| Backgrounds and objectivesIt has been reported that over 50% of patients clinically diagnosedof colorectal cancer have undergone liver metastasis,and 80%-90%ofthese patients can not proceed surgeries.In light of the urgent situations,it is imperative that we should develop new techniques and methods to subrogate the high-risk surgeries,thus prolonging patients’ survival rate.High Intensity Focused Ultrasound(HIFU) is an emerging tumor treatment method,which is characterized by its localness,non-invasive target and thermal ablation.HIFU targets tumor tissue by low-energy ultrasound transducer,transiently generating high temperature to abolishtarget tumor tissue without affecting the surrounding normal tissues. At present, the treatment has been extensively applied in diverse clinical tumor interventions,such as liver cancer,prostate cancer,breast cancer and osteosarcoma.For the moment,HIFU has been enormously explored in biological field,and investigated in areas like radiographicchanges and residual tumor tissue changes after HIFU treatment. However,there are few studies addressing if HIFU has implications intumor-related inflammation and angiogenesis,which needs to be furtherdemonstrated.Tumor angiogenesis is a complicated process requiring participation of multiple cells,such as vascular endothelial cells, stromal cells and tumor cells. And it is closely associated with tumor growth and metastasis. It’s known to us that tumor angiogenesis is regulated by a variety of active substances.For instance,Inflammatory cytokines play an important role in the process of angiogenesis.HEFU,as a non-invasive treatment,may have an influence on inflammation and angiogenesis of tumor tissue,but there is little information regarding if HIFU is involved in those processes.The efficacy of HIFU ablation targeting focused area of tumor tissue is overwhelmingly productive.Nevertheless, due to varied tumor shape,tumor location and inadequate imaging,HIFU fails to ablate tumor tissues completely,resulting in some residual tumor tissues,whose biological behaviors have a decisive impact on efficacy of HIFU treatment. Studies have shown that radiofrequency ablation and laser ablation,belonging to minimally invasive treatments,contribute to cause systematic inflammation. So we put forward the hypothesis that HIFU as a non-invasive treatment could produce the familiar effect, and a changeable tumor microenvironment might affect angiogenesis.It is very important to unravel these hypotheses in terms of HIFU treatment.In summary,our study aims to investigate biological behavior changes of mouse tumor tissue in vivo after HIFU exposure, determine if residual tumor tissue could induce inflammation, and focus on angiogenesis of residual tumor tissue, thus providing more theoretical basis for the clinical treatment of HIFU ablation.Methods1 Subcutaneous colorectal cancer murine model was constructed and HIFU ablation was performed to treat the tumor tissue,hence confirming the efficacy of HIFU.1.1 BABL/c mice were subcutaneously transplanted colorectal cancer celline to construct tumor models, which were randomly divided into HIFU-treated or sham control group, HIFU-treated group underwent systematic exposure by JC-200 instrument,while sham control group went through sham exposure.1.2 Tumor growth curve and mice survival rate were observed and recorded in both groups respectively. Blood and tumor tissue samples at day 1,3,5,7 and 14 in both groups were collected respectively,changes of tumor tissue treated by HIFU were observed with naked eye or light microscope.2 Biological behavior changes of residual tumor tissue treated by HIFU2.1 Expression levels of IL6 and IL10 in residual tumor tissue were detected by RT-PCR, while ELISA Assay was used to test the expression levels of IL6 and IL10 in peripheral blood.2.2 Endothelial marker CD31 antibody was used for IHC staining, angiogenic density was calculated with the help of observation in microscopy; VEGF and HIF1 expression patterns in residual tumor tissue were determined by Western Blot and IHC.Results1 Compared with sham control group,significant coagulation necrosis of tumor tissue was observed in HIFU-treated area, tumor growth curve significantly went down(p<0.05),and mouse survival rate was highly increased (p<0.05).2 It was observed an dull and gray area with coagulation necrosis at day 1 post HIFU exposure, necrosis tissue surrounded with congestion zone had a clear boundry against normal tissue; At day 5 it presented as a a clear red and black coagulation necrosis area, edema and congestion of surrounding tissue was diminished; At day 14, it became a fibrous necrosis with lesion area shrunk.3 It was observed that in coagulation necrosis area tumor cells had a smaller size with sparse vacuoles under a microscope, and scattered inflammatory cells could be seen in ablation zone. Necrosis area with clear boundry in tumor tissue could be seen At day 3, there was an increasing gap between cells, a large number of tumor cells with karyopyknosis and clear cell debris could be seen in residual tumor tissue, and enormous erythrocytes and inflammatory cells were infiltrated into the tumor tissue; nuclear fragementation was increased and cell debirs were absorbed by tissue in target area at day 5; Fibrosis was prevailing with fiber wrapped and inflammatory cell infiltration at day 14.4 Expression levels of IL6 and IL10 in serum initially increased but tended to decrease over time.Compared with control group,after HIFU treatment, the expression level of IL 6 at dayl,3,5 and 7 was significantly elevated (p< 0.05).5 After HIFU treatment,angiogenesis at day 1,3,5 of residual tumor tissue was significantly reduced (P<0.05); Changes in vascular density at day 7 was not statistically significant (P>0.05); At day 14 vascular density increased significantly (P<0.05).The results mentioned above suggest that HIFU ablation is effective in treating subcutaneous colorectal cancer murine model, and it can generates imflammation response as well.6 Compared to the control group, microvessel density (MVD) was significantly reduced ld-3d (P<0.05), while MVD generation significantly increased at day 7 and 14(P<0.05). During the session at day 3 to day14,MVD increased over time.7 IHC and Western Blot results showed that expression levels of both VEGF and HIF1α decreased at first but then tended to increase over time, at day 1 and 3, VEFG expression level was significantly lower that in control group(P<0,01), but it gradually increased from day 5 to day 14. The highest expression level of VEGF was at day 5, while HEF-la reached its peak at day 7.Conclusions1 HIFU treatment could effectively ablate tumor tissue of subcutaneous colorectal cancer murine model, thus prolonging mouse survival rate.2 HIFU treatment could induce imflammation response mainly presenting with inflammatory cells infiltration.3 Angiogenesis of tumor tissue after HIFU treatment may be attributed to alterations of expression levels of VEGF and HIF1α. |
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