Trastuzumab, a monoclonal anti-HER2 antibody modulates cytotoxicity against cholangiocarcinoma via multiple mechanisms.

Cholangiocarcinoma (CCA) is an aggressive and fatal cancer. The prognosis is very poor and no optimal chemotherapy has been established. Human epidermal growth factor receptor 2 (HER2, neu, and erbB2) is highly-expressed in breast cancer and is expressed in many other tumors but poorly expressed in CCA. The anti-HER2 antibody, trastuzumab, has been used for the treatment of HER2-positive breast and gastric cancer. In this study, we examined the surface expression of HER2 on seven Thai liver-fluke-associated CCA cell lines by flow cytometry, and found all of these CCA cells were weakly positive for HER2. MTT assay revealed that trastuzumab directly suppressed the growth of CCA. By using FcR-bearing recombinant Jurkat T-cell-expressing firefly luciferase gene under the control of NFAT response elements, we defined the activities of antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). ADCC was confirmed by using expanded NK cells. ADCP was confirmed by using mouse peritoneal macrophages and human monocyte-derived macrophages as effector cells. Rabbit serum was administered to test the complement-dependent cytotoxicity (CDC) activity of trastuzumab. Finally, we evaluated the efficacy of trastuzumab in in vivo patient-derived cell xenograft and patient-derived xenograft (PDX) models. Our results showed that a distinct population of CCA (liver-fluke-associated CCA) expressed HER2. Trastuzumab demonstrated a potent inhibitory effect on even HER2 weakly positive CCA both in vitro and in vivo via multiple mechanisms. Thus, HER2 is a promising target in anti-CCA therapy, and trastuzumab can be considered a promising antibody immunotherapy agent for the treatment of CCA.

Mucin-producing hamster cholangiocarcinoma cell line, Ham-2, possesses the aggressive cancer phenotypes with liver and lung metastases.

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation.

Attenuation of CD47-SIRPα Signal in Cholangiocarcinoma Potentiates Tumor-Associated Macrophage-Mediated Phagocytosis and Suppresses Intrahepatic Metastasis.

The involvement of chronic inflammation in cholangiocarcinoma (CCA) progression is well established. Cluster of differentiation 47 (CD47) is mutually expressed in various cancers and serves as a protective signal for phagocytic elimination. CD47 signaling blockage is a recent treatment strategy; however, little is known regarding CD47 in CCA. Therefore, the potential use of CD47 targeting in CCA was focused. CD47 was highly expressed in CCA compared to hepatocellular carcinoma (HCC). Disturbance of CD47-signal regulatory protein-α (SIRPα) interaction by blocking antibodies promoted the macrophage phagocytosis. The therapeutic potential of anti-CD47 therapy was demonstrated in liver metastatic model; alleviation of cancer colonization together with dense macrophage infiltrations was observed. The usefulness of anti-CD47 was emphasized by its universal facilitating macrophage activities. Moreover, increased production of inflammatory cytokines, such as IL-6 and IL-10, in macrophage exposed to CCA-conditioned media suggested that CCA alters macrophages toward cancer promotion. Taken together, interfering of CD47-SIRPα interaction promotes macrophage phagocytosis in all macrophage subtypes and consequently suppresses CCA growth and metastasis. The unique overexpression of CD47 in CCA but not HCC offers an exceptional opportunity for a targeted therapy. CD47 is therefore a novel target for CCA treatment.

Inhibition of carbonic anhydrase potentiates bevacizumab treatment in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a unique liver cancer subtype with an increasing incidence globally. The lack of specific symptoms and definite diagnostic markers results in a delayed diagnosis and disease progression. Systemic chemotherapy is commonly selected for advanced CCA even though its advantages remain unknown. Targeted therapy, especially anti-vascular endothelial growth factor (VEGF) therapy, is promising for CCA; however, improvements in the therapeutic regimen are necessary to overcome subsequent resistance. We demonstrated VEGF expression was higher in CCA cell lines than in other liver cancer cells. Secreted VEGFs played roles in the induction of peri- and intra-tumoral vascularization. VEGF neutralization by bevacizumab effectively reduced tumor growth, mainly through the suppression of angiogenesis; however, increases in the expression of hypoxia-inducible factor 1α (HIF1α) and HIF1α-responsive genes (such as VEGF, VEGFR1, VEGFR2, carbonic anhydrase (CA) IX and CAXII) indicated the potential for subsequent therapeutic resistance. Supplementation with a carbonic anhydrase inhibitor, acetazolamide, enhanced the anti-CCA effects of bevacizumab. Anti-angiogenesis and anti-proliferation were observed with the combination treatment. These results suggested a novel treatment strategy to overcome anti-angiogenesis resistance and the importance of "induced essentiality" in the treatment of CCA.