The tumour microenvironment: the effect of breast cancer cell conditioned medium on the endothelium

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dc.contributor.advisorEngelbrecht, Anna-Marten_ZA
dc.contributor.advisorFourie, Carlaen_ZA
dc.contributor.advisorMarais, Ernaen_ZA
dc.contributor.authorRass, Atarah Melanie Roseen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Physiological Sciences.en_ZA
dc.date.accessioned2023-03-07T15:48:18Z
dc.date.accessioned2023-05-18T07:09:39Z
dc.date.available2023-03-07T15:48:18Z
dc.date.available2023-05-18T07:09:39Z
dc.date.issued2023-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2023. en_ZA
dc.description.abstractENGLISH ABSTRACT: Background: Breast cancer is the most common cancer diagnosed in women and the most common cancer globally. The human mammary gland is comprised of epithelium and vascular rich stroma. It has been established that breast cancer cells interact with and alter their stroma and neighbouring cells, to establish a tumour microenvironment (TME). Mammary endothelial cells are key targets to be transformed into tumour endothelial cells (TECs). These cells are genetically and phenotypically distinct from their normal, healthy counterparts and provide various pro-tumourigenic effects. These effects are modulated by the expression of various molecules that have been classified as TEC markers based on their expression in TECs compared to normal endothelial cells. As central role players in angiogenesis, TECs are key to tumour angiogenesis. Anti-angiogenic agents have proven to be effective, yet act as a double-edged sword, as a result of downstream complications and side effects. TECs therefore serve as potential targets for therapeutic intervention. Various role players in the tumour microenvironment have been investigated, but the effect of breast cancer cells on the tumour endothelial phenotype is not well established. The aims of this study were to evaluate a TEC phenotype in breast cancer and investigate how breast cancer impacts angiogenesis. Methods: Conditioned medium (CM) was harvested from non-malignant (MCF-12A) breast epithelial cells and from malignant (MCF-7 and MDA-MB-231) breast cancer cells starved of supplements and growth factors for 24 hours. Endothelial cells (HUVECs) were then treated with CM for 24 hours. To evaluate a TEC phenotype in breast cancer, cell viability (WST-1 assay), cell morphology (phase contrast imaging), and gene (reverse transcriptase-quantitative polymerase chain reaction) and protein (Western blots) expression of markers associated with a TEC phenotype were assessed. To assess angiogenesis in breast cancer, cell migration (scratch assay) and tube formation (tube formation assay) assays were utilised. A comparative model of non-malignant versus malignant signalling was used throughout the study. Results: Breast cell CM significantly increased HUVEC cell viability in all treatment groups. Changes in morphology were observed, which included elongation and branching, and occurred to a greater degree in malignant CM groups. TEC markers were significantly upregulated in response to non- malignant signalling and tumour endothelial marker 8 was observed to contribute to the TEC phenotype in breast cancer. Significant changes in cell migration were observed in the MCF-7 CM group. Furthermore, clear qualitative differences in the tube formation of HUVECs were noted in malignant groups compared to the non-malignant group. Conclusion: Our results highlight the fact that endothelial cells are highly responsive to interactions with nutrient deprived breast cells but the interaction with non-malignant breast cells compared to malignant breast cells is significantly different. Breast cancer cells therefore do alter endothelial cells, but the characteristic TEC phenotype is not specific to a malignant response. Breast cancer cells alter the angiogenic process but the degree of hyperactivation is influenced by the breast cancer phenotype. It is therefore evident that endothelial cells and angiogenesis are altered and key to breast cancer progression, yet a TEC phenotype specific to breast cancer remains to be defined.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Inleiding: Borskanker is die mees algemene tipe kanker wat by vroue gediagnoseer word en is ook die algemeenste kanker wêreldwyd. Die menslike melkklier bestaan uit epiteel en vaskulêre ryk stroma. Dit is vasgestel dat borskankerselle in wisselwerking tree met en hul stroma en naburige selle verander om 'n tumormikro-omgewing (TME) te vestig. Bors endoteelselle word gereeld geteiken om in tumor endoteelselle (TECs) omskep te word. Hierdie selle is geneties en fenotipies onderskei van hul normale, gesonde eweknieë en verskaf verskeie pro-tumorigeniese effekte. Hierdie effekte word gemoduleer deur die uitdrukking van verskeie molekules wat as TEC-merkers geklassifiseer is, gebasseer op hul uitdrukking in TECs in vergelyking met normale endoteelselle. As sentrale rolspelers in angiogenese, speel TECs ‘n belagrike rol in tumor angiogenese. Dit is al bewys dat anti-angiogene middels effektief is, maar dien steeds as 'n tweesnydende swaard, as gevolg van stroomaf komplikasies en newe-effekte. TECs dien dus as potensiële teikens vir terapeutiese intervensie. Verskeie rolspelers in die tumor mikro-omgewing is ondersoek, maar die effek van borskankerselle op die tumor endoteel fenotipe is nie goed gevestig nie. Die doel van hierdie studie was om 'n TEC-fenotipe in borskanker te evalueer en om te ondersoek hoe borskanker angiogenese beïnvloed. Metodes: Gekondisioneerde medium (CM) is geoes van nie-kwaadaardige (MCF-12A) borsepiteelselle en van kwaadaardige (MCF-7 en MDA-MB-231) borskankerselle wat vir 24 uur lank van aanvullings en groeifaktore uitgehonger is. Endoteelselle (HUVECs) is daarna vir 24 uur met borssel CM behandel en aan ontledings onderwerp. Om 'n TEC-fenotipe in borskanker te vestig, is sellewensvatbaarheid (WST-1-toets), selmorfologie (fasekontrasbeelding), en geen- (omgekeerde transkriptase- kwantitatiewe PCR) en proteïen (Western blots) uitdrukking van merkers geassosieer met 'n TEC- fenotipe, geëvalueer. Om angiogenese in borskanker te bepaal, is selmigrasie (krap-toets) en buisvorming (buisvorming-toets) gebruik. 'n Vergelykende model van nie-kwaadaardige versus kwaadaardige seinoordrag is deur die hele studie gebruik. Resultate: Borssel-CM het HUVEC-sellewensvatbaarheid in alle behandelingsgroepe aansienlik verhoog. Veranderinge in morfologie is waargeneem, wat verlenging en vertakking ingesluit het, en het tot 'n groter mate in kwaadaardige CM-groepe voorgekom. TEC merkers is aansienlik opgereguleer in reaksie op nie-kwaadaardige sein en tumor endoteel merker 8 is waargeneem om by te dra tot die TEC fenotipe in borskanker. Beduidende veranderinge in selmigrasie is waargeneem in die MCF-7 CM groep. Verder is duidelike kwalitatiewe verskille in die buisvorming van HUVECs opgemerk in kwaadaardige groepe in vergelyking met die nie-kwaadaardige groep. Gevolgtrekking: Ons resultate beklemtoon die feit dat endoteelselle sterk reageer op interaksies met borselle waarvan die voedingstowwe ontneem is, maar die interaksie met nie-kwaadaardige borselle in vergelyking met kwaadaardige borselle is aansienlik anders. Borskankerselle verander dus endoteelselle, maar die kenmerkende TEC-fenotipe is nie spesifiek vir 'n kwaadaardige fenotipe nie. Borskankerselle verander die angiogeniese proses, maar die mate van hiperaktivering word deur die borskankerfenotipe beïnvloed. Dit is dus duidelik dat endoteelselle en angiogenese verander is en die sleutel tot borskanker bevordering is, maar 'n TEC-fenotipe wat spesifiek vir borskanker is, moet nog gedefinieer word.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxix, 124 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/127204
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshBreast -- Canceren_ZA
dc.subject.lcshEndotheliumen_ZA
dc.subject.lcshBlood-vessels -- Growthen_ZA
dc.subject.lcshAngiogenesisen_ZA
dc.subject.lcshTumour microenvironmenten_ZA
dc.subject.lcshTumor endothelial cellsen_ZA
dc.subject.lcshTumorsen_ZA
dc.subject.lcshEndothelial cellsen_ZA
dc.titleThe tumour microenvironment: the effect of breast cancer cell conditioned medium on the endotheliumen_ZA
dc.typeThesisen_ZA
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