Lung Metastasis Mouse Model Research Articles

1676P Active immunization with a multi-peptide B cell vaccine, targeting trastuzumab and pertuzumab binding sites, prevents the formation of HER-2/neu expressing lung metastases

The passive application of Trastuzumab and Pertuzumab has demonstrated remarkable success in the clinical outcome of patients with Her-2/neu positive metastatic breast cancer. However, active immunization with mimotope/B cell epitope-based vaccines can induce the production of the corresponding mAbs by the patient’s immune system. We have developed a B cell peptide-based Her-2/neu vaccine (HerVaxx) comprising Trastuzumab’s binding site. In clinical settings, the vaccine has been shown to reduce primary tumor growth by inducing polyclonal anti-tumor immune responses and immunological memory. The vaccine was also shown to result in loss of Her-2/neu expression, a phenomenon similarly observed in the clinic after treatment with Trastuzumab. Applying a Her-2/neu lung metastasis mouse model, here we tested a multi-peptide B cell vaccine containing HerVaxx and a mimotope of Pertuzumab for the prevention of lung metastases formation and investigated the loss of Her-2/neu expression. Mice were vaccinated with the multi-peptide vaccine, tail-vein injected with mammary carcinoma cells expressing human Her-2/neu, and histological evaluations of the mice lungs were carried out. A significantly reduced lung metastasis formation by active immunization with either the mimotope of Pertuzumab, HerVaxx, or the combination of both, was observed and associated with decreased lung weights and increased CD4+ and CD8+ T cells infiltration. Markedly, along with the overall reduction of lung weights and Her-2 positive metastases, the formation of Her-2/neu-negative tumors but with increased PD-L1 expression was observed. Our data might result in a multi-peptide B cell Her-2/neu vaccine serving as a secondary intervention in adjuvant settings to prevent tumor recurrence and spread. Furthermore, the loss of Her-2/neu expression suggests a combination therapy involving an anti-PD-L1 immune checkpoint inhibitor or an active immunization with a mimotope from PD-L1. Such combination therapy to alternately target Her-2/neu and PD-L1 might be adapted to the stage and progression phase of the disease and result in the remission of the metastases.

Aurantii Fructus Immaturus enhances natural killer cytolytic activity and anticancer efficacy in vitro and in vivo.

Aurantii Fructus Immaturus (AFI), extensively used in traditional herbal medicine, is known to have diverse physiological effects against various diseases, including obesity, diabetes, and cardiovascular disease. However, the effects of AFI on the immune system, especially natural killer (NK) cells, remain largely unknown. We aimed to investigate the effect of AFI on NK cell activity in vitro and in vivo and to elucidate the underlying mechanisms. Further, we verified the anticancer efficacy of AFI in a mouse lung metastasis model, underscoring the therapeutic potential of AFI in cancer therapy. Our results revealed that AFI significantly enhanced the cytolytic activity of NK cells in a dose-dependent manner, accompanied by an increase in the expression of NK cell-activating receptors, especially NKp30 and NKp46. AFI treatment also increased the expression of cytolytic granules, including granzyme B and perforin. Furthermore, the expression of CD107a, a degranulation marker, was increased upon treatment with AFI. A signaling study using western blot analysis demonstrated that the phosphorylation of extracellular signal-regulated kinase (ERK) was involved in increasing the NK cell activity following AFI treatment. In the in vivo study performed in mice, oral administration of AFI markedly enhanced the cytotoxic activity of spleen mononuclear cells against YAC-1 cells, which was accompanied by NKp46 upregulation. In addition, we confirmed that cancer metastasis was inhibited in a mouse cancer metastasis model, established using the mouse melanoma B16F10 cell line, by the administration of AFI in vivo. Collectively, these results indicate that AFI enhances NK cell-mediated cytotoxicity in vitro and in vivo via activation of the ERK signaling pathway and suggest that AFI could be a potential supplement for cancer immunotherapy.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

Transmembrane Protein 100 Inhibits the Progression of Colorectal Cancer by Promoting the Ubiquitin/Proteasome Degradation of HIF-1α.

Transmembrane protein 100 (TMEM100) is involved in embryonic cardiovascular system development. However, the biological role of TMEM100 in human cancers, particularly colorectal cancer (CRC), is unclear. In this study, tissue microarrays were stained using immunohistochemistry methods to evaluate the association between TMEM100 levels and clinic-pathological features for CRC. Kaplan–Meier and log-rank tests revealed that decreased levels of TMEM100 correlated with shorter overall survival. Cox regression revealed that reduced levels of TMEM100 was an independent prognostic factor for detrimental survival in CRC. A lentiviral vector was used to overexpress TMEM100 in HCT116 cells, and small interfering RNA was used to knockdown TMEM100 in SW480 cells. The CCK-8 assay, colony formation analysis, cell cycle analysis, cell migration assay, mouse xenograft model and mouse lung metastasis model showed that TMEM100 suppressed CRC cell proliferation and migration in vitro and in vivo. IHC scores of TMEM100 and HIF-1α were significantly negatively correlated. A half-time determination analysis in which cells were treated with cycloheximide revealed that TMEM100 shortened the HIF-1α half-life. Further immunoprecipitation experimental results showed that TMEM100 promoted the ubiquitination of HIF-1α, which caused HIF-1α degradation via the 26S proteasome pathway. Angiogenesis assay and migration assay results revealed that TMEM100 suppressed the migration and angiogenesis induction capacities of HCT116 cells, but this inhibitory effect was abolished when HIF-1α degradation was blocked by MG132 treatment. These results indicated that TMEM100 inhibited the migration and the angiogenesis induction capacities of CRC cells by enhancing HIF-1α degradation via ubiquitination/proteasome pathway.

A novel lncRNA RP11-386G11.10 reprograms lipid metabolism to promote hepatocellular carcinoma progression

ObjectiveEmerging studies suggest that long non-coding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). A rapidly increasing number of studies have shown that metabolic changes including lipid metabolic reprogramming play a significant role in the progression of HCC. But it remains to be elucidated how lncRNAs affect tumor cell metabolism. MethodsThrough analysis and screening of The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset, we found a novel lncRNA RP11-386G11.10 was overexpressed, related to prognosis, conserved and non-protein-coding in HCC and related to poor prognosis. Then, CCK-8, colony formation, Transwell invasion, wound healing assays were performed and nude mouse subcutaneous tumour formation and lung metastasis models were established to explore the effect of RP11-386G11.10 on HCC tumour growth and metastasis. Chromatography-mass spectrometry (GC-MS) and Nile red staining detected the effect of RP11-386G11.10 on lipid metabolism in HCC. Mechanistically, we clarified the RP11-386G11.10/miR-345-3p/HNRNPU signalling pathway through dual luciferase reporter, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assays and identified ZBTB7A as a transcription factor of RP11-386G11.10. ResultsRP11-386G11.10 was overexpressed in HCC and positively correlated with tumour size, TNM stage, and poor prognosis in HCC patients. RP11-386G11.10 promoted the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, RP11-386G11.10 acted as a competing endogenous RNA (ceRNA) for miR-345-3p to regulate the expression of HNRNPU and its downstream lipogenic enzymes, leading to lipid accumulation in HCC cells and promoting their growth and metastasis. In addition, we identified ZBTB7A as a transcription factor of RP11-386G11.10. Moreover, HNRNPU promoted the expression of ZBTB7A in HCC cells, thereby increasing the transcriptional activity of RP11-386G11.10, and forming a positive feedback loop, ultimately leading continuous lipid accumulation, growth and metastasis in HCC cells. ConclusionsOur results indicated that the lncRNA RP11-386G11.10 was a novel oncogenic lncRNA that was strongly correlated with the poor prognosis of HCC. The ZBTB7A-RP11-386G11.10-HNRNPU positive feedback loop promoted the progression of HCC by regulating lipid anabolism. RP11-386G11.10 may become a new diagnostic and prognostic biomarker and therapy target for HCC.

Cervical Cancer Cells-Derived Extracellular Vesicles Containing microRNA-146a-5p Affect Actin Dynamics to Promote Cervical Cancer Metastasis by Activating the Hippo-YAP Signaling Pathway via WWC2.

Application of extracellular vesicles (EVs) for cancer treatment has been well-documented. We probed into the potential role of cervical cancer cells-secreted EVs by transferring miR-146a-5p in cervical cancer. After characterization of miR-146a-5p expression in clinical cervical cancer tissue samples, gain- and loss-of-function experiments were implemented to test the effect of miR-146a-5p on the invasion, epithelial-mesenchymal transition (EMT), and anoikis in cervical cancer cells. EVs were isolated from high-metastatic cervical cancer cells, after which their effects on the malignant behaviors of low-metastatic cervical cancer cells were assessed in a co-culture system. Luciferase assay was implemented to validate the putative binding relationship between miR-146a-5p and WWC2, followed by further investigation of downstream pathway (Hippo-YAP). Finally, nude mouse lung metastasis model was developed for in vivo validation. miR-146a-5p was elevated in cervical cancer tissues and high miR-146a-5p expression promoted the metastatic potential of cervical cancer cells through enhancing their invasiveness and anoikis resistance, and inducing EMT. Furthermore, miR-146a-5p carried by EVs secreted by highly metastatic cervical cancer cells could promote the metastasis of low-metastatic cervical cancer cells. Mechanistically, miR-146a-5p targeted WWC2 to activate YAP, by which it inhibited the phosphorylation of cofilin, and promoted the process of cofilin-mediated depolymerization of F-actin to G-actin. In vivo data demonstrated that EVs-carried miR-146a-5p promoted tumor metastasis through the WWC2/YAP axis. Cancer-derived EVs delivered pro-metastatic miR-146a-5p to regulate the actin dynamics in cervical cancer, thereby leading to cancer metastasis. This experiment highlighted an appealing therapeutic modality for cervical cancer.

Anti-Angiogenetic and Anti-Lymphangiogenic Effects of a Novel 2-Aminobenzimidazole Derivative, MFB.

Background and PurposeBenzimidazoles have attracted much attention over the last few decades due to their broad-spectrum pharmacological properties. Increasing evidence is showing the potential use of benzimidazoles as anti-angiogenic agents, although the mechanisms that impact angiogenesis remain to be fully defined. In this study, we aim to investigate the anti-angiogenic mechanisms of MFB, a novel 2-aminobenzimidazole derivative, to develop a novel angiogenesis inhibitor.Experimental ApproachMTT, BrdU, migration and invasion assays, and immunoblotting were employed to examine MFB’s effects on vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation, migration, invasion, as well as signaling molecules activation. The anti-angiogenic effects of MFB were analyzed by tube formation, aorta ring sprouting, and matrigel plug assays. We also used a mouse model of lung metastasis to determine the MFB’s anti-metastatic effects.Key ResultsMFB suppressed cell proliferation, migration, invasion, and endothelial tube formation of VEGF-A-stimulated human umbilical vascular endothelial cells (HUVECs) or VEGF-C-stimulated lymphatic endothelial cells (LECs). MFB suppressed VEGF-A and VEGF-C signaling in HUVECs or LECs. In addition, MFB reduced VEGF-A- or tumor cells-induced neovascularization in vivo. MFB also diminished B16F10 melanoma lung metastasis. The molecular docking results further showed that MFB may bind to VEGFR-2 rather than VEGF-A with high affinity.Conclusions and ImplicationsThese observations indicated that MFB may target VEGF/VEGFR signaling to suppress angiogenesis and lymphangiogenesis. It also supports the role of MFB as a potential lead in developing novel agents for the treatment of angiogenesis- or lymphangiogenesis-associated diseases and cancer.

Ginsenoside Rh3 Inhibits Lung Cancer Metastasis by Targeting Extracellular Signal-Regulated Kinase: A Network Pharmacology Study.

Lung cancer has a high mortality rate and is very common. One of the main reasons for the poor prognosis of patients with lung cancer is the high incidence of metastasis. Ginsenoside Rh3, a rare ginsenoside extracted from Panax notoginseng, exhibits excellent anti-inflammatory and anti-tumor effects. Nonetheless, the inhibitory potential of Rh3 against lung cancer remains unknown. The target genes of Rh3 were screened by the PharmMapper database; the proliferation of lung cancer cells was detected by MTT assay; the migration and invasion of cells were detected by the Transwell method; and the expression of extracellular signal-regulated kinase (ERK) and EMT-related proteins in vivo and in vitro were detected by Western blotting. In addition, we established a lung metastasis model in nude mice using A549 cells to assess the effect of Rh3 on NSCLC tumor metastasis in vivo. Our findings suggest that Rh3 significantly inhibited lung cancer metastasis both in vivo and in vitro. It was determined by flow cytometry analysis that Rh3 notably inhibited cell proliferation by blocking the G1 phase. In addition, Rh3 inhibited metastasis in lung cancer cells and regulated the expression of metastasis-related proteins under hypoxia. Mechanistic studies suggested that Rh3 targeted ERK to inhibit lung cancer metastasis. The ERK inhibitor U0126 or siRNA-mediated knockdown of ERK had an enhanced effect on Rh3’s ability to inhibit lung cancer metastasis. The studies revealed that the inhibitory effect of Rh3 on the metastatic ability of lung cancer cells may be supported by ERK-related signaling pathways.

Abstract 847: Role of RHOA in diffuse gastric cancer tumors

Abstract Gastric cancer constitutes the third leading cause of cancer mortality worldwide and tumors with diffuse histology have poor prognosis and low response rates to therapy. Missense mutations in the RHOA (Ras homolog family member A) GTPase have recently been identified in 24% of patients with diffuse gastric cancer. RHOA mutations are not randomly distributed along the coding sequence, but enriched in specific and recurrent hotspots. Substitution in codon 42 (Y42C) occurs in approximately 50% of the patients in which RHOA is mutated. RHOA orchestrates cellular processes such as proliferation, differentiation, migration and invasion, whose deregulation is essential for the onset, maintenance and progression of tumors. However, the functional role of wild type RHOA and RHOA mutants remains poorly characterized in gastric cancer. We used isogenic cell lines systems and animal models to study the impact of RHOA in the gastric tumorigenesis. RHOA loss strongly increased the proliferation, migration and invasion of diffuse gastric cancer cells in vitro, but also when growing as subcutaneous xenografts in immunodeficient mice, and in experimental mouse models of peritoneal and lung metastasis. Stomach tumor burden was also increased in transgenic mice conditionally expressing a dominant negative form of RHOA in the gastrointestinal tract (RHOA T19N). Moreover, cells overexpressing wild-type RHOA or G14V RHOA (a constitutive active form of the GTPase) exhibited reduced tumorigenic features both in vitro and in vivo. Conversely, diffuse gastric cancer cells engineered for overexpressing RHOA Y42C mutant, displayed enhanced growth and invasion in vitro. Importantly, stomach cancer tumors formed more efficiently in a transgenic mouse model in which we targeted the expression of Y42C RHOA mutant to the gastric epithelium. Collectively, these results demonstrate that RHOA has tumor-suppressive activity in diffuse gastric cancer, while the recurrent Y42C hotspot RHOA mutation found in these tumors is oncogenic. Citation Format: Agueda Martinez-Barriocanal, Higinio Dopeso, Lizbeth M. Jiménez-Flores, Juliana C. Santos, Estefania Anguita, Josipa Bilic, Rocio Nieto, Elia García-Vidal, Manuel Sánchez Martín, Stefania Landolfi, Kazuto Kobayashi, Javier Hernandez-Losa, Simo Schwartz, Santiago Ramón y Cajal, Diego Arango. Role of RHOA in diffuse gastric cancer tumors [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 847.

IFI35 Promotes Renal Cancer Progression by Inhibiting pSTAT1/pSTAT6-Dependent Autophagy.

Simple SummaryInterferon-induced protein 35 (IFI35) plays an important role in virus-related immune inflammatory responses. However, the biological significance and function of IFI35 in cancer is still not well understood. Our goal is to explore whether IFI35 could be used as tumor marker or a therapeutic target for renal cell cancer (RCC). Our results showed that IFI35 expression was significantly increased in 200 RCC specimens, and its expression was negatively associated with poor overall or disease-specific 5-year patients’ survival. Next, we explored the mechanism and function of IFI35 in RCC. The results demonstrated that knockdown of IFI35 could inhibit the proliferation and invasion of renal cancer cells by enhancing the STAT1/STAT6 phosphorylation (pSTAT1/pSTAT6)-dependent autophagy. Likewise, the knockdown of IFI35 also suppressed the tumor growth or lung metastasis of renal cancer by enhancing the induction of autophagy. This study aims to provide a theoretical basis for IFI35 as a potential diagnosis and therapeutic target for RCC or other cancers with high levels of IFI35.Interferon-induced protein 35 (IFI35), is currently acknowledged to govern the virus-related immune inflammatory responses. However, the biological significance and function of IFI35 in renal cell cancer (RCC) is still not well understood. Here, IFI35 expression and function were investigated in RCC tissues, renal cancer cells, and animal models. The results showed that IFI35 expression was significantly increased in 200 specimens of RCC patients. We found that higher IFI35 levels were significantly correlated with poor RCC prognosis. In human cell lines, the knockdown of IFI35 suppressed the malignant behavior of renal cancer cells. Similarly, the IFI35 knockdown resulted in significant inhibition of tumor progression in the subcutaneous or lung metastasis mouse model. Furthermore, the knockdown of IFI35 promoted the induction of autophagy by enhancing the autophagy-related gene expression (LC3-II, Beclin-1, and ATG-5). Additionally, blockade of STAT1/STAT6 phosphorylation (pSTAT1/pSTAT6) abrogated the induced autophagy by IFI35 knockdown in renal cancer cells. The autophagy inhibitor 3-MA also abolished the prevention of tumor growth by deleting IFI35 in renal cancer models. The above results suggest that the knockdown of IFI35 suppressed tumor progression of renal cancer by pSTAT1/pSTAT6-dependent autophagy. Our research revealed that IFI35 may serve as a potential diagnosis and therapeutic target for RCC.

Liposomal β-Sitosterol Suppresses Metastasis of CT26/luc Colon Carcinoma via Inhibition of MMP-9 and Evoke of Immune System

β-sitosterol (SITO) has been reported with anticancer effects; however, with poor bioavailability. The current study aimed to investigate whether liposomal encapsulated β-sitosterol (LS) has a better inhibition effect on tumor metastasis than β-sitosterol in a CT26/luc lung metastasis mouse model and the possible underlying mechanism. LS was liposomal-encapsulated SITO and was delivered to mice by oral gavage. The cell viability was determined by the MTT assay, and invasiveness of the tumor cells and related protein expression were evaluated with the invasion assay and Western blotting. For therapeutic efficacy evaluation, male BALB/c mice were treated with PBS, SITO, and LS once a day for 7 days prior to intravenous injections of CT26/luc cells; treatments were continued twice a week post-cell inoculation throughout the entire experiment. Tumor growth inhibition was monitored by bioluminescent imaging (BLI). IL-12, IL-18, and IFN-γ in the intestinal epithelium were determined by ELISA. The results show that LS treatment had a better invasion inhibition with lower cytotoxicity than SITO when the same dose was utilized. Notably, mice treated with LS significantly exhibited fewer metastases to the lungs and other tissues/organs compared with the Control and SITO groups. Additionally, the IL-12, IL-18, and IFN-γ levels were significantly increased in the LS-treated mice compared with the Control and SITO groups. The underlying mechanism may be through the inhibition of MMP-9 and elicitation of the antitumoral Th1 immune response, such as increasing CD4+ and CD8+ T cells, IL-12, IL-18, and IFN-γ.

Reactive myelopoiesis and FX-expressing monocyte-derived macrophages triggered by chemotherapy promotes cancer lung metastasis

Abstract Lung metastasis-associated macrophages (MAMs) play a very important role in tumor metastasis. In mouse models of lung metastasis, interstitial macrophages (IMs) markedly accumulate and differentiate into MAMs. Several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis. In this study, we hypothesize that host responses to chemotherapy treatment enhance breast cancer metastasis by modulation of lung macrophage toward a pro-metastatic phenotype. Here, we show that multi-dose chemo treatment promotes breast cancer lung metastasis in the spontaneous breast cancer animal model. Pretreatment with Gemcitabine and Paclitaxel induces a significantly higher number of pulmonary metastases in the experimental metastasis model. Mechanistically, multi-dose of chemotherapy induces reactive myelopoiesis characterized by increase of BM LSK cells and multipotent progenitors in tumor-free mice, which is associated with the increase of mitochondrial ROS. Further, chemokine CCL2 expression is significantly increased in the plasma and lung tissues of chemo-treated tumor-free mice. Consequently, the percentage of BM Ly6C+ monocytes are significantly increased because of increased progenitor monocyte differentiation potential and monocyte proliferation, which results in an increase of lung monocytes and IMs with enhanced expression of coagulation factor X (FX). Inhibition of activated FX (FXa) can inhibit chemotherapy-induced lung metastasis. Together, these studies suggest a novel mechanism for chemotherapy induced metastasis via the modulation of BM-derived monocytes/macrophages and extravascular clotting in lung tissues. Supported by grants from NIH (P20GM135004)

Cordycepin Suppresses The Malignant Phenotypes of Colon Cancer Cells through The GSK3ß/ß-catenin/cyclin D1 Signaling Pathway.

Objective Cordycepin, also known as 3ˊ-deoxyadenosine, is the main bioactive ingredient of Cordyceps militaris and possesses various pharmacological effects. This study was performed to investigate the role of cordycepin in regulating the biological behaviors of colon cancer cells and the potential mechanism behind it. Materials and Methods In this experimental study, after treatment of colon cancer cells with different concentrations of cordycepin, inhibition of proliferation was detected by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Colon cancer cell migration and invasion abilities were analyzed by wound healing and Transwell assays. Flow cytometry was performed to detect cell apoptosis. A lung metastasis model in nude mice was utilized to examine the effect of cordycepin on the metastasis of colon cancer cells in vivo. Western blot was used to quantify GSK3β, β-catenin and cyclin D1 expression levels. Results Cordycepin inhibited colon cancer cell proliferation, migration and invasion, induced apoptosis in vitro, and inhibited lung metastasis of colon cancer cells in vivo. GSK-3β inhibitor (CHIR99021) treatment abolished the effects of cordycepin on cell viability, migration, invasion and apoptosis. Additionally, cordycepin promoted the expressions of GSK3β, and inhibited β-catenin and cyclin D1 in colon cancer cells, while co-treatment with CHIR99021 reversed the above effects. ConclusionCordycepin suppresses the malignant phenotypes of colon cancer through the GSK3β/β-catenin/cyclin D1 signaling pathway.

MicroRNA-15b-5p encapsulated by M2 macrophage-derived extracellular vesicles promotes gastric cancer metastasis by targeting BRMS1 and suppressing DAPK1 transcription

BackgroundExtracellular vesicles (EVs) derived from tumor-associated macrophages are implicated in the progression and metastasis of gastric cancer (GC) via the transfer of molecular cargo RNAs. We aimed to decipher the impact of microRNA (miR)-15b-5p transferred by M2 macrophage-derived EVs in the metastasis of GC.MethodsExpression of miR-15b-5p was assessed and the downstream genes of miR-15b-5p were analyzed. GC cells were subjected to gain- and loss-of function experiments for miR-15b-5p, BRMS1, and DAPK1. M2 macrophage-derived EVs were extracted, identified, and subjected to co-culture with GC cells and their biological behaviors were analyzed. A lung metastasis model in nude mice was established to determine the effects of miR-15b-5p on tumor metastasis in vivo.ResultsmiR-15b-5p was upregulated in GC tissues and cells as well as in M2 macrophage-derived EVs. miR-15b-5p promoted the proliferative and invasive potentials, and epithelial-mesenchymal transition (EMT) of GC cells. M2 macrophage-derived EVs could transfer miR-15b-5p into GC cells where it targeted BRMS1 by binding to its 3’UTR. BRMS1 was enriched in the DAPK1 promoter region and promoted its transcription, thereby arresting the proliferative and invasive potentials, and EMT of GC cells. In vivo experiments demonstrated that orthotopic implantation of miR-15b-5p overexpressing GC cells in nude mice displayed led to enhanced tumor metastasis by inhibiting the BRMS1/DAPK1 axis.ConclusionsOverall, miR-15b-5p delivered by M2 macrophage-derived EVs constitutes a molecular mechanism implicated in the metastasis of GC, and may thus be considered as a novel therapeutic target for its treatment.

Dual-aptamer-engineered M1 macrophage with enhanced specific targeting and checkpoint blocking for solid-tumor immunotherapy

Chimeric antigen receptor T (CAR-T) cell therapy has faced a series of challenges and has shown very little efficacy in solid tumors to date. Although genetically engineered macrophages have achieved definite therapeutic effect in solid tumors, heterogeneous expression of engineered proteins and the potential for toxicity limit further applications. Herein, we propose a nongenetic and simple macrophage cell engineering strategy through glycan metabolic labeling and click reaction for the treatment of solid tumors. The aptamer-engineered M1 macrophage (ApEn-M1) showed enhanced active targeting ability for tumor cells invitro and invivo, resulting in significant cytotoxicity effects. Moreover, ApEn-M1 exhibited superior antitumor efficacy in a breast cancer xenograft mouse model and a lung metastasis mouse model of breast cancer. Interestingly, the ApEn-M1 could reprogram the immunity microenvironment by increasing Tcell infiltration and enhancing Tcell activity in the tumor region. Additionally, the administration of ApEn-M1 showed no obvious systemic side effects. With glycan metabolic labeling, the macrophages could be efficiently labeled with aptamers on the cell surface via click reaction without genetic alteration or cell damage. Hence, this study serves as a proof of concept for cell-surface anchor engineering and expands the range of nongenetic macrophage cell engineering strategies.

Crumbs3 is expressed in oral squamous cell carcinomas and promotes cell migration and proliferation by affecting RhoA activity.

Despite the recent progression of treatments, the 5-year survival rate of patients with oral squamous cell carcinoma (OSCC) is still poor. One of the most critical factors affecting prognosis is tumor metastasis. Developing novel molecular targeted therapies by analyzing the molecular pathway of OSCC metastasis is an urgent issue. The present study aimed to characterize the expression and function of crumbs3 (Crb3) in OSCC cell migration. Immunohistochemistry and immunoblotting revealed that Crb3 was expressed in tissues from patients with OSCC and OSCC cell lines. The motility of OSCC cell lines was decreased by knockdown of Crb3 without affecting proliferation. However, Crb3-knockout (KO) clones exhibited decreases in both cell migration and proliferation. The expression of epithelial-mesenchymal transition markers was not altered in Crb3-KO clones compared with parent cells. A xenograft mouse model of lung metastasis revealed that the metastatic potential of Crb3-KO clones was reduced. As seen with Crb3-KO clones, the motility of OSCC cells was decreased by treatment with inhibitors of RhoA activation. Serum-induced activation of RhoA in OSCC cells was evaluated by comparing the amount of GTP-bound RhoA using affinity matrices, revealing that RhoA activation was decreased in Crb3-KO clones. To the best of our knowledge, the present study was the first to demonstrate that Crb3 was expressed in squamous cell carcinoma tissues and promoted cell migration and proliferation, which was associated with RhoA activation in OSCC cells.

Early diagnosis of breast cancer lung metastasis by nanoprobe-based luminescence imaging of the pre-metastatic niche

BackgroundEarly detection of breast cancer lung metastasis remains highly challenging, due to few metastatic cancer cells at an early stage. Herein we propose a new strategy for early diagnosis of lung metastasis of breast cancer by luminescence imaging of pulmonary neutrophil infiltration via self-illuminating nanoprobes.MethodsLuminescent nanoparticles (LAD NPs) were engineered using a biocompatible, neutrophil-responsive self-illuminating cyclodextrin material and an aggregation-induced emission agent. The chemiluminescence resonance energy transfer (CRET) effect and luminescence properties of LAD NPs were fully characterized. Using mouse peritoneal neutrophils, in vitro luminescence properties of LAD NPs were thoroughly examined. In vivo luminescence imaging and correlation analyses were performed in mice inoculated with 4T1 cancer cells. Moreover, an active targeting nanoprobe was developed by surface decoration of LAD NPs with a neutrophil-targeting peptide, which was also systemically evaluated by in vitro and in vivo studies.ResultsLAD NPs can generate long-wavelength and persistent luminescence due to the CRET effect. In a mouse model of 4T1 breast cancer lung metastasis, we found desirable correlation between neutrophils and tumor cells in the lungs, demonstrating the effectiveness of early imaging of the pre-metastatic niche by the newly developed LAD NPs. The active targeting nanoprobe showed further enhanced luminescence imaging capability for early detection of pulmonary metastasis. Notably, the targeting nanoprobe-based luminescence imaging strategy remarkably outperformed PET/CT imaging modalities in the examined mouse model. Also, preliminary tests demonstrated good safety of LAD NPs.ConclusionsThe neutrophil-targeting imaging strategy based on newly developed luminescence nanoparticles can serve as a promising modality for early diagnosis of lung metastasis of breast cancers.Graphical

Exosomal MMP-1 transfers metastasis potential in triple-negative breast cancer through PAR1-mediated EMT.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with high risk of distant metastasis, in which the intercellular communication between tumor cells also plays a role. Exosomes can be released by tumor cells and promote distant metastasis through intercellular communication or changes in tumor microenvironment, it is an optimized transportation facility for biologically active payloads. This was a hypothesis-generating research on role of exosomal payload in TNBC distant metastasis. Exosomes isolated from supernatant of MDA-MB-231 and MDA-MB-231-HM (a highly pulmonary metastatic variant of parental MDA-MB-231 cells) were characterized. MMP-1 level was detected using mass spectrometry and western blot. Transwell assay, wound healing and CCK-8 assay were employed to explore the effect of exosomal MMP-1 on the metastatic capability of TNBC cells in vitro. Human breast cancer lung metastasis model in nude mice was established to observe the effect of exosomal MMP-1 in vivo. Tissue microarray and blood samples of TNBC patients were applied to analyze the relevance between MMP-1 with metastasis. MDA-MB-231-HM cells secrete exosomes enriched MMP-1, which can be taken up and enhance invasion and migration activities of TNBC cells, including MDA-MB-231, MDA-MB-468 and BT549. After ingesting exosomes enriched with MMP-1, cells secret more MMP-1, which may interact with membrane G protein receptor protease activated receptor 1 (PAR1), thereby initiating epithelial-mesenchymal transition (EMT) to enhance capability of migration and invasion. The lung colonization model shows that the expressions of MMP-1 and PAR1 in the metastases of the 231-HM-exo treated mice were both upregulated. Clinically, the enrichment of MMP-1 can be detected in exosomes extracted from serum of patients with metastasis at higher concentration than that in pre-operative patients. Moreover, in patients with multiple distant metastases, the level of MMP-1 in exosomes is also higher than that in patients with single lesion. MMP-1 from TNBC cells of high metastasis potential can promote the distant metastasis of transform those with low metastasis potential through PAR1-mediated EMT and is likely to be a potential molecular marker.

Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2.

PurposeSmurf2, one of C2-WW-HECT domain E3 ubiquitin ligases, is closely related to the development and progression in different cancer types, including hepatocellular carcinoma (HCC). This study aims to illustrate the expression and molecular mechanism of Smurf2 in regulating the progression of HCC.MethodsThe expression of Smurf2 in human HCC and adjacent non-tumor liver specimens was detected using tissue microarray studies from 220 HCC patients who underwent curative resection. The relationships of Smurf2 and HCC progression and survival were analyzed using the chi-square test, Kaplan–Meier analysis, and Cox proportional hazards model. For Smurf2 was low expression in HCC cell lines, Smurf2 overexpression cell lines were established. The effect of Smurf2 on cell proliferation and migration was detected by Cell Counting Kit-8 and colony formation assay, and the epithelial–mesenchymal transition (EMT) markers and its transcription factors were tested by immunoblotting. The interaction and ubiquitination of Smad2 by Smurf2 were detected by co-immunoprecipitation and immunoprecipitation assay. Finally, the effect of Smurf2 on HCC was verified using the mouse lung metastasis model.ResultsSmurf2 was downregulated in HCC tissues compared to that of corresponding non-tumor liver specimens. The low expression of Smurf2 in HCC was significantly associated with macrovascular or microvascular tumor thrombus and the impairment of overall survival and disease-free survival. In vitro and in vivo analysis showed that Smurf2 overexpression decreased the EMT potential of HCC cells by promoting the ubiquitination of Smad2 via the proteasome-dependent degradation pathway.ConclusionThe expression of Smurf2 was downregulated in HCC specimens and affected the survival of patients. Smurf2 inhibited the EMT of HCC by enhancing Smad2 ubiquitin-dependent proteasome degradation.