| DEVELOPMENT
OF AN EX-VIVO PROSTATE CANCER MODEL FOR THE ASSESSMENT OF SECOND
GENERATION GENE THERAPY FOR LOCALISED PROSTATE CANCER.
Daniel Ashdown, Dr. E. Porfiri, Dr. P. Searle University of Birmingham |
 |
Background
Prostate cancer as a target for gene therapy
Prostate cancer is the most frequent male malignancy in the UK with ‑ 30,000 new cases diagnosed in 2001. 15‑20% of prostate cancer patients develop localised recurrence after treatment without metastatic spread. These men have limited curative treatment options but novel treatments for this disease state such as gene therapy could dramatically affect the outcome for this cohort of patients.
Project objective
The key objective of my research is to develop new ways to assess new gene therapy treatments, namely viruses, for prostate cancer. I have developed Conditionally Replicating Adenovirus's (CRAd's) targeted to cancer cells and expressing an enzyme (nitroreductase) which converts a harmless chemical injected into the bloodstream, into a toxic drug in the prostate. Previously either animal models or prostate cancer cells lines grown in culture were used to assess these treatments. However the predictive value of these models is unclear, in terms of efficacy in humans. It is hoped that the results of my studies will enable identification of new treatments for clinical trials.
Current work
Tissue collection
Over 20 samples of prostate tissue from patients with and without cancer have been sourced so far. This has enabled me to practice the aseptic atraumatic handling of the tissue samples to maintain viability. I had originally planned cut these samples into very thin slices prior to infection with viruses however as assessment of spread of virus within the 3D tissue structure is important I have instead used 1 sqmm cancer samples and 2 sqmm benign prostate tissue samples.
Assessment of viability of benign and malignant prostate tissue
The first experiments I performed were aimed at assessing how long tissue samples survived after removal from the patient. Variables assessed included volume and composition of media. At multiples of 24 hour time points tissue samples were embedded in paraffin, sectioned, and viability assessed microscopically by a Consultant Pathologist. The results suggest that benign prostate samples remain viable up to 7 days with fairly basic media preparations. Prostate cancer samples however need more complex media combinations including testosterone and growth factors to attain viability to this time.
Assessment of infectability of tissue with adenovirus based vectors
Before viruses are able to replicate in viable tissue they need enter the tissue by binding to the Coxsackie Adenovirus Receptor (CAR) on the surface of the cells. To ensure that CAR expression is maintained after the tissue is removed from the patient and kept in culture medium, I used immunohistochemistry to access CAR expression in benign and malignant tissue samples up to 5 days. On multiple samples, CAR expression was still evident in tissue samples up to 5 days. I will assess CAR expression at later date.
Assessment of infectability and replication of virus gene therapy vectors
The viruses I have constructed should only replicate in tumour tissue. Therefore to confirm this, using benign tissue, I assessed the replication of my viruses compared with wild type adenovirus which replicates in all prostate tissue types. Tissue samples were infected for up to 96 hours and virus particle replication assessed using quantitative PCR. Production of infective virus was assessed using plaque assays. The results confirmed that wild type adenovirus replicates in benign tissue and the amount of virus particles and infective virus particles increased over the time course of the experiments. In contrast the viruses designed not to replicate in benign tissue showed no evidence of infective virus production or significant virus particle production at 48 or 96 hours post infection. These experiments were repeated in a number of prostate cancer samples. Comparing production of virus particles and infective virus, showed a significant difference between viruses dependent on whether the viruses expressed the E3 region of adenovirus, (which encodes a number of proteins which aid virus replication and spread).
Assessment of NTR expression m* prostate samples
Having confirmed tissue viability, infectability and virus replication I have assessed expression of nitroreductase (NTR) in tissue samples. The benefit of expressing this enzyme in a replicating virus is that it allows spread of expression of the enzyme within the prostate tumour tissue. In benign prostate tissue samples NTR expression of replicating viruses expressing NTR was compared with a non replicating virus which also expressed NTR. As expected a limited amount of NTR expression was shown using immunohistochemistry but there was no difference in the amount of expression between viruses and it did not increase between 48 hours and 96 hours after infection, again suggesting that these viruses were not replicating in the benign tissue. In contrast, NTR expression in cancer tissue was significantly higher and there were marked differences between the replicating and non replicating viruses at 48 hours post infection. At 96 hours post infection NTR expression had further increased and spread through the tissue samples.
Conclusion
The results so far are encouraging and suggest tissue samples can be kept viable possibly up to 7 days and used to assess virus infection, replication and expression of enzymes. Much of the work done so far has involved perfection of tissue sample preparation and assessment. Preliminary results have identified the virus vDA012‑2C2 as having improved replication and NTR expression in prostate cancer samples compared with other viruses constructed by our laboratory members.
Future work
Over the next 3 months I will repeat the virus replication and NTR expression experiments in as many tissue samples as possible over a longer time period (of at least 7 days). I will also assess virus replication further in tissue samples using histochemistry looking at viral proteins which will show spread of virus replication in the tissue samples.
Expanding on the previous experiments the final part of the work will involve assessment of oncolysis (cell death caused by virus infection) and combined cytotoxicity by assessing cell death in tissue samples infected with virus with and without the addition of prodrug CBI 954. Cell death will be assessed and confirmed by assessment by a Consultant Pathologist. These experiments will be performed both in benign and malignant tissue.
Interim research report dated 03 November 2006
Project 2005/15