Noninvasive Real-time Monitoring Of Circulating Tumor Cells In Vivo

Cancer is one of the biggest diseases that threaten human health because of its high incidence and high mortality.Most tumors are localized diseases of the organ,but they are almost transmitted through the blood or lymph to distant organ formation in the end.The majority of cancer patients die from metastatic cancer,but not primary cancer.The metastasis of tumors has become the main reason causing the death.Circulating tumor cells(CTC),as a tumor metastasis biomarker,are gaining more and more attentions.The number of CTCs can be used to evaluate the progress of cancer metastasis.It can also be used as an important indicator for prognosis of cancer patients,rapid assessment of chemotherapy drugs,tumor recurrence etc.The current CTC detection technique is in vitro testing,and relies on blood sampling: the use of morphological features of circulating cells,the immunological characteristics of cells and flow cytometry.All of these methods have complex sample preparation and collection processes.During those processes,a series of problems such as cell contamination,missed detection,and false positive can easily occur.Due to the amount of blood drawn,the detection sensitivity is limited,while it is difficult to perform frequent blood sampling,such as in the process of evaluating the efficacy of chemotherapy for tumors.To overcome the above problems,this thesis has studied the in-vivo detectiontechnology of CTC.The CTC in-vivo detection technology combines the concept of real-time high-speed fluorescence imaging technology and flow cytometry to obtain real-time,high signal-to-noise ratio signals to monitor the presence of a targeted cell population in the blood circulation of a living small animal or human being,such as metastatic tumor cells in the blood circulation after solid tumor blood dissemination.Since no blood is needed to be drawn,the problems of cell contamination,missed detection,and false positive caused by blood sampling are effectively avoided.The same experimental subject can be monitored for a long time,while the detection sensitivity can be improved.It not only realizes real-time and dynamic non-invasive monitoring of CTCs,but also helps us understand the occurrence and development of tumors and the molecular mechanisms of metastasis,providing a new technical means for basic research and diagnostic treatment of tumor metastases.In this thesis,multi-wavelength laser in-vivo flow cytometer based on fluorescent markers is developed to detect a number of channels.We used fluorescence-labeled multi-wavelength in-vivo flow cytometry to monitor the number of CTCs in mouse prostate tumor models.The results suggest that the number of CTCs in the circulatory system of the orthotopic tumor model is much higher than the number in subcutaneous tumor model,while it grows explosively;the number of CTCs in the circulatory system of the subcutaneous tumor model showed a gradual increase;the survival rate of the subcutaneous tumor model mice was also higher than that of the orthotopic tumor model mice.This result explains the effect of tumor growth microenvironment on tumor blood-derived metastasis to some extent.At the same time,it is found that out tumor progression in orthotopic tumor models is close to clinical manifestations,indicating that the orthotopic tumor model is more suitable for studying tumor metastasis.The fluorescence label-based in-vivo flow cytometer provides a powerful tool for the detection of CTCs.However,because its detection depends on fluorescencelabeling of tumor cells,the technology is still limited to research on laboratory animals,and it is difficult to apply in clinics.In order to solve this,a label-free in vivo flow cytometer based on photoacoustic effect has been further developed.The study achieved high sensitivity and large penetration depth in vivo detection by using the absorption differences in the near-infrared light band between melanoma cells and blood background as well as the characteristics of near-infrared light/acoustic signals in the tissue with less absorption.Firstly,we theoretically calculated the boundary conditions of the photoacoustic signals generated by melanoma cells,and determined the parameters of the excitation light source according to the calculation results.The parameters of the photoacoustic signal receiving device were measured.Through the theoretical calculation and analysis of the characteristics of melanoma cells to generate photoacoustic signals,the measured cellular photoacoustic signals were denoised and analyzed by wavelet denoising and average denoising.On this basis,we developed a label-free in vivo flow cytometer based on the photoacoustic effect using a melanoma mouse model,and achieved label-free,real-time,and dynamic monitoring of circulating melanoma cells in vivo in mice.Based on the experimental data,we have summarized the results that the number of CTCs in mice increases with time,and that the arteries near the primary tumors have higher detection efficiency than the distal arteries.This result demonstrates that label-free in-vivo flow cytometry based on photoacoustic effects can achieve label-free detection of melanoma cells in the circulatory system.In this study,CTC label-free optical quantitative detection was used.The photoacoustic signal of circulating melanoma cells was used as a detection signal of an in-vivo flow cytometer to achieve CTC label-free,in vivo,real-time,and dynamic monitoring,which provides a practical method to effectively solve the biggest problem faced by flow cytometry in clinical applications.The experimental data counted the change of the number of CTCs in mice over time,and found out that the arteries in the vicinity of the primary tumor and the distalarteries were different in detecting CTCs,thus indicating that the arteries near the tumor had higher detection possibilities than the distal arteries.The monitoring of melanoma cells in the circulatory system of mice using an in vivo flow cytometer based on the photoacoustic effect demonstrated the feasibility of this technique for in vivo label-free detection.However,as the previous study was still based on animals,there are still some differences with clinical applications in a number of aspects.First of all,in the design of optical systems,clinical applications require higher stability,miniaturization,and portability of the optical system.Secondly,due to the influence of thicker human skin,the precise positioning method used for image navigation of the blood vessels to be used in the animal experimental platform is impossible to be used in the human body.Thirdly,in the clinical applications,the blood circulation velocity,blood vessel size,and the circulatory system in humans have certain differences compared to those in the animals.Finally,the laser must meet the clinical safety requirements.In order to apply the label-free detection technology to the clinics,we first miniaturized the optical path of the label-free in vivo flow cytometer based on the photoacoustic effect,and used a miniature double meniscus lens group and a double cemented lens group to shape and focus the laser spot.A photoacoustic monitoring wristband is designed to form a wearable device that can be worn on the human wrist to dynamically monitor the melanoma cells in the blood circulation system of the wrist.Secondly,we used the laser frequency doubling technology to double the 1064 nm excitation light to generate a second 532 nm wavelength laser.Using the difference in ultrasound signals generated by the skin and blood vessels irradiated by the 532 nm wavelength laser,a signal navigation system was designed to realize the human wrist,which measured the precise positioning of the blood vessels.Finally,we theoretically calculated and analyzed parameters such as blood vessel diameter,blood flow velocity,and reference laser safety requirements for the human,and finally determined the technical specifications of each component.The instrument was used to detect themelanin absorption of static tissues and the photoacoustic signals of suspended melanoma cells in vitro.The experiment verified the rationality of the system design,the selected parameter and the supporting software.The application of the device is expected to realize early clinical monitoring of melanoma cells in the human circulatory system in vivo and will have a good application prospect in the field of helping doctors to evaluate the status of melanoma metastasis,to dynamically evaluate individualized therapeutic effects in melanoma patients and to monitor melanoma recurrence.