Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

These complementary techniques evaluate and measure the permeability of tumor vasculature using high-molecular weight Dextran. The fixed-tissue protocol offers larger tumor coverage, is high throughput, and does not require multiphoton imaging equipment. On the other hand, the intravital imaging protocol offers real-time information about the kinetics, function, and dynamic interplay of the cells in the tumor microenvironment of metastasis or TMEM.

So the protocols presented here enable the direct measurement of TMEM-dependent vascular permeability which strongly correlates with cancer cell dissemination, therefore these protocols can be used to assess the metastatic potential of the tumors. To generate pieces of tumor tissue suitable for transplantation, begin with euthanasia of tumor-bearing mice as described in the text protocol. Place a Petri dish with DMEM/F12 cell culture medium on ice in the fume hood.

Bring the mouse to the fume hood and sanitize the mouse's abdomen with 70%ethyl alcohol. Using sterile gloves and surgical tools, remove the tumors and place them into the Petri dish. Cut up the tumor into small pieces, discarding any necrotic portions while they are in the Petri dish.

To transplant the tumor pieces into recipient hosts, anesthetize mice that have been raised with genetically engineered, fluorescently labeled macrophages as described in the text protocol. Reduce the anesthesia to approximately 3%isofluorane and apply ophthalmic ointment to the eyes of the mouse to prevent drying. Remove hair from over the fourth mammary gland of the mouse.

After cleaning the skin with antiseptic, use sterilized instruments to make a small incision approximately two to three millimeters just inferior to the fourth nipple. Dissect until the mammary fat pad is exposed. Take a tumor piece from the Petri dish and coat in artificial extracellular matrix.

Transplant tumors underneath the fourth mammary fat pad. Close the incision using cyanoacrylate adhesive. Finally, add one milliliter of enrofloxacin antibiotic to the animals'drinking water.

Anesthetize the mouse and insert a tail vein catheter as described in the text protocol. Make a longitudinal midline incision starting immediately superior to the genitals and carry the incision up to the level of the superior aspect of the fourth mammary gland. Carry the incision transverse to the superior aspect of the fourth mammary gland.

It is critical to avoid compromising the blood supply at this point. Stabilize the skin flap by affixing a small piece of rigid rubber measuring two by two centimeters to the skin-side of the flap. The tumors should be in the center of the area being stabilized by the rubber.

Apply a small film of cyanoacrylate to the outer rim of the custom-made imaging window frame. Now apply a small droplet of PBS to the center of the cover glass. Avoid contact between the cyanoacrylate on the window frame and the PBS on the glass to avoid premature polymerization of the cyanoacrylate.

Dry the surrounding flap tissue with a laboratory wipe. Affix the small imaging window to the tissue flap with the tumor at the center of the clear aperture. Transfer the anesthetized mouse and tail vein catheter to the microscope stage.

Use extreme caution to ensure the tail vein catheter does not fall out. At this point, perform longterm intravital imaging as described in the text protocol and in our previous JoVE publication. For higher throughput analysis and larger tumor coverage, use immunofluorescence in fixed tissue to assess the TMEM-mediated vascular permeability.

To perform immunofluorescence staining, first prepare the samples as described in the text protocol, then perform antigen retrieval by heating the sections submerged in citrate close to the boiling point for 20 minutes. After letting the samples cool to room temperature for 15 to 20 minutes, wash the samples in PBS three times for two minutes each wash. Block for 60 to 90 minutes in blocking buffer, then incubate samples with a mixture of primary rat and rapid antibodies which target endomucin and tetramethylrhodamine respectively.

Following incubation, wash the samples three times in PBST for two minutes each. Incubate samples with a mixture of secondary donkey antibodies against Rat IgG and rapid IgG, then wash the samples three times in PBST for two minutes each. Perform a routine DAPI staining and mount the slides using a glycerol-free hard mounting medium.

Store the samples in a dark place until scanning. For optimal results, scan the slides on a digital whole-slide scanner. To perform image analysis, capture 10 high-power fields per case using any software suitable for digital pathology.

Save the endomucin and TMR channels separately as TIFF. Using ImageJ, upload the TIFF files and convert them into 8-bit images. Threshold the 8-bit images to the level of the negative control and generate two binarized images showing the endomucin and TMR masks.

From the Binary tools, select Fill Holes on the endomucin mask. Generate the thresholded Dextran image and save it as Dextran ROI. Also, generate the thresholded endomucin image and save it as Vascular ROI and generate the inverted endomucin image and save it as Extravascular ROI.

Next generate the intersected Extravascular ROI and Dextran ROI image and save it as Extravascular Dextran ROI. Finally save the entire image as Tumor ROI. Divide the Extravascular Dextran ROI area by the Tumor ROI area and multiply by 100 to generate the percent area that the Extravascular Dextran covers in the entire tumor.

Repeat the process for 10 to 20 high-power fields per case and generate an average Extravascular Dextran for each case. Shown here is an image of a healthy, developing mammary gland within a transgenic animal. The vasculature is labeled by TMR Dextran in red, mammary ductile epithelium is labeled by the fluorescent protein dendra2 in green, and macrophages are labeled by a cyan fluorescent protein.

In contrast, a transplanted dendra2-labeled invasive ductile carcinoma is shown in an animal with labeled macrophages and vessels. Healthy lung tissue was visualized through a lung-imaging window within a mouse with labeled vasculature. Lung metastases are evident following injection of tumor cells into a transgenic animal with labeled macrophages and vasculature.

Shown here are stills from a time-lapsed intravital imaging movie of high-molecular weight Dextran-labeled neo-angiogenic vessels within a transplanted dendra2-labeled invasive carcinoma of the breast. The dotted yellow line denotes the outline of the transient vascular leakage area before, during, and after the leakage event. A TMEM doorway is captured in live imaging.

Multichannel immunofluorescence shows endomucin, high-molecular weight Dextran, their merged image along with DAPI, as well as the corresponding thresholded blood vessel and Extravascular Dextran masks. The corresponding sequential section of TMEM immunohistochemistry is also shown. The vascular profile away from TMEM appears as a non-leaky vascular profile, whereas the TMEM-associated vascular profile appears as a leaky vascular profile.

It is very important to avoid cutting the blood vessels supplying the tumor during the surgical exposure of the tumor. The methodology described here can be combined with drug treatment, same that inhibiting TMEM function, preventing vascular permeability, or even normalizing tumor vasculature to measure their impact and efficacy. With this protocols we have previously demonstrated that chemotherapy enhances vascular permeability and cancer cell dissemination and actually that these pro-metastatic changes can be circumvented by co-administration of inhibitors of TMEM function.