生物発光イメージングを使用して腫瘍の成長や治療に対する反応のその後の分析では頭蓋内脳の腫瘍異種移植片の確立

For many years, transplantation models using human brain tumor cells have primarily involved subcutaneous injection in immunocompromised mice. Recently, there has been a shift towards injection into the appropriate anatomical structure. The brain.

In this video article, intracranial tumors are established in mice using an injection method in which a hole is created in the skull, and a needle is used to inject tumor cells modified for bioluminescence imaging. Following surgery, the rate of tumor growth and response to treatment is monitored. Using a bioluminescence imaging station, survival analysis and histopathological and protein expression analysis, the resulting data can then be used to evaluate the efficacy of experimental brain tumor therapies.

This method can help answer key questions in the neuro-oncology field, such as whether the anti-tumor agent can access the brain. The implications of this technique extend toward therapy of brain tumors in patients because we are treating human brain tumors that are grown in an animal host Working under a tissue culture hood. Prepare subcutaneous tumor cells for transfer to the intracranial compartment.

Begin by placing excised flank tumors into tissue culture dishes. Using a scalpel, mince the tissue. Then using a pipetter mechanically disrupt the tissue to create a cell aggregate suspension.

Next, pass the cell aggregate suspension through a strainer with a 70 micrometer nylon mesh filter and collect it in a round bottom tube. Repeat this process for a total of three times to produce a single cell suspension suitable for intracranial injection.Centrifuge. The suspension at 1000 RPM for 10 minutes at four degrees Celsius following centrifugation, aspirate the sup nacent then resuspend the cell pellets in an appropriate volume of serum free medium to obtain a final working concentration as an alternative to the disaggregation of subcutaneous tumors.

Tumor cells can be harvested from cell cultures containing established brain tumor. Cell lines. Harvest the cells by trypsin in centrifugation.

Following the centrifugation Resus, suspend the cells in the appropriate volume of serum free DMEM for super tentorial injections by freehand. Prepare three to five times 10 to the five cells in three microliters. Disinfect the surgical area by spraying all surfaces with 2%chlorhexidine.

Next place the following supplies in the surgical area. A heating pad to maintain mouse body temperature. Two small petro dishes, one containing 3%hydrogen peroxide and one containing 2%Chlorhexidine sterile GREs and cotton swabs, sterile disposable scalpels, and an autoclave surgical stapler.

Once the mouse has been anesthetized with a ketamine xylazine mixture, swap the surgical area several times using sterile gores dipped in chlorhexidine. Apply ment to maintain adequate moisture during the procedure. Using a sterile scalpel, complete a sagittal incision over the par occipital bone, approximately one centimeter long.

Using a cotton swab soaked in 3%hydrogen peroxide solution, clean up the exposed skull surface. The bgma should be apparent at this point to create an opening for the injection of tumor cells. Use a sterile 25 gauge sharp needle to puncture the skull.

Two millimeters to the right of bgma and one millimeter anterior to the coronal suture. Mix the contents of the cell suspension by tapping. Load the syringe with the desired amount of cell suspension.

Avoid creating air bubbles. Clean the outside of the syringe with an alcohol swab to remove any adherence cells. This will help prevent extra cranial tumor establishment and growth as shown here to ensure that the appropriate injection depth is achieved.

When not using a small animal stereotactic apparatus, use a scalpel to cut three millimeters off the pointed end of a P 20 pipette tip. Place the tip section over the syringe to limit the injection depth to ensure that the tip of the syringe needle will be three millimeters from the underside of the skull. So the next step in the procedure is probably the single most difficult aspect of the entire method when doing the following injection, achieving the appropriate death and the delivery is of atmos importance in order to ensure the tumor is established and grows in the correct location of the brain.

Next place a syringe perpendicular to the skull in the hole and slowly inject the cell suspension. Injecting larger volumes and recommended can result in tumor cell reflux through the needle tract with results in phytic rather than intracranial tumor growth. As shown here, an inappropriate angle of syringe insertion can result in intraventricular injection of cells and subsequent spinal dissemination as shown here upon completing injection.

Leave the needle in place for another minute, then slowly withdraw. These steps will help prevent tumor reflux. Clean the skull with 3%hydrogen peroxide and dry.

Using a sterile cotton swab. Apply sterile bone wax to the hole using forceps. Draw the scalp together over the skull with the dermis of each side, pressed together, underside against underside and staple.

Then clean the scalp using chlorhexidine. Administer buprenorphine by subcutaneous injection for postoperative pain relief. Though not shown here, the buprenorphine is rapidly absorbed.

Proceed to inject additional mice. Be sure to mix the cells between injections to maintain appropriate concentration among the members of an injection series. Treatment groups of at least eight are recommended to help ensure results that are statistically meaningful.

Monitor all of the mice postoperatively until they become ambulant and regain normal activity. Typically, recovery time is around 30 minutes, beginning one week after the tumor cell injection. Quantitative bioluminescence imaging or QBLI should be performed once or twice a week.Here.

QBLI is conducted using the IVIS Lumina Imaging station and living image software version 3.2. From the Windows start menu, select the living image software program. Once the program opens, click the initialized IVIS system button in the camera control panel.

After initialization, the temperature status box should be green, indicating that the CCD camera is adequately called place the anesthetized luciferian injected mouse. On the stage of the imaging chamber, open one of the saved files from your imaging session and change the image data display from counts to photons. To determine the photons per second of the target signal, select the circle icon from the ROI dropdown menu.

A region of interest labeled ROI should appear around the target signal. The signal should be entered into an Excel file in which the animal number and day of imaging is indicated. Repeat this sequence for all animals analyzed during your imaging session.

Once photon counts have been determined for each mouse that was imaged and photon values have been transferred to an Excel file. Convert luminescence readings to normalize values by dividing each mouse's luminescence obtained during and subsequent to the completion of the therapeutic regimen by its corresponding maximal pretreatment luminescence. Reading To perform survival analysis using prism software, use the KAPLAN Mere estimator to generate survival plots and to estimate the median survival values.

Differences between survival curves are compared using a log rank test. Euthanasia of the animal should be performed if the animal displays a significant decline in health, especially if there is weight loss of greater than 20%of the maximum weight. Quick click size, the mouse brain from the euthanized mouse, then place the brain in formin for subsequent analysis of tumor morphology and immunohistochemistry.

This example shows mean normalized luminescence values for mice receiving intracranial tumor cell injection therapy with erlotinib was initiated at day 34. Error bars indicate standard error, A significant difference in mean normalized luminescence is apparent at the first imaging time point following the beginning of therapy. The difference in mean group luminescence shows further increase at subsequent time points in most cases.

Therapy induced anti-tumor activity as indicated by QBLI is accompanied by a corresponding significant difference in survival. As is the case here, hemmat and EOSIN stain sections of the paraffin embedded mouse brain shows the pattern of tumor cell distribution. P 10 is a marker that is frequently associated with tumor response to EGFR inhibition.Here.

Otin of treated tumor cells are seen to be devoid of P 10 expression as indicated by the lack of brown staining. Here, a section of the paraffin embedded mouse brain is shown. EGFR inhibitor sensitive brain tumor cells can be identified by the brown EGFR staining.

As you've seen, the most challenging aspect of this procedure is the proper injection of the tumor cells. What is the most further experiment can be performed using this approach, such as investigating the growth effects of genetic modifications to tumor cells? So that's it.

I hope you found this presentation useful. Good luck with your experiments.