{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T05:59:01Z","timestamp":1778565541264,"version":"3.51.4"},"reference-count":40,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2021,10,26]],"date-time":"2021-10-26T00:00:00Z","timestamp":1635206400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Protein Carbonic Anhydrase IX (CA IX), which is expressed in various hypoxic solid tumors in order to maintain proper pH, is also related to cancer cell adhesion, invasion, and metastasis processes. Here, we investigated whether CA IX inhibition by a highly CA IX selective agent benzenesulfonamide VD11-4-2 triggers changes in individual cell motility. We seeded breast cancer cells on an extracellular matrix-coated glass-bottomed dish and in a microfluidic device with a gradient flow of epidermal growth factor (EGF), tracked individual cell movement, calculated their migration speeds, and\/or followed movement direction. Our results showed that the inhibitor VD11-4-2 decreased the speed of CA IX positive breast cancer cells by 20\u201326% while not affecting non-cancerous cell migration. The inhibitor suppressed the cell migration velocity increment and hindered cells from reaching their maximum speed. VD11-4-2 also reduced CA IX, expressing cell movement towards the growth factor as a chemoattractant. Such a single cell-based migration assay enabled the comprehensive investigation of the cell motility and revealed that VD11-4-2 shows the ability to suppress breast cancer cell migration at a lower concentration than previously tested CA IX inhibitors.<\/jats:p>","DOI":"10.3390\/ijms222111571","type":"journal-article","created":{"date-parts":[[2021,10,26]],"date-time":"2021-10-26T23:54:33Z","timestamp":1635292473000},"page":"11571","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Inhibition of Carbonic Anhydrase IX Suppresses Breast Cancer Cell Motility at the Single-Cell Level"],"prefix":"10.3390","volume":"22","author":[{"given":"Agne","family":"Janoniene","sequence":"first","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5552-6427","authenticated-orcid":false,"given":"Linas","family":"Mazutis","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6178-6276","authenticated-orcid":false,"given":"Daumantas","family":"Matulis","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4106-5535","authenticated-orcid":false,"given":"Vilma","family":"Petrikaite","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"5574","DOI":"10.1002\/cam4.2474","article-title":"Are 90% of deaths from cancer caused by metastases?","volume":"8","author":"Rogers","year":"2019","journal-title":"Cancer Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1450","DOI":"10.1038\/nm.3391","article-title":"Tumor metastasis: Moving new biological insights into the clinic","volume":"19","author":"Wan","year":"2013","journal-title":"Nat. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1038\/s41571-018-0134-8","article-title":"A framework for the development of effective anti-metastatic agents","volume":"16","author":"Anderson","year":"2019","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2323","DOI":"10.3390\/molecules20022323","article-title":"Targeting carbonic anhydrase IX activity and expression","volume":"20","author":"Mahon","year":"2015","journal-title":"Molecules"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Mboge, M.Y., Mahon, B.P., McKenna, R., and Frost, S.C. (2018). Carbonic anhydrases: Role in pH control and cancer. Metabolites, 8.","DOI":"10.3390\/metabo8010019"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1016\/S0014-4827(03)00351-3","article-title":"Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with \u03b2-catenin","volume":"290","author":"Pastorek","year":"2003","journal-title":"Exp. Cell Res."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Debreova, M., Csaderova, L., Burikova, M., Lukacikova, L., Kajanova, I., Sedlakova, O., Kery, M., Kopacek, J., Zatovicova, M., and Bizik, J. (2019). CAIX regulates invadopodia formation through both a pH-dependent mechanism and interplay with actin regulatory proteins. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20112745"},{"key":"ref_8","first-page":"271","article-title":"The effect of carbonic anhydrase IX on focal contacts during cell spreading and migration","volume":"4","author":"Csaderova","year":"2013","journal-title":"Front. Membr. Physiol. Membr. Biophys."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Jiwa, L.S., Van Diest, P.J., Hoefnagel, L.D., Wesseling, J., Wesseling, P., and Moelans, C.B. (2014). Upregulation of Claudin-4, CAIX and GLUT-1 in distant breast cancer metastases. BMC Cancer, 14.","DOI":"10.1186\/1471-2407-14-864"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"6244","DOI":"10.1038\/onc.2017.219","article-title":"The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia\/MMP14-mediated invasion","volume":"36","author":"Swayampakula","year":"2017","journal-title":"Oncogene"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"5591","DOI":"10.1021\/jm300529u","article-title":"Antimetastatic effect of sulfamate carbonic anhydrase IX inhibitors in breast carcinoma xenografts","volume":"55","author":"Gieling","year":"2012","journal-title":"J. Med. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"5210","DOI":"10.1038\/onc.2012.550","article-title":"Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche","volume":"32","author":"Lock","year":"2013","journal-title":"Oncogene"},{"key":"ref_13","first-page":"2585","article-title":"Cellular and radiobiological effects of carbonic anhydrase IX in human breast cancer cells","volume":"41","author":"Theuerkorn","year":"2019","journal-title":"Oncol. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"24856","DOI":"10.18632\/oncotarget.4498","article-title":"Evaluation of carbonic anhydrase IX as a therapeutic target for inhibition of breast cancer invasion and metastasis using a series of in vitro breast cancer models","volume":"6","author":"Ward","year":"2015","journal-title":"Oncotarget"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1016\/j.devcel.2019.03.026","article-title":"The extracellular matrix modulates the metastatic journey","volume":"49","author":"Kai","year":"2019","journal-title":"Dev. Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1002\/med.20078","article-title":"Can anti-migratory drugs be screened in vitro? A review of 2D and 3D assays for the quantitative analysis of cell migration","volume":"27","author":"Decaestecker","year":"2007","journal-title":"Med. Res. Rev."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.addr.2015.12.017","article-title":"The extracellular matrix in breast cancer","volume":"97","author":"Oskarsson","year":"2016","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"931","DOI":"10.1016\/j.semcdb.2009.08.005","article-title":"Collagen-based cell migration models in vitro and in vivo","volume":"20","author":"Wolf","year":"2009","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"9435","DOI":"10.1021\/jm501003k","article-title":"Discovery and Characterization of Novel Selective Inhibitors of Carbonic Anhydrase IX","volume":"57","author":"Dudutiene","year":"2014","journal-title":"J. Med. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1080\/14756366.2016.1217854","article-title":"Selective inhibition of human carbonic anhydrase IX in Xenopus oocytes and MDA-MB-231 breast cancer cells","volume":"31","author":"Ames","year":"2016","journal-title":"J. Enzyme Inhib. Med. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1080\/01480545.2016.1223095","article-title":"Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide","volume":"40","author":"Aspatwar","year":"2017","journal-title":"Drug Chem. Toxicol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"13976","DOI":"10.1021\/acsami.7b04038","article-title":"A Versatile Carbonic Anhydrase IX Targeting Ligand-Functionalized Porous Silicon Nanoplatform for Dual Hypoxia Cancer Therapy and Imaging","volume":"9","author":"Janoniene","year":"2017","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1601406","DOI":"10.1002\/adhm.201601406","article-title":"Microfluidic Encapsulation of Prickly Zinc-doped Copper Oxide Nanoparticles with VD1142 Modified Spermine Acetalated Dextran for Efficient Cancer Therapy","volume":"6","author":"Zhang","year":"2017","journal-title":"Adv. Healthc. Mater."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"271","DOI":"10.3892\/ijo.2015.3253","article-title":"CA IX is upregulated in CoCl2-induced hypoxia and associated with cell invasive potential and a poor prognosis of breast cancer","volume":"48","author":"Chu","year":"2016","journal-title":"Int. J. Oncol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Ciccone, V., Filippelli, A., Angeli, A., Supuran, C.T., and Morbidelli, L. (2020). Pharmacological inhibition of CA-IX impairs tumor cell proliferation, migration and invasiveness. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21082983"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Liao, S.Y., Lerman, M.I., and Stanbridge, E.J. (2009). Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development. BMC Dev. Biol., 9.","DOI":"10.1186\/1471-213X-9-22"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1038\/sj.bjc.6603530","article-title":"Hypoxia-regulated carbonic anhydrase IX expression is associated with poor survival in patients with invasive breast cancer","volume":"96","author":"Hussain","year":"2007","journal-title":"Br. J. Cancer"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"R71","DOI":"10.1186\/bcr2916","article-title":"Prospective evaluation of serum tissue inhibitor of metalloproteinase 1 and carbonic anhydrase IX in correlation to circulating tumor cells in patients with metastatic breast cancer","volume":"13","author":"Riethdorf","year":"2011","journal-title":"Breast Cancer Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"226","DOI":"10.4161\/cam.23257","article-title":"Carbonic anhydrase IX: A hypoxia-controlled \u201ccatalyst\u201d of cell migration","volume":"7","author":"Svastova","year":"2013","journal-title":"Cell Adhes. Migr."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"26800","DOI":"10.18632\/oncotarget.25508","article-title":"Novel fluorinated carbonic anhydrase IX inhibitors reduce hypoxia-induced acidification and clonogenic survival of cancer cells","volume":"9","author":"Kazokaite","year":"2018","journal-title":"Oncotarget"},{"key":"ref_31","first-page":"3147","article-title":"The story of MCF-7 breast cancer cell line: 40 Years of experience in research","volume":"35","author":"Raica","year":"2015","journal-title":"Anticancer Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1186\/bcr2889","article-title":"Choosing correct breast cancer cell line for breast cancer research","volume":"13","author":"Holliday","year":"2011","journal-title":"Breast Cancer Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"85021","DOI":"10.18632\/oncotarget.13116","article-title":"EGF induces epithelial-mesenchymal transition through phospho-Smad2\/3-Snail signaling pathway in breast cancer cells","volume":"7","author":"Kim","year":"2016","journal-title":"Oncotarget"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1038\/onc.2012.634","article-title":"Identification of novel ROCK inhibitors with anti-migratory and anti-invasive activities","volume":"33","author":"Patel","year":"2014","journal-title":"Oncogene"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1186\/s12964-014-0054-x","article-title":"A novel small-molecule MRCK inhibitor blocks cancer cell invasion","volume":"12","author":"Unbekandt","year":"2014","journal-title":"Cell Commun. Signal."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"a018267","DOI":"10.1101\/cshperspect.a018267","article-title":"The actin cytoskeleton and actin-based motility","volume":"10","author":"Svitkina","year":"2018","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2034","DOI":"10.1016\/j.bpj.2018.09.028","article-title":"Chemotaxis Model for Breast Cancer Cells Based on Signal\/Noise Ratio","volume":"115","author":"Lim","year":"2018","journal-title":"Biophys. J."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1016\/j.yexcr.2004.06.030","article-title":"Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis","volume":"300","author":"Wang","year":"2004","journal-title":"Exp. Cell Res."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Biswenger, V., Baumann, N., J\u00fcrschick, J., H\u00e4ckl, M., Battle, C., Schwarz, J., Horn, E., and Zantl, R. (2018). Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0203040"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Stravinskiene, D., Imbrasaite, A., Petrikaite, V., Matulis, D., Matuliene, J., and Zvirbliene, A. (2019). New monoclonal antibodies for a selective detection of membrane-associated and soluble forms of carbonic anhydrase ix in human cell lines and biological samples. Biomolecules, 9.","DOI":"10.3390\/biom9080304"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/21\/11571\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:24:20Z","timestamp":1760167460000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/21\/11571"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,26]]},"references-count":40,"journal-issue":{"issue":"21","published-online":{"date-parts":[[2021,11]]}},"alternative-id":["ijms222111571"],"URL":"https:\/\/doi.org\/10.3390\/ijms222111571","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,26]]}}}