Method For Novel Anti-Cancer Drug Development Using Tumor Explants: An Essay.

1. Explain the hypothesis and rationale for this study.

2. What conclusions can the researchers from this type of experiments?

3. What are the limitations of this technique?

4. What is the difference between a Pre-Clinical Study and a Clinical Study? Are they both necessary? Why or why not?

5. Summarize the key steps of the procedure used in this experiment, and the purpose behind these steps.

6. Consider Which of the panels represent the control group? Which of the panels represent the experimental group?

7. What does the change in Ki-67 expression indicate? What is the meaning of the change in caspase-3?

8. What does indicate about temozolomide treatment?

9. Has this approach been useful to other researchers? Share the citation and abstract here. Search the PubMed Database. For help in using PubMed go to the Quick Start Guide.

Introduction to Gliomas and Malignant Gliomas

Gliomas refer to a type of tumor that begin in the glial cells of the brain and the spinal cord. They comprise of more than 30% of all kinds of tumors in the central nervous system, of which 80% are malignant. These malignant gliomas are termed as rapidly progressive brain tumors that are made up of anaplastic astrocytoma, anaplastic oligodendroglioma, and mixed anaplastic oligoastrocytoma. Furthermore, its incidence is quite high with approximately 35–45% of them representing primary brain tumors (Goodenberger & Jenkins, 2012). The research was based on the idea that existing therapies for malignant gliomas have palliative effect. This can be attributed to the fact that although malignant gliomas are lethal, chemotherapy is the mainstay of treatment for patients suffering from this condition (Ciechomska et al., 2013). The primary aim of palliative chemotherapy has been associated with prolonging the rates of disease survival. However, this treatment fails to cure the tumor (Qin et al., 2013). Another major factor that formed the rationale for the research can be attributed to recent research studies that elaborated on the drawbacks and discrepancies of drug efficacy tests on patients with malignant glioma (Miura et al., 2013).

High failure rates of drug based trials called for the need of conducting studies with greater reliability. The authors also focused on the genetic, phenotypic and epigenetic changes of carcinoma cells that are brought about in in vitro tumor cell cultures. This made them formulate the research plan for demonstrating a novel method that relied on the use of specific malignant glioma surgical specimens, in the form of undissociated tumor blocks, subjected to the use of temozolomide (TMZ), a first line chemotherapeutic agent (Joshi et al., 2011).

Conclusions drawn

The research findings suggested that in vitro cell cultures were found to specifically expand different types of tumor cells, regardless of the conditions of cell culture. Furthermore, they represent only a certain subpopulation of the complete tumor. The researchers also found that phenotypic and genetic transformation of artificial cell expansion occurs subsequently in long term cell culture experiments. They identified tumor heterogeneity or distinct phenotypic and morphological profiles of tumor cells as a major barrier that prevents proper treatment of such conditions (He et al., 2010). Thus, the researchers concluded that selectively enriching tumor cell population, regardless of their type is not a feasible and reliable method for determining the effectiveness of any chemotherapeutic agent on the tumor cells.

Existing Therapies for Malignant Gliomas

The authors were also of the view that animal models of brain tumors that have been derived from specific tumor subpopulation have the potential of illustrating the characteristic features of the subpopulation only, and not the entire tumor. Thus, it was concluded from the experiment that use of tumor tissue explant method might hold the potential of accelerating the identification of novel chemotherapeutic drugs for treating malignant tumors (Joshi et al., 2011).

Several limitations were associated with the research. One major limitations was related to the fact that the period of treatment of the tumor cells was relatively short. Owing to the fact that malignant tumor cells have been found to resistant to a range of drug therapies over time, initially controlling the short term growth of tumor cells might have not been appropriately reflected by the patient prognosis, which occurs overs a long term. This was validated by recent studies that had focused on the administration of bevacizumab, an anti-angiogenic agent (de Groot et al., 2010). Therefore, there was a need to improve the research protocol for this assay, with the aim of preserving tissues for a longer time period.

Another major limitation was related to the specific effects of the drug on the tumor. Owing to the relative resistance of SCLTC to the therapy of interest for treating malignant glioma, there was a warranty for the identification of anti-cancer drugs that eradicate SCLTC. Furthermore, the fact that small tumor sample blocks were used for the experiment might have failed in reflecting the complete tumor population, when compared to primary surgical specimens or conventional tumor lines.

In drug development studies, preclinical studies or trials refer to the stage of research that most often begin before the clinical testing on humans can start. The primary aim of such preclinical studies are based on the determination of the safety dose for the phase 1 trials or first-in-man studies, for assessing the safety profile of a particular drug (Perrin, 2014). Thus, preclinical trials involve extensive studies that help in providing exhaustive information about the toxicity, efficacy, safety and pharmacokinetic properties of a drug. Such preclinical studies are of two types namely, in vitro and in vivo experiments. While in vitro experiments involve cell culture or experiments in test tube, in vivo tests involve animal models.

On the other hand, clinical studies are researches that are performed on people who act as volunteers for evaluating the effectiveness of a particular drug or other surgical and behavioral interventions. This type of study that involves human volunteers are carried out with the intent of adding medical knowledge (Hulley et al., 2013). Such clinical studies have different phases such as, phase I, II and III. In phase I, the drugs are tested upon small population of healthy human volunteers. Larger group of patients are recruited for phase II trial for assessing the drug efficacy. On the other hand, this efficacy is tested over a period of 6-12 months on a large patient population under conditions reflecting daily life in phase III.

Limitations and Discrepancies of Existing Drug Efficacy Tests

Although preclinical studies are vital for collecting information to determine its safety before testing them on human beings, there are some ethical issues related to the choice of best species that will provide correlation to the human trials. Furthermore, the high costs of animal testing have also made several pharmaceutical industry reduce the rates of preclinical trials on animals. On the other hand, clinical studies are essential for evaluating the effect of one or more drug interventions and helps in finding ways for preventing the development or recurrence of a particular condition. Thus, both of these studies are necessary.

• Following preparation of the media, agar solution and stock solution, glioblastoma multiforma (GBM) tissues were collected after surgery, washed with ice-cold 5mL 1XPBS and sectioned into tumor blocks (10 mm in diameter) (Joshi et al., 2011).
• The blocks were transferred to 6-well plate with 1ml media and injected with 5% DMSO or 2.5 nM TMZ, followed by incubation. DMSO or TMZ was injected thrice at different places, and the blocks were washed thrice with 5 mL 1XPBS after 16 hours. They were fixed with 10 % v/v formalin and processed for paraffin embedment (Joshi et al., 2011).
• This was followed by performing immunohistochemistry assay that involved depararaffinization and rehydration. The steps followed in this part of the experiment involved antigen retrieval, internal peroxide inhibition, blocking cover and incubation with primary antibody. The blocks were also subjected to secondary antibody reactions, followed by their detection with the chromogen DAB kit. The slides were counterstained with hematoxylin and dehydrated using ethanol. The final steps involved cover slipping the slides with mounting reagent and taking images with a Olympus fluorescence microscope (Joshi et al., 2011).

An analysis of suggests that the panel containing images of GBM tissue blocks injected with TMZ with activated caspase-3 and Ki67 represent the experimental group. The aim of the research was to determine the efficacy of the oral alkylating agent, temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). The fact that the two images present immunohistochemical reactions of caspase-3 and Ki67 for TMZ, indicate that these images show the effect of the therapeutic drug on the proteins that are necessary for apoptosis and cellular proliferation, respectively (Joshi et al., 2011).

On the other hand, the panel that contains images of tumor blocks treated with DMSO represent the control group. This can be attributed to the fact that DMSO has been found to bind effectively to several chemotherapy drugs and target the cancer cells. Thus, it can be suggested that the researchers used injected the tumor blocks with DMSO as the control experiment (Joshi et al., 2011).

Performing immunohistochemistry of the GBM tissues with TMZ showed a significant reduction in the number of Ki67 positive tumor cells, upon comparison with the control blocks. Owing to the fact that Ki67 protein is considered as an integral maker for cell proliferation, a reduction in its number indicated effectiveness of the drug TMZ in reducing the proliferation or multiplication of  glioblastoma multiforme cells (Melling et al., 2016).

On the other hand, the immunohistochemistry assay failed to produce any significant difference between the number of caspase-3, the apoptosis marker, in the experimental and the control samples. Apoptosis is a well-recognized mechanism of cell death in cancer treatment, medicated by caspase-3 (Jiang et al., 2014). However, the TMZ drug did not impact the apoptotic pathway.

Thus, an analysis of the immunohistochemistry suggests that directly injecting the drug TMZ into explants containing surgical specimen of glioblastoma multiforme cells was successful in lowering the rates of proliferation of the tumor cells. In other words, the drug was effective in controlling the abnormal increase in the cell numbers, a characteristic of carcinoma. Thus, the success in this preclinical study paves the way for its implementation on patients.

Yes. The usefulness of the approach can be determined by the fact that it has been cited in 8 other research articles that focused on the treatment of proliferating glioma stem cells.

Joshi, K., Demir, H., Yamada, R., Miyazaki, T., Ray-Chaudhury, A., & Nakano, I. (2011). Method for novel anti-cancer drug development using tumor explants of surgical specimens. Journal of visualized experiments: JoVE, (53), e2846, doi:10.3791/2846

References

Ciechomska, I. A., Gabrusiewicz, K., Szczepankiewicz, A. A., & Kaminska, B. (2013). Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine a-induced cell death. Oncogene, 32(12), 1518.

de Groot, J. F., Fuller, G., Kumar, A. J., Piao, Y., Eterovic, K., Ji, Y., & Conrad, C. A. (2010). Tumor invasion after treatment of glioblastoma with bevacizumab: radiographic and pathologic correlation in humans and mice. Neuro-oncology, 12(3), 233-242.

Goodenberger, M. L., & Jenkins, R. B. (2012). Genetics of adult glioma. Cancer genetics, 205(12), 613-621.

He, J., Liu, Y., Zhu, T. S., Xie, X., Costello, M. A., Talsma, C. E., ... & Fan, X. (2010). Glycoproteomic analysis of glioblastoma stem cell differentiation. Journal of proteome research, 10(1), 330-338.

Hulley, S. B., Cummings, S. R., Browner, W. S., Grady, D. G., & Newman, T. B. (2013). Designing clinical research. Lippincott Williams & Wilkins, 1-23.

Jiang, H., Zhao, P. J., Su, D., Feng, J., & Ma, S. L. (2014). Paris saponin I induces apoptosis via increasing the Bax/Bcl?2 ratio and caspase?3 expression in gefitinib?resistant non?small cell lung cancer in vitro and in vivo. Molecular medicine reports, 9(6), 2265-2272.

Joshi, K., Demir, H., Yamada, R., Miyazaki, T., Ray-Chaudhury, A., & Nakano, I. (2011). Method for novel anti-cancer drug development using tumor explants of surgical specimens. Journal of visualized experiments: JoVE, (53), e2846, doi:10.3791/2846

Melling, N., Kowitz, C. M., Simon, R., Bokemeyer, C., Terracciano, L., Sauter, G., ... & Marx, A. H. (2016). High Ki67 expression is an independent good prognostic marker in colorectal cancer. Journal of clinical pathology, 69(3), 209-214.

Miura, Y., Takenaka, T., Toh, K., Wu, S., Nishihara, H., Kano, M. R., ... & Cabral, H. (2013). Cyclic RGD-linked polymeric micelles for targeted delivery of platinum anticancer drugs to glioblastoma through the blood–brain tumor barrier. ACS nano, 7(10), 8583-8592.

Perrin, S. (2014). Preclinical research: Make mouse studies work. Nature, 507(7493), 423-425.

Qin, S., Bai, Y., Lim, H. Y., Thongprasert, S., Chao, Y., Fan, J., ... & Lee, J. H. (2013). Randomized, multicenter, open-label study of oxaliplatin plus fluorouracil/leucovorin versus doxorubicin as palliative chemotherapy in patients with advanced hepatocellular carcinoma from Asia. J Clin Oncol, 31(28), 3501-3508.