Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

The overall goal of this procedure is to visualize the dynamic events within the tumor microenvironment via high resolution, intravital multiphoton mircrospy. This method can help answer key questions about the tumor microenvironment such as which dynamic events are involved in vascular permeability and multicellular interactions. The main advantage of this technique is that the kinetics of the events in the tumor microenvironment can be observed and measured.

Before beginning the procedure, turn on all of the microscope and laser components, including the two photon lasers and the detectors. And set the heating box to 30 degree celsius to prewarm the stage. Wipe the microscope stage and stage insert with 70%ethanol.

When the stage is dry, place a large drop of water on the 25 x 1.05 NA microscope objective to maintain optical contact with the cover glass and place a number 1.5 thickness cover glass over the insert imaging port. Then, cut a 30 centimeter piece of polyethylene tubing. Place the rubber pad on top of the cover glass.

Using forceps, break a 31 gauge needle off of its plastic fitting and insert the blunted end of the needle hub into the tubing. Insert and attached a 31 gauge needle into the other end of the PE tubing. Then, fill a one cc syringe with sterile PBS and attach the syringe to the 31 gauge needle.

Use the PBS to flush all of the air out of the tubing and needle. Then, the one one cc syringe with 200 microliters of the appropriate vascular label and another with the appropriate injectable protein. Before beginning the surgery, confirm the proper anesthesia with a lack of response to toe pinch and apply eye ointments to the anesthetized mouse.

Next, place the animal on a surgical platform under a heat lamp to increase the tail circulation. After two minutes, sterilize the tail with 70%ethanol and insert the tip of the 31 gauge catheter, two to three millimeters in to the lateral tail vein. Then, place a one centimeter length of lab tape onto the catheter to hold the needle parallel to the vein along the length of the tail.

When the catheter has been secured, swab the ventral and tumor surfaces with 70%ethanol. The, lift the skin over the abdomen and use sterile scissors to make an approximately one centimeter subcutaneous incision along the ventral midline of the abdomen, taking care to avoid puncturing the peritoneum. Gently cut the connective tissue attaching the mammary gland and tumor away from the peritoneum to expose the tumor.

Then, using the scissors and forceps, gently remove the fascia and fat from the exposed surface of the tumor. When the tumor has been exposed, transfer the mouse to the prewarmed imaging stage. Use lab tapes to secure the cover glass, then place the tumor in the hole of a rubber pad covering the cover slip over the microscope objective.

When the animal is in position, fill the rubber pad chamber with PBS to keep the tissue hydrated and to maintain optical contact with the cover slip. Place a pulse oximeter probe collar sensor around the neck to monitor the animal's vital signs and place the heating box over the animal. Then, use the microscope IPs to focus on the fluorescent tumor cells on the surface of the tumor and located area with flowing blood vessels.

Once a region of interest has been selected, switch the microscope to multiphoton imaging mode and use the focus adjuster to move the objective to the desired start location. Then, click on the Z position top button to set the upper limit of the z series and visualize the collagen fiber network at the surface of the tumor in the second harmonic generation channel. Then move the objective to the desired imaging depth and click the z position bottom button to set the lower limit of the z series.

Next, enter the desired value into the step size fields. Then, switch to the Time Lapse Panel and enter the appropriate lapse time. Once the imaging parameters had been determined, replaced the syringe attached to the catheter with a syringe of vascular labeling dye and slowly administer a maximum of 200 microliters of the dye solution, taking care that the solution enters the vein.

After the injection, return the PBS syringe to the catheter and begin the z step time lapse imaging. When all of the images have been obtained, replace the syringe of PBS with the syringe containing the injectable protein, taking care not to introduce any bubbles into the line. Then slowly inject the protein suspension into the mouse and replace the syringe with a syringe containing PBS.

Macrophages can be tracked with fluorescently labeled 70 kilodalton and dextran which acummulate in the phagocytic microphages. Dual labeling the tumor cells and the microphages allows the direct interaction with these two cell types to be visualize in real time. To visualize changes in the tumor vascular permeability, quantum dots can also be used to label the vascular space.

As they are large enough that they do not readily diffuse through the interarterial spaces. Continuous high resolution imaging facilitates the capture of the vascular permeability events and the quantification of the kinetics of the dextran extravasation and clearance. In this example field of view, the peak of the vacscular di-extravasation is observed between 29 and 34 minutes as evidenced by the extra vascular TMR signal.

Injecting additional vascular dyes or proteins to induce vascular permeability can provide insight into the signaling events regulating vascular permeability. Indeed, within the first few seconds of injection, 10 kilodaltons of dextran FITC can be observed extravasating from the vasculature, while the 155 kilodalton in dextran TMR remains confined to the vascular space. Once mastered, this technique can be completed in one hour if it is performed properly.

While attempting this procedure, it's important to remember to remove the connective tissue gently without damaging the surface of the tumor and to keep the tumor surface hydrated. Following this procedure of cellular dynamics such tumor cell migration can be visualized to answer additional questions about the directionality of tumor cell migration and intravasation. After watching this video, you should have a good understanding of how to visualize dynamic celluluar events in a tumor microenvironment by intervital multiphoton microscopy.