A Novel Approach For Detection Of Circulating Tumor Cells By Replication-selective Adenovirus In Lung Cancer

Background and ObjectiveAccording to the annual report of 2015 published by the China Cancer Registry, the incidence rate of lung cancer occupied the first place in Chinese male patients and the second in the female patients following the breast cancer. The mortality rate ranked first and the five-year survival rate is just 16.1%.The National Cancer Center also published the data of 2015 in the ?CA Cancer J Clin?in 2016,the lung cancer had the highest incidence and mortality rate in the whole. There is increasing evidence for the use of circulating tumor cells(CTCs) as a �liquid biopsy� for early detection of lung cancer recurrence, prognosticating disease and monitoring treatment response. The identification of CTCs has become more feasible with the development of increasingly sensitive detection methods. Different methods of CTC enrichment and detection have been developed that rely on physical(size, density, deformability) or biological properties(cell surface protein expression) of CTCs. For instance, the CellSearch is the only detection method approved by the FDA, it utilizes the immunomagnetic beads coupled to antibodies against the epithelial cell adhesion molecule(EpCAM) mixed with a sample of whole blood for immunomagnetic separation, but false positive results have been reported in benign and inflammatory conditions. we have created a new telomerase-specific, replication-selective adenovirus 11 expressing GFP(Ad11-Tel-GFP) by which the morphological observation and counting of CTCs could be carried out directly Under the fluorescence microscope. We aimed to detect the CTCs with different samples,try to identify the sensitivity and specificity and discuss the clinical value. MethodsThe red blood cells of the healthy volunteers was cracked and then mixed with the virus Ad11-Tel-GFP.After the incubation for 24 h at 37?,the cells would be observed under the fluorescence microscopy; Human lung cancer cell line A549 were counted after digestion,added with 10%DMEM,the virus were added and incubated for 24 hours at 37?,fixed and stained by DAPI,GFP-positive tumor cells were counted under a fluorescence microscopy;100 cells of A549 were counted after digestion and mixed with 1ml blood of healthy volunteer?After the the lysis of the red blood cells,the number of leukocytes were counted and virus were mixed for incubating for 24 hours at 37??After fixing and staining by DAPI,GFP-positive tumor cells were counted under a fluorescence microscopy;6ml blood of patients with lung cancer were obtained from the hospital. After the lysis of the red blood cells, the virus were added for 1pfu/cell according to the counted number of leukocytes. Incubated for 24 hours at 37?,the sample was fixed and stained by DAPI for observing under a fluorescence microscopy; Following the step of incubation with the virus, the leukocytes were labeled with the anti-CD45 antibody, then the sample was fixed and stained by DAPI for observing; Two repeated samples of healthy volunteers, non-neoplastic patients and patients with lung cancer were disposed with the same procedure, labeled with the anti-CD45 antibody or not. Results1. There was no positive CTCs detected in the healthy volunteers, but we found very few false positive CTCs expressing green fluorescence. They were predicted to be WBCs by the size and morphological characteristics of cells.2. Ad11-Tel-GFP could infect the human lung cancer cell line A549 and replicate massively expressing green fluorescence.3. Ad11-Tel-GFP could specifically infect tumor cells and replicate massively in A549 cells while mixed with the whole blood cells of the healthy human, the sensitivity rate is 96%.4. Ad11-Tel-GFP could specifically infect tumor cells and replicate massively in the samples of patients with lung cancer and false positive CTCs could also be found, which have the same characteristics as those in the healthy volunteers. The CTCs have a larger diameter than 10?m and even a cluster of tumor cells could be found.5. Immunocytochemistry staining of white blood cells can help to identify false positive cells, which express red fluorescence of the cell surface under the fluorescence microscopy, but the number of positive cells decreased after the step of immunohistochemical staining because of losing and so on.6. According to the current results and the theory of the ISET( isolation by size of epithelial tumor cells), the criterion for judging could be recommend as follow: The positive CTCs could be identified for those expressing green fluorescence, possessing the nucleus with a irregular shape and having a diameter over 10?m. According to the standard, false positive cells could be found in healthy donors and non-neoplastic patients and there were no positive cells, matching well with the clinical status.7. Defined by the TNM classification system, For the non small-cell lung cancer the positive rate for stage ?,?,?,? were 40%,50%,46.6%,77.8%,with no significant difference in statistics science(?2=5.541,p=0.136). The positive rate of early(stages?-?A) and advanced disease(stages ?B/?) were 43.75% and 72.73% respectively, the difference was of significance in statistics science(?2=3.893,p=0.048). As to limited disease and extensive disease of the small cell lung cancer, it was 20.0% and 76.9% respectively, the difference was of significance in statistics science(P=0.047). ConclusionThe telomerase-specific replication--selective adenovirus developed in our lab can specifically detect lung cancer cells in the circulating blood, it shows a satisfactory marker to label malignant cells in preclinical and clinical studies. It is a simple and effective new method of detecting CTCs with high sensitivity, and specificity, with a certain correlation with stage, which has a huge potential of the prognostic and predictive value and even the individual treatment.