Baocai Xing

1B50-1, a mAb Raised against Recurrent Tumor Cells, Targets Liver Tumor-Initiating Cells by Binding to the Calcium Channel α2δ1 Subunit

The identification and targeted therapy of cells involved in hepatocellular carcinoma (HCC) recurrence remain challenging. Here, we generated a monoclonal antibody against recurrent HCC, 1B50-1, that bound the isoform 5 of the α2δ1 subunit of voltage-gated calcium channels and identified a subset of tumor-initiating cells (TICs) with stem cell-like properties. A surgical margin with cells detected by 1B50-1 predicted rapid recurrence. Furthermore, 1B50-1 had a therapeutic effect on HCC engraftments by eliminating TICs. Finally, α2δ1 knockdown reduced self-renewal and tumor formation capacities and induced apoptosis of TICs, whereas its overexpression led to enhanced sphere formation, which is regulated by calcium influx. Thus, α2δ1 is a functional liver TIC marker, and its inhibitors may serve as potential anti-HCC drugs.

The properties of tumor-initiating cells from a hepatocellular carcinoma patient's primary and recurrent tumor.

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence. However, little is known about the biological characteristics of recurrent HCC cells. A single patients primary and recurrent HCC-derived cell lines, Hep-11 and Hep-12, respectively, were established by primary culture. These two cell lines have the same hepatitis B virus integration site and share many common amplifications and deletions, which suggest that they have the same clonal origin. While Hep-11 cells were non-tumorigenic at 16 weeks following injection of up to 10 000 cells, injection of only 100 Hep-12 cells was sufficient to initiate tumor growth, and all single Hep-12 clones were tumorigenic in immunodeficient mice. Compared with Hep-11, Hep-12 cells expressed the oval cell markers AFP, NCAM/CD56, c-kit/CD117, as well as multiple stem cell markers such as Nanog, OCT4 and SOX2. In addition, >90% of Hep-12 cells were aldehyde dehydrogenase positive. They were also less resistant to paclitaxel, but more resistant to doxorubicin, cisplatin and hydroxycamptothecin (HCPT), which had been administrated to the patient. Furthermore, Hep-12 cells expressed higher levels of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) than Hep-11, and PARP-1 inhibition potentiated the sensitivity to HCPT in Hep-12 cells but not in Hep-11 cells. These results indicate that a large population of the recurrent HCC-derived Hep-12 cells were tumor-initiating cells and that elevated expression of PARP-1 was related to their resistance to HCPT.

PBX3 is targeted by multiple miRNAs and is essential for liver tumour-initiating cells

Tumour-initiating cells (TICs) are advocated to constitute the sustaining force to maintain and renew fully established malignancy; however, the molecular mechanisms responsible for these properties are elusive. We previously demonstrated that voltage-gated calcium channel α2δ1 subunit marks hepatocellular carcinoma (HCC) TICs. Here we confirm directly that α2δ1 is a HCC TIC surface marker, and identify let-7c, miR-200b, miR-222 and miR-424 as suppressors of α2δ1(+) HCC TICs. Interestingly, all the four miRNAs synergistically target PBX3, which is sufficient and necessary for the acquisition and maintenance of TIC properties. Moreover, PBX3 drives an essential transcriptional programme, activating the expression of genes critical for HCC TIC stemness including CACNA2D1, EpCAM, SOX2 and NOTCH3. In addition, the expression of CACNA2D1 and PBX3 mRNA is predictive of poor prognosis for HCC patients. Collectively, our study identifies an essential signalling pathway that controls the switch of HCC TIC phenotypes.

Recurrent hepatocellular carcinoma cells with stem cell-like properties: possible targets for immunotherapy.

BACKGROUND AIMS Hepatocellular carcinoma (HCC) recurs with high frequency. Characterization of recurrent HCC cells will facilitate the design of future therapeutic strategies for recurrent HCC. METHODS Two cell lines, Hep-11 and Hep-12, were established from the same HCC patients primary and recurrent tumor tissues, respectively, and then analyzed for stem cell-like properties, immune evasion strategies and immunogenicity. RESULTS Compared with Hep-11 cells, Hep-12 cells expressed higher levels of liver progenitor cell makers and displayed persistent tumorigenic potential in the serial transplantation assay. Although Hep-12 cells down-regulated human leukocyte antigen (HLA) class I expression, they could still be recognized and killed by autologous-activated tumor-infiltrating lymphocytes (TIL) in vitro. Pre-treatment with cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) increased the expression of HLA class I molecules on Hep-12 cells, and rendered them more susceptible to CD8(+) T-cell-mediated recognition and TIL-mediated cytotoxicity in vitro. CONCLUSIONS Our results indicate that Hep-12 cells possess stem cell-like properties, are susceptible to autologous-activated TIL-mediated recognition and cytotoxicity, and pre-treatment with TNF-alpha and IFN-gamma enhances their immunogenicity. This is the first evidence to support the hypothesis that immunotherapy can be used to target recurrent HCC cells with stem cell-like properties. This strategy may be an effective therapeutic approach to prevent HCC recurrence and control recurrent HCC growth.

Abstract 5626: Investigation of T cell activation by anti-human PD-1 antibodies Nivolumab, Pembrolizumab and BGB-A317 using tumor-infiltrating lymphocytes (TILs) from colorectal cancer and colorectal liver metastasis patients

Blockade of the PD-1 pathway with anti-PD-1 antibody, such as nivolumab (nivo) and pembrolizumab (pembro), has led to remarkable clinical responses in patients with many different cancer types. In this study, we investigated the activation of tumor-infiltrating lymphocytes (TILs) from patients with colorectal cancer (CRC) and colorectal liver metastasis (CLM) by nivo, pembro and BGB-A317, a novel humanized IgG4 anti-PD-1 antibody under clinical development. BGB-A317 has a unique binding signature to PD-1 with high affinity. Additionally, it is engineered to remove Fc gamma receptor (FcγR) binding, including FcγRI, FcγRIIA, FcγRIIB and FcγRIIIA. In our studies, all three anti-PD-1 antibodies showed significant increases in IFN-γ and TILs proliferation in a 3D tumor spheroid model. In both CRC and CLM patients, BGB-A317 treatment at concentration levels of 0.1, 1 and 10 μg/mL led to higher IFN-γ production than that in nivo and pembro treated groups. The enhanced TILs function was associated with a high density of CD8+ T cells, but inversely correlated with the percentage of CD11b+ myeloid cells in CRC tumors. Of interest, BGB-A317 showed better activation of TILs in the liver metastasis tumor microenvironment (TME) where macrophages were more abundant. This observation is consistent with the hypothesis that removal of FcγR binding might offer advantage to PD-1 mAbs in tumor microenvironment enriched with macrophages. Citation Format: Lusong Luo, Xiaoran Wu, Tong Zhang, Chunyan Fu, Yanjuan Zhang, Amy Guo, Dongping Zhou, Lianhai Zhang, Kun Wang, Baocai Xing, Jiafu Ji, Lai Wang, Kang Li. Investigation of T cell activation by anti-human PD-1 antibodies Nivolumab, Pembrolizumab and BGB-A317 using tumor-infiltrating lymphocytes (TILs) from colorectal cancer and colorectal liver metastasis patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5626. doi:10.1158/1538-7445.AM2017-5626

Abstract 1666: Targeting tumor-initiating cells for the treatment of hepatocellular carcinoma

Tumor-initiating cells (TICs) or so-called cancer stem cells (CSCs) are a minor subpopulation of poor differentiated cells that are responsible for tumor initiation, maintenance, and spreading. Hence, eliminating these cells is supposed to be an effective way of the treatment of cancer. By immunizing mice with TIC-enriched hepatocellular carcinoma (HCC) cells (Xu XL, et al. The properties of tumor-initiating cells from a hepatocellular carcinoma patient9s primary and recurrent tumor. Carcinogensis. 2010; 31:167-74.) and by subsequent hybridoma technique, we have obtained an antibody 1B50-1, which recognizes the antigen of a 150 KD ion channel-composing protein. Despite the percentage of 1B50-1 positive cells varies from Funding: This study was supported by the National Basic Research Program of China (973 Program, No 2010CB529402), National Natural Science Foundation of China, and “863” Program (2008AA02Z108). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1666. doi:10.1158/1538-7445.AM2011-1666