The overall goal of this procedure is to visualize glial interactions directly during cerebral metastasis formation. This is accomplished by first preparing organotypic brain slices from mice. Next, the organotypic brain slices are co cultured with carcinoma cells.
Then stromal cells are fluorescently stained with specific markers. Finally, the level of tumor invasion is evaluated with a fluorescent microscope. Ultimately, results can be obtained that show glia cancer cell interaction and colocalization through immunofluorescence labeling and time-lapse or confocal microscopy.
The main advantage of this technique over existing methods like the intracerebral injection of carcinoma cells, is that this method is an easy alternative and offers a good platform to observe cellular interactions during tumor progression. The implications of this technique is 10 toward the discovery of a novel and the selective therapies for cancer treatment. Because the trauma cells play important roles in metastasis and also potential targets for cancer therapy, Demonstrating the technique will be Eugenia handing a postdoc of my laboratory Prior to the surgical procedure, prepare the dissection medium consisting of minimal essential medium, supplemented with 0.2 millimolar glutamine, 100 milligrams per milliliter streptomycin, and 4.5 milligrams per milliliter of glucose after decapitating mice from any mouse strain between postnatal day six and eight.
Using aseptic conditions rapidly, remove the brain from the skull and transfer it to dissection medium. Remove the frontal pole and the cerebellum from the brain. Then use cryo glue and 5%agros to fix and stabilize the remaining brain tissue composed of the cerebrum.
On a stage using a Vibram horizontally slice, 350 micron thick sections, depending on the species and age of the mouse, collect four to six whole brain slices from a single brain. After preparing the culture medium according to the text protocol, add one milliliter to each lower well of a six well trans well plate. Then place each organotypic brain slice on a 0.4 micron polycarbonate transwell membrane and insert them into the wells of the plate.
Incubate the slices overnight in a humidified atmosphere with 5%carbon dioxide and 37 degrees Celsius. The next day, embed 10 to the fifth GFP transfected tumor or other cells in 20 microliters of gel matrix consisting of 15%RPMI medium and 85%ECM gel. Place the MCF seven gel matrix mix into sterile metallic spacers directly adjacent to the cortical regions of the organotypic brain slices and incubate for two hours after the incubation, remove the spacers and allow the 3D tumor PHE to incubate with the brain slices for 24 to 96 hours.
Replace the culture medium every other day if desired. Carry out live imaging using time-lapse microscopy with 10 x magnification object to stain the brain slice co cultures. Fix them with 4%paraform aldehyde for eight hours at four degrees Celsius before washing the samples in PBST for five minutes.
Then to block the samples, use normal goat serum in PBST at room temperature for one hour. Next, stain the astrocytes by incubating the co cultures with anti glial fibrillary acidic protein or GFAP monoclonal antibody for 36 hours at four degrees Celsius, followed by goat anti mouse staining for one hour at room temperature. After washing the samples with PBST three times for five minutes each stain the microglial cells with I LB four LOR 647 for one hour at room temperature before counter staining with DPI for three minutes at room temperature.
After using a fluorescent mounting medium to mount the co-culture samples, image the samples using a fluorescent microscope with 25 x magnification. Finally, evaluate the grade of tumor invasion based on the following scoring system. That first measures the length of the contact section between the tumor plug and slice, and then the fraction of contact detectable by the invading cells.
As shown here, all tested carcinoma cell lines invaded organotypic mouse brain slices suggesting that the invasion process was species independent. This movie shows a time-lapse recording of direct glia tumor cell interactions. Stromal cells were observed invading the tumor containing cell plug and interacting with the cancer cells.
Time-lapse imaging over an extended period of time confirmed the viability of the brain slices, and allowed the visualization of microglial cells entering the three-dimensional sphere, as well as cancer cells invading the brain slices. Moreover, as we have previously shown microglia transport carcinoma cells by a still enigmatic mechanism and assist in the carcinoma invasion using immunofluorescence labeling. We demonstrated the colocalization of tumor and stromal cells both in brain tissue and in tumor cell plugs, suggesting a tight interaction between these cells during the invasion process.
As can be seen here, comparable results were obtained when mouse brain slices were co cultured with either human or urine carcinoma cells, supporting the wide range of applications for studying cells from different species, genotypes and strains. This technique paved the way for researchers in the field of cancer metastasis to explore the impact of the microenvironment during tumor colonization at distant sites. After watching this video, you should have a good understanding of how to visualize GL cancer cell reactions during cell brain metastasis formation in a direct physiologically contact system.
