Zebrafish Tumor Model Research Articles

Allosteric modulation of the CXCR4:CXCL12 axis by targeting receptor nanoclustering via the TMV-TMVI domain

CXCR4 is a ubiquitously expressed chemokine receptor that regulates leukocyte trafficking and arrest in both homeostatic and pathological states. It also participates in organogenesis, HIV-1 infection, and tumor development. Despite the potential therapeutic benefit of CXCR4 antagonists, only one, plerixafor (AMD3100), which blocks the ligand-binding site, has reached the clinic. Recent advances in imaging and biophysical techniques have provided a richer understanding of the membrane organization and dynamics of this receptor. Activation of CXCR4 by CXCL12 reduces the number of CXCR4 monomers/dimers at the cell membrane and increases the formation of large nanoclusters, which are largely immobile and are required for correct cell orientation to chemoattractant gradients. Mechanistically, CXCR4 activation involves a structural motif defined by residues in TMV and TMVI. Using this structural motif as a template, we performed in silico molecular modeling followed by in vitro screening of a small compound library to identify negative allosteric modulators of CXCR4 that do not affect CXCL12 binding. We identified AGR1.137, a small molecule that abolishes CXCL12-mediated receptor nanoclustering and dynamics and blocks the ability of cells to sense CXCL12 gradients both in vitro and in vivo while preserving ligand binding and receptor internalization.

Smart DNA Network Capturing and Destructing Tumor‐Derived Small Extracellular Vesicles at Tumor Sites for Localized Cancer Therapy

AbstractTumor‐derived small extracellular vesicles (sEVs) are proven to play important roles in accelerating the progression of tumors. Destructing the sEVs at tumor sites is therefore a promising route to inhibit tumor progression. Herein, a smart DNA network‐based sEV trap (DNET) is reported, which achieved the specific capture of tumor‐derived sEVs through recognizing sEVs by polyaptamers, and the efficient destruction of sEVs via a photodynamic process at tumor sites. The DNET is constructed through the assembly of two DNA chains generated via rolling circle amplification. The DNA chains contain polyaptamers for capturing sEVs, polyvalent G‐quadruplexes for loading photodynamic reagents, and complementary segments for forming a cross‐linked network. Upon the irradiation of the laser, the captured sEVs in DNET are destructed, causing significant inhibition effects on the migration, invasion, and proliferation of glioblastoma cells. In particular, DNET achieved tumor inhibition rates of 39.70% in the zebrafish tumor model, and 73.10% in the nude mouse tumor model, respectively, demonstrating the significant efficacy of DNET in inhibiting tumor progression.

The FLRT3-UNC5B Pathway is a Novel Regulator of T Immunosurveillance

Abstract Our understanding of molecular cell-surface interactions that control T cell responses in cancer remains limited despite many advances in the field. We hypothesized that tumor cells take advantage of developmental protein-protein interactions such as axon guidance interactions that could inhibit or repel tumor-immune interactions in the TME to evade T cell anti-tumor immunity. We used gain-of-function screening to evaluate a gene-set of axon guidance proteins for inhibitory function in human T cells and identified and validated Fibronectin and Leucine-Rich Transmembrane protein-3 (FLRT3) as an inhibitor of T cell activity. FLRT3 inhibited human T cell function and promoted tumor growth in humanized tumor cell-derived xenograft (CDX) models. FLRT3 inhibited T cells through interaction with Unc-5 Netrin Receptor B (UNC5B) expressed on activated T cells. A FLRT3 monoclonal antibody designed to specifically block FLRT3 interactions with UNC5 family proteins reduced tumor growth in CDX models that was dependent on T cells. FLRT3 blockade also enhanced CAR-T and BiTE + T cell activity and infiltration in humanized zebrafish tumor models. These studies support the notion that aberrant axon guidance protein expression in the TME act as T cell checkpoints, and the FLRT3:UNC5B pathway can be targeted therapeutically to improve T cell-based immunotherapy activity and infiltration.

A High-Throughput Microdroplet-Based Single Cell Transfection Method for Gene Knockout Based on the CRISPR/Cas9 System.

The efficient application of the newly developed gene-editing method CRISPR/Cas9 requires more accurate intracellular gene delivery. Traditional delivery approaches, such as lipotransfection and non-viral delivery methods, must contend with major problems to overcome the drawbacks of low efficiency, high toxicity, and cell-type dependency. The high-throughput microdroplet-based single-cell transfection method presented herein provides an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. This paper presents a high-throughput quantitative DNA transfection method for single cells and explores the optimal DNA transfection conditions for specific cell lines. The transfection efficiency of cells at different concentrations of DNA in microdroplets is measured. Under the optimized transfection conditions, the method is used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR/Cas9 knockout system. In a case study, the migration ability of TRIM72 knockout cancer cells is inhibited, and the tumorigenicity of cells in a zebrafish tumor model is reduced. A single-cell microfluidic chip is designed to achieve CRISPR/Cas9 DNA transfection, dramatically improving the transfection efficiency of difficult-to-transfect cells. This research demonstrates that the microdroplet method developed herein has a unique advantage in CRISPR/Cas9 gene-editing applications.

Abstract 2861: Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo

Abstract Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer lacking curative therapies, and outcomes for children with this disease have not improved in decades. The chimeric transcription factor PAX3::FOXO1 is the most common and lethal driver of fusion-positive rhabdomyosarcoma, and it consists of the DNA binding domains of PAX3 fused to the transactivation domain of FOXO1. Despite its well-established essentiality for tumorigenesis, how PAX3::FOXO1 initially generates a tumorigenic cell state has been challenging to study due to the lack of in vivo models expressing the fusion-oncogene at accessible early developmental time points. Here, we developed a novel zebrafish mRNA injection model that ubiquitously expresses PAX3::FOXO1 during gastrulation representing all three germ layers. Using this high-throughput model, we used ChIP-seq to identify initial in vivo PAX3::FOXO1 binding sites and targets. PAX3::FOXO1 has pioneering activity, where it can utilize partial-motif recognition to bind to inaccessible chromatin and alter chromatin structure. With biochemical and 2D chromatin sequencing approaches in our model, we find evidence for in vivo PAX3::FOXO1 pioneering activity. PAX3::FOXO1 consists of two DNA binding domains, a paired and homeobox domain, and we demonstrate a new mode of pioneering activity driven by its homeobox domain given a strong enrichment of homeobox-related motifs at PAX3::FOXO1 binding sites. PAX3::FOXO1 activity in developing zebrafish embryos results in an arrested development phenotype and transcriptional signatures enriched for pathways such as an inhibition of segmentation and myogenic development. Critically, we find that PAX3::FOXO1 directly activates neural-related gene targets by increasing chromatin accessibility and re-distribution of the active histone mark H3K27Ac to these loci. These discoveries are particularly striking because rhabdomyosarcoma is traditionally associated with skeletal muscle characteristics. With our findings, we hypothesize neural gene activation is a critical mechanism for fusion-positive rhabdomyosarcoma tumor initiation and could be a critical pathway across various stages of rhabdomyosarcoma tumorigenesis. This hypothesis agrees with our previous discovery of a novel PAX3::FOXO1 cooperating neural transcription factor, HES3, which we find is directly activated by PAX3::FOXO1 in our mRNA injection model. Our long-term goal is to utilize this versatile model to functionally evaluate cooperation between PAX3::FOXO1 and both canonical rhabdomyosarcoma and neural transcription factors. Our approach will employ a cross-species comparative analysis across this injection model, our zebrafish tumor models, other model systems, and patient data. This work will identify conserved targets and mechanisms of PAX3::FOXO1 tumorigenesis that could become new therapeutic vulnerabilities. Citation Format: Jack Kucinski, Cenny Taslim, Alexi Tallan, Matthew Cannon, Katherine Silvius, Benjamin Stanton, Genevieve Kendall. Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2861.

Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC.

Biodegradable fluorescent protein chromophore nanoparticles for hypoxic two-photon photodynamic therapy.

In this paper, biodegradable red fluorescent protein (RFP) chromophore analogue DPFP-SS-FA nanoparticles were synthesized for hypoxic two-photon photodynamic therapy. The maximum emission wavelength of DPFP-SS-FA is in the red-to-near-infrared region at 674 nm. Interestingly, these DPFP-SS-FA nanoparticles remain stable under physiological conditions, but deplete glutathione and disintegrate into the RFP chromophore analogue monomer in the tumor microenvironment. Meanwhile, electron paramagnetic resonance data have shown that DPFP-SS-FA produced enhanced 1O2/O2˙- signals after glutathione depletion causing an enhanced PDT effect. DPFP-SS-FA has negligible cell dark toxicity and high phototoxicity in hypoxic environments, indicating the outstanding hypoxia-overcoming ability of DPFP-SS-FA. In addition, due to its folic acid receptor and lysosome dual-targeting ability, DPFP-SS-FA is highly enriched in A-549 tumor cells. In particular, the hypoxic two-photon photodynamic therapy mediated by DPFP-SS-FA nanoparticles was validated in a zebrafish tumor model. Under 800 nm two-photon excitation, DPFP-SS-FA enabled bright two-photon fluorescence imaging and significantly inhibited the growth of tumor cells in zebrafish. The biodegradable DPFP-SS-FA nanoparticles reasonably constructed in this study can serve as excellent candidates for efficient hypoxic two-photon photosensitizers to treat deep tumor tissues.

Downregulated BIRC5 inhibits proliferation and metastasis of melanoma through the β-catenin/HIF-1α/VEGF/MMPs pathway.

Melanoma is a malignant skin tumor caused by melanocytes and associated with high mortality rates. This study aims to investigate the specific mechanism of ZWZ-3 in melanoma proliferation and metastasis. RNA sequencing was performed to identify the effect of ZWZ-3 on gene expression. siRNA was used to inhibit BIRC5 gene expression in the B16F10 cell line. A zebrafish tumor model was used to assess the therapeutic effect of ZWZ-3 in vivo. Mechanistic insights into the inhibition of tumor metastasis by ZWZ-3 were obtained through analysis of tumor tissue sections in mice. Our findings demonstrated that ZWZ-3 suppressed melanoma cell proliferation and migration. We performed RNA sequencing in melanoma cells after the treatment with ZWZ-3 and found that Birc5, which is closely associated with tumor metastasis, was significantly down-regulated. Bioinformatics analysis and the immuno-histochemical results of tissue chips for melanoma further confirmed the high expression of BIRC5 in melanoma and its effect on disease progression. Moreover, Birc5 knock-down significantly inhibited melanoma cell proliferation and metastasis, which was correlated with the β-catenin/HIF-1α/VEGF/MMPs pathway. Additionally, ZWZ-3 significantly inhibited tumor growth in the zebrafish tumor model without any evident side effects. Histological and immuno-histochemical analyses revealed that ZWZ-3 inhibited tumor cell metastasis by down-regulating HIF-1α, VEGF, and MMP9. Our findings revealed that ZWZ-3 could downregulate BIRC5 and inhibit melanoma proliferation and metastasis through the β-catenin/HIF-1α/VEGF/MMPs pathway. Therefore, BIRC5 represents a promising therapeutic target for the treatment of melanoma.

Stellettin B Sensitizes Glioblastoma to DNA-Damaging Treatments by Suppressing PI3K-Mediated Homologous Recombination Repair.

Glioblastoma (GBM) is the most aggressive type of cancer. Its current first-line postsurgery regimens are radiotherapy and temozolomide (TMZ) chemotherapy, both of which are DNA damage-inducing therapies but show very limited efficacy and a high risk of resistance. There is an urgent need to develop novel agents to sensitize GBM to DNA-damaging treatments. Here it is found that the triterpene compound stellettin B (STELB) greatly enhances the sensitivity of GBM to ionizing radiation and TMZ in vitro and in vivo. Mechanistically, STELB inhibits the expression of homologous recombination repair (HR) factors BRCA1/2 and RAD51 by promoting the degradation of PI3Kα through the ubiquitin-proteasome pathway; and the induced HR deficiency then leads to augmented DNA damage and cell death. It is further demonstrated that STELB has the potential to rapidly penetrate the blood-brain barrier to exert anti-GBM effects in the brain, based on zebrafish and nude mouse orthotopic xenograft tumor models. The study provides strong evidence that STELB represents a promising drug candidate to improve GBM therapy in combination with DNA-damaging treatments.

Apoptosis-Sensing Xenograft Zebrafish Tumor Model for Anticancer Evaluation of Redox-Responsive Cross-Linked Pluronic Micelles

A suitable animal model for preclinical screening and evaluation in vivo could vastly increase the efficiency and success rate of nanomedicine development. Compared with rodents, the transparency of the zebrafish model offers unique advantages of real-time and high-resolution imaging of the whole body and cellular levels in vivo. In this research, we established an apoptosis-sensing xenograft zebrafish tumor model to evaluate the anti-cancer effects of redox-responsive cross-linked Pluronic polymeric micelles (CPPMs) visually and accurately. First, doxorubicin (Dox)-loaded CPPMs were fabricated and characterized with glutathione (GSH)-responsive drug release. Then, the B16F10 xenograft zebrafish tumor model was established to mimic the tumor microenvironment with angiogenesis and high GSH generation for redox-responsive tumor-targeting evaluation in vivo. The high GSH generation was first verified in the xenograft zebrafish tumor model. Compared with ordinary Pluronic polymeric micelles, Dox CPPMs had a much higher accumulation in zebrafish tumor sites. Finally, the apoptosis-sensing B16F10-C3 xenograft zebrafish tumor model was established for visual, rapid, effective, and noninvasive assessment of anti-cancer effects at the cellular level in vivo. The Dox CPPMs significantly inhibited the proliferation of cancer cells and induced apoptosis in the B16F10-C3 xenograft zebrafish tumor model. Therefore, the redox-responsive cross-linked Pluronic micelles showed effective anti-cancer therapy in the xenograft zebrafish tumor model. This xenograft zebrafish tumor model is available for rapid screening and assessment of anti-cancer effects in preclinical studies.

Ethyl Acetate Fraction of Hedyotis diffusa Willd Induces Apoptosis via JNK/Nur77 Pathway in Hepatocellular Carcinoma Cells.

Background Hepatocellular carcinoma (HCC) is characterized by poor diagnosis and high mortality. Novel and efficient therapeutic agents are urgently needed for the treatment. Hedyotis diffusa Willd (HDW) is used to treat cancers, especially HCC in China. Purpose The study aimed to identify the main anti-HCC extract in HDW and to explore the mechanism of the active extract. Materials and Methods The high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was used for the simultaneous determination of main compounds in the ethyl acetate fraction of HDW (EHDW). The toxicity test of different HDW fractions was carried out on larvae at 2 day-post-fertilization (dpf) for 72 h. The in vivo anti-HCC effect of different HDW fractions was evaluated on a zebrafish tumor model by immersion administration. The antiproliferative effect of HDW fractions was determined with MTT assay, as well as hematoxylin and eosin (HE) staining assay. Hoechst 33258 staining was used to observe changes in nucleus morphology. Flow cytometry analysis was used to investigate apoptosis induction. Western blot analysis was used to examine apoptosis-related proteins, and key proteins in JNK/Nur77 signaling pathway. SP600125 was served to validate the apoptotic mechanism. Results EHDW showed the strongest tumor cell growth inhibitory effect on zebrafish tumor model. Further study revealed that EHDW induced apoptosis in zebrafish tumor model and in cultured Hep3B cells. Meanwhile, it has been shown that the levels of BCL2-associated X (Bax), cytochrome c (cyto c), cleaved-caspase 3, and poly-ADP-ribose polymerase (PARP) cells were upregulated. In contrast, the level of antiapoptotic B cell lymphoma-2 (Bcl-2) was downregulated in Hep3B cells. Additionally, EHDW activated JNK/Nur77 pathway by increasing the levels of p-JNK(Thr183/Tyr185) and p-Nur77(Ser351). Further study showed that blockage of JNK by SP600125 reversed EHDW-induced JNK/Nur77 pathway and the downstream apoptotic proteins. Conclusion In conclusion, EHDW exerted the anti-HCC effect, which may be attributed to the activation of JNK/Nur77 pathway. This study supported the rationale of HDW as an HCC therapeutic agent.

Visualization of natural killer cell-mediated killing of cancer cells at single-cell resolution in live zebrafish

Tumor immunotherapy has been an important advancement in cancer treatment in recent years. Compared with T cell-based therapy, natural killer (NK) cell-based therapy does not require human leukocyte antigen matching and has fewer side effects; thus, NK cell therapy has gradually attracted the attention of researchers and clinicians. Reliable and effective animal models are essential for evaluating the effects of NK cell therapy. NK cells kill cancer cells mainly through apoptosis. In this study, we first established a 3D coculture model using fluorescence resonance energy transfer (FRET)-based lung or breast cancer cells and tdTomato-labeled NK cells. We observed that cancer cells changed from green to blue when undergoing apoptosis induced by red NK cells. We then coinjected these green cancer cells with red NK cells into zebrafish to visualize the interaction between them and the killing process of NK cells against cancer cells in real-time and at single-cell resolution in circulation. Using this model, we found that NK cells can quickly kill cancer cells in zebrafish circulation in 40 min and the caspase-3 can be activated in 5–10 min. This FRET-based zebrafish tumor model can serve as a powerful in vivo tool that can facilitate the development of NK cell-based therapy. More importantly, cancer cells from cancer patients can be labeled with our apoptotic biosensor and then transplanted into zebrafish to evaluate the sensitivity of the cancer cells to NK cells to help clinicians make treatment plans that can benefit patients.

Abstract 6077: Mammalian adipose has many tumor features, which suggests the possible way of its evolutionary origin

Abstract Adipose expansion during obesity has remarkable similarities with the growth of solid tumors. These similarities are the following: the capability to unlimited expansion; reversible plasticity; induction of angiogenesis; chronic inflammation; remodeling and disfunction; systemic influence on the organism; hormone production; production of miRNAs that influence other tissues; immunosuppression; DNA damage and resistance to apoptosis; infiltration in other organs and tissues. The existing similarities between adipose and tumors suggest the possible evolutionary origin of mammalian adipose from some ancestral benign mesenchymal hereditary tumors. Transgenic inducible zebrafish tumor model was used to verify the above hypothesis. We described several genes, which originated in fish and were expressed in fish tumors. Their human orthologs LEP, SPRY1, PPARG, ID2, and CIDEA acquired functions connected with human adipose. They are also involved in tumor development in humans. If the hypothesis of the evolutionary origin of mammalian adipose from the ancestral benign hereditary tumors is correct, it may open new opportunities to resolve the oncological problem and the problem of the obesity epidemic. New interventions targeting LEP, SPRY1, PPARG, ID2, and CIDEA gene network, in addition to what already is going on, can be designed for treatment and prevention of both obesity and tumors. Citation Format: Andrei P. Kozlov. Mammalian adipose has many tumor features, which suggests the possible way of its evolutionary origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6077.

Ophiopogonin B alleviates cisplatin resistance of lung cancer cells by inducing Caspase-1/GSDMD dependent pyroptosis.

Drug resistance has become the main reason for the failure of tumor chemotherapy. Radix Ophiopogon Japonicus has long been used as traditional Chinese medicine to treat pulmonary disease, and Ophiopogonin B (OP-B) as a bioactive component of it has also been verified to inhibit cell proliferation of various non-small cell lung cancer (NSCLC) cells in vivo and in vitro. Therefore, we wonder whether OP-B is also effective to drug resistant lung cancer cells. Firstly, Cell Counting Kit-8 (CCK8) assay was used to compare the sensitivity of OP-B on NCI-H460, A549, cisplatin resistant A549 (A549/DDP) and paclitaxel resistant A549 (A549/PTX) cells, and A549/DDP cells were shown to be more sensitive to OP-B than other three cell lines, the results were further verified in orthotopic tumor nude mice model and zebrafish tumor model. Moreover, observation of cell morphological feature, mitochondrial membrane potential, LDH release rate, and production of IL-1β all suggested that OP-B induced pyroptosis in A549/DDP cells more significantly than that in A549 cells. Meanwhile, transcriptomic sequencing results between OP-B treated and the Mock A549/DDP group also suggested that OP-B induced more significant Caspase-1/GSDMD dependent pyroptosis in A549/DDP group, which was further verified by VX-765, the inhibitor of Caspase-1. Together, the experimental results suggested that OP-B alleviated DDP resistance of A549 cells through inducing more significant Caspase-1/GSDMD-dependent pyroptosis.

SIX6 is a TAL1-regulated transcription factor in T-ALL and associated with inferior outcome

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy driven by abnormal activity of transcription factors. Here we report an aberrant expression of the developmental transcription factor SIX6 in the TAL1-subtype of T-ALL. Our results demonstrate that the binding of TAL1 and GATA3 transcription factors into an upstream enhancer element directly regulates SIX6 expression. High expression of SIX6 was associated with inferior event-free survival within three independent patient cohorts. At a functional level, CRISPR-Cas9-mediated knockout of the SIX6 gene in TAL1 positive Jurkat cells induced changes in genes associated with the mTOR-, K-RAS-, and TNFα-related molecular signatures but did not impair cell proliferation or viability. There was also no acceleration of T-ALL development within a Myc driven zebrafish tumor model in vivo. Taken together, our results show that SIX6 belongs to the TAL1 regulatory gene network in T-ALL but is alone insufficient to influence the development or maintenance of T-ALL.

Modeling of Solid-Tumor Microenvironment in Zebrafish (Danio Rerio) Larvae.

The zebrafish larvae have emerged as a powerful model for studying tumorigenesis in vivo, with remarkable conservation with mammals in genetics, molecular and cell biology. Zebrafish tumor models bear the significant advantages of optical clarity in comparison to that in the mammalian models, allowing noninvasive investigation of the tumor cell and its microenvironment at single-cell resolution. Here we review recent progressions in the field of zebrafish models of solid tumor diseases in two main categories: the genetically engineered tumor models in which all cells in the tumor microenvironment are zebrafish cells, and xenograft tumor models in which the tumor microenvironment is composed of zebrafish cells and cells from other species. Notably, the zebrafish patient-derived xenograft (zPDX) models can be used for personalized drug assessment on primary tumor biopsies, including the pancreatic cancer. For the future studies, a series of high throughput drug screenings on the library of transgenic zebrafish models of solid tumor are expected to provide systematic database of oncogenic mutation, cell-of-origin, and leading compounds; and the humanization of zebrafish in genetics and cellular composition will make it more practical hosts for zPDX modeling. Together, zebrafish tumor model systems are unique and convenient in vivo platforms, with great potential to serve as valuable tools for cancer researches.

Aloperine induces apoptosis and G2/M cell cycle arrest in hepatocellular carcinoma cells through the PI3K/Akt signaling pathway

BackgroundHepatocellular carcinoma (HCC) ranks third among the most common causes of cancer-related deaths worldwide. The chemotherapy for HCC is still insufficient, so far. In searching for effective anti-HCC agents from traditional Chinese medicine, we discovered that aloperine (ALO), a quinolizidine alkaloid from Sophora alopecuroides L., exerts anti-HCC activities. However, the effects of ALO on HCC have been rarely studied, and its underlying mechanisms remain unknown. PurposeThis study aims to evaluate the anti-HCC activities of ALO and explore its underlying mechanisms. MethodsMTT assay and colony formation assay were used to investigate the anti-proliferative effects of ALO on human HCC Hep3B and Huh7 cells. Hoechst 33258 staining was used to observe the morphological changes of cells after ALO treatment. Flow cytometry was used to analyze apoptosis induction, the collapse of the mitochondrial membrane potential and cell cycle distribution. Western blotting was used to examine the expression levels of proteins associated with apoptosis and cell cycle arrest, and key proteins in the PI3K/Akt signaling pathway. Small interfering RNA (siRNA) transfection was used to investigate the role of Akt in ALO-induced apoptosis and cell cycle arrest. Zebrafish tumor model was used to evaluate the anti-HCC effects of ALO in vivo. ResultsALO inhibited the proliferation of Hep3B and Huh7 cells. ALO induced apoptosis in HCC cells, which was accompanied by the loss of mitochondrial potential, the release of cytochrome c into cytosol, as well as the increased cleavages of caspase-9, caspase-3 and PARP. Moreover, ALO induced G2/M cell cycle arrest by downregulating the expression levels of cdc25C, cdc2 and cyclin B1. In addition, ALO inhibited activation of the PI3K/Akt signaling pathway by decreasing the expression levels of p110α, p85, Akt and p-Akt (Ser473). Further study showed that inhibition of Akt by siRNA augmented ALO-mediated apoptosis and G2/M cell cycle arrest in HCC cells. Critically, ALO inhibited the growth of Huh7 cells in vivo. ConclusionWe first demonstrated that ALO induced apoptosis and G2/M cell cycle arrest in HCC cells through inhibition of the PI3K/Akt signaling pathway. This study provides a rationale for ALO as a potential chemotherapeutic agent for HCC.

Forkhead box C1 boosts triple‐negative breast cancer metastasis through activating the transcription of chemokine receptor‐4

The transcription factor forkhead box C1 (FOXC1) has recently been proposed as a crucial regulator of triple‐negative breast cancer (TNBC) and associated with TNBC metastasis. However, the mechanism of FOXC1 in TNBC development and metastasis is elusive. In this study, overexpression of FOXC1 in MDA‐MB‐231 cells significantly enhanced, whereas knockdown of FOXC1 in BT549 cells significantly reduced, the capabilities of TNBC cell invasion and motility in vitro and metastasis to the lung in vivo, when compared to their respective control cells. Mechanistic studies revealed that FOXC1 increased the expression of CXC chemokine receptor‐4 (CXCR4), probably through transcriptional activation. AMD3100, an inhibitor of CXCR4, could block cell migration. In a zebrafish tumor model, AMD3100 could suppress cell invasion and metastasis. In addition, overexpressing CXCR4 in FOXC1‐knockdown BT549 cells increased the capabilities of TNBC cell invasion and motility. In contrast, inhibition of CXCR4 with either AMD3100 or siRNA in MDA‐MB‐231 cells overexpressing FOXC1 reduced the capabilities of invasion and motility. Taken together, our results reveal a potential mechanism for FOXC1‐induced TNBC metastasis.

Enhancing the anti-glioma therapy of doxorubicin by honokiol with biodegradable self-assembling micelles through multiple evaluations

Combination chemotherapy is an important protocol in glioma therapy and honokiol shows synergistic anticancer effects with doxorubicin. In this paper, honokiol (HK) and doxorubicin (Dox) co-loaded Methoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) nanoparticles were prepared with a assembly method. The particle size (about 34 nm), morphology, X-ray Powder Diffraction (XRD), in vitro release profile, cytotoxicity and cell proliferation effects were studied in detail. The results indicated that honokiol and doxorubicin could be efficiently loaded into MPEG-PCL nanoparticles simultaneously, and could be released from the micelles in an extended period in vitro. In addition, honokiol and doxorubicin loaded in MPEG-PCL nanoparticles could efficiently suppress glioma cell proliferation and induce cell apoptosis in vitro. Furthermore, Dox-HK-MPEG-PCL micelles inhibited glioma growth more significantly than Dox-MPEG-PCL and HK-MPEG-PCL in both nude mice and zebrafish tumor models. Immunohistochemical analysis indicated that DOX-HK-MPEG-PCL micelles improved Dox’s anti-tumor effect by enhancing tumor cell apoptosis, suppressing tumor cell proliferation, and inhibiting angiogenesis. Our data suggest that Dox-HK-MPEG-PCL micelles have the potential to be applied clinically in glioma therapy.

Overexpression of PDGFRA cooperates with loss of NF1 and p53 to accelerate the molecular pathogenesis of malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, frequently metastatic sarcomas that are associated with neurofibromatosis type 1 (NF1), a prominent inherited genetic disease in humans. Although loss of the NF1 gene predisposes to MPNST induction, relatively long tumor latency in NF1 patients suggests that additional genetic or epigenetic abnormalities are needed for the development of these nerve sheath malignancies. To study the molecular pathways contributing to the formation of MPNSTs in NF1 patients, we used a zebrafish tumor model defined by nf1 loss in a p53-deficient background together with the overexpression of either wild-type or constitutively activated PDGFRA (platelet-derived growth factor receptor-α) under control of the sox10 neural crest-specific promoter. Here we demonstrate the accelerated onset and increased penetrance of MPNST formation in fish overexpressing both the wild-type and the mutant PDGFRA transgenes in cells of neural crest origin. Interestingly, overexpression of the wild-type PDGFRA was even more potent in promoting transformation than the mutant PDGFRA, which is important because ~78% of human MPNSTs have expression of wild-type PDGFRA, whereas only 5% harbor activating mutations of the gene encoding this receptor. Further analysis revealed the induction of cellular senescence in zebrafish embryos overexpressing mutant, but not wild-type, PDGFRA, suggesting a mechanism through which the oncogenic activity of the mutant receptor is tempered by the activation of premature cellular senescence in an NF1-deficient background. Taken together, our study suggests a model in which overexpression of wild-type PDGFRA associated with NF1 deficiency leads to aberrant activation of downstream RAS signaling and thus contributes importantly to MPNST development—a prediction supported by the ability of the kinase inhibitor sunitinib alone and in combination with the MEK inhibitor trametinib to retard MPNST progression in transgenic fish overexpressing the wild-type receptor.