Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging

Abstract

We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment.

Keywords: breast cancer; cell migration; intravital imaging; mammary imaging windows.

Figure 1.

Polydimethylsiloxane (PDMS) double-flanged mammary imaging window (MIW) and stainless steel mammary imaging window (SS MIW) in FVB-c-fms-EGFP mice. (A) A PDMS MIW designed to be surgically implanted over the fourth mammary gland in an FVB-c-fms-EGFP mouse. The MIW contains 2 PDMS double-flanged rings that form a mount for a glass coverslip, shown in the oblique photograph on the left. A 6 mm-diameter No. 1 coverglass is affixed with silicone adhesive to a PDMS double-flange configuration. The PDMS flanges can be trimmed to appropriate size and shape. The double flanges, seen clearly in the lateral photograph on the right, allow for retraction of the skin to prevent skin growth over the MIW. (B) Surgical implantation of a PDMS MIW in an FVB-c-fms-EGFP mouse. The MIW is placed above the mammary gland and skin is tucked between 2 flanges of the MIW and secured with sutures. Although the MIW retracts the skin for up to 4 days, the growth of collagen by day 4 (C) hinders the field of view for imaging. (D) To image myeloid cells in vivo, a stainless steel MIW is placed over the mammary gland in an FVB-c-fms-EGFP mouse and secured with sutures (E). A 6 mm-diameter No. 1 coverglass is affixed with silicone adhesive to a steel substructure. Holes around the periphery allow the MIW to be sutured in place. (F) Intravital image at day 0 of migration of c-fms⁺ myeloid cells in a c-fms-EGFP mouse through the stainless steel MIW. Images were acquired with an LSM META 510 inverted confocal microscope with a 10X/0.5 Plan Neofluar objective. Bars, 100 µm. The panel shows an image of GFP-expressing myeloid cells in the mammary fat pad; the vasculature is labeled with Rhodamine Dextran (red). (G) By day 5, there is collagen deposition and increased migration of myeloid cells into the wounded area. Bars, 100 µm. (H and I) By day 9, there is tissue granulation, re-epithelialization and closure of the skin.

Figure 2.

Test of stainless steel mammary imaging windows in MMTV-PyMT CFP x c-fms-EGFP mice that present with tumors in the third or fourth mammary gland. (A) The stainless steel mammary imaging window is implanted over the third mammary gland of a MMTV-PyMT-CFP x c-fms-EGFP mouse and imaged on day 1 (B and C). (B and C) The panels represent images of GFP-expressing myeloid cells in the mammary fat pad, CFP-expressing tumor cells, and the vasculature is labeled with Rhodamine Dextran (red). (D) The stainless steel mammary imaging window is implanted over the fourth mammary gland of a MMTV-PyMT-CFP x c-fms-EGFP mouse and imaged on day 1 (E and F). The panels represent images of GFP-expressing myeloid cells in the mammary fat pad, CFP-expressing tumor cells, and the vasculature is labeled with Rhodamine Dextran (red). Bars, 100 µm. Images were acquired with an LSM META 510 inverted confocal microscope with a10X/0.5 Plan Neofluar or a 20X/0.75 Plan apochromat objective. The images were processed with LSM Imaging software and Adobe Photoshop.

Figure 3.

Progression of collagen buildup and re-epithelialization of the skin under the stainless steel – double-flanged PDMS MIW in c-fms-EGFP mice in the fourth mammary gland. (A) An 8 mm-diameter No. 1 coverglass is affixed with silicone adhesive to a PDMS double-flange configuration. A central steel reinforcing ring is positioned between the flanges. The PDMS flanges can be trimmed to appropriate size and shape. The mammary imaging window is surgically implanted over the fourth mammary gland. The skin is adhered to the double-flanged PDMS with Dermabond adhesive, and progressive images of the MIW on day 0 (stereoscope image) and day 1 to day 11 (digital images) are shown. High Gelling Alginate Dressing (3M) is placed in the wound (B) to prevent collagen deposition under the mammary window in a c-fms-EGFP mouse and covered with sterile adhesive (C). The dressing was changed twice a day and monitored for collagen growth. The adhesive was removed at day 3 (D, E) and found to delay collagen formation. The arrow in (E) indicates the field of view for intravital imaging in the next panels. (F and G) Intravital images of migration of c-fms⁺ myeloid cells in the c-fms-EGFP mouse 3 days after wounding and placement of the alginate dressing. The alginate dressing was removed, and images were taken of GFP positive myeloid cells with 10x (F) and 20x Plan APO objectives (G). Bars, 100 µm. The images were processed with LSM Imaging software and Adobe Photoshop.

Figure 4.

A PDMS double-flange “open” MIW with removable glass coverslip is adhered to a stainless steel washer and placed in MMTV-PyMT CFP x c-fms-EGFP mice that present with tumors in the fourth mammary gland. (A) A PDMS double-flange configuration with central steel reinforcing ring positioned between the flanges. An 8 mm cover glass assembly (see Fig. 4B) may be temporarily affixed to the MIW. The flanges can be trimmed to appropriate size and shape. (B) The 8 mm-diameter No. 1 coverglass is affixed with silicone adhesive to a steel substructure. (C) The 8 mm-diameter No. 1 coverglass is affixed with silicone adhesive to a steel substructure (see Fig. 4B). This assembly can be temporarily affixed to a PDMS double-flange configuration with a central steel reinforcing ring positioned between the flanges, as pictured. (C and D) Digital images of surgical implantation of the mammary imaging window over the fourth mammary gland. The skin is adhered to the double-flanged PDMS with Dermabond adhesive, and the opening of the MIW is filled with a gelling alginate dressing on day 0. (D) At day 1, the gelling alginate material has become saturated with exudate from the wounded area. (E) Image of GFP-expressing myeloid cells in the mammary fat pad and CFP-expressing tumor cells, and the vasculature is labeled with Rhodamine Dextran (red). To acquire these images, the PDMS double-flange mammary imaging window is implanted over the fourth mammary gland of a MMTV-PyMT-CFP x c-fms-EGFP mouse and imaged on day 1 after the gelling alginate material was removed. Bars, 100 µm. Images were acquired with an LSM META 510 inverted confocal microscope with a 20x Plan APO objective. The images were processed with LSM Imaging software and Adobe Photoshop.