Using Multiphoton Microscopy To Assess Breast Cancer Metastasis

As a systemic disease,the main cause of death from breast cancer is the metastasis and spread of cancer cells to other organs.Studies have shown that cancer cells mainly metastasize in three ways: lymphatic vascular invasion(LVI),perineural invasion(PNI)and lymph node metastasis.In order to reduce the risk of death of breast cancer patients,it is very important to quickly and effectively evaluate the metastasis status.In this work,we attempted to evaluate the metastasis status of breast cancer using multiphoton microscopy(MPM)which is based on the two photon-excited fluorescence(TPEF)and second-harmonic generation(SHG)signals.Firstly,we imaged the LVI of breast cancer.In order to improve the accuracy of the assessment of LVI,we also studied DCIS and found that the histological characteristics of LVI and DCIS were significantly different.On this basis,we made use of quantitative analysis of collagen fibers around the vessels and ducts to quantitatively distinguish LVI from DCIS.These results showed that MPM can effectively identify LVI.Secondly,MPM was used to image the PNI.Studies have shown that MPM can clearly display the microscopic features of normal nerves.In addition,PNI and blood vessels within the epineurium were also easy to detect by MPM,and these experimental results demonstrated that MPM has the potential to identify PNI.Not only that,we also quantified the collagen fibers around normal nerves and PNI nerves to further improve the accuracy of MPM in evaluating and diagnosing PNI,which may lay the foundation for further exploration of MPM in clinical applications.Finally,we used MPM to assess lymph node status by imaging the negative and positive lymph nodes.Comparing with H&E images,we found MPM can effectively image lymphocytes,blood vessels,capsules,and so on.In order to further distinguish between negative and positive lymph nodes,we also used image processing technique to extract eight morphological features of collagen fibers.The results showed that the collagen density,the number of collagen fibers and the average length of collagen fibers in the lymph node matrix were all higher when breast cancer patients were in a positive state of lymph nodes,and there was significant difference between the negative and positive lymph nodes(P<0.05).Therefore,MPM may provide an auxiliary tool for doctors to accurately and quickly assess the status of lymph nodes.To sum up,these results indicate that multiphoton imaging has the potential ability to evaluate breast cancer metastasis,and may be helpful to further study the changes of extracellular matrix before and after breast cancer metastasis and be useful for clinicians in making rational treatment strategy.