The overall goal of this procedure is to measure longitudinal changes in the mechanical properties of the extracellular matrix in three dimensional cancer models. This is accomplished by first harvesting cancer cells and growing the tumor steroid by an appropriate method until they have reached the desired size. The second step is to embed the tumor steroid in the collagen or other appropriate extracellular matrix.
Next, the grid of sample points is constructed and a video is taken at each point. The final step is to analyze the video data and compute and coregister real logical properties at each spatial point. Ultimately, this protocol uses analysis of tracer probe trajectories to longitudinally quantify spatial changes in extracellular matrix mechanics in a non-destructive manner.
The idea is that by, by bringing together imaging based measurements of extracellular matrix radiology with in vitro 3D tumor models, that that restore extracellular matrix interactions, this approach can provide quantitative insight into time dependent changes in the, in the mechanical microenvironment that accompany tumor growth invasion processes and, and response to treatment. The method shown in this video adopts an aros overlay approach to generate non-adherent 3D OIDs from the established pancreatic cancer cell line pan one. To begin mix 10 milliliters of cell culture grade water with 0.1 gram of aros to obtain a 1%aros solution.
Heat the aros solution to above 70 degrees Celsius before Ali. Quoting 40 microliters of the solution into each well of a 96 well plate incubate the plate at 37 degrees Celsius for one hour, harvest cancer cells via trypsin and prepare a single cell suspension before diluting the cell suspension to 1000 cells per milliliter. Add 100 microliters of this dilution to the well containing the cured aros bed.
Place the sample on a shaker overnight in an incubator set at 37 degrees Celsius and 5%CO2 following incubation. Remove the sample from the shaker and add 100 microliters of cell culture media proceed to incubate the resulting steroid until the desired diameter is reached. For example, after nine days, a pan, one steroid will be approximately 450 microns in diameter.
To begin, prepare a workstation inside a laminar flow hood with two two milliliter vials. Vial one will contain the tumor steroid and vial two will be a cell-free control. Prepare a diluted mixture of carboxylate modified one micron diameter fluorescent tracer probes by adding two 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 one 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 two. Next, add approximately two microliters of one molar sodium hydroxide to vial.
One 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 steroid.
Retain this 40 microliters while conducting the next step. Check the well to see if the steroid 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 steroid to vial. One gently stir vial one 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 steroid. Then add approximately two microliters of one molar sodium hydroxide and 40 microliters of cell culture media to vial two and vortex before Ali.
Quoting 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 co-registration.
Typically, 20 sample points in each well distributed in concentric rings around the S spheroid will produce adequately detailed results. Move the stage to each desired position and use microscope interfacing software to record the XNY coordinates. Switch the microscope to a high powered objective lens and select the appropriate filter cube for the x citation 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.
To begin data analysis. Copy all the video data to an analysis folder. Import image data into MATLAB or another analysis software.
Calibrate the software by analyzing several frames of the video to appropriately determine selection and rejection parameters for identifying probe center positions. Then use the software to automatically determine each probe position for all video frames, and then link these positions into trajectories. Use the trajectory data to compute the mean square displacement or MSD as a function of lag time being sure to apply the appropriate spatial calibration factor for the specific objective lens or for any pixel benning.
Calculate G Star using the generalized Stokes Einstein relation, taking into consideration the limits of applicability of the generalized stokes Einstein relation for a particular sample. Repeat analysis steps for each field of view in the experiment. Coregister position data with viscoelastic mod I at a particular frequency of interest, depending on the manufacturer of the microscope used.
This step can be facilitated by a custom routine, which reads microscope metadata or position data can be tabulated manually. Video sequences are acquired at multiple spatial positions within a representative well containing a 3D tumor nodule and linked to provide a spatial micro radiology map of the sample when monitored longitudinally populations of cells at the periphery of the pan. One steroids used in this example will spontaneously degrade surrounding collagen fibers and invade into the extracellular matrix within a few days.
In this example, co-registration of micro ology provides a quantitative readout of the obvious invasion into the matrix occurring to the left, but not to the right of the steroid. Longitudinal monitoring of these disparate spatial regions shows a drop in the real component of the complex viscoelastic shear modulus of approximately four orders of magnitude concomitant with invasion. Full frequency dependent data is shown contrasting theological properties of the material in region one and region two at day four.
While attempting this procedure, it's important to remember that care must be taken to avoid common pitfalls during analysis of probe trajectories that lead to inappropriate conclusions. We encourage readers to refer to PTM literature cited herein, which addresses considerations such as sample drift and interpretation of heterogeneity. This procedure can be combined with additional longitudinal or terminal fluorescence imaging to answer other questions, such as how mechanical remodeling correlates with key signaling events or cytotoxic response to an intervention.
