Articles
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Almost 80% of cancer treatments entering clinical trials fail before they reach the market. The number of new cancer cases globally is predicted to double to 27 million by 2030 with the number of survivors exceeding 75 million.1 A wide variety of new oncology targets continue to be identified, many through investigations into the tumor microenvironment associated with solid tumors and tumor stem cell characteristics. Elucidation of the molecular mechanisms that enable tumor cell dissemination are providing important therapeutic strategies. Gene knockdown and fluorescent markers are important tools to enable quantification in vitro and real-time in vivo imaging in support of target validation.
Current approaches to lead validation and optimization rely on two-dimensional in vitro assays and simple xenograft and orthotopic models utilizing immortalized cell lines whose characteristics have been selected and optimized for growing on plastic. The molecular profile of these cell lines can be considerably different from primary tumor cells. Standard models will not exhibit optimal paracrine signaling due to the lack of human stroma, potentially misguiding researchers trying to determine the efficacy of novel therapeutic agents.
Using bioluminescence imaging to evaluate preclinical models is a new approach to evaluating potential therapeutic agents or regimens. It is vital to conduct research into tumor initiation and development in order to closely monitor the patient disease in terms of stromal interactions, antigenic response, and chemotherapeutic sensitivity. Tumor cell lines transfected with the reporter gene firefly luciferase emit detectable levels of light in the presence of the substrate D-luciferin, which rapidly penetrates the body. The amount of light emitted is proportional to the number of live cells present. This enables cell proliferation to be monitored in real-time, in the same subject, throughout the time course of the experiment. By using special light-detecting equipment, researchers can efficiently monitor neoplastic development and response to therapy.
Advanced cancer models
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A range of orthotopic models have been recently developed in order to more closely investigate prostate cancer. These models have been evaluated using bioluminescent imaging, which allows real time visualization of primary tumors, for example at orthotopic sites and metastatic development. A range of prostate cancer models are currently available, including a bioluminescent variant of the hormone-sensitive LNCaP, which can also be modelled orthotopically.
In addition, scientists have developed and optimized a panel of bioluminescent prostate cancer cell lines for orthotopic and metastatic modeling. A recently validated model is the orthotopic prostate model involving the bioluminescent cell line PC3M, in which cells are injected directly into the prostate of nude mice and growth is monitored in real time by whole body visualization using a bioluminescent spectrum imaging system. At termination, the prostate is removed and examined macroscopically and histologically. Bioluminescent imaging clearly indicates the location of the tumor and confirms that the tumor occupies the cuboid prostate tissue region.
A broad array of implantable, xenograft breast cancer models have been developed. Research has been undertaken involving bioluminescent imaging of the MDA-MB-231 bone metastasis model in nude mice (Figure 1). MDA-MB-231 expressing firefly luciferase was used experimentally to model bone metastasis following intracardiac injection of a cell inoculum. Cells rapidly lodged in the end plates of the long bones, jaw, ribs, and spine. Bioluminescence could be detected using an imaging system from day seven on and was monitored throughout the study. Lytic lesions were formed, similar to clinical presentation, and these could be detected and quantified post-mortem by x-ray, histology, and tomographic imaging modalities such as MRI. The standard-of-care agent pamidronate was used as a positive control and significantly reduced lytic lesions.
Bioluminescent imaging of the MDA-MB-231 orthotopic breast cancer xenograft model was also performed. Bioluminescent MDA-MB-231 was injected in the mammary fat pad and metastasized to the lungs via the lymphs. The growth of the primary tumor could be monitored in real-time using an imaging system from day seven, whereas metastatic lesions could be detected and quantified post-mortem by imaging or histology. The standard-of-care agent taxotore was used as a positive control comparison and has been shown to significantly reduce tumor size.
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A number of subcutaneous and orthotopic intracranial models of brain cancer have been recently introduced (Figure 2). Bioluminescent variants of U87MG and U373MG glioblastomas allow real-time imaging of internal tumors and treatment effects. These models have been developed with sensitivity to clinically relevant doses of chemotherapeutics which are used as positive control, such as temozolomide and lamusine (Figure 3). U87MG and U373MG can also be enriched for stem cells and neurospheres transplanted orthotopically. Development of specific therapies targeted at cancer stem cells is crucial for improvement of survival and quality of life of cancer patients, especially those with metastatic disease.
In addition, metastatic brain tumor variants from breast or melanoma primaries are being pursued for the development of relevant metastasis models. Metastasis in the brain has been seen in some mice following intracardiac injection of cells. By serial transplantation, it is aimed to select a cell line with increased metastatic ability. The development can be accelerated using an imaging system as it has been established that the substrate will pass the blood-brain barrier. A spontaneous metastasis variant of human breast cancer is also under development, whereby bioluminescent cells are injected into the mammary fat pad followed by resection of the primary tumor. Additionally, research is being undertaken to develop spontaneous metastasis from murine melanoma.
Conclusion
New prostate, breast, and brain cancer models more fully recapitulate the tumor micro-environment both in vitro and in vivo by using either human mesenchymal stem cells or patient-derived cancer-associated fibroblasts. Using advanced bioluminescent techniques can help to validate potential targets and provide accurate and reproducible patient-relevant models which ensure that preclinical efficacy assessment is predictive of how a drug is likely to behave in clinical trials with cancer patients.
About the Author
Rajendra’s expertise in cancer cell biology and model development has enabled her to implement the technology in the cancer models offered by PRECOS, through protocol management, validation of in vitro/in vivo testing and optimization, data and statistical analysis of models, and dissemination of scientific information. She manages the commercial projects which utilizes these models.
References
1. Boyles S. Global Cancer Deaths to Double by 2030. WebMD. Available from http://www.webmd.com/cancer/news/20081209/global-cancer-deaths-double-2030. Accessed on December 16, 2011.
