Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

This method can be used to evaluate the therapeutic effects of NIR-PIT on thoracic tumors in a clinically relevant tumor model. The NIR-PIT procedures using the pleural disseminated cancer model are easy to understand and to perform. To set up a mouse dissemination model, use polystyrene foam to make a stopper, and disinfect it with 70%ethanol.

Attach the needle to the stopper with the tip extending five millimeters out of the stopper. Bend the tip of a 30-gauge needle with clean forceps or against a hard object cleaned with 70%ethanol, and fill a syringe with one times 10 to the six target tumor cells in 100 microliters of PBS. Confirm a lack of response to pedal reflex in an anesthetized, 19 to 21 gram, eight to 12 week old, female, homozygote, athymic nude mouse.

Insert the needle into the chest through the intercostal space, moving the needle up and down to avoid contacting the ribs. When the tip has passed through the intercostal space, position the syringe so that it is pressed against the mouse and inject the entire volume of target cells. After the injection, roll the mouse two to three times to spread the cells throughout the thoracic cavity, and return the mouse to its cage with monitoring until full recovery.

24 hours after cell injection and every day thereafter, inject the anesthetized tumor cell-injected mice with 200 microliters of 15 milligrams per milliliter of D-luciferin. After 10 minutes, place the mice into a bioluminescence imager, and open the Acquisition Control Panel in the imager software. Select Luminescent, Photograph, and Overlay.

Set the Exposure Time to Auto, the Binning to Small, the F/Stop to one for Luminescent and to eight for Photograph, and the Field of View to C.Click Acquire to image the bioluminescence. Set the Display Format to Radians, and select the circle from the Region Of Interest Tools in the Tool Palette panel. Click Measure Regions Of Interest to measure the surface bioluminescent intensity, and use Configure Measurement to select the values relevant to the experiment.

Export this data table as a csv file. Then use the total flux values for the bioluminescent intensity quantification in the file. Before performing near-infrared phototherapy of the tumor-injected mice, use a power meter to measure the light dose of a 690-nanometer wavelength laser, and adjust the output to 100 milliwatts per square centimeter.

24 hours before the treatment, intravenously injected 100 micrograms of antibody photosensitizer conjugate in 50 to 200 microliters of PBS via the tail vein of the tumor-injected animal. On the day of the phototherapy treatment, place the anesthetized conjugate-injected, tumor-laden mouse in the supine position, and shield the non-target sites with aluminum foil. When all of the shields have been placed, use a 100-joules-per-square-centimeter laser to irradiate the thoracic cavity with near-infrared light for about 30 seconds.

When the irradiation is complete and the mouse has awoken, return the animal to its cage, and measure the bioluminescence daily as demonstrated. In this representative analysis, the conjugation of anti-podoplanin antibody with IR700 was confirmed by SDS-PAGE analysis. After tumor cell injection, bioluminescence imaging and diffuse luminescence imaging tomography should be performed to determine which mice express sufficient luciferase activity in the chest cavity for further study.

At day five after injection, anti-podoplanin antibody IR700-injected mice demonstrate high IR700 fluorescence and luciferase activity in thoracic tumors, indicating that intravenously injected IR700-conjugated antibody reaches disseminated pleural tumor sites. Notably, pleural disseminated mice treated with near-infrared photoimmunotherapy demonstrate a decreased luciferase activity, while the relative light units in the control group exhibit a gradual increase in intensity. Since the required NIR irradiation energy depends on the cell line and antibodies, be sure to check the conditions in advance in vitro.