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

Abstract

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.

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

“…30), demonstrate clearly that a TIC population with stem cell-like properties in HCC could be defined by a surface a2d1 phenotype.…”

“…The fact that most of the recurrent HCC-originated Hep-12 cells remain as TICs even with prolonged in vitro culture has led to the identification of a TIC population expressing the a2d1 subunit 29,30 . Here we developed a protocol by combining genome-wide miRNA profiling with softagar functional screening assay based on the Hep-12 cell line, to identify miRNAs that suppress the HCC TIC fate and have successfully identified a set of miRNAs, including four most effective miRNAs (let-7c, miR-200b, miR-222 and miR-424) that suppress the stemness and tumorigenicity of a2d1 þ HCC TICs.…”

“…Cells were detached from culture by 0.02% EDTA/PBS or were dissociated from primary or xenografted tumours by mechanical homogenization, and were stained by rabbit anti-a2d1 polyclonal antibody (1:100 dilution, Sigma-Aldrich, St Louis, MO, # HPA008213), or Mab1B50-1 (ref. 30; 40 mg ml À 1 ), which were pre-labeled using the LightningLink PE-Cy5 Labeling Kit (Innova Biosciences Ltd, Cambridge, UK) at 4°C for 30 min. Labeled samples were analysed or sorted on a FACSAria II flow cytometer (BD Biosciences, San Jose, CA).…”

“…On the basis of a pair of hepatocellular carcinoma (HCC) cell lines Hep-11 and Hep-12, which represent non-tumorigenic and TIC-enriched cell populations, respectively 29 , we identified a subpopulation of TICs expressing the isoform 5 of the voltagegated calcium channel a2d1 subunit (encoded by the gene CACNA2D1), which is vital for the activation of calcium signalling that controls the HCC TIC properties using an antibody, Mab1B50-1, generated against Hep-12 cells 30,31 . Here we first validate directly that a2d1 is indeed a surface marker for HCC TICs using a known a2d1 antibody and identify its prognostic role in HCC.…”

Targeting the Neurokinin-1 Receptor Compromises Canonical Wnt Signaling in Hepatoblastoma

“…In this regard, it seems that the b-catenin-mutated cell lines (HepT1 and HepG2) were more susceptible to aprepitant treatment than the b-catenin wild-type cell line HuH7. Due to the modest changes in HuH7 cells, we included gene expression analysis of previously described liver-specific CSC markers [AFP (25), CD13 (26), CD133 (27), CK19 (28), DLK1 (29), EPCAM (30), and GEP (31)] in our investigation. Here, we found downregulation of AFP and CD13 in all HB spheres upon aprepitant treatment, and decreased expression of CD133, CK19, EPCAM, and GEP in either HepT1 or HepG2 spheres (Fig.…”