Detection Of Tumor Cells By In Vivo Flow Cytometry And Stimulated Raman Scattering Microscopy

The tumor has a long history of 5000 years.It has been one of the most threatening disease since it was first recorded.According to the statistics,more than 90% mortality of cancer patients results from cancer metastasis.When tumor cells are released from the primary tumor,they enter the blood circulation and lymphatic circulation.These tumor cells could survive and grow in appropriate biological environment,then forming distant metastases,it is vital to study influence of metastasis related cancer cells on the mechanism of cancer metastasis,clinical detection and prognosis.During recent years,with the development of tumor detection methods,we have more and deeper understanding in cancer metastasis.Most of traditional detection methods of tumor cells are in vitro,meaning drawing blood or making pathological samples is needed.This processing could change the biological environment of tumor cells,thus increasing detection errors.Furthermore,they do not allow long-term detection.In this study,we set a goal to develop the in vivo flow cytometry and stimulated Raman scattering microscopy,and apply them to investigate the dynamics of circulating tumor cell clusters,the temporal distribution of circulating tumor cell in the blood,and the label free detection method to detect tumor cells in the lymph node.There is no method to detect circulating tumor cell clusters in the blood currently.Thus we set up an in vivo flow cytometer and applied it to detect circulating tumor clusters in the peripheral blood of an orthotopic liver cancer model and a subcutaneous prostate cancer model.By analyzing the data,we found out that there was great difference for the signal shape and temporal width between circulating tumor clusters and single circulating tumor cells.We set the recognition criteria of circulating tumor cell clusters.In the two tumor models,we found out that the proportion of circulating tumor cell clusters in total circulating tumor cell events increased during cancer metastasis,more than 30% finally in the orthotopic liver cancer model which is much higher than previous reports.It is important to help us understand the circulating tumor cell clusters in cancer metastasis.Then,We investigated the temporal distribution of circulating tumor cells during cancer metastasis,and found out that the distribution of neighboring circulating tumor cells was exponential,meaning the occurrence of circulating tumor cells is a Poisson event.It is important to help us decide the length of detection time and analyze the accuracy of the results.Finally,considering that the in vivo flow cytometry is a fluorescence based technique,we developed the method to detect tumor cells by stimulated Raman scattering microscopy,which is a label free technique.We imaged fresh lymph nodes from mice by this method.We found the difference in morphology and lipid of different cells in the lymph node,and found out the tumor cell area in mouse tumor models.It is the fundamental work to develop a label-free clinical detection method in the future.Furthermore,we discussed other possible application of these techniques,e.g.immunological research,aging,melanoma,etc.