Hepatocellular Carcinoma Xenograft Model Research Articles

Low energy amplitude modulated radiofrequency electromagnetic fields to show antitumor effect in combination with standard treatment or as monotherapy in patients with advanced hepatocellular carcinoma.

332 Background: Exposure to amplitude-modulated radiofrequency electromagnetic fields (EMF) in immortalized hepatocellular carcinoma (HCC) cell cultures and xenograft models demonstrated anti-tumor effect. Patients with advanced HCC exposed to systemic EMF showed objective response with potential survival benefit. Methods: An open-label, single center, prospective clinical protocol was performed in advanced HCC patients as an initial, second or third treatment modality. Systemic exposure to EMF modulated at patient-specific frequencies was applied as an add-on strategy (combination) to systemic conventional treatment or as a single therapeutic modality. A spoon-shaped antenna placed in the oral cavity delivered EMF over 90 minutes with monthly repetitions until death or consent of withdrawal. The primary objective was overall survival (OS) in comparison with historical control group of 45 advanced patients HCC from the same institution. Retrospective radiological review was conducted by two independent radiologists for objective response (OR) using RECIST1.1 criterion. Results: From March 2018 to April 2020, 55 advanced HCC patients were submitted to 373 EMF exposures. 87% were male, median age of 67, 84% were BLCL-C, 16% were Child-Pugh B, 29% had extra-hepatic metastasis, 55% had failed previous treatment and 71% had documented radiological progression. 31(56%) patients received EMF in combination with systemic therapy (25 sorafenib, 3 lenvatinib, 3 nivolumab). 24 patients received EMF as single treatment modality. The median OS for the entire patient cohort was 11.5 months. The median OS for combination treatment was 12.0 month and the median OS for single modality was 11.3 months. The median OS for the historical control was 5.3 month. The median OS from the entire cohort and the historical control were significant different (p = 0.0026) but the median OS from combination and single modality were not (p = 0.3434). Radiological images were available from 38(69%) patients. There were 6/38 (16%) documented objective responses (1 CR and 5 PR) and 30/38 (79%) patients experienced disease control. 4/21 (19%) OR were in patients using TKIs + EMF and 2/17 (12%) OR were in patients using EMF alone. Conclusions: EMF showed objective anti-tumor effect in combination with TKI or as single modality in advanced HCC patients. EMF showed improvement in overall survival in comparison with the historical control group supporting future development as a novel systemic treatment modality in advanced HCC patients. Clinical trial information: NCT 01686412.

A bispecific antibody targeting GPC3 and CD47 induced enhanced antitumor efficacy against dual antigen-expressing HCC

Glypican-3 (GPC3) is a well-characterized hepatocellular carcinoma (HCC)-associated antigen, yet anti-GPC3 therapies have achieved only minimal clinical progress. CD47 is a ubiquitously expressed innate immune checkpoint that promotes evasion of tumors from immune surveillance. Given both the specific expression of GPC3 in HCC and the known phagocytosis inhibitory effect of CD47 in liver cancer, we hypothesized that a bispecific antibody (BsAb) that co-engages with GPC3 and CD47 may offer excellent antitumor efficacy with minimal toxicity. Here, we generated a novel BsAb: GPC3/CD47 biAb. With the use of both invitro and invivo assays, we found that GPC3/CD47 biAb exerts strong antitumor activity preferentially against dual antigen-expressing tumor cells. In hCD47/human signal regulatory protein alpha (hCD47/hSIRPα) humanized mice, GPC3/CD47 biAb had an extended serum half-life without causing systemic toxicity. Importantly, GPC3/CD47 biAb induced enhanced Fc-mediated effector functions to dual antigen-expressing HCC cells invitro, and both macrophages and neutrophils are required for its strong efficacy against xenograft HCC tumors. Notably, GPC3/CD47 biAb outperformed monotherapies and a combination therapy with anti-CD47 and anti-GPC3 monoclonal antibodies (mAbs) in a xenograft HCC model. Our study illustrates a strategy for improving HCC treatment by boosting innate immune responses and presents new insights to inform antibody design for the future development of innovative immune therapies.

Homoharringtonine inhibits the progression of hepatocellular carcinoma by suppressing the PI3K/AKT/GSK3β/Slug signaling pathway.

Homoharringtonine (HHT), was first isolated from the bark of Cephalotaxus harringtonia (Knight ex J. Forbes) K. Koch and Cephalotaxus fortunei Hook trees. The bark extract is used to treat leukemia and in recent years has also been used in traditional Chinese medicine (TCM) to treat solid tumors. However, the inhibitory mechanism of HHT in the progression of hepatocellular carcinoma (HCC) is rarely studied. We aimed to evaluate the antitumor efficacy of HHT on HCC in vitro and in vivo and elucidate the underlying molecular mechanism(s). HCC cell lines, including HCCLM3, HepG2, and Huh7, were used to evaluate the antitumor efficacy of HHT in vitro. Cytotoxicity and proliferative ability were evaluated by MTT and colony formation assays. Cell cycle progression and apoptosis in HHT-treated HCC cells were evaluated by flow cytometry. To determine the migration and invasion abilities of HCC cells, wound-healing and Transwell assays were used. Finally, western blot analysis was used to reveal the proteins involved. We also established a xenograft nude mouse model for in vivo assessments of the preclinical efficacy of HHT, mainly using hematoxylin and eosin staining, immunohistochemistry, ultrasound imaging (USI), and magnetic resonance imaging (MRI). HHT suppressed the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cells, and induced cell cycle arrest at the G2 phase and apoptosis. In the HCC xenograft model, HHT showed an obvious tumor-suppressive effect. Surprisingly, Slug expression was also decreased by HHT via the PI3K/AKT/GSK3β signaling pathway at least partially suppressed the growth of HCC via the PI3K/AKT/GSK3β/Slug signaling pathway.

Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy.

Objective:Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival.Methods:DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor.Results:HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the β-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses.Conclusions:HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC.

Saringosterol acetate isolated from Hizikia fusiforme, an edible brown alga, suppressed hepatocellular carcinoma growth and metastasis in a zebrafish xenograft model

Saringosterol acetate (SSA) was isolated from an edible brown alga Hizikia fusiforme. In this study, we developed an adult zebrafish human hepatocellular carcinoma (HCC) xenograft model to confirm that SSA inhibits tumor growth and metastasis. Established Hep3B cells labeled with the fluorescent tracker CM-Dil were xenografted into the abdominal cavity of zebrafish once every three days for one month. Compared with the control group, the fish injected with Hep3B showed a significant increase in α-fetoprotein (AFP) and factors related to tumor growth and metastasis (IL-6, TNF-α, TGFβ, MMP2, and MMP9). Using the model, it was proven that SSA affected survival rate, AFP production, and the levels of factors related to tumor growth and metastasis via the PI3K/AKT/mTOR and TGFβ/Smad pathways. In conclusion, this HCC model can be used for in vivo experiments to investigate the inhibition of cancer, and SSA may be useful for the treatment of cancer.

Light-activatable liposomes for repetitive on-demand drug release and immunopotentiation in hypoxic tumor therapy

External stimuli-responsive nanomedicine with desirable repetitive on-demand drug release character is postulated to greatly accommodate patients' flexible medication regime. To this object, light-activatable liposomes (Pt/Ce6-LP) integrated with both a Ce6 photodynamic component and a tetravalent platinum prodrug (Pt(IV)) chemotherapeutic component are engineered. This multifunctional system was rationally designed using unsaturated phospholipid to achieve repetitive on-demand drug release under discontinuous light irradiation, thus performing chemo-photodynamic therapy effect and immunopotentiation in hypoxic tumor. Furthermore, glutathione (GSH) consumption during transformation from Pt(IV) prodrug to Pt(II) can avoid depletion of reactive oxygen species (ROS) in photodynamic therapy (PDT). Note this positive feedback loop appears to remodel the redox balance of H2O2 and GSH in tumors, alleviating the hypoxic tumor microenvironment. The alleviated hypoxia is found to be critical to the enhancement of PDT efficacy, reversal of cisplatin resistance in tumors, and polarization of tumor-associated macrophages (TAMs) to the immunocompetent M1-phynotype. Pt/Ce6-LP with light radiation demonstrates significant antitumor effect and persistent post-medication inhibition in patient-derived tumor xenograft model of hepatocellular carcinoma.

Indole hydrazide compound ZJQ-24 inhibits angiogenesis and induces apoptosis cell death through abrogation of AKT/mTOR pathway in hepatocellular carcinoma

Angiogenesis and the activation of AKT/mTOR pathway are crucial for hepatocarcinoma development and progression, the activation of mTORC1/2 and relevant substrates have been confirmed in clinical hepatocarcinoma samples. Therefore, AKT/mTOR pathway represents the major targets for anti-cancer drugs development. Here, we investigated the anti-proliferative activity and mechanisms of ZJQ-24 in hepatocellular carcinoma, both in vivo and in vitro. A hepatocellular carcinoma xenograft model showed that ZJQ-24 significantly inhibited tumor growth with few side effects. MTT assays, flow cytometric analysis, Western blotting and immunohistochemistry identified that ZJQ-24 effectively suppressed hepatocellular carcinoma cell proliferation via G2/M phase arrest and caspase-dependent apoptosis but had no cytotoxic on normal cells. Furthermore, ZJQ-24 significantly blocked AKT/mTOR signaling by down-regulation of mTORC1 molecules, including phospho-p70S6K (Thr389) and phospho-4EBP-1 (Ser65, Thr37/46, Thr70) and phospho-AKT (Ser473) in HCC cells. It is very important that the ZJQ-24 did not induce the mTORC1-depdent PI3K/Akt feedback activation through JNK excitation. Moreover, ZJQ-24 inhibited the cap-dependent translation initiation by impairing the assembly of the eIF4E/eIF4G complex. Immunohistochemistry further confirmed ZJQ-24 inhibited the tumor growth through suppression of VEGF and AKT/mTOR pathways in vivo. Thus, the present study is the first to illustrate that ZJQ-24 triggers antiangiogenic activity and apoptosis via inhibiting the AKT/mTOR pathway in hepatocellular carcinoma cells, providing basic scientific evidence that ZJQ-24 shows great potential function as inhibitor of angiogenesis and tumor growth in hepatocellular carcinoma.

Interaction with CD68 and Regulation of GAS6 Expression by Endosialin in Fibroblasts Drives Recruitment and Polarization of Macrophages in Hepatocellular Carcinoma.

Fibroblasts and macrophages play key roles in the development of hepatocellular carcinoma (HCC). However, cross-talk between these two kinds of cells has not been well studied. Endosialin (CD248/TEM1) is a transmembrane glycoprotein that is expressed in certain cancer cells, tumor stromal cells, and pericytes. In this study, we found that endosialin is mainly expressed in cancer-associated fibroblasts (CAF) in HCC and its expression inversely correlates with patient prognosis. Endosialin interacted with CD68 to recruit macrophages and regulated expression of GAS6 in CAFs to mediate M2 polarization of macrophages. The fully human antibody IgG78 bound glycosylated endosialin and induced its internalization in CAFs, thus weakening the cross-talk between CAFs and macrophages. In subcutaneous and orthotopic xenograft models of HCC in nude mice, treatment with IgG78 significantly inhibited tumor growth. These results indicate that endosialin-positive CAFs promote HCC progression and highlight IgG78 as a promising therapeutic candidate for HCC treatment. SIGNIFICANCE: These findings highlight CAF-expressed endosialin as a primary regulator of macrophage recruitment and polarization and demonstrate endosialin inhibition as a potential treatment strategy for HCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3892/F1.large.jpg.

Illuminating Platinum Transportation while Maximizing Therapeutic Efficacy by Gold Nanoclusters via Simultaneous Near-Infrared-I/II Imaging and Glutathione Scavenging.

Killing tumor cells with a visualized system is a promising strategy in tumor therapy to achieve minimal side effects and high efficiency. Herein, a theranostic nanomedicine (AuNCs-Pt) is developed based on nanocarrier gold nanoclusters (AuNCs) with bifunctions of both NIR-I/NIR-II imaging and glutathione-scavenging abilities. AuNCs-Pt possesses NIR-II imaging capability on a fatal high-grade serous ovarian cancer (HGSOC) model in the deep abdomen, thus facilitating it to be a promising tool for monitoring platinum transportation. Meanwhile, AuNCs-Pt depletes intracellular glutathione to minimize platinum detoxification, effectively maximizing the chemotherapeutic efficacy of platinum. AuNCs-Pt is used to eradicate the tumor burden in this study on a HGSOC model and a patient-derived tumor xenograft model of hepatocellular carcinoma, suggesting great potential for clinical visualized therapy and platinum drug sensitization.

Combined gas embolization and chemotherapy can result in complete tumor regression in a murine hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is an intractable cancer with a high mortality rate. Transarterial chemoembolization (TACE), a non-curative method, is the first line therapy for intermediate stage patients. This effectively extends patient survival but requires a complicated intraarterial catheterization procedure and is poorly suited to repeated administration. Here, we investigate gas chemoembolization, a less invasive, more easily administered transient occlusion method that circumvents these limitations. We examined the efficacy of repeated embolization combined with systemically administered doxorubicin, the most common chemotherapeutic in TACE, or tirapazamine, a hypoxia-activated cytotoxic agent, in an ectopic xenograft model of HCC. Emboli were generated in situ using acoustic droplet vaporization, the noninvasive focused ultrasound-mediated conversion of intravenously administered perfluorocarbon microdroplets into microbubbles. Gas embolization alone significantly reduced the Ki67 index and tumor viability (11.6 ± 6.71% non-necrotic vs 100% in control; p < 0.01) after 3 treatments, as assessed by histological analysis. Mice treated for three weeks exhibited significant tumor regression compared to control (23.8 ± 5.37% of initial volume vs 427 ± 49.7% in controls, p < 0.01), irrespective of the chosen chemotherapeutic agent. However, an additional three weeks of monitoring post-treatment elucidated a significant difference in the tumor recurrence rate, with combined gas embolization and doxorubicin resulting in the best treatment outcomes (60% complete regression). While doxorubicin administration resulted in significant cardiotoxicity (p < 0.01), it strongly interacted with the droplet shells, reducing the systemic dose by 11.4%. Overall, gas chemoembolization shows promise as a developmental therapy and merits further study in more complex tumor models.

Abstract 1942: AGX73 is an effective treatment for hepatocellular carcinoma (HCC) by selective inhibition of immunokinases that play crucial roles in the crosstalk between cancer and immune cells in the tumor microenvironment

Abstract HCC is the second leading cause of cancer-related death and the fifth-most-common cancer worldwide. Unlike almost all other tumor types, the vast majority (90%) of HCCs develop due to underlying chronic inflammation, the induction of fibrosis and/or subsequent cirrhosis. In established disease, persistent upregulation of inflammatory signals in the tumor microenvironment leads to suppression of antitumor immune response by various mechanisms such as reprogramming of tumor-associated macrophages (TAMs) and loss of critical immune cell subsets (e.g. tumor-infiltrating lymphocytes). We identified 6 key immunokinases (AXL, DDR1, DDR2, FMS, KIT and MER) that are frequently amplified in HCC and are significant predictive markers of poor outcome (TCGA). We design small molecule compounds that potently and selectively inhibit these kinases. AGX73, optimized for drug-like properties, is an orally bioavailable compound currently undergoing IND evaluation as potential clinical candidate for first-in-human trial. In in vitro assay, carried out at high ATP concentration (100 μM), AGX73 inhibited the activity of recombinant human AXL, DDR1, DDR2, FMS, KIT and MER with IC50 values of 30, 12, 0.63, 96, 54, and 40 nM, respectively, but was highly selective against a panel of 374 kinases. In an in vitro primary macrophage assays, AGX73 promoted M1 macrophage polarization and increased the secretion of IL-6 and TNFα. In contrast, AGX73 suppressed the activation of M2 macrophages and decreased the secretion of IL-10. Importantly, AGX73 prevented the reprogramming of human LPS/IFN-γ polarized M1 macrophages back to M2 macrophages. In contrast, AGX73 had no effect on the reprogramming of IL-4/IL-13 polarized M2 macrophages back to M1. In vivo, in the MC38 colon carcinoma model, AGX73 treatment reduced the number of TAMs, from 46 to 22% of the total CD45+ cells in the tumors. Moreover, the M1/M2 ratio was favorably increased, from 1.3 to 1.8. AGX73 demonstrated robust antitumor activity in multiple (11) patient-derived xenograft (PDX) models of HCC, with tumor growth inhibition (TGI) ranging 62-105% (Median = 84%) and was effective at multiple dose levels, from 30-150 mg/kg. In combination with anti-PD1 (10 mg/kg) in the MC38 model, AGX73 produced synergistic activity at multiple dose levels. In nonclinical ADME studies, AGX73 demonstrated favorable drug-like properties: including good bioavailability (F&amp;gt;50% in mouse, rat and monkey) and systemic exposure following oral administration in multiple species, acceptable metabolic stability, lack of significant drug-drug interaction liabilities (transporters, CYP inhibition, CYP induction) and excellent cardiac safety profile. IND toxicologic studies are currently in progress. Citation Format: Francis Y.F. Lee, Wenlian Wu, Zhiqiang Yang, John Tan. AGX73 is an effective treatment for hepatocellular carcinoma (HCC) by selective inhibition of immunokinases that play crucial roles in the crosstalk between cancer and immune cells in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1942.

Abstract 1758: Discovery of novel potent and selective inhibitors of PRMT5 with anti-tumor activity in hepatocellular carcinoma and lung pre-clinical models

Abstract Protein arginine methyltransferase-5 (PRMT5) regulates many essential biological processes by catalyzing the symmetric di-methylation of arginine (sDMA) on a variety of histones and non-histone proteins. PRMT5 activity is dysregulated across many hematological and solid tumor and its overexpression is associated with poor patient prognosis. PRMT5 is also often overexpressed in cancer cells. Genetic and pharmacological inactivation have confirmed the importance of PRMT5 activity to the survival of many cancer cells, including hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC). Thus, inhibiting PRTMT5 activity has emerged as an attractive therapeutic strategy in oncology. We used structure-based drug design to identify novel small molecule inhibitors of PRMT5. ALG-070005, ALG-070043 and ALG-070017 inhibited PRMT5 enzymatic activity in biochemical assays with half-maximal inhibitory concentrations (IC50s) values between 50-150 pM and showed high selectivity against a panel of six other PRMTs. In HepG2 cells (HCC), ALG-070005, ALG-070043 and ALG-070017 potently decreased sDMA levels and inhibited cell proliferation with IC50 values of 1, 3, and 0.2 nM, respectively. These compounds also exhibited similar antiproliferative activity in A549 NSCLC cells. The three novel PRMT5 inhibitors demonstrated favorable ADME/PK profiles with exposure levels in target organs well above IC50 values, even 24 hours after dosing. When dosed orally in mice, these Aligos compounds inhibited tumor growth in HCC and NSCLC xenograft models, with concomitant inhibition of sDMA in the tumor. In conclusion, we identified and characterized novel potent and selective inhibitors of PRMT5 with antiproliferative effects in various preclinical models of HCC and lung cancers. These drug candidates also have a broad therapeutic potential in oncology beyond HCC and lung cancer. Citation Format: Francois Gonzalvez, Koen Vandyck, Yannick Debing, Kusum Gupta, Dinah Misner, Qingling Zhang, Jyanwei Liu, Antitsa Stoycheva, Sarah Stevens, Julian Symons, Leonid Beigelman, Pierre Raboisson, Jerome Deval. Discovery of novel potent and selective inhibitors of PRMT5 with anti-tumor activity in hepatocellular carcinoma and lung pre-clinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1758.

Targeting SphK2 Reverses Acquired Resistance of Regorafenib in Hepatocellular Carcinoma.

Background: Regorafenib is a second-line therapy drug used for advanced hepatocellular carcinoma (HCC). Unfortunately, the survival benefit of the patients receiving this treatment is modest, which may be attributed to drug resistance. In the present study, sphingosine kinase 2 (SphK2) was targeted to reverse regorafenib resistance in HCC.Methods: The functions of SphK2 and sphingosine-1-phosphate (S1P), the catalytic product of SphK2 in regorafenib resistance of HCC cells, were evaluated by cell counting kit-8 assay, colony formation, cell cycle evaluation, and annexin V–fluorescein isothiocyanate/propidium iodide double-staining assay. The antitumor activity of combined treatment of regorafenib and the SphK2-specific inhibitor ABC294640 was examined in HCC cells in vitro and xenograft model in vivo. The molecular mechanisms of SphK2/S1P-mediating regorafenib resistance were investigated using cell line establishment and Western blot analysis.Results: Well-developed regorafenib-resistant HCC cells indicated high expression levels of SphK2. The sensitivity to regorafenib of regorafenib-resistant HCC cells was restored following SphK2 knockdown or pharmacological inhibition by ABC294640. In addition, ectopic expression of SphK2 and exogenous addition of S1P decreased the sensitivity of HCC cells to regorafenib. Furthermore, the combination treatment with ABC294640 sensitized resistant tumor to regorafenib in xenograft model of HCC. The phosphorylation levels of nuclear factor κB (NF-κB), as well as those of signal transducer and activator of transcription 3 (STAT3), were positively associated with SphK2 and S1P.Conclusions: SphK2/S1P mediates regorafenib resistance of HCC through NF-κB and STAT3 activation. Targeting SphK2 by ABC294640 potently reduces regorafenib resistance of HCC cells both in vitro and in vivo. The combination of ABC294640 and regorafenib could be developed as a novel potential treatment strategy for advanced HCC.

Near-infrared phototherapy for patient-derived orthotopic xenograft model of hepatocellular carcinoma in combination with indocyanine green

BackgroundHepatocellular carcinoma notably takes up and retains indocyanine green (ICG). Here, we investigated whether patient-derived orthotopic xenograft of hepatocellular carcinoma could accumulate ICG and show full remission via phototherapy. MethodsNIR light and ICG were tested for cytotoxicity in cancerous cell lines (Huh-7, Hep3B). Patient-derived orthotopic xenograft (PDoX) mice were subjected to phototherapy comprising of daily NIR exposure (0.5–1.75 W/cm2) and intravenous injection of ICG (5–20 mg/kg2). Moreover, NIR laser was flashed on individual mouse until hepatocellular carcinoma completely loss the fluorescence, as determined by NIR camera. ResultsCytotoxicity increased in response to the input energy, but insufficient energy (< 150 joule/cm2) was irresponsive at all irradiances. NIR irradiance in the range of 0.5–1.75 W/cm2 took 5–7 days to elicit complete remission from PDoX mice in combination with 20 mg/kg ICG. In contrast, phototherapy could completely ablate hepatocellular carcinoma at 5–15 mg/kg ICG. ConclusionsICG could potentiate the tumoricidal ability of NIR light in a dose-dependent manner, and vice versa. Regardless of ICG dosage, however, phototherapy treated group showed a relatively high survival rate compared to the non-treated group. Notably, real-time phototherapy could halve the effective ICG dosage for full remission of deep-seated tumor.

Sorafenib and Mesenchymal Stem Cell Therapy: A Promising Approach for Treatment of HCC.

Hepatocellular carcinoma (HCC) is the fifth most commonly diagnosed cancer and the second most common cause of cancer-related death worldwide. Sorafenib (Sora) is used as a targeted therapy for HCC treatment. Mesenchymal stem cells (MSCs) are applied as a new approach to fight malignancies. Drug resistance and side effects are the major concerns with Sora administration. The effect of using the combination of sorafenib and MSCs on tumor regression in xenograft HCC models was evaluated in this study. Methods and Materials. Human hepatocellular carcinoma cell lines (HepG2) were subcutaneously implanted into the flank of 18 nude mice. The animals were randomly divided into six groups (n = 3); each received Sora (oral), MSCs (IV injection), MSCs (local injection), Sora + MSCs (IV injection), Sora + MSCs (local injection), or no treatment (the control group). Six weeks after tumor implantation, the mice were scarified and tumoral tissues were resected in their entirety. Histopathological and immunohistochemical evaluations were used to measure tumor proliferation and angiogenesis. Apoptotic cells were quantified using the TUNEL assay. Results. No significant difference was found in the tumor grade among the treatment groups. Differentiation features of the tumoral cells were histopathologically insignificant in all the groups. Tumor necrosis was highest in the hpMSC (local) + Sora group. Tumor cell proliferation was reduced in hpMSC (local) + Sora-treated and hpMSC (IV) + Sora-treated mice compared with the other groups. Apoptotic-positive cells occupied a greater proportion in the Sora, hpMSC (IV) + Sora, and hpMSC (local) + Sora groups. Conclusion. A combination of chemotherapy and MSC can yield to more favorable results in the treatment of HCC.

SHQ1 is an ER stress response gene that facilitates chemotherapeutics-induced apoptosis via sensitizing ER-stress response

SHQ1 was reported to control the biogenesis and assembly of H/ACA ribonucleoprotein particles (RNPs). It was independently isolated as a growth suppressor, GRIM1, in a genetic screen. Recent studies have indicated that SHQ1 inhibits prostate cancer growth and metastasis. SHQ1 facilitates MYC RNA splicing to promote T-acute lymphoblastic leukemia (T-ALL) development. Thus, the mechanisms of SHQ1 in cancers remain largely unknown. We report here that SHQ1 promotes tumor apoptosis and chemo-sensitivity in hepatocellular carcinoma (HCC) cells. In HCC tissues from patients, expression of SHQ1 was significantly decreased in the tumor compared to adjacent tissues. Experiments with HCC xenograft models revealed that restoring SHQ1 levels enhanced the anti-tumor activity of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) and common chemotherapy drug paclitaxel (PTX). Mechanistically, SHQ1 is an ER-stress response gene which is regulated by p50ATF6 and XBP1s through an ER stress response like element located on the SHQ1 promoter. SHQ1 interacts with the ER chaperone GRP78 to release ER sensors PERK/IRE1α/ATF6 from GRP78/ER-sensor complexes, leading to hyper-activation of unfolded protein response (UPR). In the persistent ER stress conditions of a HepG2 xenograft tumor model, SHQ1-mediated hyper-activation of ER-sensor signaling induces apoptosis. Our study thus demonstrates a SHQ1-mediated ER-stress response feedback loop that promotes tumor sensitivity to chemotherapeutics.

Construction and function of Glypican-3-targeted fourth-generation chimeric antigen receptor T cells (secreting IL-7 and CCL19)

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.

Hemistepsin A Inhibits Cell Proliferation and Induces G0/G1-Phase Arrest, Cellular Senescence and Apoptosis Via the AMPK and p53/p21 Signals in Human Hepatocellular Carcinoma.

Hemistepsin A (HsA), a natural sesquiterpene lactone isolated from Hemistepta lyrata, has been known as a wide range of anti-tumor effects. The aim of this study was to determine whether HsA suppresses hepatocellular carcinoma (HCC) and to figure out the cellular signaling pathways involved in the anti-HCC activities by experiments using the Huh7 cells (a human HCC cell line) and a xenograft HCC model. In this study, HsA completely inhibited HCC cell proliferation, presumably because it induced G0/G1 cell cycle arrest and mitochondrial-related apoptosis. HsA up-regulated p53, p21, cleaved caspase-3 and cleaved PARP (poly (ADP-ribose) polymerase), but reduced cyclin D, CDK6 and Bcl-2 expressions, and it disrupted mitochondrial membrane potential (ΔΨm). Moreover, phosphorylation of AMP-activated protein kinase (AMPK) was increased by HsA as did the resveratrol and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, positive controls). Inhibition of AMPK by using compound C, a competent inhibitor of AMPK, attenuated the loss of ΔΨm, p53 up-regulation and cellular senescence. The efficacy of HsA to reduce HCC cell proliferation, compared to that of other known anti-HCC agents, appears to be similar or slightly better. The anti-tumor effect of HsA was also determined in mice, showing reduced growth of xenografted tumors with no weight loss. Overall, the results suggest that HsA should be considered as a candidate anti-HCC drug.

Establishment and Preclinical Therapy of Patient-derived Hepatocellular Carcinoma Xenograft Model

Abstract Hepatocellular carcinoma (HCC) is a world-wide health problem. Poor and delayed diagnoses as well as high recurrence rate resulting in high mortality rate. In this study, we established a patient-derived xenograft (PDX) model from HCC patients, and continuously maintained with subcutaneous passage more than 20 times. This HCC PDX tumor exhibited the same histological characteristics with the HCC patient and could be used to verify therapeutic effect of liver cancer. We further evaluated this PDX model by experimental chemotherapy, demonstrating that this HCC PDX model was sensitive to sorafenib treatment. Further, the potential of natural killer cell-based immunotherapy for HCC was tested using this model. We found that NK92 cells effectively suppressed the tumor growth in vivo and prolonged the survival time of HCC-bearing PDX mice. This study indicates that HCC PDX model is a good platform to testify the efficacy of preclinical chemotherapy and immunotherapy.

Establishment and Preclinical Therapy of Patient-derived Hepatocellular Carcinoma Xenograft Model

Hepatocellular carcinoma (HCC) is a world-wide health problem. Poor and delayed diagnoses as well as high recurrence rate resulting in high mortality rate. In this study, we established a patient-derived xenograft (PDX) model from HCC patient, and continuously maintained with subcutaneous passage more than 20 times. This HCC PDX tumor exhibited the same histological characteristics with the HCC patient and could be used to verify therapeutic effect of liver cancer. We further evaluated this PDX model by experimental chemotherapy, demonstrating that this HCC PDX model was sensitive to sorafenib treatment. Further, the potential of natural killer cell-based immunotherapy for HCC was tested using this model. We found that NK92 cells effectively suppressed the tumor growth in vivo and prolonged the survival time of HCC-bearing PDX mice. This study indicates that HCC PDX model is a good platform to testify the efficacy of preclinical chemotherapy and immunotherapy.