The overall goal of this procedure is to track in real time drug penetration and therapeutic response to the administration of a therapeutic drug. This is accomplished by first treating the animal with the drug. Next, the inguinal mammary gland containing the tumor is exposed by creating a skin flap.
Then the mouse is transferred to the microscopic stage and blood oxygen saturation and pulse rates are measured and an indwelling line for fluid and drug or dye administration is inserted. Finally, time-lapse images are acquired for later analyses. Ultimately, results can be obtained that show changes in cell death or tumor stroma interactions in response to a drug through live spinning disc confocal microscopy of mouse mammary tumors.
This method can help answer key questions in the tumor microenvironment field, such as how various stroma components of a tumor influence the response to therapy. Before beginning, see the written protocol for typical schedules for drug administration and imaging. Turn on a hot bead sterilizer and let it reach at least 200 degrees Celsius.
After washing the surgical tools in soap and water, including a pair of forceps with teeth, a pair of serrated forceps, and two pairs of scissors, use the hot bead sterilizer to sterilize them for at least 30 seconds. Then let them.Cool. Place a lab soaker on top of the styrofoam lid that will be used as the surgical platform.
Use laboratory tape to affix a nose cone with a line to the anesthesia system to the lab soaker and certain 18 gauge by one and a half inch needle through the tape into the styrofoam lid on either side of the nose cone to keep the nose cone secured in place. Set aside Betadine sterile gauze. Two 70%isopropanol wipes a microscope slide, super glue, and four pieces of half inch wide lab tape.
After anesthetizing the animal in an induction chamber, when it is breathing deeply and slowly transfer the mouse to the surgical platform, ventral side facing up. To avoid high pressure, open the anesthesia line to the surgical platform and then close the anesthesia line to the induction chamber. Reduce the concentration of isoflurane from 4%to 2.5%Check that the depth of anesthesia is sufficient for surgery by performing a footpad pinch.
If the animal does not react, use four pieces of lab tape to secure its limbs to the surgical platform and use an electric razor to remove the hair from the ventral surface. Use 70%isopropanol wipes, followed by Betadine applied to sterile gauze to disinfect the ventral surface of the animal using scissors and forceps with teeth. Make a subcutaneous ventral midline incision from about three millimeters above the urethra to the xiphoid process.
Take care to avoid puncturing or cutting through the peritoneum with the scissors and serrated forceps. Gently detached the skin from the peritoneal cavity with the inguinal mammary gland containing the tumor to be imaged Still attached. Position a glass microscope slide against the skin flap so that the bulk of the tumor sits flat once the mouse is placed on the stage, and so that it is not obstructed by the hind limbs.
Once the microscope slide has been properly positioned, use super glue to attach the slide to the external surface of the skin. Remove the laboratory tape securing the limbs of the animal. Open the anesthesia line to the microscope stage, and then close the anesthesia line to the surgical platform.
Quickly transfer the animal to the microscope stage Once the animal has been transferred, position and secure the anesthesia line and nose cone to ensure that the mouse remains anesthetized and is in a comfortable position. Reduce the concentration of isoflurane to 1.5%Expose the tumor so that it is positioned on top and in the center of one of the glass covered imaging ports on the microscope stage. Insert the indwelling intraperitoneal line with winged infusion set attached to the one milliliter syringe containing sterile PBS saline or saline with propidium iodide.
When the intraperitoneal line is inserted, inject the animal with 100 microliters saline with propidium iodide and continue to inject 50 microliters of saline with propidium iodide every hour until the imaging session is complete. Using the eye pieces, verify that the tumor is correctly positioned and gently taped down the microscope slide and the intraperitoneal line. The slide should be secured loosely enough that blood flow to the tissue is not obstructed.
Taping down the microscope slide helps to minimize imaging artifacts introduced by the animal's breathing to acquire time-lapse images. Use the open source software micromanager, compile and analyze the raw data using IRIS or other image analysis software. Fluorescently conjugated DExT strands can be used as a surrogate marker for drug delivery into tissues.
Shown here is the arrival of fitzy conjugated two megadalton dextran IV injected into a mouse expressing blue fluorescent protein in breast cancer cells. Following the arrival of the drug into the tumor, extravasation and diffusion can be imaged to determine intravascular half-life and drug distribution. Here, the extravasation and distribution of LOR conjugated 10 kilodalton dextran IV injected into a mouse expressing blue fluorescent protein in breast cancer cells and green fluorescent protein in myeloid cells is shown.
Imaging can provide information regarding the type of cell death that cancer cells undergo. Here, structural nuclear changes indicative of necrosis or apoptosis are observed in a mouse expressing green fluorescent protein conjugated to hisone two b. After doxorubicin treatment, chemotherapy induced cancer cell death frequently results in a reactive recruitment of immune cells into tumors.
Here is an example of this stromal response to acute treatment with doxorubicin in a mouse expressing blue and green fluorescent proteins in breast cancer and myeloid cells respectively. In addition to visualizing the overall stromal response to chemotherapy, specialized stromal responses can also be visualized here. The phagocytosis of necrotic cell material by a neighboring cell is shown Once mastered.
This technique can be done in 30 to 60 minutes and animals can be imaged for 12 to 40 hours if it is performed properly.
