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Utilizing Biopolymers in 3D Tumor Modeling and Tumor Diagnosis
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology, Fibre Technology.ORCID iD: 0000-0002-6544-9955
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancer represents a significant global public health challenge and ranks as the second mostcommon cause of death in the United States. The onset of cancer entails an initial phasewhere cells lose their polarity and disconnect from the normal basement membrane, allowingthem to form distinct three-dimensional (3D) configurations that interact with adjacent cellsand the surrounding microenvironment. Cells grown in 2D monolayers demonstrate differentgene expression patterns and different activation of signaling pathways compared to cellscultivated within the natural structure of tumor tissue of the same cell type. Multicellulartumor spheroids (MCTS) are extensively investigated as a well-studied model of organotypiccancer. These spheroids are formed by tumor cells, either alone or in combination with othercell types, and they can be created with or without the application of supportive scaffolds.The MCTSs are also considered promising models for preclinical assessments of chemosensitivity.However, the creation of these tumor spheroids presents challenges, as not alltumor cell lines can consistently form regular spheroids.Cellulose nanofibrils (CNF) have become essential as a sustainable and environmentallyfriendly material. For example, thin films, with inherent mechanical properties, and flexibility,offer versatility across various applications. Also known for its biocompatibility and non-toxicnature, native CNF is a natural option to use. Its fibrous structure closely mimics the collagenmatrix in human tissue, showing potential as an effective scaffold for 3D cell culture. In thisregard, an innovative Layer-by-Layer (LbL) coating technique using CNF-polyelectrolytebilayers was investigated to generate spheroids. This method constructs bilayers of CNFand polyelectrolytes and can coat various surfaces. In this thesis, the first focus was ondemonstrating the spheroid formation capability using low molecular weight polyelectrolytesin LbL assembly. Secondly, an investigation was conducted involving embedding of LbLgrownspheroids in a decellularized extracellular matrix (ECM) aiming to determine howECM, possessing suitable mechanical characteristics, could influence the cancer stem celltraits in spheroids. Thirdly, the thesis demonstrated the utilization of LbL for capturing andreleasing of circulating tumor cells. Lastly, the shift from using low molecular weightpolyelectrolytes in the LbL assembly to high molecular weight counterparts and analyzingthe differences in spheroid formation abilities to assess the underlying differences inmolecular weights of the polyelectrolytes was explored. All-in-all, employing the CNF-basedLbL surface coating strategy explored in the thesis has proven to be promising for thedevelopment of spheroid models closely resembling in vivo conditions and holds significantpotential for applications in drug development.

Abstract [sv]

Cancer utgör en betydande global utmaning inom folkhälsan och rankas som den nästvanligaste dödsorsaken i USA. Cancer börjar med en initial fas där celler förlorar sin polaritetoch lossnar från basalmembranet, vilket tillåter dem att bilda distinkta tredimensionella (3D)kluster som interagerar med intilliggande celler och den omgivande mikromiljön. Celler somodlas i 2D-monolager visar olika genuttryck och olika signalvägar jämfört med celler somodlas som mer naturlig 3D struktur likt tumörvävnad. Multicellulära tumörsfärer (MCTS) ärväl studerade som modell för organotypisk cancer, dessa sfärer bildas av tumörceller,antingen av samma typ eller i kombination med andra celltyper, och de kan skapas medeller utan användning av underliggande stödjande strukturer. MCTS betraktas även somlovande modell för preklinisk bedömning av cellernas kemokänslighet. Dock är skapandetav dessa tumörsfärer utmanande, eftersom alla tumörcellinjer inte verkar kunna bildaregelbundna sfärer.Cellulosananofibriller (CNF) är ett alternativ som hållbart och miljövänligt material. Sombland annat kan forma till tunna filmer, med inbyggda mekaniska egenskaper, flexibilitet ,erbjuder mångsidighet över olika tillämpningsområden, till följd av dess flexibilitet och dessinbyggda mekaniska egenskaper. Känd för sin biokompatibilitet och ofarliga natur fungerarCNF som ett bra alternativ för användning även inom biomedicinska tillämpningar. CNFstrukturen liknar kollagenmatrisen i mänsklig vävnad och visar potential som ett effektivtunderlag för 3D-cellodling. Inom detta område undersöktes en innovativ lager-på-lager (LbL)beläggningsmetod innehållande CNF och polyelektrolyt bilager för att skapa sfärer. Metodbygger bilager av CNF och polyelektrolyter som kan belägga ytor av olika material. Dennaavhandling fokuseradar först på att demonstrera sfärformationsförmågan vid användning avpolyelektrolyter med lågmolekylvikt i de tillverkade LbL-modifieringarna. Däreftergenomfördes en undersökning som innefattar inbäddning av sfärerna som bildades pådessa LbL-ytor i extracellulärmatris (ECM) dels i form av collagen samt decellulariseradextracellulärmatris. För att undersöka om ECMs mekaniska egenskaper kan påverkacancercellernas egenskaper i sfärerna samt om EMC med liknande mekaniska egenskaperlikt naturlig vävnad är lämplig. Därefter demonstrerar avhandlingen användningen av LbLför att fånga cirkulerande tumörceller som sedan kunde släppas i en liten volym. Till sistutforskades övergången från användning av polyelektrolyter med låg molekylvikt vid LbLuppbyggnadentill samma typ av polyelektrolyter men med högre molekylvikt, samtanalysera skillnaderna i möjligheten att bilda sfärer. Sammantaget har användningen avCNF-baserad LbL-ytbeläggningsstrategi som utforskats i avhandlingen visat sig varalovande för utvecklingen av multicellulära sfäroidmodeller och som kan ha en betydandepotential för tillämpningar inom läkemedelsutveckling.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2023. , p. 64
Series
TRITA-CBH-FOU ; 2023:57
Keywords [en]
3D tumor modeling, Spheroids, Cellulose Nano Fibrils, Layer by Layer, Extracellular matrix, Circulating Tumor Capturing, Pancreatic ductal adenocarcinoma (PDAC)
Keywords [sv]
3D-tumörmodellering, Sfärer, Cellulosa, Lager-på-lager, Extracellulärmatris, Infångning av cirkulerande tumörceller, Mänskliga Duktala Adenocarcinom-Hepatocyter
National Category
Biomaterials Science
Research subject
Fibre and Polymer Science
Identifiers
URN: urn:nbn:se:kth:diva-339964ISBN: 978-91-8040-788-5 (print)OAI: oai:DiVA.org:kth-339964DiVA, id: diva2:1814193
Public defence
2023-12-15, F3 (Flodis), Lindstedtsvägen 26, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20231124

Available from: 2023-11-24 Created: 2023-11-23 Last updated: 2024-01-08Bibliographically approved
List of papers
1. Layer-by-Layer Cellulose Nanofibrils: A New Coating Strategy for Development and Characterization of Tumor Spheroids as a Model for In Vitro Anticancer Drug Screening
Open this publication in new window or tab >>Layer-by-Layer Cellulose Nanofibrils: A New Coating Strategy for Development and Characterization of Tumor Spheroids as a Model for In Vitro Anticancer Drug Screening
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2022 (English)In: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, Vol. 22, no 10, article id 2200137Article in journal (Refereed) Published
Abstract [en]

Three-dimensional multicellular spheroids (MCSs) are complex structure of cellular aggregates and cell-to-matrix interaction that emulates the in-vivo microenvironment. This research field has grown to develop and improve spheroid generation techniques. Here, we present a new platform for spheroid generation using Layer-by-Layer (LbL) technology. Layer-by-Layer (LbL) containing cellulose nanofibrils (CNF) assemble on a standard 96 well plate. Various bi-layer numbers, multiple cell seeding concentration, and two tumor cell lines (HEK 293 T, HCT 116) are utilized to generate and characterize spheroids. The number and proliferation of generated spheroids, the viability, and the response to the anti-cancer drug are examined. The spheroids are formed and proliferated on the LbL-CNF coated wells with no significant difference in connection to the number of LbL-CNF bi-layers; however, the number of formed spheroids correlates positively with the cell seeding concentration (122 ± 17) and (42 ± 8) for HCT 116 and HEK 293T respectively at 700 cells ml−1. The spheroids proliferate progressively up to (309, 663) µm of HCT 116 and HEK 293T respectively on 5 bi-layers coated wells with maintaining viability. The (HCT 116) spheroids react to the anti-cancer drug. We demonstrate a new (LbL-CNF) coating strategy for spheroids generation, with high performance and efficiency to test anti-cancer drugs.

Place, publisher, year, edition, pages
Wiley, 2022
Keywords
anti-cancer drug screening, cellulose nanofibrils, layer-by-layer, tumor spheroids, Cell culture, Cellulose, Diagnosis, Diseases, Nanofibers, Tumors, Anticancer drug, Bi-layer, Cell seeding, Drug-screening, In-vitro, Layer by layer, New coatings, Tumor spheroid, Cells, cellulose nanofiber, irinotecan, Article, atomic force microscopy, cell proliferation, cell viability, coating (procedure), drug screening, fluorescence intensity, human, human cell, in vitro study, Hardiness, Spheres, Wells
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-326459 (URN)10.1002/mabi.202200137 (DOI)000835450100001 ()35899862 (PubMedID)2-s2.0-85135253200 (Scopus ID)
Note

QC 20230522

Available from: 2023-05-22 Created: 2023-05-22 Last updated: 2023-11-29Bibliographically approved
2. Influence of Decellularized Extra Cellular Matrix on 3D spheroids formed on Layer-by-Layer cellulose nanofibril/Polyelectrolytes coating as an in-vitro model for Hepatocellular Carcinoma
Open this publication in new window or tab >>Influence of Decellularized Extra Cellular Matrix on 3D spheroids formed on Layer-by-Layer cellulose nanofibril/Polyelectrolytes coating as an in-vitro model for Hepatocellular Carcinoma
(English)Manuscript (preprint) (Other academic)
National Category
Pharmacology and Toxicology Biomaterials Science
Identifiers
urn:nbn:se:kth:diva-339927 (URN)
Note

QC 20231127

Available from: 2023-11-22 Created: 2023-11-22 Last updated: 2023-11-29Bibliographically approved
3. Multi-layer assembly of cellulose nanofibrils in a microfluidic device for the selective capture and release of viable tumor cells from whole blood
Open this publication in new window or tab >>Multi-layer assembly of cellulose nanofibrils in a microfluidic device for the selective capture and release of viable tumor cells from whole blood
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2020 (English)In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 12, no 42, p. 21788-21797Article in journal (Refereed) Published
Abstract [en]

According to reports by the World Health Organization (WHO), cancer-related deaths reached almost 10 million in 2018. Nearly 65% of these deaths occurred in low- to middle-income countries, a trend that is bound to increase since cancer diagnostics are not currently considered a priority in resource-limited settings (RLS). Thus, cost-effective and specific cancer screening and diagnostics tools are in high demand, particularly in RLS. The selective isolation and up-concentration of rare cells while maintaining cell viability and preventing phenotypic changes is a powerful tool to allow accurate and sensitive downstream analysis. Here, multi-layer cellulose nanofibril-based coatings functionalized with anti-EpCAM antibodies on the surface of disposable microfluidic devices were optimized for specific capture of target cells, followed by efficient release without significant adverse effects. HCT 116 colon cancer cells were captured in a single step with >97% efficiency at 41.25 mu L min(-1) and, when spiked in whole blood, an average enrichment factor of similar to 200-fold relative to white blood cells was achieved. The release of cells was performed by enzymatic digestion of the cellulose nanofibrils which had a negligible impact on cell viability. In particular, >80% of the cells were recovered with at least 97% viability in less than 30 min. Such performance paves the way to expand and improve clinical diagnostic applications by simplifying the isolation of circulating tumor cells (CTCs) and other rare cells directly from whole blood.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2020
National Category
Biomaterials Science
Identifiers
urn:nbn:se:kth:diva-286608 (URN)10.1039/d0nr05375a (DOI)000585977000027 ()33103175 (PubMedID)2-s2.0-85095799323 (Scopus ID)
Note

QC 20201201

Available from: 2020-12-01 Created: 2020-12-01 Last updated: 2023-12-05Bibliographically approved
4. Evaluating the Impact of Positively Charged Polyelectrolyte Molecular Weightand Bilayer Number on Tumor Spheroid Formation in the Interaction with Negatively Charged Cellulose Nanofibrils in layer by layer assembly
Open this publication in new window or tab >>Evaluating the Impact of Positively Charged Polyelectrolyte Molecular Weightand Bilayer Number on Tumor Spheroid Formation in the Interaction with Negatively Charged Cellulose Nanofibrils in layer by layer assembly
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2023 (English)Manuscript (preprint) (Other academic)
National Category
Biomaterials Science
Identifiers
urn:nbn:se:kth:diva-339926 (URN)
Note

QC 20231127

Available from: 2023-11-22 Created: 2023-11-22 Last updated: 2023-11-29Bibliographically approved
5. Assessing the Layer-by-Layer Assembly of Cellulose Nanofibrils and Polyelectrolytes in Pancreatic Tumor Spheroid Formation
Open this publication in new window or tab >>Assessing the Layer-by-Layer Assembly of Cellulose Nanofibrils and Polyelectrolytes in Pancreatic Tumor Spheroid Formation
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2023 (English)In: Biomedicines, E-ISSN 2227-9059, Vol. 11, no 11Article in journal (Refereed) Published
Abstract [en]

Three-dimensional (3D) tumor spheroids are regarded as promising models for utilization as preclinical assessments of chemo-sensitivity. However, the creation of these tumor spheroids presents challenges, given that not all tumor cell lines are able to form consistent and regular spheroids. In this context, we have developed a novel layer-by-layer coating of cellulose nanofibril–polyelectrolyte bilayers for the generation of spheroids. This technique builds bilayers of cellulose nanofibrils and polyelectrolytes and is used here to coat two distinct 96-well plate types: nontreated/non-sterilized and Nunclon Delta. In this work, we optimized the protocol aimed at generating and characterizing spheroids on difficult-to-grow pancreatic tumor cell lines. Here, diverse parameters were explored, encompassing the bilayer count (five and ten) and multiple cell-seeding concentrations (10, 100, 200, 500, and 1000 cells per well), using four pancreatic tumor cell lines—KPCT, PANC-1, MiaPaCa-2, and CFPAC-I. The evaluation includes the quantification (number of spheroids, size, and morphology) and proliferation of the produced spheroids, as well as an assessment of their viability. Notably, our findings reveal a significant influence from both the number of bilayers and the plate type used on the successful formation of spheroids. The novel and simple layer-by-layer-based coating method has the potential to offer the large-scale production of spheroids across a spectrum of tumor cell lines.

Place, publisher, year, edition, pages
MDPI AG, 2023
Keywords
pancreatic ductal adenocarcinoma, three-dimensional tumor model, layer-by-layer, cellulose nanofibrils
National Category
Biomaterials Science
Identifiers
urn:nbn:se:kth:diva-339943 (URN)10.3390/biomedicines11113061 (DOI)001107899000001 ()2-s2.0-85178372456 (Scopus ID)
Note

QC 20231215

Available from: 2023-11-22 Created: 2023-11-22 Last updated: 2023-12-29Bibliographically approved

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