A Combinatory Strategy For Detection Of Live CTCs Using Microfiliteration And A New Telomerase-Selective Adenovirus

Cancer has becoming more and more common nowdays, although we have made progress in therapying tumor. There are still some tumors becoming malignant tumor. Once malignant tumors metastasis, the survival rate of patients are so low. According to the reasons above, it is demanding to find an effective way to early diagnosis. Circulating tumor cells(CTCs) have been detected in patients with many types of cancers, and are clinically relevant to cancer early detection and prognosis. Because the numbers of CTCs are as low as one CTC per 105–107 nucleated blood cells and the detection methods nowdays have so many defects(such as low specificity and sensitivity) that could not be used widely, CTC detection and capture are technically demanding. Telomerase expression is a hallmark of cancer and is required for limitless proliferation of tumor cells. The catalytic subunit of human telomerase, hTERT, is silenced in normal human somatic cells but activated in the majority of cancers. Recently, a replicationcompetent recombinant adenovirus(OBP-401) driven by a human telomerase gene(hTERT) promoter was shown to detect live CTCs in blood samples of cancer patients. Because OBP-401 could not be used for research and infect white blood easily. But the clinically relevant numbers of CTCs are as low as one CTC per 105–107 nucleated blood cells, which OBP-401 result in many false positive cells. Here, our research reports a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared to the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays(FMSAs) and adenovirus imaging. And here we get several results listed in following:1. Our research constructed four recombinant adenoviruses. Recombinant adenoviruses were generated from the BAC construct pAdZ5. The the E3 gene of pAdZ5, dispensable for virus replication in cell culture, was replaced by a GFP cassette to generate pAd5 G. Three telomerase-specific adenoviral vectors were engineered from pAd5 G. The four adenoviruses have been confirmed by fluorescent imaging. In pAd5 GTS and pAd5 GTL, the 179-bp E1 promoter is replaced by a 450-bp and a 1.4-kb h TERT promoter fragment, respectively, with the h TERT ATG codon serving as the E1 A initiation condon. Compared with p Ad5 GTS, pAd5 GTL has an additional 1 kb core h TERT promoter involved in h TERT repression in normal cells. pAd5 GTSe contains the short h TERT promoter fragment and three extra copies of a repressor elment, which repress the h TERT in normal but not cancer cells.2. The research seperated tumor cells from human normal cells by detecting the expression of GFP. We detected the relationship between the hTERT expression and GFP signals post infection in cancer cell lines and human normal cell lines by q RT-PCR and microplate reader, respectively. Here with the purpose of homogeneity, we used the mCherry-marked cells and the GFP signals were normalized by mCherry. Finally all of the recombinant adenoviruses replicated more efficiently in telomerase-positive cancer cells than innormal cells, while low infection efficiency in telomerase-negative cells. The result showed the expression of GFP could be used to separate tumor cells from human normal cells.3. The cancer cell lines, the human normal cell lines and the white blood cells were infected by four adenoviruses separately. And we determined the best infection concentration. And we further infected the mixture of cancer cell/human normal cell and white blood cells. Finally we chose the best adenovirus-- Ad5 GTSe. Because it could infect less white blood cells and more cancer cells, which reduced the false positive signal.4. The adenovirus, Ad5 GTSe, was put into clinical practice combined with the FMSA microfilters. We firstly detect the feasibility of FMSA microfilter by spiking the mCherry-marked cancer cells into 7.5 ml of blood from healthy donors. After going through the FMSA microfilter, the left cells on the microfilter were infected by Ad5 GTSe for 24 hours and the data showed the Ad5 GTSe could detect live cancer cells following their enrichment on FMSA. We further applied this combinatory strategy in the analyses of blood samples from patients with breast and pancreatic cancers. And the results demonstrated proof of principle that the combinatory strategy of microfiltration and adenoviral imaging using Ad5 GTSe is an extremely sensitive and specific method for CTC detection.All the results above showed our research got a new method to capture and detect the live CTCs. The new method was a combinatory of microfilteration(FMSA) and telomerase selective-adenovirus. The initial analyses of blood samples demonstrated the method is an extremely sensitive and specific method for CTC detection and offer a preliminary detection for early diagnosis. And we still need more fresh blood sampes for the clinical detection.