Oseltamivir phosphate monotherapy ablates tumor neovascularization, growth, and metastasis in mouse model of human triple-negative breast adenocarcinoma

Figures & data

Figure 1 EGF induces sialidase activity in live (A) TNBC MDA-MB-231 cells, (B) MDA-MB-231 cells and its resistant clones against 5 μM and 10 μM tamoxifen, and (C) MCF-7 cells and its resistant clones against 5 μM and 10 μM tamoxifen.

Notes: Cells were allowed to adhere on 12 mm circular glass slides in media containing 10% FCS for 24 hours. After removing media, 0.318 mM 4-MUNANA substrate (2′-[4-methlyumbelliferyl]-α-N-acetylneuraminic acid) in Tris-buffered saline, pH 7.4, was added to live cells alone (control) or with EGF alone and in combination with OP, anti-Neu1 neutralizing antibodies, and specific inhibitor of MMP-9 at the indicated dosage. Fluorescent images were taken at 1–2 minutes after adding substrate using Zeiss Imager M2 epi-fluorescence microscopy (20× objective). The mean fluorescence of 50 multipoint replicates was quantified using Image J software. The data are a representation of one out of three independent experiments showing similar results.
Abbreviations: EGF, epidermal growth factor; FCS, fetal calf serum; MMP-9, matrix metalloproteinase-9; OP, oseltamivir phosphate; TNBC, triple-negative breast cancer.

Figure 2 Cell viability of (A) MCF-7 and (C) MDA-MB-231 cells treated with tamoxifen at indicated doses, (B) MCF-7 and (D) MDA-MB-231 cells and their resistant clones against 5 μM and 10 μM tamoxifen treated with OP at indicated doses using the WST-1 assay.

Notes: Cells were incubated in 96-well plates (5,000 cells/well) and allowed to adhere for 24 hours in 1× DMEM media containing 10% FCS. The media were replaced with fresh DMEM media containing 5% FCS with or without various concentrations of tamoxifen or OP for indicated time periods. Cell viability was expressed as a percent of control ± SEM of triplicate values. The data are a representation of one out of three independent experiments showing similar results. LD₅₀ value is given as μM of drug concentration as determined by WST-1 assay after 72 hours for the indicated cell lines.
Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; FCS, fetal calf serum; IC₅₀, half maximal inhibitory concentration; LD₅₀, lethal dose to kill 50% of viable cells; OP, oseltamivir phosphate; SEM, standard error of the mean.

Figure 3 Cell viability of MDA-MB-231 cells treated with OP at indicated doses in combination with 1 μM of either cisplatin, 5-FU, paclitaxel, gemcitabine, or tamoxifen using the WST-1 assay.

Notes: Cells were incubated in 96-well plates (5,000 cells/well) and allowed to adhere for 24 hours in 1× DMEM media containing 10% FCS. The media were replaced with fresh DMEM media containing 5% FCS with or without various concentrations of OP or chemodrugs for 24, 48, and 72 hours as predetermined optimally. Cell viability was expressed as a percent of control ± SEM of three independent experiments. Statistical analysis was carried out using GraphPad Prism, and results were compared by unpaired t-test.
Abbreviations: 5-FU, 5-fluorouracil; DMEM, Dulbecco’s Modified Eagle’s Medium; FCS, fetal calf serum; OP, oseltamivir phosphate; SEM, standard error of the mean.

Figure 4 OP treatment of RAGxCγ double mutant mice bearing heterotopic xenograft of MDA-MB-231 tumors.

Notes: Cells at 0.5×10⁶ in 0.2 mL were implanted cutaneously in the right back flank of mice. Twice a week following implantation of the cancer cells, each mouse was monitored for tumor volume ([width squared/2] × length) at the site of implantation. Mice were treated with 30 mg/kg and 50 mg/kg of OP in sterile saline ip daily at day 10 postimplantation, when the tumor volume reached ~10–20 mm³. (A) Necropsy tumors, H&E staining of tumors, paraffin-embedded tumor sections (5 μm) on glass slides were processed for immunohistochemistry using primary DyLight 488-conjugated rat monoclonal anti-mouse CD31/PECAM-1 antibody, primary anti-E-cadherin, and N-cadherin antibodies, followed with polyclonal goat anti-rabbit Alexa Fluor^® 488 secondary antibody in 1% BSA in PBS blocking solution and Entellan^® rapid mounting medium. Background control (2nd Ab ctrl) sections were prepared without the primary antibodies. Stained tissue sections were photographed using an AxioCamMRm3-2 fluorescence camera attached to a Zeiss Imager M2 fluorescence microscope at 200× magnification. Images are representative of at least five fields of view from two tumor sections. (B) Quantitative analysis was done by assessing the density of tumor staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the figure represents the mean (± SEM) corrected density of tumor staining within the respective images; (C) Enlarged visualized image of live necropsy tumors; (D) Tumor growth rates for individual mice (mouse label A1, A2, and A4 for the control cohort; B1–4, 30 mg/kg OP cohort; and C1 and C3, 50 mg/kg cohort); (E) Mean ± SEM of live necropsy tumor weight per mouse body weight; Statistical analysis was carried out using GraphPad Prism, and results were compared by unpaired t-test.
Abbreviations: BSA, bovine serum albumin; H&E, hematoxylin and eosin; ip, intraperitoneally; ns, not significant; OP, oseltamivir phosphate; PBS, phosphate-buffered saline; SEM, standard error of the mean.

Figure 5 (A) Percent survival of indicated cohorts of mice taken from Figure 4D and (B) mean ± SEM of live necropsy spleen, pancreas, heart, and liver weight per mouse body weight for each of the cohorts in Figure 4.

Note: Statistical analysis was performed using the Log-rank (Mantel–Cox) test.
Abbreviations: OP, oseltamivir phosphate; SEM, standard error of the mean.

Figure 6 (A) H&E staining of necropsy lung for number of metastatic clusters per lung (B) in paraffin-embedded tissues taken from xenograft A2780 tumor-bearing RAGxCγ double mutant mice.

Notes: Mice were implanted with 0.5×10⁶ MDA-MB-231 TNBC cells cutaneously on the rear back flank, and OP treatment began daily (ip) 10 days postimplantation, when tumors reached 10–20 mm³. Paraffin-embedded tissue sections (5 μm) on glass slides were processed for H&E staining for each mouse necropsied at indicated necropsy day postimplantation. Stained tissue sections were photographed using AxioCam MRc5 digital color camera attached to a Zeiss Imager M2 fluorescence microscope at 400× magnification. Images are representative of at least five fields of view from two tissue sections. (B) Metastatic lung clusters as representative in the insert were microscopically counted per tissues sections (5 μm) and plotted in the graph. Statistical analysis was carried out using GraphPad Prism and results were compared by unpaired t-test. (C) Mean ± SEM of live necropsy lung weight per mouse body weight for each of the cohorts.
Abbreviations: H&E, hematoxylin and eosin; ip, intraperitoneally; OP, oseltamivir phosphate; SEM, standard error of the mean; TNBC, triple-negative breast cancer.