The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

The overall goal of this procedure is to perform a chick choal toic membrane or CAM assay to study sarcoma, cell, or graft behavior. This is accomplished by first making a window in the shell of an egg on incubation day three. In the second step on incubation day nine, the tumor material is prepared.

Then after removing the epithelial layer, the tumor is added to the cam During the final step on incubation day 16, the CAM is photographed, harvested, and fixed in formalin for histological evaluation. Ultimately, classical h and d histology and immunohistochemistry are used to demonstrate sarcoma, cell proliferation and invasion vascularization, and host reactions. The main advantage of this technique of existing methods like xenograft most models, is that it is simple to learn and relatively cheap.

It also allows you to study a myriad of metastasis associated activities in a very short time period, and it does not require an ethical committee. This method can help answer key questions in the sarcoma research field, such as the effect of the tumor host interactions on tumor survival, the reconstitution of the tumor microenvironment, and the recruitment of stroma cells. The implications of this technique extend toward personalized therapy for sarcoma as the patient's own material can be studied.

All This new technique can provide us new insights into SAR neurogenesis. It's also very applicable to other systems such as oncogenesis in general. Begin by using a sterile scalpel to cut the tumor sample into small pieces, no larger than one cubic millimeter, removing all calcified areas.

Then weigh the sample and transfer two to four grams of the tissue into a dissociating tube. Digest the tissue in 2.5 milliliters of collagenase two and 2.5 milliliters of DN a's solutions. After processing the tissue, according to the dissociation H tumor protocol, filter the resulting suspension through a 150 micron cell strainer.

Now centrifuge the suspension at an appropriate centrifugal force, such as 100 to 500 times G for five minutes at four degrees Celsius. After carefully removing the supernatant with a pipette, incubate the pellet in 10 milliliters of erythrocyte lysis, buffer at room temperature with occasional tation. After 10 minutes, add 25 milliliters of culture medium to stop the reaction after spinning down the suspension again, the pellet should be white, discard the supernatant and add five milliliters of medium to the cells, and then filter the suspension through a 70 micron cell strainer.

Use an automatic cell counter to determine the number of live cells per milliliter on development. Day three, insert an 18 gauge needle into the tip of the egg and remove two milliliters of the albumin to lower the level of the OVO chorio all toic membrane or cam. Now apply a semi-permeable adhesive film at the marked upper side of the shell to prevent the spilling of shell particles onto the cam while cutting the window.

Then make an introduction hole with a small all at the surface of the egg. Not damaging the cam during the opening of the shell is the trickiest part of the procedure. We use sharp scissors holding them horizontally in one hand while keeping the egg in the other hand.

Now, use a pair of sterile, sharp pointed surgical scissors to cut a one centimeter square window in the shell. The pulsating heart and adjoining vessels of the embryo should now be visible at the surface of the egg yolk. Remove any non fertilized eggs or dead embryos.

Then seal the window with more semi-permeable adhesive film. After spinning down the tumor suspension again, resuspend the pellet in cooled extracellular matrix gel at one times 10 to the six cells per 100 microliters of gel. Keep this solution on melting ice.

Next, under laminar flow, use a pair of sterile surgical scissors to excise part of the semi-permeable adhesive film. Remove the epithelial cell layer by touching the cam lightly with a sterile glass rod. Then add 4 25 microliter drops of the cell ECM gel suspension to the cam, allowing the ECM gel to polymerize in between the applications.

In this way, an adherent plaque containing the cancer cells is created. Then seal the shell window again with semi-permeable adhesive film. After the appropriate experimental incubation period, use a pair of surgical scissors to enlarge the shell window, making sure not to cut too low.

Next, use a pipette to add 300 to 500 microliters of PBS onto the CAM section containing the inoculated material. Then photograph the cam through a stereo fluorescence microscope. Afterwards, use a pair of sterile scissors to cut the membrane at the border lying against the shell and place the harvested cam in a sterile Petri dish filled with PBS or buffered formaldehyde.

Then photograph both the upper and the lower sides of the cam, both with and without a filter as seen. In this first figure, the tumor grafts become adherent to the cam. Here, a single cell suspension from patient material displaying a dried, slightly raised plaque can be observed in these two images.

The marked wrinkling of the membrane that occurs after the excision of the cam is shown. The SAO two line forms a spreading plaque over the CAM with a distinct increase of the surface from day seven. After inoculation, the GFP signal remains strong indicating viable cells.

These plaques containing SW 1 3 53 cells show a decrease in surface and tend to have a contracted cam, which results in a wrinkled membrane. After harvesting from day seven, bleeding is frequently observed. The DSR signal decreases as the fluorescent probe becomes diluted after many cell divisions, as well as if bleeding occurs.

In most cases, vascular reaction of the CAM is observed as new tortuous vessels enter the sample. For example, branching as indicated by the arrowheads and anastomosing as indicated by the asterisks in this punctiform bleeding as indicated by the arrows, can also be observed throughout the plaque. Note the migration of the SAOs two cells parallel to the vessels of the cam.

This vascular reaction can be visualized after excision of the cam at the lower view showing tortuous vessels surrounding the graft or plaque. Vascular reaction of the CAM is observed in the tumor graft group and the single cell suspension group. Histological evidence shows that SAOs two cells maintain a single cell appearance throughout the complete volume of the extracellular matrix gel causing an increased thickness and diameter of the plaque.

The tumor cells invade the mesodermal layer of the cam as indicated here by the arrow, thereby enlarging the distance between the endodermal and the dermal layers of the cam like an opening zipper. As indicated by the double arrow, the growth pattern of SW 1 3 5 3 cells and of a single cell suspension of a patient's conscious sarcoma is more clustered with islands of cells in an expanse of the extracellular matrix gel. As indicated by the arrow, not withstanding their ectopic site of growth, we found that the essential features and immunohistochemical characteristics of the original sarcoma tumors were maintained in the cam.

In addition, the tumor grafts became revascularized as evidenced by the appearance of the dark pink blue nucleated chick erythrocytes as indicated by the arrowheads and found in the vessels cell counting of KI 67 positive and KI 67 negative cells shows a steady increase in the total number of cells from day four for both cell lines. After this day, the total number of cells remain stable seven days after inoculation, the number of KI 67 positive cells and the total number of cells starts to decline. A larger proportion of KI 67 positive cells are observed close to the cam and a larger proportion of KI 67 negative cells at the surface of the plaque are observed.

Once masters, the cam SA technique can be used to analyze 50 x in a 14 hour spread over three days if performed properly. Additional h and e stainings and evaluation can be performed and require three hours per 10 conditions Following this procedure. Additional methods like lifestyle imaging can be performed in order to address additional issues like extravasation or ization of flu labeled cancer cells.

So by the development of this new technique, it allows researchers in the field of preclinical testing to explore new prognostic and therapeutic factors in sarcoma patients. After watching this video, you should have a good understanding of how to apply tumor material in the cmaa and how to translate your microscopic and microscopic observations toward a better understanding of tumor host interactions.