Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

The overall goal of this procedure is to perform ex vivo validation of in vivo near infrared fluorescence xenograft imaging experiments in mice using Fluor four labeled nano bodies or conventional antibodies. This is accomplished by first administering near infrared fluoro, four labeled antigen specific antibodies and conventional antibodies to mice bearing both antigen positive and negative xenografts. The second step is to perform in vivo imaging.

After imaging, the mice are sacrificed to remove the xenografts, which are processed for ex vivo analyses. Ultimately, ex vivo flow cytometry and fluorescence microscopy are used to validate in vivo imaging results. The advantage of this method over existing methods like nuclear imaging, is that the use of a near infrared fluorescent dye allows for a comprehensive in vitro, in vivo and ex vivo multimodality imaging comparison with a single probe for flow cytometry, fluorescence microscopy, and near infrared fluorescence imaging.

This method can help answer key questions in the tumor imaging field, such as localizing metastases during surgery and monitoring the efficiency of anti-tumor therapies. The labeling of antibodies with fluoro force allows the exact ex vivo quantification of cell bound antibodies. This allows us to distinguish whether specific singles that we see in vivo are due to antibodies, bound to cell surface antigens, or unspecific due to the so-called enhanced permeability and retention effect.

Seven to nine days after the tumor cells were injected when the tumors should be around eight millimeters in diameter. Prepared to image the mice first, apply ophthalmic ointment and initialize the imaging system. Then position the anesthetized mice on the heated stage with their tumors directed to the camera.

Maintain one to 2%ISO fluorine for the duration of the imaging procedure using the isof fluorine manifold housed in the imaging chamber. Monitor the respiration rates. If any are too fast, then the anesthesia is too light, and if it is slow or irregular, then the anesthesia is too deep.

Then image the mice after getting the baseline image. Inject the mice with 50 micrograms of LOR six 80 labeled antibody constructs. Do this in a volume of 200 microliters saline into the tail vein.

After imaging euthanize the mouse and mount it to a styrofoam block and clean it with 70%ethanol. Then cut open the skin to expose the tumors. Remove the tumors with a scalpel and keep the tumors intact.

Once removed, cut the tumors in half and transfer one half to a dish of P-B-S-A-E-B-S-F solution on ICE for fax analysis and transfer the other half to twice cold, 4%PFA for immunohistochemistry. After 24 hours in fixative at four degrees Celsius, transfer the tumors to PBS with 30%sucrose and keep them at four degrees Celsius until they fully sunk into the solution. Then dice the tumors into pieces that will fit into cryo molds.

Fill the molds with OCT compound and transfer the tumor chunks to the molds. The tissue must be fully immersed in OCT. Then transfer the cryo molds to dry ice and when they are completely frozen, store them at minus 80 degrees Celsius until they can be processed.

To prepare the cells, transfer the tumor half to a cell, strainer in the strainer. Cut it into three to four pieces, and then take the plunger out of a two milliliter syringe and use the plunger to mash the tissue against the strainer. Collect the solution that flows through the strainer After the tissues have been mashed up, flush the strainer with PBS.

Then transfer the collected suspension to a new tube and spin it down at 500 G for five minutes. We suspend the cell pellet in 10 milliliters of PBS with 0.2%BSA. Then count the cells aliquot one to 5 million lymphoma cells to a five milliliter fax tube, and spin the tube down at 500 G and discard the supernatant.

Then suspend the cells in 100 microliters of PBS with 0.2%BSA. At this point, it is possible to block FCR using an antibody to bind FC gamma R.Wait 10 minutes and wash the cells once with PBS with 0.2%BSA. Now add an anti CD 45 monoclonal antibody to identify the leukocytes.

Incubate the cells for 20 minutes in the dark with this antibody, and then use two washes to remove it just before fax analysis. Stain the cells with propidium iodide for 15 minutes on ice to identify dead cells. Then wash them clean with plain PBS.

Follow this by Resus suspending the cells in 150 microliters of PBS and proceed the facts. Mice were injected with 50 micrograms of nano body and monoclonal antibody to evaluate the specificity of the fluorescently labeled constructs. For in vivo imaging.

All imaging was performed six hours after injection flow. Cytometric analysis of tumor cell suspensions showed specific labeling of antigen positive tumor cells with both AF six 80 conjugates. There was no non-specific labeling of antigen negative cells with either construct.

The tumor. Cryo sections showed a strong and almost homogenous labeling of antigen positive cells with the nano body. However, the monoclonal antibody gave a much weaker in homogeneous stain as expected antigen.

Negative tumors showed no staining of the antibody antigen negative tumors showed non-specific scattered staining in the interstitial space after injection of the conventional antibody. This was very similar to the staining scene in the antigen. Positive tumors after injection of the conventional antibody Once mastered.

The imaging part of this technology can be performed in a single day Following this procedure. Other methods like radio labeling of nano bodies can be performed in order to investigate the biodistribution of the injected conjugates into different tissues or other questions. After watching this video, you should have a good understanding of how to perform multimodality ex vivo validation of your in vivo, near infrared fluorescence imaging experience with a fluoro four labeled probe.

Thus providing you with a technique for evaluation of new antibody constructs for preclinical molecular imaging.