Methods to Enable Spatial Transcriptomics of Bone Tissues

Our goal is to find better therapeutic approaches to treat a human bone cancer called osteosarcoma. In order to do so, we want to uncover the biological processes responsible for the onset of this tumor, and above all, for the development of its metastasis, which is the leading cause of patient's death. By employing spatial transcriptomic analysis on several matching specimens of human primary osteosarcoma and lung metastasis, we located in multiple patient common cell populations residing within the tumoral areas, and by comparing them, we identified several key genes, which may represent valuable therapeutic targets to prevent lung metastasis.

Spatial transcriptomic analysis of our tissue, such as bone, have been very challenging because of the inability to preserve good quality RNA and tissue morphology, while at the same time processing the heart tissue for sectioning. Our protocol addresses this gap, allowing researchers to successfully apply spatial transcriptomic analysis also to our tissues. To begin, spray the mouse skin with 70%ethanol.

Then using sterile dissecting scissors, open the skin and expose the leg muscle. Cut the muscle alongside the leg to view the bone clearly and gently remove the bone without damaging it. Immediately place the bone into a tube containing 4%paraformaldehyde, and place the tube on ice.

After collecting all bone samples, place the tubes at four degrees Celsius in a cold room on an orbital shaker for a minimum of 24 hours. After fixation, retrieve the bone tissue from the orbital shaker and wash with 1X PBS twice for three minutes each. Then use sterile dissecting scissors to remove any remaining muscle attached to the bone.

Wash the bone again with 1X PBS for three minutes. Place the bone in a new container containing 20%EDTA at pH 8, and incubate on an orbital shaker at 140 RPM for 30 minutes at room temperature. Then replace the EDTA solution with fresh 20%EDTA and repeat the agitation for another 30 minutes.

After the final EDTA treatment, add fresh EDTA and incubate the container at four degrees Celsius on an orbital shaker overnight. Next, retrieve the samples from the EDTA container and wash them two times with 1X PBS to remove EDTA residues. Initiate the dehydration process by placing the bone in 70%ethanol and incubating it for 15 minutes at 140 RPM.

Then discard the 70%ethanol solution and add 90%ethanol solution. Incubate for 15 minutes at 140 RPM. Next, initiate the clearing process by placing the dehydrated bone tissue in a new container with xylene.

Incubate for 20 minutes at 140 RPM. Replace the used xylene with fresh xylene and incubate for another 20 minutes. Next, place the cleared bone sample in an embedding cassette and begin wax infiltration.

Incubate for 30 minutes at 60 degrees Celsius. Discard the used wax. Add new wax and incubate for another 30 minutes.

Then replace the wax and incubate for another 45 minutes. Carefully position the bone in a mold in the desired orientation. Allow the wax to solidify on a cold surface.

Store the block at four degrees Celsius until sectioning. Next, prepare the equipment for sectioning. Spray all work surfaces and instruments with decontaminating solutions to remove RNases.

Also, set up a water bath with double distilled water at 42 degrees Celsius. Clean a microtome blade with 70%ethanol to remove oil, then decontaminate to remove RNases. Secure the blade on the microtome and ensure the clearance angle is set to 10 degrees.

Spray tweezers, brushes, and probes with decontaminating solutions and store them in one ice bucket. Prepare an ice bath by adding double distilled water to another ice bucket. Start the sectioning process by placing the FFPE block on the microtome.

Set the microtome to trim or set a scroll thickness of 14 micrometers. Begin trimming the sample and continue until the tissue is visible. Hydrate the FFPE block by placing it on an ice bath.

Secure the hydrated sample in the microtome specimen clamp. Align it with the blade and set the scroll thickness to five micrometers. Start sectioning the tissue.

Collect the sectioned tissue using cold tweezers and brushes, and place it in a 42 degree Celsius water bath. Place the tissue on a glass slide and incubate at 42 degrees Celsius for three hours. Dry the slide overnight in an oven at room temperature.

Store the prepared slide in a slide box at room temperature. Using this method, demineralized FFPE bone samples were prepared from mirroring femurs. The decalcification time course was optimized by comparing undecalcified femurs with those decalcified for three and 24 hours.

H&E staining showed that shorter decalcification time led to fractures and damage in the sections, whereas the 24-hour process resulted in superior histological quality. RNA quality assessment using RNA fragment distribution value DV200 revealed that decalcified samples maintained DV200 scores above 50%suitable for analyses like scRNA-seq or spatial transcriptomics. However, extended incubation times resulted in a decline in RNA integrity and hence are not recommended.