Number Of Lung Metastatic Nodules Research Articles

PAD4 Inhibitor-Functionalized Layered Double Hydroxide Nanosheets for Synergistic Sonodynamic Therapy/Immunotherapy Of Tumor Metastasis.

Sonodynamic therapy (SDT) is demonstrated to trigger the systemic immune response of the organism and facilitate the treatment of metastatic tumors. However, SDT-mediated neutrophil extracellular traps (NETs) formation can promote tumor cell spread, thus weakening the therapeutic effectiveness of metastatic tumors. Herein, the amorphous CoW-layered double hydroxide (a-CoW-LDH) nanosheets are functionalized with a peptidyl arginine deiminase 4 (PAD4) inhibitor, i.e., YW3-56, to construct a multifunctional nanoagent (a-LDH@356) for synergistic SDT/immunotherapy. Specifically, a-CoW-LDH nanosheets can act as a sonosensitizer to generate abundant reactive oxygen species (ROS) under US irradiation. After loading with YW3-56, a-LDH@356 plus US irradiation not only effectively induces ROS generation and immunogenic cell death, but also inhibits the elevation of citrullinated histone H3 (H3cit) and the release of NETs, enabling a synergistic enhancement of anti-tumor metastasis effect. Using 4T1 tumor model, it is demonstrated that combining a-CoW-LDH with YW3-56 stimulates an anti-tumor response by upregulating the proportion of immune-activated cells and inducing polarization of M1 macrophages, and inhibits immune escape by downregulating the expression of PD-1 on immune cells under US irradiation, which not only arrests primary tumor progression with a tumor inhibition rate of 69.5% but also prevents tumor metastasis with the least number of lung metastatic nodules.

Kinesin Family Member-18A (KIF18A) Promotes Cell Proliferation and Metastasis in Hepatocellular Carcinoma

Background & AimsKinesin family member 18A (KIF18A) is notable for its aberrant expression across various cancer types and its pivotal role is driving cancer progression. In this study, we aim to investigate the intricate molecular mechanisms underlying the impact of KIF18A on the progression of HCC.MethodsWestern blotting assays, a quantitative real-time PCR and immunohistochemical analyses were performed to quantitatively assess KIF18A expression in HCC tissues. We then performed genetic manipulations within HCC cells by silencing endogenous KIF18A using short hairpin RNA (shRNA) and introducing exogenous plasmids to overexpress KIF18A. We monitored cell progression, analyzed cell cycle and cell apoptosis and assessed cell migration and invasion both in vitro and in vivo. Moreover, we conducted RNA-sequencing to explore KIF18A-related signaling pathways utilizing Reactome and KEGG enrichment methods and validated these critical mediators in these pathways.ResultsAnalysis of the TCGA-LIHC database revealed pronounced overexpression of KIF18A in HCC tissues, the finding was subsequently confirmed through the analysis of clinical samples obtained from HCC patients. Notably, silencing KIF18A in cells led to an obvious inhibition of cell proliferation, migration and invasion in vitro. Furthermore, in subcutaneous and orthotopic xenograft models, suppression of KIF18A sgnificantly redudce tumor weight and the number of lung metastatic nodules. Mechanistically, KIF18A appears to facilitate cell proliferation by upregulating MAD2 and CDK1/CyclinB1 expression levels, with the activation of SMAD2/3 signaling contributing to KIF18A-driven metastasis.ConclusionOur study elucidates the molecular mechanism by which KIF18A mediates proliferation and metastasis in HCC cells, offering new insights into potential therapeutic targets.

Natural polyphenol-loaded cross-linked lipoic acid vesicles treat triple-negative breast cancer by cancer cell killing and metastasis inhibition

The high incidence of metastases remains a major hurdle for triple negative breast cancer (TNBC) treatment. Herein, we developed a fairly safe anticancer nanodrug for TNBC treatment by loading Epigallocatechin-3-gallate (EGCG) in cross-linked lipoic acid vesicles (EGCG@cLAVs), which cannot only directly kill TNBC cells through inducing apoptosis, but also significantly suppress the TNBC metastasis by metalloproteinases (MMPs) inhibition, a key event in tumor extracellular matrix (ECM) remodeling. The cytotoxicity assay revealed that the ICEGCG50 of EGCG@cLAVs (85.1 μM) fell in the range of common cytotoxic drugs. The in vitro antimetastasis results disclosed that the EGCG@cLAVs effectively suppressed the migration and invasion of TNBC cells (4T1) with low wound healing rate and relative invasion rate of 19.10 ± 2.12% and 15.0 ± 1.63%, 4.0 and 6.7 times lower than that of control, respectively. The outcomes of animal models revealed that EGCG@cLAVs not only achieved the comparable antitumor effect to that of first-line chemotherapeutic drug DOX, but also reduced the number of lung metastatic nodules from 31.4 (DOX) to 1.4. This nanodrug provides a promising candidate for TNBC therapy.

Local radiotherapy for murine breast cancer increases risk of metastasis by promoting the recruitment of M-MDSCs in lung

BackgroundRadiotherapy is one of the effective methods for treatment of breast cancer; however, controversies still exist with respect to radiotherapy for patients with TNBC. Here, we intend to explore the mechanism by which local radiotherapy promotes the recruitment of M-MDSCs in the lung and increases the risk of lung metastasis in TNBC tumor-bearing mice.MethodsA single dose of 20 Gy X-ray was used to locally irradiate the primary tumor of 4T1 tumor-bearing mice. Tumor growth, the number of pulmonary metastatic nodules, and the frequency of MDSCs were monitored in the mice. Antibody microarray and ELISA methods were used to analyze the cytokines in exosomes released by irradiated (IR) or non-IR 4T1 cells. The effects of the exosomes on recruitment of MDSCs and colonization of 4T1 cells in the lung of normal BALB/c mice were observed with the methods of FCM and pathological section staining. T lymphocytes or 4T1 cells co-cultured with MDSCs were performed to demonstrate the inhibitory effect on T lymphocytes or accelerative migration effect on 4T1 cells. Finally, a series of in vitro experiments demonstrated how the exosomes promote the recruitment of M-MDSCs in lung of mice.ResultsEven though radiotherapy reduced the burden of primary tumors and larger lung metastatic nodules (≥ 0.4 mm2), the number of smaller metastases (< 0.4 mm2) significantly increased. Consistently, radiotherapy markedly potentiated M-MDSCs and decreased PMN-MDSCs recruitment to lung of tumor-bearing mice. Moreover, the frequency of M-MDSCs of lung was positively correlated with the number of lung metastatic nodules. Further, M-MDSCs markedly inhibited T cell function, while there was no difference between M-MDSCs and PMN-MDSCs in promoting 4T1 cell migration. X-ray irradiation promoted the release of G-CSF, GM-CSF and CXCl1-rich exosomes, and facilitated the migration of M-MDSCs and PMN-MDSCs into the lung through CXCL1/CXCR2 signaling. While irradiated mouse lung extracts or ir/4T1-exo treated macrophage culture medium showed obvious selective chemotaxis to M-MDSCs. Mechanistically, ir/4T1-exo induce macrophage to produce GM-CSF, which further promoted CCL2 release in an autocrine manner to recruit M-MDSCs via CCL2/CCR2 axis.ConclusionsOur work has identified an undesired effect of radiotherapy that may promote immunosuppressive premetastatic niches formation by recruiting M-MDSCs to lung. Further studies on radiotherapy combined CXCR2 or CCR2 signals inhibitors were necessary.

Abstract 1076: The peptide-drug conjugate TH1902 inhibits growth of subcutaneous melanoma xenografts and formation of lung metastases in a syngeneic mouse model

Abstract Syngeneic mouse models provide an effective approach for studying how cancer therapies perform in the presence of a functional immune system. Here TH1902, a peptide-drug conjugate (PDC) which consists of two molecules of docetaxel attached by a cleavable succinyl linker to a peptide (TH19P01) that targets the scavenger receptor sortilin (SORT1), was investigated using subcutaneous implantation of murine B16-F10 melanoma cells within the flanks of syngeneic C57BL/6 mice. Tumor growth was at most inhibited by half upon weekly administration of docetaxel compared to vehicle-treated mice, while administration of TH1902 was well tolerated and led to tumor regression. The B16-F10 SORT1-expressing melanoma cell line is also known to produce distant lung metastases when injected intravenously into the mice. Tail vein injection of these cells (1.25x105 cells per implantation) created a model system for evaluating TH1902’s effects on metastatic nodules formation. Each implantation produced 70 to 100 visible, black surface nodules on the lungs at 13 days following cancer cell implantation, appropriate for nodule quantification. No black nodules were detected elsewhere within the mice tissues other than those detected in the lungs. This model was next used to demonstrate that the formation of lung metastases could be sharply curtailed by bi-weekly intravenous administration of either 10 mg/kg docetaxel or 23 mg/kg TH1902 (equivalent to the docetaxel injection in terms of docetaxel content) during this period. Administration of docetaxel routinely decreased the number of metastatic lung nodules by about 50%, whereas administration of TH1902 decreased the number of nodules by more than 85%. Similar results were obtained whether the test articles were administered i) one time as a bolus at one hour prior to implantation of the cells, ii) as a bolus twice weekly beginning 3 days post-implantation, or iii) both prior to and following implantation as described. Although little weight loss was recorded over the 13-day treatment duration, all TH1902-treated mice showed better tolerability profile in terms of weight compared to docetaxel for all conditions tested. In a subsequent experiment using prolonged administration at the same dosage (up to 24 days of treatment), TH1902 was able to inhibit metastatic formation over a longer period compared to docetaxel. In fact, after six cycles of treatment, TH1902-treated mice significantly sustained its inhibitory effect on melanoma lungs metastasis formation (-77%) whereas docetaxel was unable to maintain its effect (-7%). Moreover, considerable weight loss was associated with docetaxel treatment over 24 days while TH1902 treatments resulted in no net change in mouse body weights. In this syngeneic model, TH1902 is more tolerated and effective than docetaxel at inhibiting both melanoma xenograft growth and metastatic formation. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Larocque, Alain Zgheib, Sophie Kozelko, Richard Béliveau, Christian Marsolais, Borhane Annabi. The peptide-drug conjugate TH1902 inhibits growth of subcutaneous melanoma xenografts and formation of lung metastases in a syngeneic mouse model [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 1076.

Inhibiting ATR Modulates Response to Radiation and Stimulates Immune Responses

Radiotherapy (RT) has been shown, albeit rarely, to stimulate anti-tumor immune responses. Recent evidence suggests this can, in part, be attributed to the accumulation of cytoplasmic DNA in the form of micronuclei (MN) following RT, which can stimulate IRF3 and NF-κB immune signaling that recruits various immune components to the tumor microenvironment. We hypothesized this effect is increased by combining RT with AZD6738, an inhibitor of ATR, which is a key protein in DNA repair and cell cycle regulation. Furthermore, we propose that the more clustered DNA damage caused by protons results in a higher level of MN than photons when these radiation types are combined with AZD6738.H1299 and PANC-1 cells were treated with 6 MV x-rays and 9.9 keV/mm (dose-weighted LET in water) protons alone or with AZD6738 (1 µM) to assess DNA repair, cell cycle distribution, MN and c-GAS positive MN (MN-cGAS+) 1 to 72 h after RT+AZD6738. We then irradiated a syngeneic 4T1 tumor model in BALB/c (hind leg tumors) with 6 MV x-rays to 3 × 6 Gy fractions with and without AZD6738 to assess tumor growth delay and the status of tumor infiltrating immune cells and immune cells in the draining lymph nodes (DLN).Protons+AZD6738 and photons+AZD6738 increased gH2AX foci, abrogated G2 arrest and amplified MN and MN-cGAS+ compared to photons or protons alone for both cell lines (H1299 and PANC-1). Compared to photons or AZD6738 alone, photons+AZD6738 delayed tumor growth, increased survival time and decreased the number of metastatic lung nodules. Photons+AZD6738 and photons alone increased T-regs and decreased T helper 1 cells in the tumor compared to vehicle and ATRi alone. Photons+AZD6738 increased and decreased CD8+ T cells in the tumor and DLN compared to the other treatments, respectively. Photons alone increased CD8+ PD-1+ T cells and photons+AZD6738 decreased that population back to baseline for both tumor and DLN. CD8+ GranzB+ T cells increased for photons+AZD6738 and photons alone in the tumor. Photons+AZD6738 and AZD6738 alone increased type 1 macrophages (M1) and decreased M2. Photons+AZD6738 increased the total population of dendritic cells (DCs) and non-significantly increased type 1 and 2 classical DCs compared to other treatments.Our data show that RT+AZD6738 initiates intracellular immune signaling, likely attributable to disruption of DNA repair and abrogation of cell cycle arrest. We also observed this effect was amplified with proton RT. Our findings of increased immune signaling were corroborated in vivo with the observation that RT+AZD6738 increased CD4+, CD8+ and macrophage populations within the tumor, however after an initial delay, tumors continued to progress. Additional investigation is warranted to determine the balance of regulatory elements such as T-regs, immune checkpoints, and antitumor immune signaling.

Evaluation of the potential benefits of alkaline drinking water on tumor development reveals vascular protective effects

IntroductionThe proposed benefits of alkaline water generally lack experimental supporting evidence.Material and methodsWe tested the effects of alkaline water on tumor development in C57BL/6 mice. To recapitulate early tumor development, mice were fed a high-fat diet and treated with DEN (50 µg/l) and TCPOBOP (0.5 µg/g) for 24 weeks.ResultsCell proliferation was significantly lower in animals receiving alkaline water, and VEGFR2 staining was higher in the vasculature, suggesting less advanced disease stages. B16-V5 cells were injected subcutaneously or through the tail vein to generate primary tumors or lung metastatic nodules, respectively. Subcutaneous tumors of mice exposed to alkaline water showed a lower proliferative index and vasculature with a better-preserved intima layer and structure. The number of lung metastatic nodules was lower in mice exposed to filtered water. The vascular effects were tested in a rat model of hypertension (spontaneously hypertensive rat – SHR). Following 12 weeks of alkaline water consumption, the aortic rings had an enhanced vasodilatory response to a nitric oxide donor (nitroprusside – NTP), and several inflammatory markers were reduced in blood and heart tissue.ConclusionsThese results indicate that alkaline water could have a protective effect on vascular function, reduce inflammation, and in the context of tumor development, reduce the incidence of metastasis.

Loss of sphingosine kinase 1 increases lung metastases in the MMTV-PyMT mouse model of breast cancer.

Breast cancer is a very heterogeneous disease, and ~30% of breast cancer patients succumb to metastasis, highlighting the need to understand the mechanisms of breast cancer progression in order to identify new molecular targets for treatment. Sphingosine kinase 1 (SK1) has been shown to be upregulated in patients with breast cancer, and several studies have suggested its involvement in breast cancer progression and/or metastasis, mostly based on cell studies. In this work we evaluated the role of SK1 in breast cancer development and metastasis using a transgenic breast cancer model, mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT), that closely resembles the characteristics and evolution of human breast cancer. The results show that SK1 deficiency does not alter tumor latency or growth, but significantly increases the number of metastatic lung nodules and the average metastasis size in the lung of MMTV-PyMT mice. Additionally, analysis of Kaplan-Meier plotter of human disease shows that high SK1 mRNA expression can be associated with a better prognosis for breast cancer patients. These results suggest a metastasis-suppressing function for SK1 in the MMTV-PyMT model of breast cancer, and that its role in regulating human breast cancer progression and metastasis may be dependent on the breast cancer type.

Melittin inhibits lung metastasis of human osteosarcoma: Evidence of wnt/β-catenin signaling pathway participation

Melittin is a major active peptide component of bee venom that has been demonstrated to show anti-tumor effects. Osteosarcoma is a type of bone tumor with a high degree of malignancy, and metastasis is the main challenge of osteosarcoma therapy. This study aimed to investigate the role of melittin in the lung metastasis of osteosarcoma. 143 B cells were treated with different concentrations of melittin in vitro. Wound-healing and transwell assays were performed to determine the cell migration and invasion potential. Quantitative real-time PCR and Western blot experiments were performed to evaluate the expression levels of Wnt/β-catenin signaling pathway-related factors after treatment with melittin. The orthotopic implantation model and hematoxylin-eosin staining were used to investigate the effect of melittin treatment on tumor formation and lung metastasis. Immunohistochemical staining and Western blot experiments were performed to indicate the melittin-mediated expression changes in Wnt/β-catenin signaling pathway-related factors. The cell migration and invasion potential were observed to be inhibited in a dose-dependent manner upon treatment with melittin. Treatment with medium and high concentrations of melittin attenuated the mRNA and protein expression of LRP5, β-catenin, MMP-2, cyclin D, c-Myc, survivin, MMP-9, and VEGF genes in vitro. Melittin significantly inhibited the growth of tibia xenografts in nude mice and decreased the number of lung metastatic nodules. Consistent with the results observed in vitro, treatment with melittin at medium and high concentrations attenuated the expression of Wnt/β-catenin signaling pathway-related factors in vivo. In vitro, Wnt/β-catenin signaling pathway was involved in Melittin-mediated -migration and invasion potential of 143 B cells. Similarly, as observed in the in vivo experiments, Wnt/β-catenin signaling pathway was also associated with the role of melittin on lung metastasis of osteosarcomas.

Lumican Inhibits In Vivo Melanoma Metastasis by Altering Matrix-Effectors and Invadopodia Markers.

It was reported that lumican inhibits the activity of metalloproteinase MMP-14 and melanoma cell migration in vitro and in vivo. Moreover, Snail triggers epithelial-to-mesenchymal transition and the metastatic potential of cancer cells. Therefore, the aim of this study was to examine the effect of lumican on Mock and Snail overexpressing melanoma B16F1 cells in vivo. Lung metastasis was analyzed after intravenous injections of Mock-B16F1 and Snail-B16F1 cells in Lum+/+ and Lum−/− mice. At day 14, mice were sacrificed, and lungs were collected. The number of lung metastatic nodules was significantly higher in mice injected with Snail-B16F1 cells as compared to mice injected with Mock-B16F1 cells confirming the pro-metastatic effect of Snail. This effect was stronger in Lum−/− mice as compared to Lum+/+, suggesting that endogenous lumican of wild-type mice significantly inhibits metastasis to lungs. Scanning electron and confocal microscopy investigations demonstrated that lumican inhibits the development of elongated cancer cell phenotypes which are known to develop invadopodia releasing MMPs. Moreover, lumican was shown to affect the expression of cyclin D1, cortactin, vinculin, hyaluronan synthase 2, heparanase, MMP-14 and the phosphorylation of FAK, AKT, p130 Cas and GSK3α/β. Altogether, these data demonstrated that lumican significantly inhibits lung metastasis in vivo, as well as cell invasion in vitro, suggesting that a lumican-based strategy targeting Snail-induced metastasis could be useful for melanoma treatment.

FYN promotes gastric cancer metastasis by activating STAT3-mediated epithelial-mesenchymal transition.

Gastric cancer is one of the most lethal cancers worldwide. FYN, a gene that is differentially expressed in gastric cancer, is considered a critical metastasis regulator in several solid tumors, but its role in gastric cancer is still unclear. This study aimed to evaluate the role of FYN and test whether FYN promotes migration and invasion of gastric cancer cells in vitro and in vivo via STAT3 signaling. FYN was overexpressed in gastric cancer and positively correlated with metastasis. FYN knockdown significantly decreased cancer cell migration and invasion, whereas FYN overexpression increased cancer migration and invasion. Genetic inhibition of FYN decreased the number of metastatic lung nodules in vivo. Several epithelial-mesenchymal transition markers were positively correlated with FYN expression, indicative of FYN involvement in this transition. Furthermore, gene set enrichment analysis of a Cancer Genome Atlas dataset revealed that the STAT3 signaling pathway was positively correlated with FYN expression. STAT3 inhibition reversed the FYN-mediated epithelial-mesenchymal transition and suppressed metastasis. In conclusion, FYN promotes gastric cancer metastasis possibly by activating STAT3-mediated epithelial mesenchymal transition and may be a novel therapeutic target for gastric cancer.

Sequentially-targeted biomimetic nano drug system for triple-negative breast cancer ablation and lung metastasis inhibition

As a breast cancer subtype with high mortality in women, the efficient treatment of Triple-negative breast cancer (TNBC) is still a challenge due to its unique metastatic mode and poor prognosis. In this study, we developed a biomimetic nanodelivery system (denoted as GTDC@M-R NPs) based on erythrocyte membrane (M)-camouflaged graphene oxide quantum dots (GOQDs, G) for TNBC therapy. The TAT (T) and RGD (R) peptides were used to endow targeting accumulation ability of Gamabufotalin (CS-6, C) and doxorubicin (DOX, D) in tumor tissue. In vitro assay indicated good biocompatibility, prolonged blood circulation time (3-fold longer than GT NPs), and effectively enhanced cell and nucleus targeting capability of this nanosystem. Fluorescence activated cell sorter (FACS) analysis indicated that the combination of DOX and CS-6 induced TNBC cell apoptosis more than 89 % under the ratio of 10:1. In vivo assay indicated that the accumulation of GTDC@M-R NPs in tumor sites increased about 2-fold compared to naked GTDC NPs, which was accompanied by high tumor apoptosis rates through blocking chemotherapy-activated cyclooxygenase-2 (COX-2) and enhancing DOX's anti-tumor activity of chemical drugs (85%). Moreover, comparing with the control, the average number of lung metastatic nodules in tumor-bearing mice reduced 84%, the molecular mechanism of which is related to the down expression of COX-2, matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF). Taken together, our results proved that the developed GTDC@M-R NPs can inhibit the growth and suppress metastasis of TNBC, which broaden our insights into the application of combinational strategy for efficient TNBC therapy. Statement of SignificanceIn this study, we developed a biomimetic nanodelivery system (denoted as GTDC@M-R NPs) based on erythrocyte membrane (M)-camouflaged graphene oxide quantum dots (GOQDs, G) for TNBC therapy. The TAT (T) and RGD (R) peptides were used to endow targeting accumulation ability of Gamabufotalin (CS-6, C) and doxorubicin (DOX, D) in tumor tissue. These GTDC@M-R NPs indicated synergistic chemotherapy against TNBC cells through the precise cell and nuclear targeting, immune escape, and improved DOX sensitivity. A effective inhibition of tumor growth and metastasis was achieved by inhibiting Bcl-2/BAX, COX-2 and VEGF related signal pathways. Our finding suggests that the developed GTDC@M-R NPs present great treating effects in the preclinical models of TNBC, which broaden our insights into the application of combinational strategy for efficient TNBC therapy.

Suppression of KIF3A inhibits triple negative breast cancer growth and metastasis by repressing Rb-E2F signaling and epithelial-mesenchymal transition.

Triple negative breast cancer (TNBC) displays higher heterogeneity, stronger invasiveness, higher risk of metastasis and poorer prognosis compared with major breast cancer subtypes. KIF3A, a member of the kinesin family of motor proteins, serves as a microtubule‐directed motor subunit and has been found to regulate early development, ciliogenesis and tumorigenesis. To explore the expression, regulation and mechanism of KIF3A in TNBC, 3 TNBC cell lines, 98 cases of primary TNBC and paired adjacent tissues were examined. Immunohistochemistry, real‐time PCR, western blot, flow cytometry, short hairpin RNA (shRNA) interference, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), colony formation techniques, transwell assays, scratch tests, and xenograft mice models were used. We found that KIF3A was overexpressed in TNBC and such high KIF3A expression was also associated with tumor recurrence and lymph node metastasis. Silencing of KIF3A suppressed TNBC cell proliferation by repressing the Rb‐E2F signaling pathway and inhibited migration and invasion by repressing epithelial‐mesenchymal transition. The tumor size was smaller and the number of lung metastatic nodules was lower in KIF3A depletion MDA‐MB‐231 cell xenograft mice than in the negative control group. In addition, KIF3A overexpression correlated with chemoresistance. These results suggested that high expression of KIF3A in TNBC was associated with the tumor progression and metastasis.

Down-regulation of SEPT9 inhibits glioma progression through suppressing TGF-β-induced epithelial-mesenchymal transition (EMT)

Malignant glioma is a highly aggressive cancer, known as one of the most dangerous types of primary brain tumor occurring in the central nervous system (CNS). Septin 9 (SEPT9) has been involved in tumor growth. However, its exact roles in regulating glioma development have not been fully understood. In the present study, we found that SEPT9 expression levels were markedly up-regulated in glioma tissues and cell lines. High expression of SEPT9 predicted a poor overall survival in patients with glioma. SEPT9 knockdown significantly reduced the proliferation, migration and invasion of glioma cells. Moreover, epithelial-mesenchymal transition (EMT) markers, including N-cadherin, matrix metalloproteinase-9 (MMP9), Vimentin and Twist, were significantly reduced by SEPT9 knockdown; however, the expression of E-cadherin was elevated by SEPT9 silence. This EMT process in glioma cells was dependent on the expression transforming growth factor (TGF)-β1. In addition, the clinical analysis suggested that SEPT9 gene expression had a positive correlation with TGF-β1 in patients with primary glioma at different grades. Furthermore, knockdown of SEPT9 significantly reduced the glioma progression in vivo. The anti-metastasis regulated by the knockdown of SEPT9 was further confirmed in mouse model, as evidenced by the reduced number of lung metastatic nodules. Our results supported that reducing SEPT9 expression could inhibit glioma progression through the suppression of EMT induced by TGF-β1.

Mycophenolic acid is a drug with the potential to be repurposed for suppressing tumor growth and metastasis in osteosarcoma treatment.

Our previous review of proteomics data showed that in osteosarcoma, some overexpressed proteins were targets of FDA-approved immunosuppressive and anti-arrhythmic drugs, including mycophenolate mofetil (MMF), ribavirin, leflunomide, azathioprine and digoxin. Here, these drugs were screened for growth inhibitory effects in human osteosarcoma cell lines, including MNNG/HOS, U2OS, SaOS-2, MG-63 and 143B cells. Only mycophenolic acid (MPA), an active metabolite of MMF, efficiently inhibited osteosarcoma cell growth with IC50 values of 0.46-7.3 μM; these values are in the therapeutic range for organ transplant patients. At a therapeutic dose (10 μM), MPA significantly inhibited colony formation, caused cell cycle arrest in the S phase, and induced apoptosis. Moreover, the in vitro invasion of osteosarcoma cells was reduced by MPA by inhibiting cell migration capability. The in vivo antitumor effect of MMF was determined in nude mice harboring 143B cell xenografts. Daily oral administration of 200 mg/kg/day MMF for 2 weeks significantly suppressed tumor growth in treated mice, achieving 57.4 ± 11.1% tumor growth inhibition. Compared with the vehicle group, the MMF group treated with 50-200 mg/kg/day for 3 weeks had a significant reduction in the number of lung metastatic nodules in a tail vein-lung metastasis model of 143B cells. MMF doses of 50, 100 and 200 mg/kg/day are approximately equivalent to the non-toxic doses of 0.25, 0.5 and 1 g/day in humans, respectively. These findings indicate that MPA/MMF can effectively control osteosarcoma tumor growth and metastasis. Thus, the potential to repurpose MPA/MMF for use in osteosarcoma chemotherapy is of great interest.

Repurposing of the anti-helminthic drug niclosamide to treat melanoma and pulmonary metastasis via the STAT3 signaling pathway

The incidence of melanoma is increasing rapidly worldwide. Additionally, new and effective candidates for treating melanoma are needed because of the increase in drug resistance and the high metastatic potential of this cancer. The STAT3 signaling pathway plays a pivotal role in pathogenesis of melanoma, making STAT3 a promising anticancer target for melanoma therapy. Niclosamide, an FDA-approved anti-helminthic drug, has been identified as a potent STAT3 inhibitor that suppresses STAT3 phosphorylation at Tyr705 and its transcript activity. In this study, we evaluated the biological activities of niclosamide in melanoma in vitro and in vivo. Niclosamide potently inhibited the growth of four melanoma cell lines and induced the apoptosis of melanoma cells via the mitochondrial apoptotic pathway. Further, western blot analysis indicated that cell apoptosis was correlated with activation of Bax and cleaved caspase-3 and decreased expression of Bcl-2. Moreover, niclosamide markedly impaired melanoma cell migration and invasion, reduced phosphorylated STAT3Tyr705 levels, and inhibited matrix metalloproteinase-2 and −9 expression. Additionally, in a xenograft model of A375, intraperitoneal administration of niclosamide inhibited tumor growth and tumor weight in a dose-dependent manner without obvious side effects. Histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells and p-STAT3Try705-positive cells and increase in cleaved caspase-3-positive cells. Notably, niclosamide significantly inhibited pulmonary metastasis in a B16-F10 melanoma lung metastasis model, including the number of lung metastatic nodules and lung/body coefficient. Importantly, a marked reduction in myeloid-derived suppressor cells (Gr1+CD11b+) infiltration in the pulmonary metastasis tissue was observed. Taken together, these results demonstrate that niclosamide is a promising candidate for treating melanoma.

Core needle biopsy promotes lung metastasis of breast cancer: An experimental study.

Core needle biopsy (CNB) may be used to diagnose early-stage breast cancer, but it may increase the risk of distant metastasis of tumor cells. The aim of the present study was to explore the effect of CNB on the distant metastasis of breast cancer. A total of 30 BALB/c mice were divided into two groups, namely biopsy and non-biopsy groups. The biopsy-related lung metastasis model (biopsy group) was established by the inoculation in the mammary fat pad of the mouse breast cancer cell line 4T1 combined with CNB. Flow cytometry, quantitative polymerase chain reaction analysis, morphological analysis, as well as other techniques, were used to evaluate the biological behavior of the tumors in the mouse model. A stable and reliable lung metastasis model of breast cancer was successfully established. The number of metastatic lung nodules in the biopsy group was significantly higher compared with that in the non-biopsy group (P<0.05). Compared with the non-biopsy group, the mRNA expression of transforming growth factor (TGF)-β1, SOX4 and Ezh2 in the biopsy group was significantly upregulated (P<0.05) and the number of natural killer (NK) cells detected by flow cytometry was increased, but the difference was not statistically significant (P>0.05). Therefore, CNB was found to promote the lung metastasis of breast cancer, and the underlying mechanism may be associated with epithelial-to-mesenchymal transition (EMT) mediated by the TGF-β1 signaling pathway.

Huaier extract exerts immunomodulatory effects by regulating serum cytokines in breast cancer

Background: Huaier extract, a traditional Chinese medicine (TCM) with a 1,600-year history, has recently become a complementary anticancer drug in China. Previous studies have demonstrated that Huaier extract has multiple anticancer effects. However, whether Huaier extract has immunomodulatory effects on serum cytokine profiles in breast cancer patients remains unknown. Methods: Thirty-eight serum specimens were collected from 19 female breast cancer patients who were treated with Huaier extract before surgery in Qilu Hospital, and serum cytokines were analyzed by Bio-Plex ProTM cytokine assays. Murine breast cancer model was built to further verify the immunomodulatory effects of Huaier extract, and serum cytokine levels were detected by enzyme linked immunosorbent assay (ELISA) assay and pulmonary metastasis were counted. Results: In this study, we found that Huaier extract significantly decreased the MCP-1, MIP-1β levels (P Conclusions: The results of this study showed that Huaier extract regulated serum cytokines levels and decreased the number of metastatic lung nodules in breast cancer. Our study also identified a serum cytokine profile, which potentially could predict disease outcome and be used to monitor the efficacy of Huaier extract treatment in breast cancer.

Immunosuppression Induced by Perioperative Peritonitis Promotes Lung Metastasis.

Perioperative intra-abdominal infection has been reported as a risk factor for metastasis. The aim of this study was to investigate the mechanism by which peritonitis induces immunosuppression in the lung, which in turn promotes lung metastasis. C57BL/6 mice were intravenously administered B16F10 melanoma cells to induce lung metastasis and subsequently subjected to cecal ligation and puncture (CLP) to induce peritonitis or sham surgery. The number of lung metastatic nodules was evaluated. Cell fractions in lungs and serum cytokines after CLP were investigated. CLP mice showed an increased number of lung metastases compared to sham-treated mice. The fraction and number of natural killer (NK) cells in lungs of CLP mice were significantly reduced in early post-CLP phase. Myeloid-derived suppressor cells (MDSCs) in lungs were significantly decreased in CLP mice. Serum IL-6 and TNF levels were significantly elevated in CLP mice. Peritonitis promoted lung metastasis in a murine model, which may be attributable to the impact of NK cells and MDSCs in the lungs.

Abstract 4726: Novel intravenous injectable TLR7 agonist, DSP-0509, synergistically enhanced antitumor immune responses in combination with anti-PD-1 antibody

Abstract Toll-like receptors (TLRs) are a family of pattern-recognition receptors (PRR) that recognize pathogen-associated molecular patterns (PAMPs). TLR7 is mainly expressed in plasmacytoid dendritic cells (pDCs) and recognizes virus derived single-stranded RNA (ssRNA). TLR7 stimulation in pDCs induces type I interferon secretion, which results in innate immune activation. Clinical studies have shown that TLR7 agonist induced anti-tumor immune activation. Despite these signs of clinical activity, administration of TLR7 agonist is generally limited to topical application, due to the potential of immune related adverse effects. In this study, we present a novel small molecule TLR7 agonist DSP-0509 formulated for intravenous administration. TLR7 agonistic activity was evaluated by in vitro reporter assay systems and TLR7 knock out mice. Induction of interferon alpha (IFNα) and inflammatory cytokines in mice and human blood was measured as PD response with ELISA. Anti-tumor effect was evaluated in LM8-bearing syngeneic mouse model. Combination effect of DSP-0509 with anti-PD-1 antibody was evaluated in CT26-bearing mice, along with flow cytometric analysis of tumor infiltrating lymphocytes (TILs). DSP-0509 had agonistic activity on human TLR7 (EC50= 316 nM), but not on human TLR8 (EC50&amp;gt; 10 μM). DSP-0509 had high water solubility and rapid elimination from the body (T1/2:0.69h), partly explained by excretion via organic anion transporting peptide (OATP) transporters. Intravenous administration of DSP-0509 induced IFNα secretion in wild type mice, but not in TLR7 knock out mice. Minimum cytokine induction dose of DSP-0509 in human whole blood was lower than that of a well-known TLR7/8 agonist 852A. Intravenous administration of DSP-0509 suppressed the primary tumor growth and the number of lung metastatic nodules in LM8-bearing model. Combination of DSP-0509 with anti-PD-1 antibody significantly suppressed the tumor growth compared to treatments with each monotherapy (P&amp;lt;0.05). The ratio of CD8+ T cells and effector memory T cell populations (CD8+CD62L-CD127+) in TILs and the surface expression of MHC class I molecule on tumor cells was significantly increased in the combination group (P&amp;lt;0.05). All mice responded to combination therapy rejected re-challenged tumor growth. The study showed that DSP-0509 is a novel TLR7 agonist with an intravenous injectable profile and rapid elimination from the body (T1/2: 0.69h). DSP-0509 showed anti-tumor effect against primary tumor growth and metastasis. In addition, anti-PD-1 antibody furthermore enhanced the effect of DSP-0509. We also reported that the combination of DSP-0509 with anti-PD-1 antibody significantly induced effector memory T cells (p&amp;lt;0.05 vs each monotherapy) and showed durable response. Further evaluations of DSP-0509 are warranted. Citation Format: Yosuke Ota, Takeshi Otsubo, Junya Koroki, Yuko Hirose, Erina Koga-Yamakawa, Masashi Murata, Masashi Goto, Yasushi Matsuki. Novel intravenous injectable TLR7 agonist, DSP-0509, synergistically enhanced antitumor immune responses in combination with anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4726.