Summary of optimal cell number determination in the development of the metastatic mouse model.

<p>(<b>A</b>) Chart indicating the benefits of optimizing cell number for primary tumor take and metastatic progression in the 4T1 breast cancer model. (<b>B</b>) Percent tumor take in immune-proficient BALB/cJ mice implanted with 4T1-Luc2GFP cells into the number four mammary fat pad. Data are actual percent tumor take based on number of animals indicated above each bar from no fewer than nine independent experiments. Micrometastasis (<b>C</b>) and macrometastasis (<b>D</b>) in lung six weeks after implantation. Data are mean ± <i>SEM</i> from two independent experiments with <i>n</i> = 5 for experiment one, <i>n</i> = 10 for experiment two.</p

Review of literature on the 4T1 model being used for metastasis evaluation.

<p>Review of literature on the 4T1 model being used for metastasis evaluation.</p

Growth of 4T1-Luc2GFP primary tumors: Timecourse.

<p>Primary tumor growth was recorded over a five week period from BALB/cJ mice implanted with 7,500 4T1-Luc2GFP cells into the mammary fat pad. (<b>A</b>) Tumor volume (mm³). (<b>B</b>) Tumor weight (mg). (<b>C</b>) Animal weights (g). Tumor volumes were calculated using the ellipsoidal method, volume (mm³) = 0.52×length × width². All data are mean ± <i>SEM</i> from two independent experiments with 5–8 mice for experiment one and 7–11 mice for experiment two and are fitted to a Gomperzian growth curve by Prism 6.0 software.</p

Efficacy of traditional chemotherapeutic agent in the 4T1 Luc2GPF mouse model.

<p>BALB/cJ mice implanted with 7,500 4T1-Luc2GFP cells were treated with the maximal tolerated dose (MTD) of doxorubicin, the dose shown not to result in any gross toxicity to the animal but shown to result in a small but insignificant loss in weight over the course of the experiment; or with carrier (5% Pharmasolve and 5% Solutol HS in saline), or with injectable saline as control once a week after visible primary tumor formation, for five weeks. (<b>A</b>) Flowchart indicating timing of cell implantation, doxorubicin treatments, and experimental determinations. (<b>B</b>) Tumors volume (mm³) over time measured by calipers. (<b>C</b>) End weight of tumors (mg), (<b>D</b>) Lung micrometastases. (<b>E</b>) Lung large micrometastases. (<b>F</b>) Lung macrometastases. Data representative of two independent experiments <i>n</i> = 15 mice for first experiment, and <i>n</i> = 10 mice for second experiment. Tumor volumes were calculated using the ellipsoidal method, volume (mm³) = 0.52 × length × width². Data are mean ± <i>SEM</i> and a two way ANOVA/Tukey post-test was performed (*p<0.05 and **p<0.01).</p

Development of lung metastasis in the 4T1 Luc2GFP mouse model: Timecourse.

<p>(<b>A</b>) Fluorescent image of a fully vascularized primary tumor removed five weeks after 7,500 4T1-Luc2GFP cells (green in all images) implanted into BALB/cJ mammary fat pad. Vasculature (red) in primary tumor (<b>A</b>) and five weeks lung metastasis (<b>F</b>) are indicated by arrows and was achieved by retro-orbital injection of tetramethylrhodamine labeled 2×10⁶ MW dextran. At weeks one and two (<b>B</b> and <b>C</b>) micrometastases, which are defined as single-to small clusters of cells, are present in lungs. Large micrometastases lacking blood vessels are present in lungs by weeks three and four (<b>D</b> and <b>E</b>), and by week five (<b>F</b>) macrometastases containing visible blood vessels are present in the lungs.</p

Discovery of Antitubulin Agents with Antiangiogenic Activity as Single Entities with Multitarget Chemotherapy Potential

Antiangiogenic agents (AA) are cytostatic, and their utility in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of vascular endothelial growth factor receptor-2 (VEGFR2) inhibitors with antitubulin agents have been particularly successful. We have discovered a novel, potentially important analogue, that combines potent VEGFR2 inhibitory activity (comparable to that of sunitinib) with potent antitubulin activity (comparable to that of combretastatin A-4 (CA)) in a single molecule, with GI₅₀ values of 10^–7 M across the entire NCI 60 tumor cell panel. It potently inhibited tubulin assembly and circumvented the most clinically relevant tumor resistance mechanisms (P-glycoprotein and β-III tubulin expression) to antimicrotubule agents. The compound is freely water-soluble as its HCl salt and afforded excellent antitumor activity <i>in vivo</i>, superior to docetaxel, sunitinib, or Temozolomide, without any toxicity

Development and quantification of lung and brain metastases: Timecourse.

<p>Micrometastases examination in lungs and brain of BALB/cJ mice implanted with 7,500 4T1-Luc2GFP cells into mammary fat pad. Lungs and brain excised, examined for metastases, degree of vascularity, and imaged at weeks two-five. (<b>A</b>) Lung micrometastases. (<b>B</b>) Lung large micrometastases. (<b>C</b>) Lung macrometastases. (<b>D</b>) Brain micrometastases. (<b>E</b>) Brain large micrometastases. All data are mean ± <i>SEM</i> from two independent experiments with 5–8 mice for experiment one and 7–11 mice for experiment two.</p