Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

The overall goal of the following protocol is to generate prostate cancer. Patient derived xenograft from circulating tumor cells. This is achieved by first collecting peripheral blood from patients with advanced prostate cancer.

As a second step, the blood mononuclear cell compartment is isolated, which contains the circulating tumor cells. Next, the mononuclear cells are stained with a CD 45 ZI antibody In order to select the circulating tumor cells using flow cytometry, the cells are then injected into immunocompromised mice. Results show the generation of prostate cancer xenografts based on the injection of circulating tumor cells isolated by flow cytometry from the peripheral blood of prostate cancer patients.

This method can help answer key questions in the prostate cancer field by generating new experimental models that can be used for the molecular characterization of prostate cancer and the development of new biomarkers. Demonstrating the procedure will be Williams, a student from the laboratory and Omada, a researcher from the oncology department here at Mount Sinai To begin collect whole blood from select patients with metastatic prostate cancer as they have the potential to have high numbers of circulating tumor cells in their peripheral blood. Using a 10 milliliter serological pipette transfer the whole blood into a 50 milliliter polystyrene conical tube, along with Hank's balance salt solution.

In a one one-to-one ratio, gently pipette the mixture to homogenize it. Next, add 15 milliliters of a FI call pack solution to an empty 50 milliliter polystyrene conical tube. Gently pipette 20 milliliters of the diluted whole blood, using the lowest setting on top of the solution to form a distinct top layer.

Then centrifuge the tube at 400 Gs for 30 minutes. At room temperature, set the centrifuge's deceleration to the lowest setting to prevent mixing of solutions after separation. Following centrifugation, identify the thin, white, gray stripe of peripheral blood mononuclear cells and circulating tumor cells sandwiched between the plasma top layer and the separation solution.

At the bottom of the tube, carefully collect the cells from the white gray stripe and transfer them into a 50 milliliter polystyrene tube. Using a plastic transfer pipette, then add Hank's. Balance salt solution to the isolated cells for a total volume of 10 milliliters.

Centrifuge the mixture again at 400 Gs for 10 minutes at room temperature. In order to wash the cells, remove the supernatant and repeat this wash. Step once more.

Following the second wash, discard the supernatant. Suspend the remaining pellet in five milliliters of red blood cell lysis buffer, and incubate the solution for five minutes at room temperature. Next, centrifuge the sample at 400 Gs for three minutes at room temperature and remove the lysis buffer by discarding the supernatant.

Finally, resuspend the pellet in one milliliter of PBS supplemented with 10%fetal bovine serum prior to staining. Quantify the number of viable cells using the standard trian blue exclusion method on a hemo, cytometer, or automated cell counter. Then dilute the cells to 1 million cells per milliliter in PBS with 10%FBS, and place them on ice for one hour.

To block nonspecific binding, distribute the cell suspension into two separate tubes. Label one tube for control cells and one for CD 45 stain cells in the control tube. Add IgG one Kappa Zi at a dilution of one to 250 to a final concentration of 10 nanograms per milliliter.

In the CD 45 staining tube. Add CD 45 ZI conjugated primary antibody at the same concentration of 10 nanograms per milliliter and incubate the cell suspensions on ice for 30 minutes. Next, centrifuge the cells at 400 Gs for three minutes at four degrees Celsius, and then discard the snat.

Wash the cells twice by suspending each pellet in sterile PBS supplemented with 10%FBS followed by centrifugation at 400 Gs for three minutes at four degrees Celsius. After washing resus, suspend the cells in a one milliliter solution of PBS containing 10 micrograms per milliliter of dappy stain. Filter the final solution through 35 micrometer strainer caps into 12 millimeter by 75 millimeter polystyrene tubes In order to exclude any cell clumps or debris.

Then exclude the CD 45 positive cells and dead cells from the suspension using fluorescence activated cell sorting to remove them as described in the accompanying text protocol. Mix the sorted prostate tumor cell suspension with extracellular matrix proteins in a one-to-one ratio and place the mixture on ice. Next, anesthetize an eight to 10 week old male immunodeficient mouth in accordance with institutional guidelines using 5%inhaled iso fluorine in one liter per minute of oxygen.

Ensure appropriate anesthesia by checking for loss of corneal and toe reflex in the mouse. Draw up 250 microliters of the cell suspension using a 25 gauge needle and a one milliliter syringe. Then inject the entire volume of the extracellular matrix cell suspension subcutaneously into both the upper flanks of the mouse monitor tumors by performing weekly palpation of mouse injection sites for growth of subcutaneous nodular densities.

Based on the negative selection method used in this protocol, it is necessary to exclude dead cells using DAPI stain. As shown here, the percentage of CD 45 negative cells from the remaining viable cells is variable and depends on the tumor burden of the patient, but are easily separated from the CD 45 positive cells. When proper gating is used, following implantation of the prostate tumor cell suspension, subcutaneous nodular densities will become visible on both flanks of the mouse.

Each nodular density represents xenograft growth and can be appreciated as distinct from healthy tissue as it protrudes from the dorsal musculature of the mouse and is firmer in texture. Patient derived xenograft models generated from circulating tumor cells recapitulate human prostate tumors as seen by hemat, toin and eosin staining, and by immunohistochemistry for prostate specific cellular markers such as androgen receptor and prostate specific membrane antigen After generating the xenografts. Other protocols like gene or protein expression profiling can be done to explore the cellular and molecular mechanisms that contribute to the aggressiveness of prostate cancer.