In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

The overall goal of this procedure is to demonstrate the use of standard techniques to quantitate ovarian cancer colonization of peritoneal adipose depots. This is accomplished through intraperitoneal injection of ovarian cancer cells into mice following sacrifice of the mice at specific time points. Peritoneal fat depots are identified and isolated from the mice.

Finally, the number of ovarian cancer cells present in specific adipose depots is quantitated using flow cytometry. In addition for mechanism based studies, ovarian cancer cell colonization can also be studied in ex vivo co-culture, followed by appropriate quantitative and or molecular analyses. Ultimately, these in vivo and ex vivo approaches enable the interrogation of processes involved in ovarian cancer cell colonization of peritoneal adipose via qualitative or quantitative methods.

The main advantage of this approach is that we are able to implement standard techniques to evaluate specific steps in ovt cancer metastatic colonization of peritoneal adipose. The implications of the technique extend the metastasis prevention. Specifically, it enables the identification of cancer cell microenvironment interactions that regulate the outgrowth of ovarian cancer cells within immune cell containing structures known as milky spots.

Generally, individuals new to this method will find it helpful to have experience in handling mice. Although intraperitoneal injections are seemingly straightforward, lack of a good technique can lead to inaccurate data. Furthermore, tissue dissociation to single cell suspension requires practice and meticulous planning prior to the start of the experiment.

This particular technique does not require the animals to be under anesthesia. Under approved protocols, perform this technique on live animals. Load 500 microliters of the fluorescently tagged single cell suspension into the syringe.

Then put the syringe into a sterile capped 25 gauge needle to reduce cell shearing prior to injection. Restraining the mouse with the other hand, pick the mouse up by the scruff of the neck and hold the tail using the palm and forefinger. Then fix the left hind leg between the ring and little finger to avoid traumatizing abdominal organs.

Restrain the mouse well so that it cannot move during the injection. Imagine a line across the abdomen and locate a point on the animal's right side and close to the midline. The point of entry is cranial two and slightly medial of the last nipple.

Prepare to insert the needle on the mouse's right side to avoid the cecum and reduce the risk of puncturing the intestines. Insert the needle at the lower lateral region of the mouse's abdomen to a depth of approximately 0.5 centimeters. Pull back on the plunger to confirm that the needle has not penetrated a blood vessel or other peritoneal organs.

If no fluid is aspirated, inject the sample using slow, steady pressure. Then withdraw the needle and return the mouse to its cage. Do not recap syringe before disposal in the sharps container.

Sacrifice the mice at specific time points post injection for vital organ removal. As described in the text protocol as harvesting peritoneal fat depots constitutes internal organ removal. Make a midline incision close to the inguinal pape through the peritoneal wall to expose the internal organs to excise the omental fat.

Expose the omental pancreatic complex by extending the spleen from the peritoneal cavity with forceps for the gonadal fat. Use forceps to lift the gonadal fat surrounding the ovaries and excise it by cutting tissue connections.Immediately. Place the tissues in ice cold PBS.

Next, remove the uterine fat by using forceps to lift the uterine fat surrounding the uterine horns and excise by cutting the tissue connections before immediately placing the tissues in ice cold PBS. When obtaining the mesenteric fat, cut the junction between the small intestine and the pylorus. Use forceps to firmly grip the free end of the small intestine and slowly peel it away from the mesenteric fat.

Release the mesenteric fat from the mesenteric root using dissecting scissors. Then immediately place the tissues in ice cold PBS. Finally, to remove the pleo portal fat, lift the distal end of the spleen using forceps to expose the thin fatty band of tissue, connecting the hilum of the spleen to the pancreas.

Excise the pleo portal fat by first releasing it from the pancreas and then carefully dissecting it from the spleen. Then immediately place the tissues in PBS to begin weight. Match all the adipose tissues to the omentum.

Transfer the tissues to separate five milliliter tubes containing 1.5 milliliters of serum free ECCOs, modified eagles medium or DMEM and 0.1%bovine serum albumin. Pool the tissues harvested from three independent mice to ensure a sufficient yield of cells for flow cytometry. Next, mince the tissues using surgical scissors and add 1.5 milliliters of serum free DMEM containing 0.4%collagenase one.

Incubate the tissue suspension at 37 degrees Celsius for 30 minutes with rotational mixing as an optional step. Further dissociate the tissue by mastication. In this case, transfer the tissue collagenase suspension to a micro stomach or bag and masticate for 10 minutes on low, rotating the orientation of the bags after five minutes.

Next, filter the samples through a nylon mesh filter to remove large debris. Collect the cells via centrifugation at 250 times G for five minutes at four degrees Celsius and discard the supernatant fraction. Resuspend the cell pellet in 100 microliters of PBS combined with 900 microliters of ammonium chloride, potassium lysis, buffer, and incubate at room temperature for one minute.

Then centrifuge the cells again as before and discard the supernatant after resus, suspending the cell pellet in 250 microliters of ice cold PBS filter the samples through a 60 micron pour nylon mesh to ensure a single cell suspension. Then rinse the filter with 250 microliters of ice cold PBS grow and prepare fluorescently tagged cells and resus. Suspend at a concentration of 2 million cells per milliliter.

Apply approximately six microliters of the tissue adhesive to the membrane of the culture. Insert and allow it to air dry. Then wash the membrane twice with sterile water to remove any excessive adhesive Before air drying the membranes under a laminar hood, carefully excise the OA and attach it to the adhesive coated membrane using sterile forceps.

After allowing the tissue to adhere to the membrane for one minute, add 500 microliters of the cell suspension on top of each omentum in each culture insert. Then fill the area around the transwell chamber with 2.5 milliliters of DMEF 12 media. Incubate the OA with cell suspension for six hours at 37 degrees Celsius in a 5%CO2 environment.

Carefully remove and wash the OA with about 10 milliliters of PBS. Finally, visualize fluorescent cancer cell foci using an appropriate fluorescent imaging system. The relative locations of peritoneal adipose depots can be seen via gross anatomic dissection.

Shown here is a closer look at Pleo portal and omental adipose. The relative sizes of the excised adipose depots are shown here. Milky spots are observed in omental and pleo portal adipose using standard histology.

Quantitative results from the flow analysis of various peritoneal fat depots are shown. The gating criteria for the analyses are shown here. Ideate parental cells were used as a negative control.

Omental tissue preparations contained a significant population of TD tomato positive cells relative to uterine mesentary and gonadal fat. At the quantified flow cytometry data are expressed as mean fold change increase of TD tomato positive events relative to PBS injected mice in vivo and ex vivo colonization of OA by SKV three, IP one GFP ovarian cancer cells is shown here. Fluorescence imaging of whole OA demonstrated similar colonization patterns of cancer cell localization in both in vivo and ex vivo assays.

Histologic examination of these tissues confirmed the presence of cancer cells localized to the milky spots. Finally, immunohistochemical detection of cytokeratin expressed by SKO V three IP one GFP cells confirms these histologic findings. While attempting this procedure, it's important to remember to identify and carefully excise peritoneal adipose tissue and prevent contamination by other tissue types, which will then qu skew the quantitative data obtained from flow cytometry.

Second thing, it's important to remember to choose appropriate my strains for the study in question. The technique can be used to identify interactions of well-established ovarian cancer cell lines in the omentum microenvironment that are important to metastasis formation. It can also be used to evaluate the malignant potential of cell lines that model early lesions seen in the fallopian tubes.