1Department of Neurological Surgery, The Ohio State University Medical Center, 2Department of Pathology, The Ohio State University Medical Center
Joshi, K., Demir, H., Yamada, R., Miyazaki, T., Ray-Chaudhury, A., Nakano, I. Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens. J. Vis. Exp. (53), e2846, doi:10.3791/2846 (2011).
The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.
We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.
1. Preparation of media
2. Tissue processing
Immunohistochemistry was performed as described previously. 1
4. Representative Results:
We collected surgical specimens of glioblastoma multiforme (GBM). The patients underwent the first surgery without prior chemotherapies including temozolomide (TMZ). The T1-weighted image of MRI with Gadolinium enhancement in Figure-1a demonstrated an enhanced tumor in the right frontal lobe before surgery. The postoperative image (Figure-1b) confirmed subtotal removal of the lesion after surgery. The surgical tissues were sent to the Department of Pathology for the clinical diagnosis purpose, and the remaining specimens were processed to the tissue procurement under the approved Institutional Review Board (IRB) protocol at the Ohio State University Medical Center. Hematoxylin and Eosin (H&E) staining demonstrated the presence of necrosis, pseudopallisading cells, and microvascular proliferation (Figure 1-c). Thus, these tumors were histopathologically diagnosed as GBM. Of note, tumor explants following incubation without TMZ up to 48 hours maintained the cytoarchitecture of GBM .In contrast, with incubation for 72 hours or longer, we noticed the increased number of condensed nuclei in the specimens, suggesting some of the tumor cells starting to undergo apoptosis. As a result, tumor tissues following longer incubation contained patchy regions that lost tumor cells, which were preferentially observed at and around necrotic areas (data not shown).
In order to investigate if these tumor explant samples can reliably be utilized for the purpose of drug efficacy test, we tested the effect of TMZ treatment. Figure-2 represents the flow of the experiments in this study. A recent study indicated that intratumoral injection of TMZ has more potent effect on growth control of malignant glioma than that of the systemic administration of this drug 2. Following incubation for 16 hours, these explants were embedded with paraffin and serial sections were prepared for staining. Immunohistochemistry of TMZ-treated GBM tissues demonstrated a substantial reduction of Ki-67-positive tumor cells in comparison with the control samples. In turn, immunohistochemistry with an apoptosis marker, Caspase-3, did not yield any significant difference between TMZ-treated glioma specimens and the control samples (Figure-3).
Treatment effect with TMZ was further characterized by combining this explant assay together with either in vitro culture or flow cytometry. Specifically, we sought to determine the effect on stem cell-like tumor cells (SCLTC). Therefore, following intratumoral treatment with TMZ, we performed sphere forming assay and flow cytometry of these tumor explants. The TMZ-treated specimens were dissociated into individual single cells and these single cells were seeded in a low density (1 cell per microliter or lower) in 96-well plates in serum-free medium supplemented with bFGF and EGF. In the control group, formation of tumor spheres from the tumor explants was observed after 7 day incubation. Given that sphere formation is a property of SCLTC3 4, alterations in the number of tumor spheres by a drug treatment indicates the effect on SCLTC. Similarly, flow cytometry of the dissociated tumor explants with a cell surface marker, CD133, was carried out to verify the effect on SCLTC. If SCLTC in heterogeneous tumor cells in GBM are selectively eradicated by a drug treatment, one might predict that flow cytometry should detect decrease of the CD133-positive fraction by treatment (data not shown).
Figure.1 Magnetic resonance images (MRI) of a patient GBM. Representative figures of pre-operation Figure-1a and post-operation Figure-1b. Figure-1c is H&E staining of fresh GBM tissue sample. Original magnification, 40X.
Figure. 2 The experimental flow of block culture experiment. Figure-2a. Image of fresh surgical GBM received from department of pathology. Figure-2b shows Dissection of tumor block into 10mm small pieces with the help surgical blade. Figure-2c is the drug (TMZ) treatment of tumor blocks using insulin syringe.
Figure. 3 Immunohistochemistry. Immunohistochemistry of GBM tissue blocks injected with DMSO or TMZ with activated Caspase-3 and Ki67. Hematoxylin was used for nuclear staining. Original magnification, 40X.
There is a gap between the drug efficacy in the current pre-clinical experimental models and the efficacy in patients. More specifically, in the brain tumor research field, novel and reliable methods are required that would help filling the existing gap between the drug evaluation with the current methods and the efficacy in patients. The described assay in this study is an additional asset, if not a solution, to facilitate filling the discrepancy of the experimental data and outcome of affected patients.
In vitro cell cultures preferentially expand certain tumor cell types regardless of the culture conditions, and represent only a subpopulation of the entire tumor. 5 In addition, genetic and phenotypic transformation of the artificial cell expansion occurs and is inevitable with the long-term cell cultures. One of the major hurdles to treat malignant glioma is the heterogeneity of tumor cells, both within a single tumor and between tumor samples 6, 7. Therefore, selective enrichment of a certain population of tumor cells, regardless of one type or another, may not be an appropriate means to determine efficacy of any chemotherapeutic agents on heterogeneous tumor cells. 8 9 Any animal model of brain tumors derived from a subpopulation of tumor cells shed lights on drug efficacy on a subpopulation, but not the entire tumor.
Several limitations, on the other hand, still remain in this tumor explant method. One such limitation is the relatively short period of treatment. Since malignant tumor cells are considered to acquire resistance to most, if not all, therapies over time, initial control of the short-term tumor cell growth may not be reflected by the long-term patient prognosis, as was recently reported with an anti-angiogenic agent, bevacizumab 10. Thus, improvement of the protocol for this explant assay to preserve tissues for a longer period is required with further investigations. Another question is a specific effect of a tested drug on SCLTC in the tumor. Given that SCLTC are relatively resistant to the current therapies for malignant glioma, identification of novel anti-cancer drugs that potently eradicate SCLTC is warranted. In this regard, we currently seek to combine this explant assay with the subsequent in vitro experiments, such as neurosphere forming assay (data not shown). We expect to learn more about the effects, if any, of a drug candidate on SCLTC through these combined assays. Lastly, using small blocks of tumor samples may not reflect the entire tumor cell populations, as is the case with the conventional tumor cell lines or primary cultures from surgical specimens. These issues aside, preserving tumor stroma, including the vascular niche, is helpful in characterizing the environmental factors for tumor cells. Therefore, this assay may have potential to evaluate any therapeutic strategies under a more physiologically relevant condition.
Here, we established an assay for drug efficacy testing with explants of surgical specimens of GBM. In fact, with the tested surgical specimens, we found that a direct injection of TMZ reduces proliferation in the GBM samples. This tissue explant method is theoretically applicable to other solid cancers and provides asset to determine drug efficacy on a patient's tumor, which will hopefully help us avoiding another failure in clinical trials for cancers.
No conflicts of interest declared.
The American Cancer Society (MRSG-08-108-01), Vincent J. Sgro/The American Brain Tumor Association, and the Khan Family Foundation for I Nakano.
|Fetal Bovine Serum, Qualified, Heat-Inactivated||GIBCO, by Life Technologies||16140-071|
|D-MEM/F-12 (1X) Liquid 1:1||Invitrogen||10565-042|
|Agar-Agar Purified, Granulated||EMD Millipore||1.01614.1000|
|DPBS for staining||Invitrogen||14190|
|10% w/v formalin Caspase-3||Ricca Chemical Company||3810|
|Envision system- anti-mouse||Dako||2012-07|
|Envision system- anti-rabbit||Dako||2011-12|
|Table 1. Materials.|
|6 Well Plate||Greiner Bio-One||657160|
|Petri dish||VWR international||25384-302|
|Serological pipette||Axygen Scientific|
|Filter tips||USA Scientific, Inc.|
|500 Ám polyester mesh Netwell insert||Costar||3480|
|Matrigel Matrix||BD Biosciences||354230|
|BD 10 mL syringe||BD Biosciences||309604|
|Hypodermic needle Aluminum Hub.||Tyco Healthcare, Covidien||2000029|
|Tissue culture flask||Corning|
|Thin wall tubes||Eton||1107A|
|Surgical Blade stainless steel||Feather Safety Razor Co, Ltd.||2976#10|
|Insulin Syringe||Comfort point||26027|
|50mL flat top screw cap tube||Basix||5539802|
|Dissection microscope||Tritech Research, Inc.|
|Fluorescence microscope||Olympus Corporation||DP-72|
|Table 2. Equipment.|