PTM Based Tumor Microenvironment Monitoring: A Method for Tracking Changes in ECM Rigidity Using Tumor Spheroids

1. Preparing 3D Tumor Spheroids Embedded in ECM

1. Prepare a work station with needed materials inside a laminar flow hood.
2. Prepare a diluted mixture of carboxylate-modified 1 μm diameter fluorescent tracer probes by adding 2 parts stock probes (2% solids) to 25 parts sterile water.
3. Remove a bottle of 3.1 mg/ml bovine collagen from the refrigerator and place it on ice.
4. Aliquot 125 μl of collagen into an empty 2 ml vial.
5. Add 50 μl of diluted tracer probe solution to the vial containing the collagen and vortex briefly to distribute probes.
6. Add 235 μl of appropriate cell culture media containing phenol red (which will turn yellow) for a total volume of 410 ml and vortex briefly before removing 205 ml (half the total volume) and placing it in a new 2 ml vial. Vial 1 will contain the tumor spheroid and Vial 2 will be a control mixture.
7. Add ~2 μl of 1 M NaOH to Vial 1 to bring the solution back to neutral pH (culture media containing phenol red should return to red). Note that this will cause the mixture to begin curing if it is not kept on ice. Vortex briefly to mix, then return to ice rack immediately.
   NOTE: The spheroid is barely visible to the naked eye after 9 days of culture and its removal from the agarose bed is a delicate task. Use of a dissection microscope may be helpful for some users. Maintaining the integrity of the spheroid during transfer is paramount.
8. Using a wide-mouth pipette tip, gently remove 40 μl of media from the well containing the tumor spheroid. Retain this 40 μl while conducting the next step.
9. Check the well to see if the spheroid was removed in the previous step. If it was, add the 40 μl containing the spheroid to Vial 1. If not, place the 40 μl back into the well and repeat the previous step.
10. Gently stir Vial 1 (do not risk vortexing, it may damage the spheroid) before transferring the mixture in 60 μl portions into three separate wells of a 96-well plate. Inspect each well with a microscope after adding mixture to determine which well contains the spheroid.
11. Add ~2 μl 1 M NaOH and 40 μl of cell culture media to Vial 2 and vortex before aliquoting 60 μl of this mixture to an empty well in the 96-well plate and labeling as a control.
12. Place the plate in a 37 °C incubator to cure for at least 1 hr.

2. Construct Grid of Sample Points and Take a Video at Each Point

1. Transfer the sample plate from the incubator to the microscope stage. Allow 10 min for the sample to equilibrate with the room temperature if a heated stage is not available.
2. Observe the sample with low powered objective lenses to make sure it is intact and ready for imaging. Determine the tumor position within the well.
3. Decide how many sample points to take. Typically, 20 sample points in each well, distributed in concentric rings around the spheroid will produce adequately detailed results.
4. Move the stage to each desired position and use microscope interfacing software to record the x and y coordinates (or record coordinates manually if interfacing software is unavailable).
5. Switch the microscope to a high-powered objective lens (typically 100X), and select the appropriate filter cube for the excitation wavelength of the tracer probes. Use the list of points created in step 2.4 to move to the first point in the grid.
6. Adjust the focus to find the bottom of the well, then move up to find a field of view (fov) containing several in-focus tracer probes.
7. Observe the intensity histogram and adjust the exposure intensity and time to give the greatest dynamic range possible while ensuring that the image does not become saturated.
8. Obtain a video sequence at a frame rate of 20-30 msec per frame (approximately 800 frames or 16-24 sec in length is recommended to provide sufficient statistics for robust MSD calculation balanced with the need for minimizing acquisition time at each spatial grid point) and save with an appropriate convention. During the recording, do not touch the microscope or table.
9. Repeat steps 2.6 to 2.9 for each sample point in the grid.
10. Repeat steps 2.3 to 2.10 for each well in the experiment.