The overall goal of this procedure is the efficient preclinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia. This is accomplished by first performing Western blood analysis to confirm target inhibition in cultured leukemia cells. The second step is to evaluate functional activity using a clon genic assay in Methylcellulose or soft dagger.
Next pharmacodynamic studies are used to evaluate target inhibition in leukemic blasts in the bone marrow of mice transplanted with a human leukemia. The resulting data are used to determine the dose and schedule of administration required for effective target inhibition in vivo. Ultimately, this strategy is used to assess the therapeutic efficacy of the TKIs in vivo using bioluminescent imaging.
This strategy is designed to find the best candidate tyrosine kinase inhibitor for advancement to the clinic. The Procedures described in this video can be used to effectively evaluate tyrosine kinase inhibitors in vitro and in vivo to identify targeted agents with therapeutic potential and to compare multiple compounds thereby answering key questions in drug development workflows. The protocols presented here are focused on the development of small molecule inhibitors against receptor tyrosine kinases.
However, these protocols can be easily adapted for the preclinical evaluation of other molecular targeted agents for the treatment of acute leukemia. In each well plate five times 10 of the six cultured leukemia cells in 400 microliters of growth medium and incubate at 37 degrees Celsius in 5%carbon dioxide for two to three hours. Next, add 100 microliters of tyrosine kinase inhibitor in medium and culture for another 60 minutes.
Make sure that the tyrosine kinase inhibitor is in solution During the 60 minute incubation of the culture. Prepare the lysis buffer if the cultures will not be treated with pate prior to harvest. Add phosphatase inhibitors to the lysis buffer.
Also prepare the proven vanadate phosphatase inhibitor solution. Now dilute 60 microliters of proven vanadate into 940 microliters of complete medium. Add 100 microliters of diluted proven vanadate per well.
Then incubate the samples in a cell culture incubator. After three minutes, place the plate on ice. Collect the cells and centrifuge in cold micro fuge tubes at 2, 500 RPM for five minutes.
Aspirate the supernatants, then resuspend the cell pellets in 120 microliters of lysis buffer, and place the samples on ice for 15 minutes. To clarify the lysates centrifuge, the samples, transfer the supernatants to fresh cold tubes. Store the sample lysates at minus 80 degrees Celsius using a sterile, large bore blunt end needle Aliquot four milliliters of Methylcellulose human base medium into 50 milliliter conical tubes.
Then add 500 microliters of 10 XTKI or vehicle in growth medium, and fourex the mixture for 10 seconds. Now harvest the cells. Prepare a tenfold concentrated cell suspension in growth medium.
Add 500 microliters of cell suspension to the methylcellulose mixture. Then vortex for 10 seconds. And let's sit for 10 minutes.
To remove bubbles, draw up four milliliters of Methylcellulose mixture into a five milliliter syringe with a sterile large bore blunt end needle. Then dispense one milliliter into the center of 3 35 millimeter tissue culture plates. Swirl the dishes until the bottom is completely covered with methyl cellulose.
For each methyl cellulose plate, prepare a 10 centimeter sterile tissue culture plate with two additional 35 millimeter tissue culture plates filled with sterile water. To ensure a humid environment, place the cultures in a water jacketed incubator at 37 degrees Celsius in 5%carbon dioxide for 10 to 14 days. Count colonies using a microscope stage with a grid, or using an automated colony, counter colonies should be more than 20 cells and not overlapping.
Begin the next step by harvesting log face cells by centrifugation. After one PBS wash, re suspend the pellets at an appropriate concentration for the particular cell line. After disinfecting gloves, inject 100 microliters of the cells into the tail vein of six to 12 week old knot skid gamma mice.
Transplant at least three animals per group. Maintain the mice on gentamicin sulfate. When the mice have developed leukemia, weigh the animals and administer the freshly prepared TKI or vehicle treatment by injection or by oral gavage.
If prevent date treatment is desired, prepare PV solution as detailed in the text protocol at the appropriate times. Post treatment, harvest the femur for each femur. Flush the bone marrow with one milliliter of cold PBS or diluted room temperature PV solution using a 27 gauge needle.
If treating with PV incubated room temperature in the dark for 10 minutes, prepare the lysates and analyze phosphoprotein and total protein levels as described earlier. Then using dent cytometry, quantitate, relative phosphoprotein and total protein levels for each sample, including the controls. To begin the evaluation of anti leukemia activity, Allah, at least six mice per group.
Identifying the mice by ear punch. Add enrichment to the cages to reduce the potential for cage made aggression. After transplanting mice with a leukemia cell line of interest, treat the mice with the tyrosine kinase inhibitor or vehicle.
As per experimental design, monitor the health status of the mice daily via physical examination and weight determination. Expected symptoms of leukemia include reduced activity level weight loss, hind limb paralysis and eye infections. The engraftment and development of leukemia is monitored up to twice weekly using an in vivo bioluminescence imaging system.
To prepare for imaging, make a 15 milligram per milliliter working delucci solution and filter, sterilize it through a 0.2 micrometer filter. After anesthetizing the mouse administer 150 milligrams per kilogram body weight Lucifer by IP injection. Capture two series of images with sequential 30, 60 and 92nd exposures, and a final image with a 122nd exposure.
After imaging, return the mice to their cages and monitor for recovery from anesthesia. Depending on the intensity of bioluminescence, exposure times can be varied. Optimal exposure times should be predetermined for a given model and kept consistent such that the signal intensities for individual time points are comparable across the entire study.
Data analysis includes determining the bioluminescence intensity for each mouse using living image 3.2 acquisition and analysis software determine total flux values measured in photons per second by drawing regions of interest of identical size over each mouse. At the end of the study, dissect the mice and record observations such as enlarged liver, spleen, or lymph nodes and pale bone marrow. Collect the cells by centrifugation resus suspend cells in PBS containing 2%FBS and incubate for five minutes at room temperature.
After collecting the cells stain with 20 milligrams per milliliter, DPI and ZI conjugated anti-human CD 45 or mouse IgG one isotype control antibody. Then evaluate leukemia engraftment using flow cytometry gait on the DAPI negative viable population and determine the percentage of CD 45 positive cells as percent bone marrow blasts. Initially, Western blood analysis is used to confirm target inhibition in cultured leukemia cells.
In this example, TKIC appears the most potent TKIB is less potent and TKIA has no effect. Functional activity is then evaluated using clon genic assays in Methylcellulose or soft agricultures treatment with the TKI significantly reduced colony number in Methylcellulose. In this a LL cell line.
Treatment of this A ML cell line with TKI results in a trend toward decreased colony number. Accurate counting of viable cells prior to plating on soft agar is important for consistency between experiments and also to avoid standard errors only include soft aerates with optimal distribution of colonies. In the analysis of the experiment here, pharmacodynamic analysis is used to determine whether TKIs can inhibit the target protein in vivo.
A decrease in phosphoprotein levels is observed in leukemic blasts isolated from mice treated with TKI relative to vehicle only. In this example, TKIE is more effective than TKID. An orthotopic mouse xenograft model of acute leukemia can be generated in NSG mice injected with a luciferase expressing a LL cell line and can be used to assess the effects of treatment with A TKI.
On oncogenic potential in vivo mice treated with TKI had a significantly lower level of bioluminescence indicating a reduced leukemia burden relative to mice treated with vehicle only. After 10 days of treatment, all mice had a low level of bioluminescence that was not significantly different between animals treated with vehicle and animals treated with TKI. In contrast, by 19 days post-treatment vehicle treated mice had significantly higher bioluminescence intensity.
While mice treated with a high dose of TK, I had much lower signal intensity. After watching this video, you should have a good understanding of how to effectively evaluate novel tyrosine kinase inhibitors for the treatment of acute leukemia. Using this approach, biochemical and anti leukemia activities are determined first in cell-based assays in vitro, and then in xenograft models in vivo After its development.
This strategy paved the way for researchers in the field of drug development to explore more effective and interesting compounds for the treatment of acute leukemia and other types of cancer.
