Values represent the mean SD, = 3. T-DM1 when combined with DHA is more effective in killing breast cells compared to the effect induced by any single agent. In an orthotopic breast malignancy xenograft model (HCC1954), the growth of the tumour cells resumes after having achieved a complete response to T-DM1 treatment. Conversely, DHA and T-DM1 treatment induces a severe and irreversible cytotoxic effect, even after treatment interruption, thus, improving the long-term efficacy of T-DM1. These results suggest that DHA increases the effect of T-DM1 as poison for microtubules and supports the clinical development of the combination of DHA and T-DM1 for the treatment of aggressive HER2-overexpressing breast cancer. site of pBABE-Puro retroviral vector to obtain FLAG-TCTP-pBABE and FLAG-AA-TCTP-pBABE. All constructs were confirmed by DNA sequence analysis. 2.17. Cell Transfection Retroviruses were produced by transfection of Phoenix-Ampho packaging cells with pBABE-puro, AA-TCTP-pBABE, and WT-TCTP-pBABE using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 48 h after transfection, supernatants made up of the retroviral particles were collected and frozen at ?80 C until use. MCF10A cells were infected with diluted supernatant in the presence of 8 g/mL Polybrene (Sigma-Aldrich) overnight, and cells made up of the pBABE, AA-TCTP-pBABE, and WT-TCTPpBABE constructs were selected with puromycin (1 g/mL) (Sigma-Aldrich) 48 h after contamination. After 10 days in selective medium, the three pools referred to vacant vector (MCF10A-pBABE), the wild type TCTP protein (WT-TCTP), the Ser46Ala Ser64Ala double mutant TCTP (AA-TCTP), were isolated. The puromycin selective pressure was removed 24 h before experimental procedures. 2.18. Evaluation of Cell Sensitivity to Combined Treatment Cells were plated in triplicate in PIK3R4 96-well and treated with DHA, T-DM1, and with the DHA/T-DM1 combination. Growth inhibition was calculated as the percentage of viable cells compared to untreated cells by the CellTiter-Glo Luminescent Cell Viability assay (Promega, Madison, WI, USA) The CompuSyn software program has been used to calculated synergistic, additive or antagonistic effects. This program is based on the Median-Effect Theory (Chou) and the Combination IndexCIsobologram Taltirelin Theorem (Chou-Talalay) . Because all terms in the equations are ratios, all the dose models become dimensionless quantities. Drug can be different models. The combination index (CI) indicates a quantitative measure of the degree of drug conversation in terms of synergistic (CI 1), additive (CI = 1) or antagonistic effect (CI 1). DRI is the dose-reduction index and it is a measure of how many-fold the dose of each drug in a synergistic combination may be reduced at a given effect level compared with the doses of each drug alone. 2.19. Immunodeficient Mice Study We generated HCC1954 cells expressing luciferase in order to implement bioluminescent imaging analysis to follow breast tumour growth in small animal models in vivo. Briefly, HCC1954 cells were transduced at multiplicity of contamination MOI 10 with a third-generation self-inactivating lentiviral vector expressing firefly luciferase . Six-week-old CB17SCID female mice were purchased from Charles River (Calco, Italy) and housed with laboratory chow and water available ad libitum. A cell-line derived orthotopic xenograft model of breast cancer was established by mammary gland implantation of 5 105 HCC1954 luciferase-expressing cells. Mice were regularly palpated and tumour sizes were measured once a week using a digital calliper. Moreover, tumour cell engraftment and early detection of tumour growth was assessed by longitudinal bioluminescent analysis (BLI). BLI analysis has been performed using the IVIS? Lumina II equipped with the Living Image? software for data quantification (PerkinElmer). Animals were sedated and D-luciferin (PerkinElmer) Taltirelin dissolved in PBS (150 mg/kg body weight) was administered i.p. 10 min before analysis . Photons emitted from luciferase expressing HCC1954 cells implanted into the animals were collected with final accumulation times ranging from of 1 1 s to 1 1 min, depending on the intensity of the bioluminescence emission. All animal experiments were conducted in accordance with institutional guidelines, in the full observation of the Directive 2010/63/UE. 2.20. Statistical Analysis All experiments were carried out at least three times unless normally indicated. The results are offered as means SD. Results were analysed using a MannCWhitney test. One-way ANOVA followed by the Bonferroni test using the PRISM GraphPad software was used in the analysis of three or more data sets. Differences were considered significant for 0.05 and highly significant for 0.01 and 0.001 3. Results 3.1. DHA Affects Mitosis of HER2+ BC Cell Lines with Aberrant PI3K/AKT Signalling We investigated the effect of DHA on Taltirelin HER2+ breast malignancy cells resistant to trastuzumab. Since PI3KCA mutations and/or loss of phosphatase Taltirelin and tensin homolog (PTEN) have been associated with a lower response to trastuzumab and chemotherapy [1,48,49], we chose the HCC1954 cell collection.