| Metastasis derives from tumor cells detach from their primary site,circulate in the vasculature and colonize a new site.The primary tumor releases a large number of tumor cells into the circulation,which are termed as circulating tumor cells(CTCs).CTC is a powerful biomarker for monitoring cancer metastasis and treatment efficiency.Monitorinng CTCs during operation is helpful for monitoing operation-induced tumor dissemination.In clinical examinations,the current CTC detection methods are based on transient blood sampling.However,the detection accuracy is affected by the temporal distribution of CTCs due to their rare number.Besides,the dynamic changes of CTCs over minutes and housrs cannot be acquired due to the limitation of blood volume and sampling frequency.In vivo flow cytometry(IVFC),which uses animal superficial blood vessels as a natural flow system,has the capability to measure CTCs in a real time and continuous way.IVFC provides temporal information for each CTC and provides CTC dynamic changes within a short time and over time.Exploring the circulating dynamics of CTCs is important for understanding the mechanisms underlying tumor dissemination and improving the detection rate of CTCs.Studying the dynamic changes of CTCs in the course of treatment is helpful for understanding operationinduced tumor cell release and designing adjuvant therapeutic strategies to eliminate this unintentional effect.Here,we perform in vivo flow cytometry(IVFC)to monitor the temporal distribution of CTCs during cancer progression and the dynamic changes of CTCs during operation.It is unclear whether CTC release occurs at a constant rate or in a certain rhythm.By utilizing the temporal information on CTCs provided by IVFC,we observe a short-time variation of CTCs that exhibit bursting activity when they are rare.Therefore,use of random small blood samples for CTC estimation is not accurate.To investigate whether circadian clock regulates CTC counts,we perform IVFC to monitor CTCs over 24 hr and analyze the circadian rhythm of CTCs.The results indicate that the numbers of CTCs are regulated by endogenous circadian rhythm.Our results thus can help understand the shedding pattern of CTCs and optimize treatment timing to improve the results of chronotherapy.Besides,previous studies have proposed that radiofrequency ablation(RFA)can unintentionally cause tumor cells to spread from the primary tumor into adjacent vessels and disseminate throughout the body.However,this hypothesis has not yet been validated.We established a GFP transfected hepatocellular carcinoma model in nude mice and analyzed dynamic changes of CTCs using IVFC while performing RFA.We demonstrate for the first time that the CTC count significantly increases during ablation.According to the IVFC data,81.2% of CTCs increased during RFA are CTC clusters.Combining with the intravital confocal images of the lung,we demonstrate that the CTCs stimulated by RFA eventually translate to increased metastasis and decreased survival.It is found out that CTC shedding and metastasis caused by RFA are significantly reduced by using ethanol injection before RFA,which provides an easy-to-use method to decrease CTC release caused by RFA in the clinic.The immunohistochemical results indicate that the ethanol injection leads to complete vascular occlusion and block the way through which CTC clusters disseminate.IVFC data also confirms that the proportion of CTC clusters during RFA are significantly reduced by ethanol injection.In this study,we also demonstrate that RFA can induce CTC release in an orthotopic model of prostate cancer.Besides,RFA may promote the infiltration of tumor-associated macrophages in the lung.Taken together,our study ultilizes IVFC to monitor the temporal distribution of CTCs during cancer progression and monitor CTC dynamics during RFA.Our results provide guideline for understanding the mechanism underlying tumor dissemination,improving detection rate of early metastasis and reducing RFA-induced tumor dissinination. |
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