Co-culture of primary human tumor hepatocytes from patients with hepatocellular carcinoma with autologous peripheral blood mononuclear cells: study of their in vitro immunological interactions

Abstract

BackgroundMany studies have suggested that the immune response may play a crucial role in the progression of hepatocellular carcinoma (HCC). Therefore, our aim was to establish a (i) functional culture of primary human tumor hepatocytes and non-tumor from patients with hepatocellular carcinoma (HCC) and (ii) a co-culture system of HCC and non-HCC hepatocytes with autologous peripheral blood mononuclear cells (PBMCs) in order to study in vitro cell-to-cell interactions.MethodsTumor (HCC) and non-tumor (non-HCC) hepatocytes were isolated from the liver resection specimens of 11 patients operated for HCC, while PBMCs were retrieved immediately prior to surgery. Four biopsies were obtained from patients with no liver disease who had surgery for non malignant tumor (normal hepatocytes). Hepatocytes were either cultured alone (monoculture) or co-cultured with PBMCs. Flow cytometry measurements for MHC class II expression, apoptosis, necrosis and viability (7AAD) were performed 24 h, 48 h and 72 h in co-culture and monocultures.ResultsHCC and non-HCC hepatocytes exhibited increased MHC-II expression at 48h and 72h in co-culture with PBMCs as compared to monoculture, with MHC II-expressing HCC hepatocytes showing increased viability at 72 h. PBMCs showed increased MHC-II expression (activation) in co-culture with HCC as compared to non-HCC hepatocytes at all time points. Moreover, CD8+ T cells had significantly increased apoptosis and necrosis at 48h in co-culture with HCC hepatocytes as compared to monocultures. Interestingly, MHC-II expression on both HCC and non-HCC hepatocytes in co-culture was positively correlated with the respective activated CD8+ T cells.ConclusionsWe have established an in vitro co-culture model to study interactions between autologous PBMCs and primary HCC and non-HCC hepatocytes. This direct interaction leads to increased antigen presenting ability of HCC hepatocytes, activation of PBMCs with a concomitant apoptosis of activated CD8+ T cells. Although, a partially effective immune response against HCC exists, still tumor hepatocytes manage to escape.

Androgen receptor roles in hepatocellular carcinoma, fatty liver, cirrhosis and hepatitis

“…Although hepatocytes comprise more than 85% of all cells in normal livers, the cancerous liver presents with an increased number of infiltrating immune cells (Doumba, et al 2013; Guo, et al 2013), including dendrocytes (Butterfield 2004), monocytes/macrophages (Shirabe, et al 2012), CD4/8+ T cells (Guo et al 2013), neutrophils (Motomura, et al 2013), B cells (Chen, et al 2012) and mast cells (Ju, et al 2009). Other cells in the liver, such as endothelial cells (Sato and Mori 2011) or BM-MSCs (Garcia, et al 2011) may also be altered due to chronic inflammation or tumorigenesis.…”

IL-17 and TNF-α co-operation contributes to the proinflammatory response of hepatic stellate cells

“…This ratio was chosen according to data from the literature [10,11]. To block the IL-6 and TGF-β1 pathways, tocilizumab (Roche, Welwyn, UK) and anti-TGF-β1 (R&D Systems) were used at 10 μg/ml.…”

“…For the co-culture assays, cells were maintained in DMEM supplemented with 2% fetal bovine serum (Life Technologies) and 2 mM L-glutamine (Eurobio) at a ratio of 25 PBMCs for five hepatocytes for one HSC. This ratio was chosen according to data from the literature [10,11]. HSC-hepatocyte co-cultures were stimulated or not with the combination of IL-17A 50 ng/ml and TNF-α 0·5 ng/ml.…”

Hepatoma cell‐derived leptin downregulates the immunosuppressive function of regulatory T‐cells to enhance the anti‐tumor activity of CD8+ T‐cells

“…Recently the interactions between cancer cells and immune cells have been broadly studied to disclose the alterations of immune response during carcinogenesis. Hepatoma cells are able to increase CD8 + T‐cell apoptosis 13 . Hepatic stellate cells promote tumor progression by enhancement of immunosuppressive cells 14 .…”

“…Hepatoma cells are able to increase CD8 + T-cell apoptosis. 13 Hepatic stellate cells promote tumor progression by enhancement of immunosuppressive cells. 14 Hepatoma cells activate natural killer cells to produce IFN-γ.…”