The overall goal of the following experiment is to perform a detailed analysis of the infiltrating leukocytes in a murine subcutaneous tumor model. This is accomplished by first inoculating murine B 16 F zero melanoma cells from female C 57 BL six mice to establish the growing tumor in vivo. Next B 16, tumor bearing mice are euthanized and the subcutaneous tumor is resected on block, including overlying and surrounding skin.
Then a single cell suspension of both tumor cells and tumor infiltrating leukocytes is isolated, which can then be analyzed by flow cytometry. Finally, the viable cell yield per volume is determined and the number of cells to be stained and analyzed by facts is calculated. Ultimately, flow cytometry is used to show the frequency and type of leukocyte subsets within the tumor microenvironment.
The implications of this technique extend towards the therapy or diagnosis of cancer because one can evaluate the effects of a therapy on the leukocytes within the tumor microenvironment. It is this balance of effector and suppressor cells that help determine whether or not the tumor grows or regresses To induce tumor growth. Subcutaneously inoculate murine B 16 F zero melanoma cells in a physiologic buffer such as phosphate buffered saline at or near the midline of the abdomen in female C 57 BL six mice according to the text protocol, seven to 14 days later, just prior to resecting tumors set up one 50 milliliter conical tube per tumor and place on ice.
Insert a 40 to 70 micron cell suspension filter into each tube. Prepare surgical forceps and find scissors for resecting and 70%ethanol to clean instruments between tumor samples after euthanizing a B 16 tumor bearing mouse, according to the text protocol, secure the mouse on a surgical stage, use 70%ethanol to spray the tumor area and wipe off the access to prevent spreading hair. Ensure topical ethanol is fully wiped and evaporated prior to resecting the tumor using forceps and scissors, resect the subcutaneous tumor on block, including overlying skin and approximately two to three millimeters of surrounding skin.
Weigh the resected tumors with and without the surrounding skin and place each section in a filter in the conical tube. To prepare a single cell tumor suspension, use forceps and scissors to mince the tumor and overlying and surrounding skin until all large sections of tissue are processed into one to two millimeter sections. Next, using a plunger from a disposable five or 10 milliliter syringe, mechanically disaggregate the minced tumor tissue against the secured filter.
Then with a pipette man and five to 10 milliliters of cold, medium, or buffer. Wash the processed tumor tissue. This will flush the single cells through the filter.
Repeat the disaggregation and washing steps several times, ensuring that the total number and volumes of washings are approximately the same For similarly sized tumors. After washing the tissue, verify that small fragments of skin and or other connective tissue are left behind in the filter to wash and pellet the cells add medium and centrifuge at 500 Gs and four degrees Celsius for five minutes. To carry out fax analysis, use ice cold fax buffer to re suspend the cells.
Then with trian blue and a hemo cytometer count, the viable cells determine the total number of viable cells per volume to be used to extrapolate absolute numbers of tumor infiltrating leukocytes or till in combination with facts. Data from the trian stained counting data calculate the volume of cells to be stained for facts. For example, if TIL typically constitute 5%of total tumor cells, factor this into the total number of tumor cells in suspension to be stained.
To stain the cells for live dead discrimination. Use a fixable or non fixable stain such as propidium iodide. Then use FBS in PBS with 1%rat serum and FC block to block the cells for 10 minutes before using a standard fact staining protocol to stain the cell suspension for specific leukocyte subsets.
After staining, use 4%paraform aldehyde to fix the samples and store at four degrees Celsius in the dark until time for collection and analysis. Use a flow cytometer to collect the samples with facts. Data analysis software begin by gating out dead cells, then with an antibody against a leukocyte specific marker such as anti CD 45, identify leukocytes within the live cells.
Next, use various gating schemes to identify specific subsets according to the text protocol. Analyze data and present in a number of ways to ensure consistency and identify patterns or trends as shown here. Forced overexpression of kein in murine B 16 tumors resected day 17 resulted in a significant increase in total CD 45 positive cells compared to controls in this figure using immunofluorescence on cryo sectioned B 16 tumors.
An increase in infiltrating CD 45 positive cells from tumors was confirmed. Further analysis of fax data shows a relative increase on average of NK cells, T cells, and conventional CD 11 C positive dendritic cells. In the tumors where kein was over expressed compared to the control tumors, there was also a relative decrease in the percentage of immune suppressing leukocytes, including myeloid derived suppressor cells and plasmacytoid dendritic cells.
Studies have shown that the ratio of leukocytes subsets within the tumor may be an important factor in terms of tumor growth and clinical outcomes, and in support of this, the number of CD three positive T cells and NK cells per tumor were increasingly represented in the kein expressing tumors compared to controls. This figure shows that in a direct comparison of effector and suppressor cell populations within the tumor microenvironment, significant increases in the ratios of effector to suppressor cell populations were evident within the kein expressing tumors. Compared to controls Follow this procedure.
Other methods like cell sorting or cell isolation can be performed in order to answer additional questions that address tumor infiltrating leukocyte functionality.
