Macrophage-derived MMP-9 And MMP-2 Are Closely Related To The Rupture Of The Fibrous Capsule Of Hepatocellular Carcinoma Leading To Tumor Invasion

Background : Hepatocellular carcinoma(HCC)is a type of liver cancer that originates in hepatocytes.It accounts for the vast majority of primary liver cancers(80%-90%)and is the third leading cause of cancer-related deaths.Hepatocellular carcinoma(HCC)is an aggressive tumor with a poor clinical prognosis.HCC is characterized by rapid proliferation,easy metastasis,a high rate of recurrence and an insidious onset,with most patients already in an advanced stage by the time they are found,all of which contribute to the high mortality rate of HCC.Rupture of the fibrous capsule(FC)is a vital clinical phenomenon in the invasion and metastasis of HCC.FC is mainly composed of type I collagen(COL1A1).However,the cause of FC rupture,which leads to further tumor invasion and metastasis,is currently unknown.FC rupture of HCC may become a promising and challenging research direction for HCC.therefore,it is significant to explore the rupture of the fibrous capsule of HCC for the treatment and prevention of HCC.Purpose: In humans,MMP-9 and MMP-2 are considered to be a family of zinc-containing enzymes closely associated with collagen and extracellular matrix degradation.In this study,we aimed to determine whether the rupture of FC in HCC patients was related to macrophage-derived MMP-9 and MMP-2,and their clinical diagnostic value for FC rupture.Methods:(1)The clinicopathological data of all patients were retrospectively analyzed and the chi-squared test(χ2)was used to calculate the possible correlation between whether the FC was ruptured or not and the clinicopathological data and a P value < 0.05 was considered statistically significant.(2)To verify the pathway of tumor invasion into the paracancerous liver tissue,we retrospectively collected magnetic resonance imaging(MRI)data from HCC patients(n=6)and performed H&E staining on tissue sections from HCC patients(n=6).(3)To verify whether macrophages and hepatic stellate cells are associated with FC rupture,we used COL1A1 immunohistochemical staining to determine the location of the ruptured and intact capsule,and CD163 and α-SMA immunohistochemical staining was performed on serial tissue slides(n=6).(4)To verify whether MMP-9 and MMP-2 was associated with FC rupture,we performed MMP-9 and MMP-2 immunohistochemical staining of all consecutive slides and western blot analysis of FC rupture regions and their relative intact FC regions in patients with FC rupture,which was further confirmed by q RT-PCR at the m RNA level.(5)To verify whether MMP-9 and MMP-2 was derived from macrophages,We performed immunohistochemical staining for CD163,MMP-9 and MMP-2 as well as immunofluorescence staining of the FC ruptured areas on all serial slides.(6)To determine the potential clinical diagnostic value of MMP-9 and MMP-2 for FC rupture,we measured protein levels in ruptured FC tissue(n=19)and intact FC tissue(n=19)by ELISA and used ROC curves to determine the diagnostic value of MMP-9 and MMP-2 for FC rupture.Results:(1)By retrospectively analyzing the clinicopathological data of all patients,the results showed that FC rupture was significantly associated with vascular invasion,TNM stage,AFP,and tumor recurrence(P<0.05).(2)By retrospectively collecting magnetic resonance imaging(MRI)data from HCC patients(n=6)and performing HE staining of their tissue slides,the results showed that HCC cells invaded the paracancerous liver tissue through the area of capsule rupture.(3)The results of CD163 immunohistochemical staining of the ruptured FC area and the intact FC area showed that macrophages were associated with FC rupture.(4)By immunohistochemical staining of MMP-9 and MMP-2 in the ruptured FC and intact FC areas,it was concluded that a large amount of MMP-9 and MMP-2accumulated in the ruptured FC area,followed by Western blot analysis and q RT-PCR,which showed that MMP-9 and MMP-2 were differently expressed,and then concluded that MMP-9 and MMP-2 were the key molecule causing the rupture of the capsule.(5)By performing immunohistochemistry and immunofluorescence staining,the results showed substantial co-localization of macrophages and MMP-9 and MMP-2 in the area of capsule rupture,and it was concluded that macrophage-derived MMP-9and MMP-2 caused FC rupture in HCC.(6)Finally,it was verified by ELISA that MMP-9 and MMP-2 can effectively distinguish between ruptured and intact FC and has clinical diagnostic value for FC rupture.Conclusion: Taken together,macrophage-derived MMP-9 and MMP-2 were closely related to the rupture of the FC of HCC and subsequently led to the migration and invasion of the tumor cells into the ruptured area of FC to the para cancer.therefore,it is suggested that when performing surgical resection,it is necessary to expand the range of tumor resection for patients with ruptured FC and hence reduce the possibility of recurrence and metastasis in HCC patients.