Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

The goal of this procedure is to study breast cancer cell proliferation, colonization, and migration in a human bone tissue explan model system. This is accomplished by isolating trabecular bone tissue fragments from femoral head surgical specimens and culturing them with breast cancer cells expressing luciferase and green fluorescent protein. Bioluminescence imaging is used to measure proliferation of cells and verify the colonization of breast cancer cells in the bone tissue fragments.

Patterns of breast cancer cell colonization are imaged by fluorescence microscopy. The marrow compartment can be flushed from the fragments for analysis by flow cytometry by luminescence and staining assays. Breast cancer cell migration towards tissue culture, supernatants and bone tissue fragments is measured in Transwell migration chambers with bioluminescence imaging bone is the most frequent site of breast cancer metastasis, and ultimately this model provides a new window for studying breast cancer cells within the bone metastatic niche.

A big advantage of this model is that it provides direct access to the microenvironment of human bone tissue fragments, allowing us to easily observe and analyze breast cancer cell behavior during short term co culture experiments. This method can be used to study key questions about mechanisms underlying breast cancer, metastas to bone with the goal of identifying therapeutic strategies to effectively prevent and treat it action. Although this model can provide insight into breast cancer metastasis, it can also be used to study other bone seeking malignancies, such as prostate cancer.

Bone tissue fragments must be harvested at various points in each assay. This begins with collecting and transporting a femoral specimen in saline and preparing the workstation in a biosafety cabinet wearing a surgical glove. Take hold of the femur head in one hand and use a surgical raw to extract trabecular bone.

Fragments show about two to five millimeters in size. Forceps won't work for this step. You need a good quality surgical raw.

For this experiment, prepare a breast cancer cell suspension with 100, 000 cells per 50 microliters of DMEM plus 10%FBS. In addition, prepare plugs of bone wax for mobilizing the bone fragments using the cutoff ends of a micro pipette tip, store the plugs in a Petri dish. Then with forceps, transfer the plugs into a six well plate and using a sterile glove, press the plugs into the 12 o'clock position.

Next pipette 50 microliters of the cell suspension into the center of each. Well place the plate in a tissue culture incubator for 45 minutes to promote cell attachment while the plate incubates extract the bone fragments with the cells attached to the plate. Place a bone tissue fragment onto the bone wax in three wells and press each down using the GER three wells without fragments servers controls.

Next, slowly add five milliliters of DMEM plus 10%FBS to the wall of each well. The bone wax and bone fragments mustn't be dislodged. Then incubate the culture for 20 to 24 hours.

The next day image the cells first. Add 300 micrograms per milliliter of Lucifer to each well, and then immediately image the plate with an Ivis imaging platform. This experiment requires a breast cancer cell suspension like the previous one.

After extracting bone fragments, use forceps to transfer each fragment to an empty well of a 24 well plate, then pipette 50 microliters of cell suspension directly onto each bone fragment, and transfer the plate to an incubator for 45 minutes to promote cell attachment. Once the cells have attached gently add one to two milliliters of medium to each well enough to submerge the bone fragment. Then place the plate in the tissue culture incubator for 20 to 24 hours.

In preparation for Ivis imaging, fluorescence imaging or collecting marrow cells for analysis for Ivis imaging, transfer the bone fragments to a 24 well plate with one milliliter of fresh medium per well. Add 300 micrograms per milliliter Lucifer to each well and proceed as previously described for fluorescence microscopy. Pipette five microliters of fluorescent bisphosphate labeling solution into each well to label the bony spicules of the bone fragment and incubate the plate for another 24 hours.

24 hours later, use forceps to transfer the fragments to plates containing phenol red free medium. Then view the plates using a fluorescence microscope configured to image at 485 nanometers to localize colonize GFP expressing breast cancer cells and a 680 nanometers. To identify bisphosphate labeled bone spicules to flush the bone marrow compartment for analysis of cell numbers or properties.

Transfer a fragment to a 70 micron strainer over a 50 milliliter chronicle tube tube. Then use a 10 milliliter syringe with a 25 gauge needle to vigorously flush the fragment with 10 milliliters of PBS centrifuge. The suspension at 300 G for three minutes at room temperature to pellet the cells aspirate and discard the supinate reus.

Bend the pellet of marrow cells in PBS or other desired solutions per flow cytometry or viability assays. Begin by generating bone tissue snet. Place bone fragments into the wells of a 12.

Well plate containing 2.2 milliliters of DMEM 10%FBS per well and leave some control wells without bone fragments. Culture the plate for 24 hours. Then aspirate and replace the medium and continue the culture for another 24 hours, 48 hours into the culture transfer 0.9 milliliters of supinate to a receiver plate while temporarily incubating the receiver plate.

Place transwell inserts into an empty 24 well plate. Then iPet 100, 000 breast cancer cells in 350 microliters onto each insert. Then culture the plates for 45 minutes to promote cell attachment After 45 minutes, use forceps to transfer the inserts to a receiver plate.

Be sure to angle the inserts when placing them into the receiver. Well supinate so bubbles do not form. Then incubate the plate with inserts for 20 hours at 37 degrees Celsius the next day.

Use forceps to remove the inserts and add 300 micrograms per milliliter Lucifer in to each. Well perform bioluminescence imaging on the receiver plate to detect breast cancer cells that have migrated through the membranes and attached to the bottom of the receiver plate wells. First, load a receiver plate with medium and place it in the tissue culture incubator.

To seed the inserts. Invert them in an empty micro fused tube box with a lid. Then add 100, 000 cells in 50 microliters to the outer bottom surface of each insert membrane.

Cover the box leaving the lid cracked and incubate the inserts for 45 minutes in the tissue culture incubator. Then use forceps to invert and transfer the inserts, the wells of a receiver plate into each insert cup at 0.45 milliliters of DMEM with 10%FBS. Then add a three millimeter bone fragment or glass bead to each insert and cut to the plate for 20 hours.

The next day use forceps to transfer the fragments and beads to a plate with wells containing a milliliter of fresh medium. Add 300 micrograms per milliliter Lucifer per well and proceed with bioluminescence imaging. MDA MB 2 31 F.Luke EGFP.

Breast cancer cells were co cultured adjacent to immobilized bone fragments to measure breast cell proliferation. Bioluminescence imaging after 24 hours of culture showed enhanced breast cancer cell proliferation in the presence of bone fragments in the top three wells compared to the absence of bone fragments in the bottom three wells. The results of experiments using fragments from three separate surgical specimens showed enhanced proliferation in the presence versus the absence of bone.

In all three cases, colonization of MCF seven F Luke EGFP cells seated directly under bone tissue fragments was observed with bioluminescence imaging after 24 hours. Decreased signal was associated with declining seeding density after 48 hours. Directly seeded fragments labeled with bisphosphonate reagent revealed colonization of the GFP positive breast cancer cells within the mineralized and marrow compartments.

Migration of the breast cancer cells across the porous migration membrane toward bone tissue culture, supernatants seen in the top three wells was robust compared to migration into control. Medium seen in the bottom three wells. Migration into three bone fragments from a given THR specimen was also observed, whereas no migration onto the beads was observed.

After watching this video, you should have a good understanding of how to extract human bone tissue fragments from a femoral head specimen and initiate them into co culture assays to measure breast cancer cell behaviors including migration, colonization and proliferation.