Platelet lysates in Hepatocellular Carcinoma patients after radiofrequency ablation facilitate tumor proliferation, invasion and vasculogenic mimicry

Abstract

Background: Platelets play important roles in tumorigenesis, angiogenesis and metastatic dissemination of tumor cells. Radiofrequency ablation (RFA) could increase the circulating tumor cells in patients with primary or metastatic lung tumors. Whether platelet lysates in hepatocellular carcinoma (HCC) after RFA promote tumor progression has not been elaborated. Methods: HCC patients within Milan Criteria and without taking anti-platelet drugs were selected in the study. MTT assay, colony formation assay, transwell assay, tube formation and western blot were used to evaluate the effect of platelet lysates on HCC cells in vitro. Lung metastatic assay was performed in vivo. Results: Platelet lysates from patients after RFA promoted cell proliferation, colony formation, migration, invasion and vasculogenic mimicry in Hep3B and HCCLM3 cells compared with those from patients before RFA. Platelet lysates after RFA significantly increased the expression of p-Akt, p-Smad3 and snail, and decreased the expression of E-cadherin compared with those before RFA in Hep3B and HCCLM3 cells. Hep3B-Luc2-tdT cells incubation with platelet lysates from patients after RFA displayed enhanced lung metastasis compared with those before RFA. Conclusions: Platelet lysates from HCC patients after RFA promoted the proliferation, migration, invasion and vasculogenic mimicry of HCC cells, which indicated that RFA in combination with anti-platelet drug may be used to improve the prognosis of HCC.

Keywords: Hepatocellular carcinoma; Metastasis; Platelets; Radiofrequency ablation; Vasculogenic mimicry.

Figure 1

Platelet lysates from patients after RFA promote cell proliferation and colony formation of HCC cells. We selected HCC patients within Milan Criteria, acquired the platelet lysates before and after RFA, and examined the effect of platelet lysates on HCC cells proliferation. (A) Hep3B and HCCLM3 cells were seeded at 3 × 10³ cell per well into a 96-well plate, and incubated with platelet lysates. On 24 h, 48 h and 72 h, MTT was added and OD was measured. (B) Hep3B and HCCLM3 were seeded at 1 × 10³ cells per well into a 6-well plate, and incubated with platelet lysates. On 14 days, cells were stained with crystal violet. Data are presented as mean ± SD. ns: no significance; ^**P<0.01; ^***P<0.001; ns, no significance.

Figure 2

Platelet lysates from patients after RFA accelerate cell migration and invasion in HCC cells. Transwell assay was used to examine the effect of platelet lysates on HCC cells migration and invasion. Hep3B and HCCLM3 cells were seeded into the upper chamber, and incubated with platelet lysates. On 24 h or 48 h, migrating or invading tumor cells were examined. (A, C) Representative migration and invasion of HCC cells treated with platelet lysates from patients before or after RFA in Hep3B and HCCLM3 cells. (B, D) Quantification of tumor cell migration and invasion is shown in Hep3B cells and HCCLM3 cells. Data are presented as mean ± SD. ^* P<0.05; ^** P<0.01.

Figure 3

Platelet lysates from patients after RFA facilitate vasculogenic mimicry of HCC cells. Tube formation was performed to evaluate the effect of platelet lysates on vascular mimicry in HCC cells. (A, C) Representative in vitro capillary network formation of HCC cells treated with platelet lysates from patients before or after RFA in Hep3B and HCCLM3 cells. (B, D) Quantitative analysis of the mean number of tube-like structures formed using ImageJ in Hep3B and HCCLM3 cells. Data are presented as mean ± SD. ^* P<0.05.

Figure 4

Platelet lysates activate the Akt, ERK1/2 and Smad3 signaling pathways in HCC cells. We also investigated the associated mechanism involved in the process. The expression of p-Akt, p-ERK1/2, snail and E-cadherin was examined in Hep3B (A) and HCCLM3 cells (B) after treatment with platelet lysates from patients before and after RFA using western blot. (C, D) The expression of p-Smad3 was examined in Hep3B and HCCLM3 cells. (E) The statistical results in the expression of E-cadherin, p-Akt, snail and p-ERK1/2 were shown. (F) The statistical results in the expression of p-Smad3 were shown. Data are presented as mean ± SD. ^* P<0.05; ^** P<0.01; ns, no significance.

Figure 4

Platelet lysates activate the Akt, ERK1/2 and Smad3 signaling pathways in HCC cells. We also investigated the associated mechanism involved in the process. The expression of p-Akt, p-ERK1/2, snail and E-cadherin was examined in Hep3B (A) and HCCLM3 cells (B) after treatment with platelet lysates from patients before and after RFA using western blot. (C, D) The expression of p-Smad3 was examined in Hep3B and HCCLM3 cells. (E) The statistical results in the expression of E-cadherin, p-Akt, snail and p-ERK1/2 were shown. (F) The statistical results in the expression of p-Smad3 were shown. Data are presented as mean ± SD. ^* P<0.05; ^** P<0.01; ns, no significance.

Figure 5

Platelet lysates from patients after RFA promote tumor lung metastasis. To evaluate the effect of platelet lysates on tumor metastasis, Hep3B-Luc2-tdT cells after the treatment of platelet lysates from patients before or after RFA were injected through vein tail into mice. (A) In vivo Imaging System was used to quantify metastatic nodules. Pictures of lung metastatic nodules were shown at the end of the experiment. (B) The number of lung metastatic nodules was counted. (C) Mice weight was examined every three days. Data are presented as mean ± SD. ^**P<0.01.