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Establishment of a co-culture system to study the interactions between major immune effector cells and hepatocellular carcinoma
Establishment of a co-culture system to study the interactions between major immune effector cells and hepatocellular carcinoma
The immune system plays an essential role in protecting humans from cancer, especially the major immune effector cells such as CD8+ T lymphocytes and NK cells. In recent years, immunotherapy has developed greatly, especially with regard to checkpoint blockade and cellular therapies. As the fourth leading cause of cancer death, hepatocellular carcinoma (HCC) is of public health concern. Surgery remains the most effective method for treating HCC, but immunotherapy has become a hotspot in the management of refractory and recurrent HCC and might therefore serve as adjuvant treatment in the future. However, the interactions between major immune effector cells and HCC cells and related mechanisms remain unclear which is quite essential to the study of immunotherapy for HCC. To date, there are insufficient studies describing the direct cell-cell interactions with an appropriate model in a physiologically and clinically relevant manner. In our research work, a detailed co-culture system was set up with primary HCC cells and autologous tumor infiltrating lymphocytes (TILs) by establishing co-culture of HCC cell line and PBMC initially, to study the direct interactions between major immune effector cells and HCC cells. First, a systematic review was conducted to assess the interactions between HCC cells and CD8+ T lymphocytes as well as NK cells. It was found that most of these studies use cell line and PBMC as co-cultured cell source. There’s no study conducting co-culture with primary HCC cells and TILs to investigate the interactions between HCC cells and major immune effector cells. The co-culture system we established with primary HCC cells and TILs holds the potential to give a more complete picture of the immune response against HCC. In the co-culture of HepG2 and PBMC with target: effect (T:E) ratio of 1:10, no obvious difference is detected between mono-cultured group and co-cultured group regarding the viability of HCC cells (mono-H-24h vs. co-H-24h: 77.20 ± 16.13% vs. 76.97 ± 13.84%, p=0.997). Compared to mono-cultured group, the degranulation of CD8+ T96 lymphocytes and NK cells in co-cultured group increased to some extent but without statistical significance (mono-CTL-24h vs. co-CTL-24h: 1.10 ± 0.95% vs. 7.03 ± 3.47%, p=0.101; mono-NK-24h vs. co-NK-24h: 0.90 ± 0.89% vs. 9.83 ± 6.73%, p=0.259). In the co-culture of HepG2 and PBMC with T:E ratio of 1:25, compared to mono-cultured group, the viability of HCC cells in co-cultured group declines obviously (mono-H-24h vs. co-H-24h: 74.70 ± 10.28% vs. 65.10 ± 10.61%, p=0.020). The degranulation of CD8+ T lymphocytes and NK cells in co-cultured group increased significantly (mono-CTL-24h vs. co-CTL-24h: 3.50 ± 1.50% vs. 19.80 ± 0.44%, p=0.005; mono-NK-24h vs. co-NK-24h: 5.53 ± 6.21% vs. 27.40 ± 3.66%, p=0.042). From the results mentioned above, we can conclude that HCC cells could activate major immune effector cells into degranulation state, and activated major immune effector cells could in turn kill or inhibit the viability of HCC cells. The T: E ratio must be bigger enough to got positive results. In the co-culture of primary HCC cells and TILs, no difference was found regarding cytotoxicity and degranulation (mono-HCC-24h vs. co-HCC-24h: 94.40 ± 5.20% vs. 95.47 ± 0.74%, p=0.930; mono-CTL-24h vs. co-CTL-24h: 10.43 ± 5.73% vs. 12.13 ± 2.99%, p=0.569; mono-NK-24h vs. co-NK-24h: 39.10 ± 28.14% vs. 55.80 ± 14.05%, p=0.934). Several possible reasons maybe involved, but the most obvious reason may lie in the dysfunction of major immune effector cells resulted from tumor microenvironment in HCC patients. Taken together, a novel direct contact co-culture (DCC) system with primary HCC cells and TILs has been established in this study. The DCC system could study the direct interactions between major immune effector cells and HCC cells in a physiologically and clinically relevant manner. It can be used as a powerful tool to study the mechanisms related to the target/effector cell interactions and provide more evidence for HCC relevant immunotherapy.
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Li, Xiaokang
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Li, Xiaokang (2022): Establishment of a co-culture system to study the interactions between major immune effector cells and hepatocellular carcinoma. Dissertation, LMU München: Faculty of Medicine
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Abstract

The immune system plays an essential role in protecting humans from cancer, especially the major immune effector cells such as CD8+ T lymphocytes and NK cells. In recent years, immunotherapy has developed greatly, especially with regard to checkpoint blockade and cellular therapies. As the fourth leading cause of cancer death, hepatocellular carcinoma (HCC) is of public health concern. Surgery remains the most effective method for treating HCC, but immunotherapy has become a hotspot in the management of refractory and recurrent HCC and might therefore serve as adjuvant treatment in the future. However, the interactions between major immune effector cells and HCC cells and related mechanisms remain unclear which is quite essential to the study of immunotherapy for HCC. To date, there are insufficient studies describing the direct cell-cell interactions with an appropriate model in a physiologically and clinically relevant manner. In our research work, a detailed co-culture system was set up with primary HCC cells and autologous tumor infiltrating lymphocytes (TILs) by establishing co-culture of HCC cell line and PBMC initially, to study the direct interactions between major immune effector cells and HCC cells. First, a systematic review was conducted to assess the interactions between HCC cells and CD8+ T lymphocytes as well as NK cells. It was found that most of these studies use cell line and PBMC as co-cultured cell source. There’s no study conducting co-culture with primary HCC cells and TILs to investigate the interactions between HCC cells and major immune effector cells. The co-culture system we established with primary HCC cells and TILs holds the potential to give a more complete picture of the immune response against HCC. In the co-culture of HepG2 and PBMC with target: effect (T:E) ratio of 1:10, no obvious difference is detected between mono-cultured group and co-cultured group regarding the viability of HCC cells (mono-H-24h vs. co-H-24h: 77.20 ± 16.13% vs. 76.97 ± 13.84%, p=0.997). Compared to mono-cultured group, the degranulation of CD8+ T96 lymphocytes and NK cells in co-cultured group increased to some extent but without statistical significance (mono-CTL-24h vs. co-CTL-24h: 1.10 ± 0.95% vs. 7.03 ± 3.47%, p=0.101; mono-NK-24h vs. co-NK-24h: 0.90 ± 0.89% vs. 9.83 ± 6.73%, p=0.259). In the co-culture of HepG2 and PBMC with T:E ratio of 1:25, compared to mono-cultured group, the viability of HCC cells in co-cultured group declines obviously (mono-H-24h vs. co-H-24h: 74.70 ± 10.28% vs. 65.10 ± 10.61%, p=0.020). The degranulation of CD8+ T lymphocytes and NK cells in co-cultured group increased significantly (mono-CTL-24h vs. co-CTL-24h: 3.50 ± 1.50% vs. 19.80 ± 0.44%, p=0.005; mono-NK-24h vs. co-NK-24h: 5.53 ± 6.21% vs. 27.40 ± 3.66%, p=0.042). From the results mentioned above, we can conclude that HCC cells could activate major immune effector cells into degranulation state, and activated major immune effector cells could in turn kill or inhibit the viability of HCC cells. The T: E ratio must be bigger enough to got positive results. In the co-culture of primary HCC cells and TILs, no difference was found regarding cytotoxicity and degranulation (mono-HCC-24h vs. co-HCC-24h: 94.40 ± 5.20% vs. 95.47 ± 0.74%, p=0.930; mono-CTL-24h vs. co-CTL-24h: 10.43 ± 5.73% vs. 12.13 ± 2.99%, p=0.569; mono-NK-24h vs. co-NK-24h: 39.10 ± 28.14% vs. 55.80 ± 14.05%, p=0.934). Several possible reasons maybe involved, but the most obvious reason may lie in the dysfunction of major immune effector cells resulted from tumor microenvironment in HCC patients. Taken together, a novel direct contact co-culture (DCC) system with primary HCC cells and TILs has been established in this study. The DCC system could study the direct interactions between major immune effector cells and HCC cells in a physiologically and clinically relevant manner. It can be used as a powerful tool to study the mechanisms related to the target/effector cell interactions and provide more evidence for HCC relevant immunotherapy.