Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Cancer cells have a remarkable ability to internalize nutrients, such as ATP, from the extracellular environment. Our protocol permits us to demonstrate ATP internalization and to visualize ATP localization within cells. This high resolution imaging of internalized ATP, within macropynozomes, is ideal for understanding especial localization and completing quantity of analysis of internalization.

The protocol describes different experimental applications to study ATP internalization. This method can be used to study different mechanisms of cellular internalization, including macropinocytosis and exosome-mediated endocytosis. For metabolic diseases, ATP internalization in non-cancer cells can be studied with this protocol.

To begin, seed the cancer cells in a 225 square centimeter flask, containing DMEM supplemented with FBS and antibiotics. Allow the cells to grow at 37 degrees Celsius and 5%carbon dioxide in the cell culture incubator. Once the 80%confluence is achieved, detach the cells and pellet down the detach cells by centrifugation, and resuspend the cells in ice cold PBS to achieve the cell density of 5 times 10 to the 6 cells per 100 microliters of PBS.

Transfer the cell suspension to a 1.5 milliliter microcentrifuge tube. Clean the injection site on the flank of an immunodeficient mouse, with 75%ethanol, and wipe excess ethanol with a delicate task wipe. Draw the cells into a one milliliter latex-free syringe, equipped with a 27 gauge precision glide needle.

For subcutaneous injection, hold the needle at about a 10 degree angle to the skin and insert the needle tip with the bevel facing up underneath the skin, keeping only one to two millimeters of the needle outside the skin. Dispense the cells from the syringe slowly over approximately 10 seconds. After injecting the entire volume, hold the needle in place for three to five seconds and then withdraw the needle.

Apply gentle but firm pressure to the injection site for three to five seconds with fingers, to prevent leaking of the injected content. Monitor and measure the tumor growth using Vernier calipers, until the tumors reach a volume of 200 to 500 cubic millimeters. Dissolve 300 microliters of 16 milligrams per milliliter high molecular weight fluorescent dextran and serum-free DMEM, and incubate in a water bath, tempered at 37 degrees Celsius for 30 minutes, then, centrifuge the dextran solution and transfer the supernatant to a new 1.5 milliliter microcentrifuge tube.

At 40 microliters, a one millimolar non hydrolyzable fluorescent ATP analog stock to 160 microliters of serum-free DMEM, to make a 0.2 millimolar nonhydrolyzable fluorescent ATP solution. Prepare the treatment solutions of DMEM or eight milligrams per milliliter's high or low molecular weight fluorescent dextran, with or without 100 micromolar nonhydrolyzable fluorescent ATP in DMEM, by mixing the stock solutions prepared previously. Use a one milliliter syringe to collect 50 microliters of one treatment solution.

Inject the solution directly into the xenograft tumor and repeat for four biological replicates of each treatment. After euthanizing the mouse and isolating the tumor tissue, use a perforated spoon to scoop up the resected tumor tissue and immediately place the tissue into a freezing medium and roll the tissue, ensuring that the tissue remains submerged in the medium bathes, all the tissue surfaces. Carefully place the tissue into the embedding mold containing the freezing medium.

Place the corresponding label tag vertically into the freezing medium or mold, ensuring that the written label is visible outside the medium. When the freezing medium turns opaque white, remove the tissue block from the mold, place the tissue block on dry ice and repeat freezing for each tumor section. Store the tissue blocks at minus 80 degrees Celsius for several months before the cryosectioning procedure.

After making the slides of the tissue blocks, fix the tissue section slides in 95%ethanol at minus 18 degrees Celsius for five minutes. Wash the fixed section with PBS for five minutes. Add 10 to 50 microliters of an aqueous mounting medium with DAPI to the tissue sections, according to the size of the sections, and place a glass cover slip over the tissue section.

After 12 to 24 hours of mounting, identify regions of interest of the fixed tumor sections and acquire the images using fluorescence microscopy as demonstrated previously. After two to 24 hours of the human tumor cell fixation and standing with DAPI, capture the images using an epifluorescence imaging system and data acquisition software. Place the slide on the stage of an upright epiflorescence microscope in binocular mode.

Open the imaging program, select the 10 times objective and adjust the stage to define focus. Scan the slide from left to right in a serpentine manner to identify the regions of interest. Select the 40 times objective, and use the toggle on the microscope to switch from binocular to image capture mode.

Click on the live quality icon to view and subsequently acquire the images. Use the OC panel on the acquisition toolbar to define the exposure parameters for each filter cube or fluorescent channel, then, use the multi-channel acquisition toolbar to acquire a three channel image with the defined exposure settings. Alternatively, multi-channel images can be acquired manually by toggling between the filter cubes, setting the exposure time, closing or opening the shutter between image acquisition for each channel and overlaying each image taken for individual channels.

Save the image as nd2 file. Save the TIF files, including the merged channel image and individual channel images. Use the object count feature on the annotations and measurements toolbar to count the number of NHF-ATP, TMR high molecular weight fluorescent dextran and TMR low molecular weight fluorescent dextran positive cells on a saved nd2 image file and export the data to a spreadsheet through the analysis program.

This protocol was developed for spatial, temporal and quantitative analysis of the cellular internalization of nonhydrolyzable ATP. The intracellular internalization of nonhydrolyzable fluorescent ATP was demonstrated by colocalization of nonhydrolyzable fluorescent ATP with high or low molecular weight fluorescent dextran in vitro, ex vivo and in vivo. To ensure that tumor cells retained internalized ATP, limited experimental time for all intratumoral injection to cryo-embedding.

Also account for intratumoral variation by imaging tissue throughout the tumor. Future iterations of our method could involve real-time visualization of the E-ATP internalizing process, revealing information about macropynocytotic kinetics and trafficking in specific tissues.