The overall goal of the following experiment is to observe the morphology of cancer cells and their interaction with the extracellular matrix in 3D cultures, this is achieved first by labeling rat tail collagen, one with the red fluorescent DTRA to allow visualization of the 3D collagen networks. In the second step, cancer cells grown as single cells or multicellular Steroids are embedded in the 3D Tamra labeled collagen matrices. The cells are then allowed to disperse throughout the collagen for one to three days, mimicking the invasion and migration of cancer cells into the surrounding three DS stroma in vivo.
Next, the cells are fixed and immuno stain with the appropriate antibodies and Phin to visualize the cell morphology and localization of the proteins of interest. Ultimately, the distinctive morphology of cancer cells cultured in 3D and their interaction with the 3D extracellular matrix can be analyzed through the acquisition of deep Zacks of the samples by confocal microscopy. This method can help answer key questions in the cell migration field by contributing to a better description of the molecular composition function and localization of cellular components in 3D environments that for many years were analyzed in 2D.
Demonstrating this procedure with me will be Anton Smo, a technician in our laboratory To label collagen one with Tamara. Begin by slowly filling a one milliliter disposable syringe with one milliliter of highly concentrated rat tail collagen, one solution avoiding air bubbles. Attach a 21 gauge hypodermic needle and then inject the collagen into a pre soaked three milliliter dialysis cassette with a 10, 000 Dalton protein molecular weight cutoff, taking care not to damage the membrane with the needle.
Pull back the plunger to remove all the air from the cassette, and then dialyze the collagen overnight against one liter of labeling buffer. The next morning, carefully remove the collagen from the dialysis cassette and then mix one milliliter of the dialyzed collagen solution with one milliliter of cold freshly diluted Tamra solution in a two milliliter micro centrifuge tube. Wrap the tube in foil and incubate overnight with rotation.
The next morning transfer the TAMRA labeled collagen into a pre soaked dialysis cassette and dialyze the collagen overnight against one liter of labeling buffer to remove the excess dye. Then to preserve the Tamara labeled collagen, place the dialysis cassette into one liter of 0.2%volume per volume, acetic acid solution with a pH of four and dialyze the cassette overnight. Again, the next morning transfer the Tamara labeled collagen from the dialysis cassette into a micro centrifuge tube using a micro pipette measure the final volume of the collagen, then calculate the final concentration of the Tamara labeled collagen.
Considering the initial volume and concentration of the collagen solution used and store the DY collagen at four degrees Celsius, protected from light for embedding single cells. Determine the volume of two milligrams per milliliter. Tamara collagen mix necessary for the experiment, and then mix the appropriate volumes of 10 XPBS and one normal sodium hydroxide.
Then mix the appropriate volumes of both Tamara labeled collagen and unlabeled collagen in a one to six ratio to achieve a final total collagen concentration of two milligrams per milliliter. Now suspend the cells of interest in the appropriate volume of chilled cell medium without FBS, and then add the cells to the Terah collagen mix to obtain a final cell density of one times 10 to the fifth cells per milliliter. Confirm the pH by testing 10 microliters of the mix on a pH test strip, and then pipette 100 microliter drops of the Tamara collagen cell suspension onto glass bottom dishes, allowing the mix to polymerize at room temperature until the collagen turns into a white-ish gel about 30 to 45 minutes.
Then carefully add sufficient culture medium to cover the collagen embedded cell drops and keep the drops at 37 degrees Celsius in 10%carbon dioxide for enough time for the cells to migrate throughout the matrix. Typically one to three days for embedding cell steroids. Pipette 100 microliter drops of freshly diluted teric collagen.
Mix onto glass bottom dishes and allow the collagen to initiate polymerization for two to five minutes to slightly increase the gel viscosity. Then collect a cell steroid and place it on a fresh Petri dish. Remove any excess liquid and resuspend the steroid in 10 microliters of Tamara collagen mix to prevent dilution of the collagen within the cell media.
Then use a P 20 pipette to collect the collagen suspended steroid and place it in the center of the top of the 100 microliter TEMA collagen mix drop. Letting the collagen polymerize briefly for two to five minutes is usually enough to ensure that your OID is more than a hundred microns away from both the top and the bottom of the collagen drop. But if your steroid still sinks, try inverting the dish for about two minutes and then turning it to the upright position.
Allow the collagen to polymerize as just demonstrated, and then carefully add sufficient culture medium to cover the collagen steroid drops. Then incubate the drops to allow the cells to migrate throughout the matrix for immunofluorescent staining. Begin by carefully removing the cell media and rinsing the steroid embedded collagen matrices with PBS.
Next, incubate the cells with extraction fixation buffer. After five minutes further, fix the cells with PFA and sucrose in PBS for 30 minutes. Then rinse the cells.
Now incubate the cells with the primary antibodies of interest diluted in PBS. Then wash the matrices three times for 30 minutes each time. After the third wash, incubate the matrices with the secondary antibodies, Alexa conjugated Phin and DPI diluted in PBS.
Then after washing the matrices again, remove the excess liquid and add about 500 microliters of mounting media filling the bottom of the dishes. Now seal each drop with a 24 millimeter cover slip, being careful not to press down on the drops and damage the structure of the matrices. To image the samples.
Position the 40 x objective at the middle of the collagen drop. Then using the fluorescent lamp, obtain an overview of the sample both in the XY and the xz axi. Next, switch to the confocal live imaging mode.
Using the 561 nanometer laser, start focusing from the bottom of the glass until the collagen fibers start to appear. Consider this depth Z equals zero micrometers. Now raise the objective until it reaches 100 micrometers of above Z equals zero, and then image the cells found at z equals 100 micrometers or above to avoid any tension effects from the rigid glass bottom labeling rat tail collagen one with Tamara allows an easy preparation and visualization of 3D collagen networks.
The slow room temperature polymerization allows the formation of these networks visualized here in red with a comparable organization to those found in vivo cytoskeleton proteins such as tubulin, which appear here in blue or a actin shown here in green, can be visualized by immuno staining as demonstrated in these images when viewed in 3D matrices, colon adenocarcinoma CT 26 cells present a typical mesenchymal morphology characterized by an elongated cell body tipped with f actin rich cellular protrusions that resemble Filip Podia and lam podia. This elongated morphology is even more evident in cells attempting to escape cellular steroid by invading the collagen matrix. After two days in culture, the cells start invading the collagen 3D matrix moving away from the cell steroid.
It is crucial to analyze the collected Z stacks from all the planes of view to ensure a uniform 3D distribution of the cells in these images. For example, when visualized as a maximal XY projection, CT 26 cells grown as steroids appear to extensively invade the 3D collagen matrix. However, the x XZ view reveals that the cells are actually invading a restricted Z interval compared to the steroid volume suggesting a preferential region of invasion.
This representative steroid was in fact placed too close to the glass bottom of the dish, resulting in the cells migrating away from the steroid, not by invading the 3D collagen matrix, but by crawling on the 2D rigid glass Following this procedure. Other methods like live imaging of GFP expressing cells embedded in TAMRA collagen matrixes can be used to closely follow dynamic interactions between cell and matrix or cell induced matrix deformation.
