Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The overall goal of this procedure is to establish a novel method for drug efficacy testing with surgical specimens of brain tumors termed the tumor X explan method. This is accomplished by first securing freshly removed surgical specimens of malignant gliomas during surgery for preclinical experiments. Next, the samples are transported to the tissue procurement core facility to separate them for clinical diagnoses and research purposes.

Then in the research laboratory, tumor explants are dissected into multiple smaller tissues and transferred into optimized medium. Finally, intratumoral injections with drug candidates are performed and the explants are incubated for 16 hours. Ultimately, results can be obtained that reveal the efficacy of treatment through hematin and ein staining, as well as immunohistochemistry with cell type specific markers.

We can also evaluate drug efficacy in vitro using flow cytometry and neuros sphere forming assays. The majority of the current drug efficacy assays leave a gap between the drug efficacy in the current preclinical experimental models and the efficacy in inpatients. In particular, when we test drug candidates on solid cancers such as malignant gliomas, we cannot underestimate intercellular as well as intracellular signaling exchanges.

In order to avoid breaking microenvironment for solid tumors, we established anty culture method with our tumor explan. After tumors are removed from the patients, the samples are transferred from or to Department of Pathology, We divide the tumor chunks into two pieces. Tumor sample from one piece is used for frozen sectioning and paraffin embedding for clinical diagnosis, and the rest of the tumor tissue is sent to the research laboratories to be used by the researchers.

Using the method, we can preserve the microenvironment of the tumor with adjacent tumor endothelial and immune cells, as well as normal brain cells that are either surrounding the tumor or migrating into tumor cavities. Prior to processing glioblastoma tissue, prepare liquid, medium, and agar solution. Mix them in a one-to-one ratio.

Then add one milliliter to each well of six well plates. After receiving fresh glioblastoma multiform tissue, transfer it to a Petri dish using forceps and carefully wash it three times with five milliliters of ice cold one times PBS with the surgical blade and forceps. Cut serial sections around 10 millimeters in diameter of the specimens from the dissected samples to create tumor blocks, transfer each block into one well of the six well plate containing agar medium, allowing it to have enough exposure to air to keep it viable.

Next, using a one milliliter insulin syringe and a 28 gauge half inch needle, inject tumor blocks three times with 5%DMSO or with 2.5 nanomolar temozolomide or a novel anti-cancer drug candidate and incubate for 16 hours at 37 degrees Celsius in humidified air containing 5%carbon dioxide. After the incubation, wash the blocks three times with five milliliters of one times PBS. Then in 50 milliliter tubes, fix the blocks with 10 milliliters of 10%volume per volume, four milli for 24 hours.

Paraffin embed the tissue and cut four micron sections to depa, anize, and rehydrate the sections. Place the slides in a rack and incubate them in the following solutions. Silene three times for five minutes each.

100%ethanol three times for five minutes each. 95%ethanol once for five minutes and 70%ethanol once for five minutes. Finally, rinse and running tap water for three minutes.

Please see the accompanying written protocol for details on antigen retrieval and inhibition of internal peroxide. To block the sections, cover the tissues with 10%normal goat serum trans transfer the slides into a humid box and incubate for one hour of room temperature. Wash the slides three times in one times PBS for five minutes each.

Next, add primary antibodies such as human specific caspase three or KI 67 diluted with one times PBS then incubate overnight at four degrees Celsius the following day. Rinse the slides in one times PB S3 times for 10 minutes each. Next, cover the tissues with two to three drops of HRP labeled secondary antibody.

Put the slides in a humid box and incubate for one hour at room temperature. Wash with one times PB S3 times for five minutes each. Develop the samples using a chromogen DAB kit following the manufacturer's instructions to counter stain.

Immerse the slides in hematin for 10 to 15 seconds. Then rinse with running tap water for three minutes. Dehydrate the samples by immersing the slides in the following solutions at a mounting reagent and a cover slip.

Then use a fluorescence microscope to image the slides. The T one weighted MRI image with gadolinium enhancement in this figure demonstrates an enhanced glioblastoma multiform or GBM tumor in the right frontal lobe of a patient before surgery. The postoperative image confirmed subtotal removal of the lesion after surgery.

Hematin and eosin staining demonstrates the presence of necrosis, pseudo palisade cells, and microvascular proliferation. Thus, these tumors were histopathologically diagnosed as GBM to investigate if these tumor explan samples could be reliably utilized. For the purpose of a drug efficacy test, we tested the effect of the drug temozolomide or TMZ immunohistochemistry of TMZ treated GBM tissues demonstrates a substantial reduction of KI 67 positive tumor cells in comparison to the control samples.

In turn, immunohistochemistry with an apoptosis marker caspase three did not yield any significant difference between TMZ treated glioma specimens and the control samples. Treatment effect with temozolomide can be further characterized by combining this ex explan assay together with either in vitro culture or flow cytometry. We can compare this ex explan method with xenograft transplantation and in vitros sphere forming assay for screening of novel anti-cancer agents.

Once you obtain the tumor tissues in the laboratory, the procedure for this experiment can be completed within one hour. This new assay is relatively easy to perform and reproducible after dissociation of treated tumor implants. We can combine with in vitro experiments.

A major advantage of this assay is that it preserves the tumor microenvironment. We obtain positive data with Temodar treated tumor explan, which were consistent with the outcome of the same patients who underwent Temodar treatment following surgeries. Lastly, this is applicable to other solid cancers and therefore may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.