentage of positive cells. Tumors were grouped into: less than 25% (low); 25 to 50% (moderate) and more than 50% (high). Then association hypotheses were tested, using Chi-square test for discrete variables. SPSS software (version 22.0) was used for statistical analysis. […]

Pipeline specifications

Software tools ImageJ, ImmunoRatio
Applications Bright-field microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Canis lupus familiaris
Diseases Breast Neoplasms, Influenza, Human, Neoplasms
Chemicals Oseltamivir