Invasive progression is a crucial determinant of clinical outcome in patients with bladder cancer. Here we will describe a simple in vitro method, which utilizes tumor spheroids generated using cell lines or primary tumors to study invasion. The main advantage of this technique is that we are able to monitor the process of cancer invasion in a real-time fashion, and also use immunofluorescent staining to interrogate the samples at the end of the experiment.
This technique can be tailored to incorporate other cell types, such as fibro blast or immune cells. It can also be used to test compounds which might be useful to block invasion. Critical factors for success include optimization of tumor spheroid formation and embedding in collagen use of appropriate microscopy with correct working distance and appropriate immunostaining procedures.
To begin generating spheroids from cell lines, culture human bladder cancer cells under conventional adherence cell culture conditions. One day prior to the experiment, trypsinize the cells and distribute 1, 000, 000 cells in three milliliters of cell culture media to each well of a six well plate. Next, incubate the cells in low attachment conditions at 37 degrees Celsius for at least 16 hours.
To generate spheroids from primary tumors, collect and wash 0.5 millimeter cubed tumor pieces in five milliliters of ice-cold PBS. Centrifuge the tumor pieces at 200 times G for five minutes at four degrees Celsius. After this, collect the tumor pieces via centrifugation at 200 times G for 10 minutes at four degrees Celsius.
Then remove the supernatant and add five milliliters of DMEM supplemented with 10%FBS. Transfer the tumor spheroid media mixture to a six-well ultra low attachment plate. Then incubate the plate at 37 degrees Celsius for at least 16 hours.
First, dilute type one collagen in DMEM supplemented with 10%FBS to make a two milligrams per milliliter mixture. Then quickly coat the wells of a chamber slide with the mixture. Keep the chamber slide stationary at room temperature for 15 minutes.
After this, gently pipette 500 microliters of spheroid media from the ultra-low attachment plate into a 1.5 milliliter micro centrifuge tube. Then allow the spheroids to settle at the bottom of the tube for two minutes. Carefully remove the supernatant from the tube.
Then quickly add 500 milliliters of fresh collagen DMEM mixture to the spheroids. Pipette up and down to gently mix the spheroids and collagen. Next add 250 microliters of the spheroid collagen mixture to the wells of the collagen coated chamber slide.
Allow the collagen matrix containing the spheroid to solidify completely. After the collagen matrix solidifies, add one milliliter of DMEM supplemented with FBS to each well of the chamber slides. Then incubate the chamber slide at 37 degrees Celsius with 5%CO2.
Prepare the confocal microscope according to the manufacturer's instructions and ensure that the climate chamber reaches 37 degrees Celsius and is supplied with 5%CO2. Then, transfer the chamber slide to the microscope slide adapter. Using a low power objective, locate the spheroids of interest then start imaging with high power objective and perform time lapse imaging for 24 to 72 hours.
First, use small forceps to lift the block of collagen gel and spheroids from the chamber slide. Then place the collagen gel block in a plastic histology mold. Rinse the collagen gel block with 1x PBS briefly.
And fix it with 4%PFA and PBS for 30 minutes at room temperature. After this, wash the collagen gel block with 1.5 milliliters of 1x PBS on a shaker for 15 minutes. Apply a thin layer of OTC compound to the bottom of a new plastic histology mold.
Then place the fixed and washed collagen gel block on top of the OTC compound before carefully filling the rest of the mold with OTC compound. Keep the mold at four degrees Celsius for one hour. After this, flash freeze the mold on a 100 millimeter petri dish floating on liquid nitrogen and store the samples at minus 80 degrees Celsius.
Transfer the frozen sample block to the minus 20 degrees Celsius chamber of a cryostat. Then perform conventional frozen sectioning with a seven micrometer section interval. After this, air dry the slides for one hour at room temperature.
Then permeabilize the samples for 15 minutes with PBS containing 0.5%Triton X-100. Next, wash the samples with PBS three times for 10 minutes each. Then encircle the samples with a hydrophobic barrier pen.
Treat the samples with blocking solution for one hour at room temperature. Then apply 40 microliters of primary antibody containing solution to each sample. Incubate the slides for one hour at 37 degrees Celsius.
After this, wash the sample three times with PBS for 15 minutes per wash. Next apply 40 microliters of secondary antibody containing solution to each sample. Then wash the samples three times with PBS for 15 minutes per wash.
Stain the sample with Hoechst 33342 solution at room temperature for 10 minutes. Then wash the samples with PBS for five minutes. Mount the samples with mounting medium and cover them with cover slips.
Finally, leave the slides in the dark at room temperature for 24 hours before performing confocal microscopy. In this protocol, invasive bladder cancer tumor spheroids were formed from cell lines and primary tumors. Representative time lapse video demonstrates bladder cancer tumor spheroid invasion into the collagen matrix.
Representative time lapse video demonstrates invasive behavior of GFP labeled bladder cancer tumor spheroids alone or co-cultured with RFP labeled fibroblasts. To demonstrate the feasibility of using immunofluorescent staining of protein markers, the spheroids were fixed, sectioned and stained at 24 or 72 hours. The higher magnification view of the UM-UC-14 spheroids, shows filamentous staining for ataxia telangiectasia group D associated protein.
The highly invasive cells disseminated from UM-UC-18 spheroids after 24 hours of invasion, express a high level of Vimentin a marker of Epithelial-to-Mesenchymal transition. This assay is useful for limited screens of pharmacologic inhibitors of bladder cancer invasion. Cytochalasin D is used here to represent pharmacologic inhibition of invasion.
Here we describe a model that allows real-time observation of bladder cancer invasion. This system is amenable to incorporation of various stromal and cellular components, which allows investigators to better recapitulate the tissue microenvironment where bladder cancer invasion takes place. This system not only provides a useful tool for studying the invasion in 3D environment, but also is suitable for limited screening of pharmalogic compounds that have potential to block cancer cell invasion.
