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

- In tumor invasion, the peripheral tumor cells undergo epithelial-to-mesenchymal transition or EMT, resulting in the local degradation of the extracellular matrix or ECM. We can study the interaction of tumor cells with ECM in vitro.

For studying these interactions, transfer a preformed spheroid derived from pancreatic tumor cells to a plate containing collagen scaffold along with embedded fluorescent tracer probes and culture media. Tumor spheroid releases proteases, which degrade collagen and reduce ECM rigidity, resulting in the free movement of tracer probes in the ECM.

Incubate the plate for desired time duration. Observe the sample under a light microscope to look at the grid of sample points in the collagen scaffold and record video data from them. The region of the matrix near the spheroid shows maximum collagen degradation. Therefore, it has increased probe motion. In contrast, the area farther from the spheroid shows less collagen degradation, resulting in restricted movement of probe molecules.

This technique of examining the rheological properties of ECM by measuring the trajectories of tracer particles is known as particle-tracking microrheology or PTM. In the following protocol, we will perform a sphere formation assay to identify pancreatic cancer stem cells and evaluate the effect of metformin.

To begin, prepare a workstation inside a laminar flow hood with two 2-milliliter vials. Vial 1 will contain the tumor spheroid, and vial 2 will be a cell-free control. Prepare a diluted mixture of carboxylate-modified, 1-micron-diameter fluorescent tracer probes by adding 2 parts stock probes to 25 parts sterile water.

Remove a bottle of 3.1 milligrams per milliliter bovine collagen from the refrigerator and place it on ice. Aliquot 125 microliters of collagen followed by 50 microliters of diluted tracer probe solution into vial 1. Vortex briefly to distribute the probes.

Then, add 235 microliters of appropriate cell culture media containing phenol red for a total volume of 410 microliters and vortex briefly before removing 205 microliters and placing it into vial 2. Next, add approximately 2 microliters of 1 molar sodium hydroxide to vial 1 to bring the solution back to neutral pH. Vortex briefly to mix. Then, return to the ice rack immediately so that the mixture does not begin curing.

Gently remove 40 microliters of media from the well containing the tumor spheroid. Retain this 40 microliters while conducting the next step. Check the well to see if the spheroid was removed in the previous step. If not, place the 40 microliters back into the well and repeat the previous step.

If it was, add the 40 microliters containing the spheroid to vial 1. Gently stir vial 1 before transferring the mixture in 60-microliter portions into three separate wells of a 96-well plate. Inspect each well with a microscope after adding the mixture to determine which well contains the spheroid.

Then add approximately 2 microliters of 1 molar sodium hydroxide and 40 microliters of cell culture media to vial 2, and vortex before aliquoting 60 microliters of this mixture to an empty well in the 96-well plate and labeling it as a cell-free control. Place the plate in a 37 degree Celsius incubator to cure for at least one hour.

To construct a grid of sample points, first, transfer the sample plate from the incubator to the microscope stage. Allow 10 minutes for the sample to equilibrate to room temperature if a heated stage is not available. 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, and document this with a transmitted light image for subsequent spatial coregistration.

Typically, 20 sample points in each well distributed in concentric rings around the spheroid will produce adequately detailed results. Move the stage to each desired position and use microscope interfacing software to record the x- and y-coordinates.

Switch the microscope to a high-powered objective lens, and select the appropriate filter cube for the excitation wavelength of the tracer probes. Using the list of created points, move to the first point in the grid. Adjust the focus to find the bottom of the well before moving up to find a field of view containing several in-focus tracer probes.

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. Obtain a video sequence at a frame rate of 20 to 30 milliseconds per frame and save with an appropriate naming convention. During the recording, do not touch the microscope or table.

Repeat the recording for each sample point in the grid. Then proceed to repeat the process to construct a grid for each well in the experiment and repeat the entire process for each time point over the duration of longitudinal monitoring.