Matrigel Invasion Assay

An increase in the risk of cancer is one of the consequences of obesity. The predominant cancers associated with obesity have a hormonal basis and include breast, prostate, endometrium, colon and gall-bladder cancers. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease states. Therefore, it is plausible that leptin acts to promote cancer growth by acting as a mitogenic agent. However, a direct role for leptin in endometrial cancer has not been demonstrated. In this study, we analyzed the proliferative role of leptin and the mechanism(s) underlying this action in endometrial cancers which express both short and long isoforms of leptin receptors. Treatment with leptin resulted in increased proliferation of ECC1 and Ishikawa cells. The promotion of endometrial cancer cell proliferation by leptin involves activation of STAT3 and ERK2 signaling pathways. Moreover, leptin-induced phosphorylation of ERK2 and AKT was dependent on JAK/STAT activation. Therefore blocking its action at the JAK/STAT level could be a rational therapeutic strategy for endometrial carcinoma in obese patients. We also found that leptin potently induces invasion of endometrial cancer cells in a Matrigel invasion assay. Leptin-stimulated invasion was effectively blocked by pharmacological inhibitors of JAK/STAT (AG490) and phosphatidylinositol 3-kinase (LY294002). Taken together these data indicate that leptin promotes endometrial cancer growth and invasiveness and implicate the JAK/STAT and AKT pathways as critical mediators of leptin action. Our findings have potential clinical implications for endometrial cancer progression in obese patients.

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Lysophosphatidic Acid Promotes Epithelial to Mesenchymal Transition in Ovarian Cancer Cells by Repressing SIRT1

Cellular Physiology and Biochemistry ◽

10.1159/000458744 ◽

2017 ◽

Vol 41 (2) ◽

pp. 795-805 ◽

Cited By ~ 16

Author(s):

Upasana Ray ◽

Sib Sankar Roy ◽

Shreya Roy Chowdhury

Keyword(s):

Ovarian Cancer ◽

Lysophosphatidic Acid ◽

Epithelial To Mesenchymal Transition ◽

Ovarian Tissue ◽

Cancer Therapeutics ◽

Ovarian Cancer Cells ◽

Matrigel Invasion Assay ◽

Mesenchymal Transition ◽

Sirt1 Expression ◽

Matrigel Invasion

Background/Aims: Epithelial-to-mesenchymal transition (EMT) plays an essential role in the transition from early to invasive phenotype, however the underlying mechanisms still remain elusive. Herein, we propose a mechanism through which the class-III deacetylase SIRT1 regulates EMT in ovarian cancer (OC) cells. Methods: Expression analysis was performed using Q-PCR, western blot, immunofluorescence and fluorescence-IHC study. Matrigel invasion assay was used for the invasion study. Morphological alterations were observed by phalloidin-staining. Co-immunoprecipitation study was performed to analyze protein-protein interaction. Results: Overexpression of SIRT1-WT as well as Resveratrol-mediated SIRT1 activation antagonized the invasion of OC cells by suppressing EMT. SIRT1 deacetylates HIF1α, to inactivate its transcriptional activity. To further validate HIF1α inactivation, its target gene, i.e. ZEB1, an EMT-inducing factor was found to attenuate upon SIRT1 activation. To uncover the regulatory factor governing SIRT1 expression, lysophosphatidic acid (LPA), a highly enriched oncolipid in ascites/serum of OC patients, was found to down-regulate SIRT1 expression. Importantly, LPA was found to induce the mesenchymal switch in OC cells through suppression of SIRT1. Decreased level of SIRT1 was further validated in ovarian tissue samples of OC patients. Conclusion: We have identified a mechanism that relates SIRT1 down-regulation to LPA-induced EMT in OC cells and may open new arenas on developing novel anti-cancer therapeutics.

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Biobanked Glioblastoma Patient-Derived Organoids as a Precision Medicine Model to Study Inhibition of Invasion

International Journal of Molecular Sciences ◽

10.3390/ijms221910720 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10720

Author(s):

Emilie Darrigues ◽

Edward H. Zhao ◽

Annick De Loose ◽

Madison P. Lee ◽

Michael J. Borrelli ◽

...

Keyword(s):

Precision Medicine ◽

Ex Vivo ◽

Model Systems ◽

Established Cell Line ◽

Matrigel Invasion Assay ◽

Tubulin Inhibitor ◽

Matrigel Invasion ◽

Established Cell ◽

Inhibitor Compounds ◽

Invasion Inhibition

Glioblastoma (GBM) is highly resistant to treatment and invasion into the surrounding brain is a cancer hallmark that leads to recurrence despite surgical resection. With the emergence of precision medicine, patient-derived 3D systems are considered potentially robust GBM preclinical models. In this study, we screened a library of 22 anti-invasive compounds (i.e., NF-kB, GSK-3-B, COX-2, and tubulin inhibitors) using glioblastoma U-251 MG cell spheroids. We evaluated toxicity and invasion inhibition using a 3D Matrigel invasion assay. We next selected three compounds that inhibited invasion and screened them in patient-derived glioblastoma organoids (GBOs). We developed a platform using available macros for FIJI/ImageJ to quantify invasion from the outer margin of organoids. Our data demonstrated that a high-throughput invasion screening can be done using both an established cell line and patient-derived 3D model systems. Tubulin inhibitor compounds had the best efficacy with U-251 MG cells, however, in ex vivo patient organoids the results were highly variable. Our results indicate that the efficacy of compounds is highly related to patient intra and inter-tumor heterogeneity. These results indicate that such models can be used to evaluate personal oncology therapeutic strategies.

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MPPa-PDT suppresses breast tumor migration/invasion through inhibiting AKT-NF-κB-dependent MMP-9 expressions via ROS

10.21203/rs.2.11559/v1 ◽

2019 ◽

Author(s):

Liyi Huang ◽

Haidan Lin ◽

Qing Chen ◽

Lehua Yu ◽

Dingqun Bai

Keyword(s):

Breast Cancer ◽

Tumor Metastasis ◽

Migration And Invasion ◽

Therapeutic Treatment ◽

Oxygen Scavenger ◽

Akt Inhibitor ◽

Matrigel Invasion Assay ◽

Matrigel Invasion ◽

Mcf 7

Abstract Background: breast cancer is the most commonly women cancer and most breast cancer deaths are related to tumor metastasis. Therefore, inhibiting metastasis may provide a therapeutic treatment for breast cancer. In the present study, pyropheophorbide-α methyl ester mediated photodynamic therapy (MPPa-PDT) was used to inhibit metastasis in breast cancer cells MCF-7. Methods: Uptake of MPPa was detected by fluorescence microscope. Cell viability was evaluated by CCK-8. Generation of ROS were detected by DCFH-DA. Migration of cells was assessed by wound healing assay and invasion ability was assessed by Matrigel invasion assay. Levels of MMP2 and MMP9 were measured by PCR. Akt, Phospho-Akt, Phospho-NF-kB p65 and NF-kB p65 were measured by western blotting. F-actin cytoskeleton was observed by immunofluorescence. Lung organs were stained with Hematoxylin and Eosin. Results: Following MPPa-PDT, migration and invasion were decreased in the MCF-7 cells. MPPa-PDT down-regulated expression of MMP2 and MMP9 which is responsible for metastasis. MPPa-PDT reduced the phosphorylation of AKT and NF-κB. MPPa-PDT also destroyed cytoskeleton F-actin in MCF-7 cells. These effects were blocked by the reactive oxygen scavenger NAC or AKT activator SC79 while PI3K inhibitor LY294002 or AKT inhibitor Triciribine increased these effects. Moreover, MPPa-PDT inhibited tumor metastasis and destroyed F-actin in vivo. Conclusion: taken together, these results demonstrated that MPPa-PDT inhibits metastasis of MCF-7 cells both in vitro and vivo, and that may involve in AKT-NF-κB-dependent MMP-9 signaling pathway. Thus, MPPa-PDT may be a promising therapeutic treatment to inhibit metastasis.

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A Novel Oral Arginase 1/2 Inhibitor Enhances the Antitumor Effect of PD-1 Inhibition in Murine Experimental Gliomas by Altering the Immunosuppressive Environment

Frontiers in Oncology ◽

10.3389/fonc.2021.703465 ◽

2021 ◽

Vol 11 ◽

Author(s):

Paulina Pilanc ◽

Kamil Wojnicki ◽

Adria-Jaume Roura ◽

Salwador Cyranowski ◽

Aleksandra Ellert-Miklaszewska ◽

...

Keyword(s):

Tumor Cells ◽

Rna Sequencing ◽

Checkpoint Inhibitors ◽

Oral Treatment ◽

Experimental Studies ◽

Myeloid Cells ◽

Antitumor Effects ◽

Matrigel Invasion Assay ◽

Matrigel Invasion ◽

Arginase 1

Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.

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Effect of the serine proteases inhibitor gabexate mesylate (GM) on the activity of gemcitabine (G) in cell lines of pancreatic cancer (PC)

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e15645 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e15645-e15645

Author(s):

G. Brandi ◽

P. Paterini ◽

S. Tavolari ◽

G. Da Pozzo ◽

E. Nobili ◽

...

Keyword(s):

Cell Cycle ◽

Pancreatic Cancer ◽

Extracellular Matrix ◽

Cell Line ◽

Serine Proteases ◽

Combined Treatment ◽

Pancreatic Cancer Cell Line ◽

Matrigel Invasion Assay ◽

Matrigel Invasion ◽

Cell Vitality

e15645 Background: Negligible advances for PC treatment have been done over the last decade and G remains the standard. Proteolitic degradation of extracellular matrix (ECM) is essential for early local invasion, metastasis and desmoplastic reaction characterizing PC. Differently from MMPs, the serine proteases (uPA and TAT) action is earlier and larger, degradating not only ECM, but also basement membrane, and activating trypsin, plasmin, angiogenesis via TGF-β1 and proliferation via PAR-2. GM is an inhibitor of u-PA,TAT, trypsin and plasmin, used in Italy and Japan for prophylaxis of acute pancreatitis after ERCP. In a previous study, GM demonstrated antinvasion and antimetastatic activity. Study aim: to evaluate if GM increases G efficacy on pancreatic cancer cell line. Methods: In vitro study of phenotypic effects of GM and G in poor differentiated PANC-1 PC cell line using:1) Cell vitality test (Trypan blu);2) Invasion test (Matrigel invasion assay);3) Cell cycle analysis (cytofluorimeter);4) Antiangiogenic test (tube formation assay in extracellular matrix using E.A.hy926 endothelial cells with matrigel). Different doses of G and GM (100,200,250,500 μM;1mM) alone or combined (concomitant or sequential) have been tested vs controls (PANC-1 without any treatment). All tests have been done in triplicate. Results: G alone (250 μM) decreases invasion by 40% (±5,6%) and cell vitality by 15% (±1,3%.). GM alone (100 μM) decreases invasion by 30% (±4,6%.) but 1mM is needed for similar vitality decrease. GM+G together are detrimental vs G alone while sequential treatment (GM before G with or without 24 hours of interval) enhances G activity. GM (200 μM) and G (250 μM) in immediate sequence show better results decreasing ability of invasion by 75% (±8,3%). Cell vitality is better inhibited from GM (100)/G (250) 24 h-delayed sequence by 28% (±3,8%). Combined treatment mainly blocks cells in G1 phase of cell cycle (5%±0,5%) vs controls. Concerning antiangiogenic assay, the administration of G alone is ineffective to inhibit angiogenesis, while pre-treatment with GM results in a strong anti-angiogenic effect. Conclusions: Association of GM to G could represent a new effective approach to inhibit invasion, angiogenesis and growth in pancreatic cancer. No significant financial relationships to disclose.

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Effect of Wnt signaling protein (Wisp2/CCN5) on angiogenesis and invasion in prostate cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.5_suppl.227 ◽

2012 ◽

Vol 30 (5_suppl) ◽

pp. 227-227 ◽

Cited By ~ 2

Author(s):

Yasunobu Hashimoto

Keyword(s):

Extracellular Matrix ◽

Vascular Endothelial Cells ◽

Gelatin Zymography ◽

Metastatic Potential ◽

Tube Formation ◽

Vegf Mrna ◽

Matrigel Invasion Assay ◽

Signaling Protein ◽

Matrigel Invasion ◽

Endothelial Tube Formation

227 Background: The Wnt-Induced Signaling Protein-2 (Wisp-2 /CCN5) is a secreted protein implicated in modification of extracellular matrix, invasion, and angiogenesis. The regulation of Wisp-2 and its function in CaP is poorly explored although it is highly expressed in advanced CaP cells. We discovered recently that a pro-inflammatory chemokine, Interleukin-8 (IL-8) strongly modulates Wisp2 expression in CaP cells. Since IL-8 is a major effecter of inflammatory pathway, we investigated the physiological consequence of Wisp-2 modulation in CaP cells. We hypothesised that Wisp-2 is a down-stream effector of IL-8 induced angiogenesis and invasion. Methods: Quantitative PCR (qPCR) was used to determine the expression of Wisp-2 mRNA and other mRNA in prostate epithelial and cancer cells. Wisp-2 mediated-VEGF secretion was determined by ELISA and endothelial tube formation assay using human vascular endothelial cells (HUVEC). Invasive activity and their attributes were determined using gelatin-zymography for matrix metalloproteinases (MMPs), and Matrigel invasion assay. Results: Wisp2 RNA was strongly expressed in two highly metastatic CaP cell lines, DU145 and PC-3 and moderately in less invasive LAPC4 and LNCaP cells. Constitutive autocrine expression or external addition of IL-8 increased Wisp-2 mRNA by ≥15.2 times in LNCaP, and 7.4 times in LAPC4 cells. Depleting Wisp-2 by RNA interference caused 82% reduction of Wisp2 in DU145, and 75% in PC-3. Depletion of Wisp2 did not affect cell proliferation in DU145 and PC3 but it reduced expression of VEGF mRNA by 64% in DU145 and 43% in PC3. Secreted VEGF protein in cultures of DU145 and PC-3 cells showed a decrease of 62% in DU145 and 30% in PC3. Condition medium of Wisp-2 depleted DU145 cell cultures showed 20% decrease in proliferation and vessel formation activity in HUVEC cells. Further, Wisp-2 depletion in DU145 cells resulted in significant decrease in chemoinvasion activity (28%) and secretion of MMP-9 (25%). Conclusions: These results show that Wisp2 is down stream effector of IL-8 and is an important modulator, affecting extracellular matrix to stimulate angiogenesis and invasiveness in CaP cells. Suppression of Wisp-2 in CaP may reduce their metastatic potential.

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Effect of Wnt-1 induced signaling protein-2 (Wisp-2/CCN5) on angiogenesis and invasion in prostate cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.6_suppl.164 ◽

2013 ◽

Vol 31 (6_suppl) ◽

pp. 164-164

Author(s):

Yasunobu Hashimoto ◽

Rajendra Singh ◽

Bal L. Lokeshwar

Keyword(s):

Extracellular Matrix ◽

Vascular Endothelial Cells ◽

Gelatin Zymography ◽

Metastatic Potential ◽

Tube Formation ◽

Vegf Mrna ◽

Matrigel Invasion Assay ◽

Signaling Protein ◽

Matrigel Invasion ◽

Endothelial Tube Formation

164 Background: The Wnt-Induced Signaling Protein-2 (Wisp-2/CCN5) is a secreted protein implicated in modification of extracellular matrix, invasion, and angiogenesis. The regulation of Wisp-2 and its function in CaP is poorly explored although it is highly expressed in advanced CaP cells. We discovered recently that a pro-inflammatory chemokine, Interleukin-8 (IL-8) strongly modulates Wisp2 expression in CaP cells. Since chronic inflammation is thought to be significant in CaP metastasis and IL-8 is a major effecter of inflammatory pathway, we investigated the physiological consequence of Wisp-2 modulation in CaP cells. Methods: Quantitative PCR was used to determine the expression of Wisp-2 mRNA and other mRNA in prostate epithelial and cancer cells. Wisp-2 mediated-VEGF secretion was determined by ELISA and endothelial tube formation assay using human vascular endothelial cells. Invasive activity and their attributes were determined using gelatin-zymography for matrix metalloproteinases (MMPs), Matrigel invasion assay and chemotaxis assay. Results: Wisp2 RNA was strongly expressed in two highly metastatic CaP cell lines, DU145 and PC-3 and moderately in less invasive LAPC4 and LNCaP cells. Constitutive autocrine expression or external addition of IL-8 increased Wisp-2 mRNA by ≥15.2 times in LNCaP, and 7.4 times in LAPC4 cells. Depleting Wisp-2 by RNA interference caused 82% reduction of Wisp2 in DU145, and 75% in PC-3. Depletion of Wisp2 did not affect cell proliferation in DU145 and PC3 but it reduced expression of VEGF mRNA by 64% in DU145 and 43% in PC3. Secreted VEGF protein in cultures of DU145 and PC-3 cells showed a decrease of 62% in DU145 and 30% in PC3. Condition medium of Wisp-2 depleted DU145 cell cultures showed 20% decrease in proliferation and vessel formation activity in HUVEC cells. Further, Wisp-2 depletion in DU145 cells resulted in significant decrease in chemoinvasion activity (28%) and secretion of MMP-9 (25%). Conclusions: These results show that Wisp2 is down stream effector of IL-8 and is an important modulator, affecting extracellular matrix to stimulate angiogenesis and invasiveness in CaP cells. Suppression of Wisp-2 in CaP may reduce their metastatic potential.

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Blocking pro-invasive signaling and inflammatory activation in triple-negative breast cancer with nucleic-acid scavengers (NASs).

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e13096 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e13096-e13096

Author(s):

Elias Eteshola ◽

Karenia Landa ◽

Eun-Sil Shelley Hwang ◽

Smita Nair ◽

Bruce Sullenger

Keyword(s):

Breast Cancer ◽

Nucleic Acid ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Chemotherapeutic Agents ◽

Metastatic Progression ◽

Matrigel Invasion Assay ◽

Matrigel Invasion ◽

Insight Into

e13096 Background: Breast cancers remain the most lethal malignancies amongst women worldwide and the second leading cause of cancer-related mortalities in the US. Subtype heterogeneity and aggressive invasive potential are believed to be the major contributors of these outcomes. Triple-negative breast cancer (TNBC) are notoriously aggressive, difficult-to-treat, and metastatic. Inflammation-driven tumorigenesis has been shown to correlate with cell-free DNA (cfDNA) and other damage-associated molecular patterns (DAMPs) in cancer patient sera. We showed that nucleic-acid scavengers (NAS) can block pro-inflammatory signals elicited by DAMP-activation of innate immune sensors (e.g. toll-like receptors). Treatment with the NAS PAMAM-G3 drastically reduced liver metastatic burden in an immunocompetent murine model of pancreatic cancer. Methods: TNBC cells lines were treated with a cocktail of standard-of-care chemotherapeutic agents and the conditioned media (CM) from these cells served as an in vitro DAMP source. Downstream function of TLR activation was tested via a HEK293-TLR reporter cell line measuring absorbance at 655nm. The in vitro invasive phenotype was tested and quantified using a Transwell-Matrigel invasion assay. Cytokine secretion was measured using a BioLegend cytokine array. Results: TNBC CM greatly increased TNBC cell invasion in vitro and that treatment with the NAS PAMAM-G3 significantly inhibits this effect. Treatment of human monocytes (THP-1) with TNBC CM elicited a strong pro-inflammatory response with elevated levels of IL-8, IL-6, CCL2, and IL-1β. Other biologically immune responders including human PBMCs will be tested to determine the potential impact on the tumor immune microenvironment during tumorigenesis and treatment. Conclusions: To elucidate the mechanism by which this NAS works in these tumor settings, our lab has developed several PAMAM-G3 derivatives, including biotin, IR-, and near-IR fluorophore labeled molecules. These molecules will allow us to capture and characterize DAMPs and do in vivo live imaging experiments to gain insight into NAS PK/PD properties. This insight into NAS capabilities will enhance our understanding of metastatic progression and its interplay with the immune system. Moreover, these principles will aid in the development of novel of anti-metastatic therapies to improve TNBC patient outcomes.

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Nonlethal Levels of Zeaxanthin Inhibit Cell Migration, Invasion, and Secretion of MMP-2 via NF-κB Pathway in Cultured Human Uveal Melanoma Cells

Journal of Ophthalmology ◽

10.1155/2016/8734309 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Ming-Chao Bi ◽

Nicole Hose ◽

Cai-Lian Xu ◽

Chen Zhang ◽

Jodi Sassoon ◽

...

Keyword(s):

Cell Migration ◽

Uveal Melanoma ◽

Melanoma Cells ◽

Boyden Chamber ◽

Dependent Manner ◽

Wound Healing Assay ◽

Matrigel Invasion Assay ◽

Matrigel Invasion ◽

Nuclear Extracts ◽

Dose Dependent

Zeaxanthin at nonlethal dosages (3–10 μM) significantly inhibited the cell migration of cultured uveal melanoma cells (C918 cell line) as determined by wound healing assay and Boyden chamber assay. Matrigel invasion assay showed that cell invasion of uveal melanoma cells could be significantly inhibited by zeaxanthin. Secretion of MMP-2 by melanoma cells was significantly inhibited by zeaxanthin in a dose-dependent manner as measured by ELISA kit. Zeaxanthin also significantly inhibited the NF-κB levels in nuclear extracts of the UM cells, which is the upstream of the MMP-2 secretion. These results suggest that zeaxanthin might be a potentially therapeutic approach in the prevention of metastasis in uveal melanoma.

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