2.1 Patients demographic characteristics and histopathology
A total of 45 HCC patients were reviewed at Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital from January 2017 to December 2019, of which, 45 patients with primary HCC met inclusion criteria. The demographics and clinic-pathological parameters of the enrolled 45 patients including age, gender, ethnic group, pathological grade, metastasis, presence of HBV were shown in Table 1. All the 45 patients were Han nationality people diagnosed with primary HCC by pathology for the first time and did not have other malignancies. The proportion of above 50 years and male in patients was 55.6% and 91.1%, respectively.
The HE staining results suggested that differently differentiated HCC lesions were detected in 45 patients. The typical cases of HCC with poor differentiation, moderate differentiation and high differentiation are shown in Figure 1. In highly differentiated HCC, most of the cancer cells with increased nucleo-cytoplasmic ratio were arranged trabecularly and accompanied by alveolar structure, fat changes could be seen as well (Figure 1A). In moderately differentiated HCC, cells rich in cytoplasm and had clear nucleolus were arranged into small trabecular structure or cell cord (Figure 1B). In poorly differentiated HCC, the nucleo-cytoplasmic ratio of cancer cells increased significantly, and the tumor cells were with obvious pleomorphism, varied size and distinct shape (Figure 1C).
Additionally, the majority (86.7%, 39/45) of HCC patients were HBV positive. For pathological grade, high differentiation, moderate differentiation and low differentiation accounted for 20% (9/45), 20% (9/45) and 60% (27/45), respectively. Among these patients, 11 patients (24.4%) did not occur metastases, 34 patients (75.6%) suffered from metastases. The focal liver lesions located at right lobe liver or left three lobe liver. Correspondingly, these 45 patients received right hepatectomy or left trisegmentectomy according to the focal liver lesion location and did not receive radiotherapy, chemotherapy, or immunotherapy.
2.2 Localization of SEPCs in HCC patients
The SEPCs with CD31, CD45 and CD133 expressions was consider as progenitor cells of LSECs, therefore, we used double staining of CD133/CD45 and CD133/CD31 at the same location to prove that the staining was triple positive so as to determine phenotypic signature for SEPCs. The SEPCs were found within abundant of blood vessels in tumor nodules from all 45 patients, and we didn’t observe any SEPC in the tumor-adjacent tissues (Figure 2). SEPCs were significantly more in tumorous than in adjacent nontumorous liver tissues (P<0.01) (Figure 2).
2.3 Liver functional data
As shown in Table 2, liver functional data including total protein, albumin, direct bilirubin, indirect bilirubin, ALT and AST. Total protein, albumin, direct bilirubin, indirect bilirubin and ALT were normal in most cases, and AST exhibited approximately equal ratio of high and normal cases (23 versus 22 cases). A linear regression analysis revealed there was no correlation between SEPCs expressions and total protein, albumin, direct bilirubin, indirect bilirubin, ALT and AST levels (Figure 3).
2.4 Tumor markers
As shown in Table 3, HCC tumor markers including AFP, CA199, CEA and CA125 were tested. Most patients were with high levels of AFP, while CA199, CEA and CA125 were normal in most HCC patients. Furthermore, linear regression analysis indicated there was a strong positive correlation between SEPCs markers’ expression and AFP or CA199 levels (Figure 4A, B). But this was no correlation between SEPCs markers’ expression and CEA and CA125 level (Figure 4C, D).
2.5 The relationship between SEPCs and the size of HCC
The diameter of HCC tumors (the largest diameter of tumor) in highly differentiated, moderately differentiated, and poorly differentiated specimens were measured respectively to explore the relationship between SEPCs and the size of HCC lesion in the different differentiated cases. According to the diameter of tumors measured, samples were divided into three groups, i.e. 1.5-5 cm group, 5-9 cm group and 9-16 cm group. Then, we observed the SEPCs contents in each HCC specimen (Figure 5 A, B). The linear regression analysis revealed there was a positively strong correlation between SEPCs contents and HCC tumor diameter in highly differentiated, moderately differentiated, and poorly differentiated specimens, respectively (P<0.01) (Figure 5 C).
2.6 The relationship between SEPCs contents and the differentiation grades of HCC
In order to explore the relationship between SEPCs and the differentiation of HCC, we observed the SEPCs contents in highly differentiated, moderately differentiated, and poorly differentiated specimens. The SEPCs contents in poorly differentiated HCC patients were significantly higher than those in the moderately and highly differentiated HCC patients (P<0.01) (Figure 6B). Meanwhile, compared with highly differentiated HCC, the expression of SEPCs in moderately differentiated HCC was also statistically different (P<0.05).