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Minimal dosing of leukocyte targeting TRAIL decreases triple-negative breast cancer metastasis following tumor resection

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Science Advances  24 Jul 2019:
Vol. 5, no. 7, eaaw4197
DOI: 10.1126/sciadv.aaw4197
  • Fig. 1 ETL kills 4T1 breast carcinoma cells more efficiently than ST under static conditions.

    (A) ETL formulation schematics. (B) Size distribution analysis of ETL formulation using a Zetasizer. (C) To assess the ability of ETL to target and kill 4T1 cancer cells under static cell culture conditions, 300 ng of TRAIL per milliliter of ETL was added to 1 × 105 4T1 cells/ml for 24 hours. Cells were then washed and assessed for their viability. Percentage of annexin V and propidium iodide (PI) double-negative 4T1 cells after 24 hours of ST, NL, or ETL treatment at 300 ng/ml TRAIL concentration is plotted (mean ± SEM; n = 3). **P < 0.005.

  • Fig. 2 Fluid shear stress significantly increases TRAIL-induced 4T1 breast carcinoma cell death in vitro.

    4T1 breast cancer cells (5 × 105) labeled with CellTracker dye were spiked into 0.5 ml of whole blood and treated with ST, NL or, ETL (300 ng/ml of TRAIL per milliliter) following exposure to shear flow in a cone-and-plate viscometer, at a shear rate of 150 s−1for 2 hours. Buffy coat containing 4T1 cells was isolated from whole blood by Ficoll density centrifugation, washed, and resuspended in calcium-saturated phosphate-buffered saline (PBS). (A) Representative annexin V/PI fluorescence-activated cell sorting (FACS) plots of 4T1 cancer cells sheared with ST, NL, or ETL (300 ng of TRAIL per milliliter) after 2 hours. The ETL-treated samples showed a marked loss of intact CellTracker-positive cancer cells, contributing to the notable reduction in cell numbers included in the analysis. (B) Percentage of annexin V and PI double-negative 4T1 cells after treatment with ST, NL, or ETL at a TRAIL concentration of 300 ng/ml under static (for 24 hours) or shear flow (2 hours) conditions (mean ± SEM; n = 3). (C) Representative micrographs of cells in the isolated buffy coat after treatment with ST, NL, or ETL at a TRAIL concentration of 300 ng/ml under shear flow for 2 hours [blue represents 4′,6-diamidino-2-phenylindole (DAPI)/nucleus; red represents 4T1 cells marked with CellTracker dye; scale bars, 50 μm]. *P < 0.05 and **P < 0.005.

  • Fig. 3 Minimal treatment of ETL decreases metastatic burden and prolongs survival in the 4T1 breast carcinoma model after tumor resection.

    (A) Timeline of in vivo experiment. i.v., intravenous; i.p., intraperitoneal. (B) Photomicrographs (H&E stain) of the isolated primary tumors taken (scale bars, 1 mm) to show the tumor size and central necrosis in ST-, NL-, and ETL-treated mice followed by (C) graphical representation of the ratio of necrotic area to tumor area after digitization and image analysis. (D) Antimetastatic effect of ETL in vivo, as shown by IVIS imaging of bioluminescence in 4T1 metastatic breast carcinoma mice that received mammary fat pad transplant of 4T1-luc cells (4T1 cells stably expressing luciferase) and were treated with three injections of ST, NL, or ETL (50 μg per injection; before, during, and after tumor resection within a period of 6 days; n = 10). IVIS imaging was performed at day 13 and day 8 after the first and last injections, respectively. Graphical representation of quantitation of IVIS imaging of (E) overall luciferase signal. (F) Mouse body weight during the course of the in vivo study at indicated time points (Fig. 4A; mean ± SEM; n = 10). (G) Survival of 4T1 metastatic breast carcinoma mice that received mammary fat pad transplant of 4T1-luc cells and were treated with three injections of ST, NL, or ETL (50 μg per injection), designating the transplantation date as day 0 (n = 10). (H) Photomicrographs of the lung (scale bar, 500 μm) and liver (scale bar, 100 μm) to show metastatic foci in NL and ETL treatment groups, respectively, with a focus of vascular space invasion in lung compared to the small metastatic foci in the liver (T) with extramedullary hematopoiesis (asterisks). (I) Photomicrographs of distal femur and proximal tibia (scale bars, 250 μm) to show bone metastasis. *P < 0.05 and **P < 0.005.

  • Fig. 4 ETL treatment reduces disseminated 4T1 metastatic cells in circulation in vivo.

    To investigate whether ETL treatment can target metastatic cancer cells flowing in the circulation of mice, 30,000 4T1-mCherry cells were injected in the fourth mammary fat pad of 12-week-old healthy BALB/c mice (n = 4 per group) followed by treatment with three injections of ST, NL, or ETL (50-μg TRAIL dose). The time and mode of injections were identical to those specified for Fig. 3A. Tail bleeding was performed at day 15 (before treatment), day 20 (after surgery and two injections), and day 25 (after treatment) to collect peripheral blood from mice. (A) Representative FACS plots of peripheral blood cells from the ST, NL, or ETL treatment group at different time points. The presence of disseminating cancer cells in mouse circulation is shown by mCherry-positive cells in the plots. (B) Representative micrographs of cells in the isolated peripheral blood at day 20 from mice with 30,000 cells injected in the fourth mammary fat pad at day 0 (blue represents DAPI/nucleus; red represents mCherry-positive disseminated cancer cells). (C) Graphical representation of quantitation of 4T1-mCherry–positive cells in peripheral blood using flow cytometry in the ST, NL, or ETL treatment group (n = 4) at different time points. (D) Graphical representation of fold change in the percentage of mCherry-positive cancer cells in the circulation in the ST, NL, or ETL treatment group (n = 4) at different time points.

  • Fig. 5 Minimal administration of ETL is tolerable and safe in mice.

    To monitor the safety profile of ETL administration in mice, 12-week-old healthy BALB/c mice (n = 3 per group) were treated with three injections of PBS/CTRL or 50 μg of ETL. The time and mode of injections were identical to those specified for Fig. 3A. The mice were monitored and euthanized 24 hours after the final injection for the following analysis: (A) total mouse body weight, (B) relative liver weight, (C) relative organ weight (spleen, kidney, and heart), (D) white blood cell count, (E) total neutrophil count, (F) total lymphocyte count, (G) hemoglobin levels, and (H) platelet count in peripheral blood of mice (mean ± SEM; n = 3). (I) Serum biochemical profile in mice (serum was collected at 24 hours following the last injection; ALT, alanine aminotransferase; Creat, creatinine; BUN, blood urea nitrogen; AST, aspartate aminotransferase; Amyl, amylase; mean ± SEM; n = 3). Representative photomicrographs of spleen histology sections of (J) the splenic white pulp area and (K) the splenic red pulp area from the mice (n = 3). *P < 0.05.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw4197/DC1

    Fig. S1. ETL kills 4T1 breast carcinoma cells in a dose-dependent and more efficient manner than ST under static conditions.

    Fig. S2. PL, Cur, and ASP do not sensitize 4T1 cells to liposomal TRAIL treatment under static conditions.

    Fig. S3. Tumor resection efficiently removes 4T1 primary tumor burden in mice.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. ETL kills 4T1 breast carcinoma cells in a dose-dependent and more efficient manner than ST under static conditions.
    • Fig. S2. PL, Cur, and ASP do not sensitize 4T1 cells to liposomal TRAIL treatment under static conditions.
    • Fig. S3. Tumor resection efficiently removes 4T1 primary tumor burden in mice.

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