Identification and functional characterization of potential oncofetal targets in human hepatocellular carcinoma.

Here, we present a detailed protocol for the identification of potential oncofetal targets for hepatocellular carcinoma (HCC) patients through a hepatocyte differentiation model and a sorafenib refractory cell-line-derived xenograft model. We describe the procedures of tumor sphere formation, organoid generation, and subcutaneous tumor formation for functional studies. We then detail the procedures of immunohistochemistry and immunofluorescence for examination of changes in lineage-specific markers. Finally, we describe the development of antibody-based therapeutics targeting tumor lineage plasticity in HCC. For complete details on the use and execution of this protocol, please refer to Kong et al. (2021).1.

An NK cell line (NK92-41BB) expressing high levels of granzyme is engineered to express the high affinity chimeric genes CD16/CAR.

Natural killer (NK) cells are known to play a role in mediating innate immunity and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) based on the reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, devoid of CD16 and derived from a lymphoma patient, has been well characterized. The adoptive transfer of irradiated NK-92 cells demonstrated safety and showed preliminary evidence of clinical benefit for cancer patients. The molecules 41BB and CD3 are commonly used as stimulators in the CAR structure, and their expression in NK cells can promote the activation of NK cells, leading to the enhanced perforin- and granzyme-mediated lysis of tumor cells. This study showed that genetically modified NK-92 cells combined with antibody-mediated ADCC using rituximab and trastuzumab monoclonal antibodies lysed tumor cells more efficient than the NK-92 cell lines. It also showed that the anti-tumor activity of chimeric stimulator molecules of the CAR-modified CD16 receptor was stronger than that of CD16 (allotype V158). These studies provide a rationale for the use of genetically modified NK-92 cells in combination with IgG1 anti-tumor monoclonal antibodies. We also provide a rationale for the chimeric modified CD16 receptor that can improve the anti-tumor effect of NK92 cells via ADCC. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s10616-021-00476-1) contains supplementary material, which is available to authorized users.

Long non-coding RNA DPP10-AS1 exerts anti-tumor effects on colon cancer via the upregulation of ADCY1 by regulating microRNA-127-3p.

Herein we hypothesized that DPP10-AS1 could affect the development of colon cancer via the interaction with miR-127-3p and adenylate cyclase 1 (ADCY1). After sorting of CD133 positive cells, sphere formation, colony formation, proliferation, invasion, migration, and apoptosis were detected to explore the involvement of DPP10-AS1 and miR-127-3p in the colon cancer stem cell (CCSC) properties through gain- and loss-of function approaches. Furthermore, tumor xenograft in nude mice was conducted to investigate the effect of DPP10-AS1 and miR-127-3p on tumor growth in vivo. Poorly expressed DPP10-AS1 and ADCY1, while highly expressed miR-127-3p were found in CCSCs. Low expression of DPP10-AS1 was correlated with TNM stage, lymphatic node metastasis, and tumor differentiation. Upregulation of DPP10-AS1 increased ADCY1 protein expression, decreased the protein expression of CCSC-related factors, inhibited sphere formation, colony formation, proliferation, invasion and migration, and accelerated apoptosis of HT-29 and SW480 cells by suppressing the expression of miR-127-3p. Further, the above in vitro findings were also confirmed by in vivo assays. Taken together, this study demonstrates that DPP10-AS1 inhibits CCSC proliferation by regulating miR-127-3p and ADCY1, providing fresh insight into a promising novel treatment strategy for colon cancer.

Targeting tumor lineage plasticity in hepatocellular carcinoma using an anti-CLDN6 antibody-drug conjugate.

Tumor lineage plasticity is emerging as a critical mechanism of therapeutic resistance and tumor relapse. Highly plastic tumor cells can undergo phenotypic switching to a drug-tolerant state to avoid drug toxicity. Here, we investigate the transmembrane tight junction protein Claudin6 (CLDN6) as a therapeutic target related to lineage plasticity for hepatocellular carcinoma (HCC). CLDN6 was highly expressed in embryonic stem cells but markedly decreased in normal tissues. Reactivation of CLDN6 was frequently observed in HCC tumor tissues as well as in premalignant lesions. Functional assays indicated that CLDN6 is not only a tumor-associated antigen but also conferred strong oncogenic effects in HCC. Overexpression of CLDN6 induced phenotypic shift of HCC cells from hepatic lineage to biliary lineage, which was more refractory to sorafenib treatment. The enhanced tumor lineage plasticity and cellular identity change were potentially induced by the CLDN6/TJP2 (tight junction protein 2)/YAP1 (Yes-associated protein 1) interacting axis and further activation of the Hippo signaling pathway. A de novo anti-CLDN6 monoclonal antibody conjugated with cytotoxic agent (Mertansine) DM1 (CLDN6-DM1) was developed. Preclinical data on both HCC cell lines and primary tumors showed the potent antitumor efficiency of CLDN6-DM1 as a single agent or in combination with sorafenib in HCC treatment.

Sequential delivery of dual drugs with nanostructured lipid carriers for improving synergistic tumor treatment effect.

To improve synergistic anticancer efficacy and minimize the adverse effects of chemotherapeutic drugs, temozolomide (TMZ) and curcumin (CUR) co-loaded nanostructured lipid carriers (NLCs) were prepared by microemulsion in this study. And the physicochemical properties, drug release behavior, intracellular uptake efficiency, in vitro and in vivo anticancer effects of TMZ/CUR-NLCs were evaluated. TMZ/CUR-NLCs showed enhanced inhibitory effects on glioma cells compared to single drug loaded NLCs, which may be owing to that the quickly released CUR can sensitize the cancer cells to TMZ. The inhibitory mechanism is a combination of S phase cell cycle arrest associated with induced apoptosis. Notably, TMZ/CUR-NLCs can accumulate at brain and tumor sites effectively and perform a significant synergistic anticancer effect in vivo. More importantly, the toxic effects of TMZ/CUR-NLCs on major organs and normal cells at the same therapeutic dosage were not observed. In conclusion, NLCs are promising nanocarriers for delivering dual chemotherapeutic drugs sequentially, showing potentials in the synergistic treatment of tumors while reducing adverse effects both in vitro and in vivo.

High-throughput single-cell analysis of exosome mediated dual drug delivery, in vivo fate and synergistic tumor therapy.

Exosomes could serve as delivery platforms, owing to their good biocompatibility, stability, and long blood circulation time. Tracking the biological fate of exosomes in vivo is essential for evaluating their functions, delivery efficacy, and biosafety, and it is invaluable for guiding exosome-based therapy. Here, we merged a single-cell technique, mass cytometry, with in vivo uptake analysis to comprehensively reveal the fate of exosomes at the single-cell level. In tandem with multivariate cellular phenotyping, in vivo uptake of exosomes labeled with heavy metal-containing tags was quantified in a high-throughput manner. Interestingly, an organ-dependent uptake landscape of exosomes by diverse cell types was distinctly demonstrated, which implied that cancer cells seemed to preferably take up more released drugs from the exosomes. Using these cellular insights, the administration method of drug-loaded exosomes was optimized to elevate their accumulation in tumor sites and minimize their spread into healthy organs. Dual drug-loaded exosomes were locally administered and superior synergistic tumor treatment effects were achieved in a solid tumor model. The disclosure of exosome cellular distribution, together with the successful engineering of exosomes with multiple anticancer capacities, provides a new level of insight into optimizing and enhancing exosome-based drug delivery and synergistic tumor therapy.