N6-methyladenosine modification of circMARK2 enhances cytoplasmic export and stabilizes LIN28B, contributing to the progression of Wilms tumor.

BACKGROUND: The potential involvement of circular RNAs (circRNAs) and N6-methyladenosine (m6A) modification in the progression of Wilms tumor (WT) has not been fully elucidated. This study investigates the regulatory mechanisms and clinical significance of m6A-modified circMARK2 and its role in WT progression. METHODS: We identified dysregulated circRNAs through deep sequencing and validated their expression by qRT-PCR in WT tissues. The biological functions of circMARK2 were assessed using clone formation, transwell migration, and orthotopic animal models. To dissect the underlying mechanisms, we employed RNA immunoprecipitation, RNA pull-down, dual-luciferase reporter assays, Western blotting, and immunofluorescence and immunohistochemical staining. RESULTS: CircMARK2, upregulated in WT tissues, was found to be m6A-modified and promoted cytoplasmic export. It facilitated WT progression by stabilizing LIN28B mRNA through the circMARK2/IGF2BP2 interaction. In vitro and in vivo studies demonstrated that circMARK2 enhances the malignant behavior of WT cells. Clinically, higher circMARK2 levels in tumor tissues of WT patients were linked to increased tumor aggressiveness and reduced survival rates. CONCLUSIONS: Our study provides the first comprehensive evidence that m6A-modified circMARK2 contributes to WT progression by enhancing LIN28B mRNA stability, promoting cellular aggressiveness. CircMARK2 emerges as a potential biomarker for prognosis and a promising target for therapeutic intervention in WT, underscoring the clinical relevance of m6A modification in pediatric renal cancer.

Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

Partial Acquisition of Spectral Projections Accelerates Four-dimensional Spectral-spatial EPR Imaging for Mouse Tumor Models: A Feasibility Study.

PURPOSE: Our study aimed to accelerate the acquisition of four-dimensional (4D) spectral-spatial electron paramagnetic resonance (EPR) imaging for mouse tumor models. This advancement in EPR imaging should reduce the acquisition time of spectroscopic mapping while reducing quality degradation for mouse tumor models. PROCEDURES: EPR spectra under magnetic field gradients, called spectral projections, were partially measured. Additional spectral projections were later computationally synthesized from the measured spectral projections. Four-dimensional spectral-spatial images were reconstructed from the post-processed spectral projections using the algebraic reconstruction technique (ART) and assessed in terms of their image qualities. We applied this approach to a sample solution and a mouse Hs766T xenograft model of human-derived pancreatic ductal adenocarcinoma cells to demonstrate the feasibility of our concept. The nitroxyl radical imaging agent 2H,15N-DCP was exogenously infused into the mouse xenograft model. RESULTS: The computation code of 4D spectral-spatial imaging was tested with numerically generated spectral projections. In the linewidth mapping of the sample solution, we achieved a relative standard uncertainty (standard deviation/| mean |) of 0.76 µT/45.38 µT = 0.017 on the peak-to-peak first-derivative EPR linewidth. The qualities of the linewidth maps and the effect of computational synthesis of spectral projections were examined. Finally, we obtained the three-dimensional linewidth map of 2H,15N-DCP in a Hs766T tumor-bearing leg in vivo. CONCLUSION: We achieved a 46.7% reduction in the acquisition time of 4D spectral-spatial EPR imaging without significantly degrading the image quality. A combination of ART and partial acquisition in three-dimensional raster magnetic field gradient settings in orthogonal coordinates is a novel approach. Our approach to 4D spectral-spatial EPR imaging can be applied to any subject, especially for samples with less variation in one direction.

Speeding up Glioblastoma Cancer Research: Highlighting the Zebrafish Xenograft Model.

Glioblastoma multiforme (GBM) is a very aggressive and lethal primary brain cancer in adults. The multifaceted nature of GBM pathogenesis, rising from complex interactions between cells and the tumor microenvironment (TME), has posed great treatment challenges. Despite significant scientific efforts, the prognosis for GBM remains very poor, even after intensive treatment with surgery, radiation, and chemotherapy. Efficient GBM management still requires the invention of innovative treatment strategies. There is a strong necessity to complete cancer in vitro studies and in vivo studies to properly evaluate the mechanisms of tumor progression within the complex TME. In recent years, the animal models used to study GBM tumors have evolved, achieving highly invasive GBM models able to provide key information on the molecular mechanisms of GBM onset. At present, the most commonly used animal models in GBM research are represented by mammalian models, such as mouse and canine ones. However, the latter present several limitations, such as high cost and time-consuming management, making them inappropriate for large-scale anticancer drug evaluation. In recent years, the zebrafish (Danio rerio) model has emerged as a valuable tool for studying GBM. It has shown great promise in preclinical studies due to numerous advantages, such as its small size, its ability to generate a large cohort of genetically identical offspring, and its rapid development, permitting more time- and cost-effective management and high-throughput drug screening when compared to mammalian models. Moreover, due to its transparent nature in early developmental stages and genetic and anatomical similarities with humans, it allows for translatable brain cancer research and related genetic screening and drug discovery. For this reason, the aim of the present review is to highlight the potential of relevant transgenic and xenograft zebrafish models and to compare them to the traditionally used animal models in GBM research.

Evaluation of the EMulate Therapeutics Voyager's ultra-low radiofrequency energy in murine model of glioblastoma.

BACKGROUND: Glioblastoma (GBM) presents as an aggressive brain cancer, notorious for its recurrence and resistance to conventional treatments. This study aimed to assess the efficacy of the EMulate Therapeutics Voyager®, a non-invasive, non-thermal, non-ionizing, battery-operated, portable experimental medical device, in treating GBM. Using ultra-low radiofrequency energy (ulRFE) to modulate intracellular activity, previous preliminary results in patients have been encouraging. Now, with a focus on murine models, our investigation seeks to elucidate the device's mechanistic impacts, further optimizing its therapeutic potential and understanding its limitations. METHODS: The device employs a silicone over molded coil to deliver oscillating magnetic fields, which are believed to interact with and disrupt cellular targets. These fields are derived from the magnetic fluctuations of solvated molecules. Xenograft and syngeneic murine models were chosen for the study. Mice were injected with U-87 MG or GL261 glioma cells in their flanks and were subsequently treated with one of two ulRFE cognates: A1A, inspired by paclitaxel, or A2, based on murine siRNA targeting CTLA4 + PD1. A separate group of untreated mice was maintained as controls. RESULTS: Mice that underwent treatments with either A1A or A2 exhibited significantly reduced tumor sizes when compared to the untreated cohort. CONCLUSION: The EMulate Therapeutics Voyager® demonstrates promising potential in inhibiting glioma cells in vivo through its unique ulRFE technology and should be further studied in terms of biological effects in vitro and in vivo.

Development and in-depth characterization of BRAFi-resistant melanoma cell lines in vitro and in vivo.

Regardless of the clinical response and improved patient survival observed following treatment with BRAFi like Vemurafenib (Vem), rapid development of resistance still remains as a major obstacle in melanoma therapy. In this context, we developed and characterized two acquired Vem-resistant melanoma cell lines, A375V and SK-MEL-28V, and an intrinsically Vem-resistant cell line, RPMI-7951. Altered morphology and growth rate of the resistant cell lines displayed spindle-shaped cells with filopodia formation and enhanced proliferation rate as compared to parental cells. Further in vitro characterization in 2D models confirmed the emergence of a resistant phenotype in melanoma cells. To mimic the in vivo tumor microenvironment, spheroids were developed for both parental and resistant cell lines to recognize materialization of invadopodia structures demonstrating elevated invasiveness and proliferation of resistant cells-based spheroids, especially A375V. Importantly, we validated A375V cell line in vivo to prove its tumorigenicity and drug resistance in tumor xenograft model. Taken together, our established clinically relevant Vem-resistant tumor model could be beneficial to elucidate drug resistance mechanisms, screen and identify novel anticancer therapies to overcome BRAFi resistance in melanoma.

Development of Orthotopic Patient-Derived Xenograft Models of Pediatric Intracranial Tumors.

The development of clinically relevant and reliable models of central nervous system tumors has been instrumental in advancing the field of Neuro-Oncology. The orthotopic intracranial injection is widely used to study the growth, invasion, and spread of tumors in a controlled environment. Orthotopic models are performed to examine tumor cells isolated from a specific region in a patient in the same site or location in an animal model. Orthotopic brain tumor models are also utilized for preclinical testing of therapeutics as they closely recapitulate the behavior of such cancer and the brain environment of patients. Below, we describe our experiences in the development of murine models of pediatric brain tumors including diffuse midline glioma (DMG), glioblastoma (GBM), and medulloblastoma. The method provides an overview of intracranial stereotactic injections in mice.

AS1411 aptamer/RGD dual functionalized theranostic chitosan-PLGA nanoparticles for brain cancer treatment and imaging.

Conventional chemotherapy and poor targeted delivery in brain cancer resulting to poor treatment and develop resistance to anticancer drugs. Meanwhile, it is quite challenging to diagnose/detection of brain tumor at early stage of cancer which resulting in severity of the disease. Despite extensive research, effective treatment with real-time imaging still remains completely unavailable, yet. In this study, two brain cancer cell specific moieties i.e., AS1411 aptamer and RGD are decorated on the surface of chitosan-PLGA nanoparticles to improve targeted co-delivery of docetaxel (DTX) and upconversion nanoparticles (UCNP) for effective brain tumor therapy and real-time imaging. The nanoparticles were developed by a slightly modified emulsion/solvent evaporation method. This investigation also translates the successful synthesis of TPGS-chitosan, TPGS-RGD and TPGS-AS1411 aptamer conjugates for making PLGA nanoparticle as a potential tool of the targeted co-delivery of DTX and UCNP to the brain cancer cells. The developed nanoparticles have shown an average particle size <200 nm, spherical in shape, high encapsulation of DTX and UCNP in the core of nanoparticles, and sustained release of DTX up to 72 h in phosphate buffer saline (pH 7.4). AS1411 aptamer and RGD functionalized theranostic chitosan-PLGA nanoparticles containing DTX and UCNP (DUCPN-RGD-AS1411) have achieved greater cellular uptake, 89-fold improved cytotoxicity, enhanced cancer cell arrest even at lower drug conc., improved bioavailability with higher mean residence time of DTX in systemic circulation and brain tissues. Moreover, DUCPN-RGD-AS1411 have greatly facilitated cellular internalization and higher accumulation of UCNP in brain tissues. Additionally, DUCPN-RGD-AS1411 demonstrated a significant suppression in tumor growth in brain-tumor bearing xenograft BALB/c nude mice with no impressive sign of toxicities. DUCPN-RGD-AS1411 has great potential to be utilized as an effective and safe theranostic tool for brain cancer and other life-threatening cancer therapies.

PKM2 promotes lymphatic metastasis of hypopharyngeal carcinoma via regulating epithelial-mesenchymal transition: an experimental research.

BACKGROUND: Patients with hypopharyngeal carcinoma (HPC) have a poor prognosis mainly because of lymphatic metastasis. This research aimed to determine the PKM2 role in lymphatic metastasis in HPC and the underlying molecular mechanism contributing to this phenomenon. METHODS: PKM2 in HPC was studied for its expression and its likelihood of overall survival using TCGA dataset. Western blotting, qRT-PCR, and IHC were employed to confirm PKM2 expression. Methods including gain- and loss-of-function were used to examine the PKM2 role in HPC metastasis in vitro and in vivo. In vitro and in vivo studies also confirmed lymphatic metastasis's mechanism. RESULTS: Prominent PKM2 overexpression was seen in patients with lymphatic metastasis of HPC, and there was an inherent relationship between a high PKM2 level and poor prognosis. In vitro research showed that knocking down PKM2 decreased tumor cell invasion, migration, and proliferation while promoting apoptosis and inhibiting epithelial-mesenchymal transition, but overexpressing PKM2 had the reverse effect. Animal studies suggested that PKM2 may facilitate tumor development and lymphatic metastasis. CONCLUSIONS: Our findings suggest that PKM2 may be a tumor's promoter gene of lymphatic metastasis, which may promote lymphatic metastasis of HPC by regulating epithelial-mesenchymal transition. PKM2 may be a biomarker of metastatic potential, ultimately providing a basis for exploring new therapeutic targets.

The generation of glioma organoids and the comparison of two culture methods.

BACKGROUND: The intra- and inter-tumoral heterogeneity of gliomas and the complex tumor microenvironment make accurate treatment of gliomas challenging. At present, research on gliomas mainly relies on cell lines, stem cell tumor spheres, and xenotransplantation models. The similarity between traditional tumor models and patients with glioma is very low. AIMS: In this study, we aimed to address the limitations of traditional tumor models by generating patient-derived glioma organoids using two methods that summarized the cell diversity, histological features, gene expression, and mutant profiles of their respective parent tumors and assess the feasibility of organoids for personalized treatment. MATERIALS AND METHODS: We compared the organoids generated using two methods through growth analysis, immunohistological analysis, genetic testing, and the establishment of xenograft models. RESULTS: Both types of organoids exhibited rapid infiltration when transplanted into the brains of adult immunodeficient mice. However, organoids formed using the microtumor method demonstrated more similar cellular characteristics and tissue structures to the parent tumors. Furthermore, the microtumor method allowed for faster culture times and more convenient operational procedures compared to the Matrigel method. DISCUSSION: Patient-derived glioma organoids, especially those generated through the microtumor method, present a promising avenue for personalized treatment strategies. Their capacity to faithfully mimic the cellular and molecular characteristics of gliomas provides a valuable platform for elucidating tumor biology and evaluating therapeutic modalities. CONCLUSION: The success rates of the Matrigel and microtumor methods were 45.5% and 60.5%, respectively. The microtumor method had a higher success rate, shorter establishment time, more convenient passage and cryopreservation methods, better simulation of the cellular and histological characteristics of the parent tumor, and a high genetic guarantee.

Anti-proliferative and apoptotic effect of cannabinoids on human pancreatic ductal adenocarcinoma xenograft in BALB/c nude mice model.

Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti-tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti-proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.

GFP Transfection Alters Protein Expression Patterns in Prostate Cancer Cells: A Proteomic Study.

PURPOSE: Green Fluorescent Protein is widely used as a cellular marker tool, but its potential influence on cells has been questioned. Although the potential off-target effects of GFP on tumor cells have been studied to some extent, the findings at the molecular level are insufficient to explain the effect of GFP expression on the tumorigenic capacity of cancer cells. Here, we aimed to investigate the effect of GFP expression on the tumorigenicity of PC3 prostate cancer cells. METHODS: Using GFP-expressing and wild-type PC-3 cells, xenograft models were generated in athymic BALB/C mice. To identify differentially expressed proteins, the change in cells proteome was investigated by label-free quantification with nano-high performance liquid chromatography to tandem mass spectrometry (nHPLC-MS/MS). Proteins that showed significantly altered expression levels were evaluated using the bioinformatics tools. RESULTS: Unlike the wild-type PC-3 cells, GFP-expressing cells failed to develop tumor. Comparative proteome analysis of GFP-expressing cells with WT PC-3 cells revealed a total of 216 differentially regulated proteins, of which 98 were upregulated and 117 were downregulated. CONCLUSION: Upon GFP expression, differential changes in several pathways including the immune system, translational machinery, energy metabolism, elements of cytoskeletal and VEGF signaling pathway were observed. Therefore, care should be taken into account to prevent reporting deceitful mechanisms generated from studies utilizing GFP.

Ononin triggers ferroptosis-mediated disruption in the triple negative breast cancer both in vitro and in vivo.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is difficult to treat due to a lack of targeted therapies. In this study, we aimed to investigate whether a natural flavonoid compound called ononin could be effective in treating TNBC by triggering ferroptosis in MDA-MB-231 and 4 T1 cell lines, and MDA-MB-231-xenograft nude mice model. Ononin inhibited TNBC through ferroptosis, which was determined by MTT assay, flow cytometry, RT-PCR, immunofluorescence, transmission electron microscopy, histological analysis, western blot and bioluminescence assay. Our results showed that treatment with ononin led to increased levels of malondialdehyde and reactive oxygen species and decreased activity of superoxide dismutase, which are indicatives of ferroptosis. We also found that ononin downregulated two key markers of ferroptosis, SLC7A11 and Nrf2, at both the transcriptional and translational level. Additionally, the administration of ononin resulted in a notable decrease in tumor size and weight in the mouse model. Furthermore, it was observed to enhance the rate of apoptosis in TNBC cells. Importantly, ononin did not induce any histological changes in the kidney, liver, and heart. Taken together, our findings suggest that ononin could be a promising therapeutic strategy for TNBC, and that it works by disrupting the Nrf2/SLC7A11 axis through ferroptosis. These results are encouraging and may lead to the development of new treatments for this challenging cancer subtype.

Dynamic evolution of bone marrow adipocyte in B cell acute lymphoblastic leukemia: insights from diagnosis to post-chemotherapy.

Adipocyte is a unique and versatile component of bone marrow microenvironment (BMM). However, the dynamic evolution of Bone Marrow (BM) adipocytes from the diagnosis of B cell Acute Lymphoblastic Leukemia (B-ALL) to the post-treatment state, and how they affect the progression of leukemia, remains inadequately explicated. Primary patient-derived xenograft models (PDXs) and stromal cell co-culture system are employed in this study. We show that the dynamic evolution of BM adipocytes from initial diagnosis of B-ALL to the post-chemotherapy phase, transitioning from cellular depletion in the initial leukemia niche to a fully restored state upon remission. Increased BM adipocytes retards engraftment of B-ALL cells in PDX models and inhibits cells growth of B-ALL in vitro. Mechanistically, the proliferation arrest of B-ALL cells in the context of adipocytes-enrichment niche, might attribute to the presence of adiponectin secreted by adipocytes themselves and the absence of cytokines secreted by mesenchymal stem cell (MSCs). In summary, our findings offer a novel perspective for further in-depth understanding of the dynamic balance between BMM and B-ALL.

In Vitro and In Silico of Cholinesterases Inhibition and In Vitro and In Vivo Anti-Melanoma Activity Investigations of Extracts Obtained from Selected Berberis Species.

Berberis species have a long history of use in traditional Chinese medicine, Ayurvedic medicine, and Western herbal medicine. The aim of this study was the quantification of the main isoquinoline alkaloids in extracts obtained from various Berberis species by HPLC, in vitro and in silico determination of anti-cholinesterase activity, and in vitro and in vivo investigations of the cytotoxic activity of the investigated plant extracts and alkaloid standards. In particular, Berberis species whose activity had not been previously investigated were selected for the study. In the most investigated Berberis extracts, a high content of berberine and palmatine was determined. Alkaloid standards and most of the investigated plant extracts exhibit significant anti-cholinesterase activity. Molecular docking results confirmed that both alkaloids are more favourable for forming complexes with acetylcholinesterase compared to butyrylcholinesterase. The kinetic results obtained by HPLC-DAD indicated that berberine noncompetitively inhibited acetylcholinesterase, while butyrylcholinesterase was inhibited in a mixed mode. In turn, palmatine exhibited a mixed inhibition of acetylcholinesterase. The cytotoxic activity of berberine and palmatine standards and plant extracts were investigated against the human melanoma cell line (A375). The highest cytotoxicity was determined for extract obtained from Berberis pruinosa cortex. The cytotoxic properties of the extract were also determined in the in vivo investigations using the Danio rerio larvae xenograft model. The obtained results confirmed a significant effect of the Berberis pruinosa cortex extract on the number of cancer cells in a living organism. Our results showed that extracts obtained from Berberis species, especially the Berberis pruinosa cortex extract, can be recommended for further in vivo experiments in order to confirm the possibility of their application in the treatment of neurodegenerative diseases and human melanoma.

Dclre1c-Mutation-Induced Immunocompromised Mice Are a Novel Model for Human Xenograft Research.

Severe combined immunodeficient (SCID) mice serve as a critical model for human xenotransplantation studies, yet they often suffer from low engraftment rates and susceptibility to graft-versus-host disease (GVHD). Moreover, certain SCID strains demonstrate 'immune leakage', underscoring the need for novel model development. Here, we introduce an SCID mouse model with a targeted disruption of the dclre1c gene, encoding Artemis, which is essential for V(D)J recombination and DNA repair during T cell receptor (TCR) and B cell receptor (BCR) assembly. Artemis deficiency precipitates a profound immunodeficiency syndrome, marked by radiosensitivity and compromised T and B lymphocyte functionality. Utilizing CRISPR/Cas9-mediated gene editing, we generated dclre1c-deficient mice with an NOD genetic background. These mice exhibited a radiosensitive SCID phenotype, with pronounced DNA damage and defective thymic, splenic and lymph node development, culminating in reduced T and B lymphocyte populations. Notably, both cell lines and patient-derived tumor xenografts were successfully engrafted into these mice. Furthermore, the human immune system was effectively rebuilt following peripheral blood mononuclear cells (PBMCs) transplantation. The dclre1c-knockout NOD mice described herein represent a promising addition to the armamentarium of models for xenotransplantation, offering a valuable platform for advancing human immunobiological research.

Antitumor Effects of Intravenous Natural Killer Cell Infusion in an Orthotopic Glioblastoma Xenograft Murine Model and Gene Expression Profile Analysis.

Despite standard multimodality treatment, containing maximum safety resection, temozolomide, radiotherapy, and a tumor-treating field, patients with glioblastoma (GBM) present with a dismal prognosis. Natural killer cell (NKC)-based immunotherapy would play a critical role in GBM treatment. We have previously reported highly activated and ex vivo expanded NK cells derived from human peripheral blood, which exhibited anti-tumor effect against GBM cells. Here, we performed preclinical evaluation of the NK cells using an in vivo orthotopic xenograft model, the U87MG cell-derived brain tumor in NOD/Shi-scid, IL-2RɤKO (NOG) mouse. In the orthotopic xenograft model, the retro-orbital venous injection of NK cells prolonged overall survival of the NOG mouse, indirectly indicating the growth-inhibition effect of NK cells. In addition, we comprehensively summarized the differentially expressed genes, especially focusing on the expression of the NKC-activating receptors' ligands, inhibitory receptors' ligands, chemokines, and chemokine receptors, between murine brain tumor treated with NKCs and with no agents, by using microarray. Furthermore, we also performed differentially expressed gene analysis between an internal and external brain tumor in the orthotopic xenograft model. Our findings could provide pivotal information for the NK-cell-based immunotherapy for patients with GBM.

Killer cell lectin-like receptor G2 facilitates aggressive phenotypes of gastric cancer cells via dual activation of the ERK1/2 and JAK/STAT pathways.

BACKGROUND: Advanced gastric cancer (GC) has a poor prognosis. This study aimed to identify novel GC-related genes as potential therapeutic targets. METHODS: Killer cell lectin-like receptor G2 (KLRG2) was identified as a candidate gene by transcriptome analysis of metastatic GC tissues. Small interfering RNA-mediated KLRG2 knockdown in human GC cell lines was used to investigate KLRG2 involvement in signaling pathways and functional behaviors in vitro and in vivo. Clinicopathological data were analyzed in patients stratified according to tumor KLRG2 mRNA expression. RESULTS: KLRG2 knockdown in GC cells decreased cell proliferation, migration, and invasion; caused cell cycle arrest in G2/M phase; induced apoptosis via caspase activation; suppressed JAK/STAT and MAPK-ERK1/2 pathway activities; and upregulated p53 and p38 MAPK activities. In mouse xenograft models of peritoneal metastasis, the number and weight of disseminated GC nodules were decreased by KLRG2 knockdown. High tumor levels of KLRG2 mRNA were significantly associated with lower 5-year overall survival (OS) and relapse-free survival (RFS) rates in patients with Stage I-III GC (5-year OS rate: 64.4% vs. 80.0%, P = 0.009; 5-year RFS rate: 62.8% vs. 78.1%, P = 0.030). CONCLUSIONS: KLRG2 knockdown attenuated the malignant phenotypes of GC cells via downregulation of JAK/STAT and MAPK-ERK1/2 pathway activity and upregulation of p38 MAPK and p53. Targeted suppression of KLRG2 may serve as a new treatment approach for GC.

Synthesis and Selective Anticancer Activity Evaluation of 2-phenylacrylonitrile Derivatives as Tubulin Inhibitors.

OBJECTIVE: This study aimed at synthesizing 13 series of novel derivatives with 2-phenylacrylonitrile, evaluating antitumor activity both in vivo and in vitro, and obtaining novel tubulin inhibitors. METHOD: The 13 series of 2-phenylacrylonitrile derivatives were synthesized by Knoevenagel condensation and the anti-proliferative activities were determined by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometer. Quantitative cell migration was performed using 24-well Boyden chambers. The proteins were detected by western blotting. in vitro kinetics of microtubule assembly was measured using ELISA kit for Human ß-tubulin (TUBB). Molecular docking was done by Discovery Studio (DS) 2017 Client online tool. RESULTS: Among the derivatives, compound 1g2a possessed strong inhibitory activity against HCT116 (IC50 = 5.9 nM) and BEL-7402 (IC50 = 7.8 nM) cells. Compound 1g2a exhibited better selective antiproliferative activities and specificities than all the positive control drugs, including taxol. Compound 1g2a inhibited proliferation of HCT116 and BEL-7402 cells by arresting them in the G2/M phase of the cell cycle, inhibited the migration of HCT116 and BEL-7402 cells and the formation of cell colonies. Compound 1g2a showed excellent tubulin polymerization inhibitory activity on HCT116 and BEL-7402 cells. The results of molecular docking analyses showed that 1g2a may inhibit tubulin to exert anticancer effects. CONCLUSION: Compound 1g2a shows outstanding antitumor activity both in vivo and in vitro and has the potential to be further developed into a highly effective antitumor agent with little toxicity to normal tissues.

High Engraftment and Metastatic Rates in Orthotopic Xenograft Models of Gastric Cancer via Direct Implantation of Tumor Cell Suspensions.

Although the implantation of intact tumor fragments is a common practice to generate orthotopic xenografts to study tumor invasion and metastasis, the direct implantation of tumor cell suspensions is necessary when prior manipulations of tumor cells are required. However, the establishment of orthotopic xenografts using tumor cell suspensions is not mature, and a comparative study directly comparing their engraftment and metastatic capabilities is lacking. It is unclear whether tumor fragments are superior to cell suspensions for successful engraftment and metastasis. In this study, we employed three GC cell lines with varying metastatic capacities to stably express firefly luciferase for monitoring tumor progression in real time. We successfully minimized the risk of cell leakage during the orthotopic injection of tumor cell suspensions without Corning Matrigel by systematically optimizing the surgical procedure, injection volume, and needle size options. Comparable high engraftment and metastatic rates between these two methods were demonstrated using MKN-45 cells with a strong metastatic ability. Importantly, our approach can adjust the rate of tumor progression flexibly and cuts the experimental timeline from 10-12 weeks (for tumor fragments) to 4-5 weeks. Collectively, we provided a highly reproducible procedure with a shortened experimental timeline and low cost for establishing orthotopic GC xenografts via the direct implantation of tumor cell suspensions.