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Identification of Changes in Biomarkers Relevant for Breast Cancer Biology Occurring in a Novel 3D-Biosilk Model

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Identification of Changes in Biomarkers Relevant for Breast Cancer Biology Occurring in a Novel 3D-Biosilk Model DEGREE PROJECT IN BIOTECHNOLOGY, SECOND CYCLE, 30 CREDITS STOCKHOLM, SWEDEN 2021 Identification of changes in biomarkers relevant for breast cancer biology occurring in a novel 3D-Biosilk model EMMY STÅHL KTH ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH Identification of changes in biomarkers relevant for breast cancer biology occurring in a novel 3D-Biosilk model Emmy Ståhl Supervisor: My Hedhammar Co-supervisor: Caterina Collodet Kungliga Tekniska Högskolan, KTH Royal Institute of Technology School of Engineering Sciences in Chemistry, Biotechnology and Health Stockholm 2021 Abstract Breast cancer is the most common cancer among women. It is a heterogenous and complex disease composed of several subtypes, each with distinct morphological and clinical implications [1]. To model and study cell biology, tissue morphology, molecular mechanisms and drug actions, cell cultures are canonically used [2]. Today two-dimensional (2D) models are still widely the preferred method for culturing cells in vitro [3]. A drawback with 2D models is that the microenvironment in these models does not mimic the in vivo structure of tumors and tissues, lacking three-dimensional (3D) cell-cell and cell-extracellular matrix (ECM) interactions [2]. Due to the disadvantages of 2D models, 3D cultures have become an increasingly interesting alternative to solve the need for a reliable preclinical model for drug testing and the study of cancer biology. To develop a relevant tool for cancer research, the laboratory of professor My Hedhammar is currently establishing a 3D model of breast cancer. In such novel model, Biosilk is used as scaffold to grow immortalized cell lines representative of the three major classes of breast cancer (i.e. MCF-7 (luminal-like), SKBR-3 (HER2-overexpression) and MDA-MB-231 (triple- negative)). Since transcriptional signatures can be used to classify and study breast cancers, it is important to investigate if and how growth in 3D-Biosilk can impact gene expression profiles. The hypothesis tested in this study was that cells cultured in 3D-Biosilk have differences in expression of biomarkers relevant to breast cancer biology, when compared to the same cell lines cultured in 2D. To examine this, 3D-Biosilk models were created and evaluated to ensure their quality and reproducibility, for instance, the scaffold structure was monitored by brightfield microscopy, the construct’s area was measured with ImageJ, staining with phalloidin confirmed the presence of cells as well as their attachment to the construct, and Alamar blue was used to assess the cellular metabolic activity. Differences in gene expression of target genes were investigated using reverse transcription quantitative PCR (RT- qPCR), which revealed statistically significant changes depending on whether the cells were cultivated in 2D or a 3D-Biosilk model. For cell line MDA-MB-231 three genes were found, for SKBR-3 two genes were found and for MCF-7 four genes were found. The expression of one gene which was found downregulated in MCF-7 cultured in 3D-Biosilk (i.e. ZO-1) was validated at protein level by immunofluorescence. In conclusion, cultivating cells in 3D-Biosilk indicates a more aggressive phenotype. Keywords: 3D-Biosilk, breast cancer, 3D cell culture, recombinant spider silk, ZO-1 1 Sammanfattning Bröstcancer är den vanligaste formen av cancer som drabbar kvinnor. Det är en heterogen och komplex sjukdom som består av flera undergrupper, var och en med distinkt morfologi och kliniska implikationer [1]. För att modellera och studera cellbiologi, vävnadsmorfologi, molekylära mekanismer och läkemedels effekter används cellkulturer [2]. Idag är två- dimensionella (2D) modeller fortfarande den mest använda metoden för att odla celler in vitro [3]. En nackdel med 2D-modeller är att mikromiljön i dessa modeller inte imiterar in vivo strukturen av tumörer och vävnader, då de saknar tre dimensionella (3D) cell-cell och cell- extracellulär matrix (ECM) interaktioner [2]. På grund av nackdelarna med 2D-modeller, har 3D-modeller blivit mer intressanta som alternativ för att lösa behovet av en pålitlig preklinisk modell för läkemedelstestning och för studier av cancerbiologi. För att utveckla ett redskap som är relevant för cancerforskning etablerar professor My Hedhammars laboratorium en 3D-modell av bröstcancer. I en sådan ny modell används Biosilk som byggnadsställning för att odla odödliga cellinjer som är representativa för de tre huvudklasserna av bröstcancer (i.e. MCF-7 (luminal-lik), SKBR-3 (HER2-överuttryckt) och MDA- MB-231 (trippel-negativ)). Eftersom transkriptions signaturer kan användas för att klassificera och studera bröstcancer är det viktigt att undersöka om och hur tillväxt i 3D-Biosilk kan påverka genuttrycksprofiler. Hypotesen som testades i denna studie var om cellkulturer i 3D- Biosilk kan ha signifikanta skillnader i uttryck av biomarkörer, relevanta för bröstcancerbiologi, vid jämförelse av samma cellinje kultiverad i 2D. För att testa detta utvärderades kvalitén och reproducerbarheten av 3D-Biosilk konstruktionen med hjälp av olika kvalitetstester. Strukturen granskades med brightfield mikroskopi, arean av konstruktionen mättes med ImageJ, infärgning med phalloidin bekräftade cellnärvaro och cellvidhäftning till modellen. Alamar blue utfördes för att bedöma den cellulära metaboliska aktiviteten i modellen. Förändringarna av målgenernas genuttryck undersöktes med kvantitativ omvänd transkription PCR (RT-qPCR) och detta påvisade en statistiskt signifikant skillnad i genuttrycket beroende på om cellerna odlats i 2D- eller 3D-Biosilk modeller. I cellinje MDA-MB-231 hittades tre gener, i cellinje SKBR-3 hittades två gener och i cellinje MCF-7 hittades fyra gener. Genuttrycket för en av dessa gener i cellinje MCF-7, som var kultiverad i 3D-Biosilk, var ned- reglerad (i.e. ZO-1). Detta kunde valideras på proteinnivå med immunofluorescens. Sammanfattningsvis, celler odlade i 3D-Biosilk visar på en mer aggressiv fenotyp. Nyckelord: 3D-Biosilk, bröstcancer, 3D cellkultur, rekombinant spindeltråd, ZO-1 2 Table of Contents Abstract ............................................................................................................................ 1 Sammanfattning ............................................................................................................... 2 1 Introduction .............................................................................................................. 4 1.1 How breast cancer affects society .................................................................................. 4 1.2 Breast cancer classification ............................................................................................ 4 1.2.1 Breast cancer cell lines .................................................................................................................. 4 1.3 Why 3D models? ............................................................................................................ 5 1.3.1 Different types of models ............................................................................................................. 6 1.3.2 Biosilk ............................................................................................................................................ 6 1.3.3 Comparison between Biosilk and hydrogel .................................................................................. 6 2 Materials & Methods ................................................................................................ 8 2.1 Cell culture .................................................................................................................... 8 2.2 3D-Biosilk model ........................................................................................................... 8 2.2.1 Creating the 3D-Biosilk model ...................................................................................................... 8 2.2.2 Alamar blue viability assay ............................................................................................................ 9 2.2.3 3D-Biosilk area .............................................................................................................................. 9 2.2.4 Staining with phalloidin and DAPI ................................................................................................. 9 2.2.5 Immunofluorescence staining .................................................................................................... 10 2.3 Sample preparation and RT-qPCR ................................................................................. 10 2.3.1 RNA extraction and cDNA synthesis ........................................................................................... 10 2.3.2 Quantitative RT-PCR ................................................................................................................... 11 2.3.3 Primer design .............................................................................................................................. 12 3 Results .................................................................................................................... 14 3.1 Evaluating the quality of 3D-Biosilk breast cancer constructs ........................................ 14 3.1.1 Brightfield images of the 3D-Biosilk structure ............................................................................ 14 3.1.2 Area measurement ....................................................................................................................
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