Precision cancer medicine with small tyrosine kinase inhibitors (TKIs) directed toward oncogenic drivers, are important treatment regimens for solid tumours. The epidermal growth factor receptor (EGFR)-TKI osimertinib is a preferred therapy for patients with non-small cell lung cancer (NSCLC) driven by activating mutations in EGFR, unfortunately responses are heterogeneous. This calls for non-invasive methods to predict or monitor treatment response, for example, via biomarker analyses in blood. To reveal such putative biomarkers, we analysed the proteome of extracellular vesicles (EVs) from osimertinib resistant or responsive NSCLC cells in vitro and from EVs isolated from serum samples of NSCLC patients treated with osimertinib in second line within the phase II clinical trial TREM. The protein cargo of the EVs was analysed by mass spectrometry (MS) and proximity extension assay (PEA). Western blotting, ELISA and single vesicle analysis was performed to validate and further confirm the expression of certain proteins. MS profiling of the NSCLC cells and their released EVs revealed a protein signature associated with osimertinib refractoriness. Among them were CSPG4, HSPG2, MCAM, L1CAM, TAGLN, THBS1 and TNC. GO-pathway analysis related several of these proteins to the focal adhesion and proteoglycan in cancer pathways. Some of these proteins, including CSPG4, which when suppressed by transient siRNA transfection in NSCLC cells resulted in reduced cell viability, were expressed also in EVs from serum of the NSCLC patients. Moreover, PEA profiling of the serum-isolated EVs revealed signatures associated with immune cells, best response and/or progression-free survival, including PD-L1, CD73/NT5E, FR-alpha/FOLR1, LAMP3, FASLG1 and ANXA1. In summary, we demonstrate that protein profiling of EVs in relation to osimertinib refractoriness has the potential to identify possible biomarkers that can indicate osimertinib treatment resistance, for example, CSPG4, HSPG2, TAGLN, TNC, THBS1, ANXA1 and CD73/NT5E. Studies in expanded cohorts should be conducted to further validate these putative osimertinib biomarkers.

The elastic properties of nanoscale extracellular vesicles (EVs) are believed to influence their cellular interactions, thus having a profound implication in intercellular communication. However, accurate quantification of their elastic modulus is challenging due to their nanoscale dimensions and their fluid-like lipid bilayer. We show that the previous attempts to develop atomic force microscopy-based protocol are flawed as they lack theoretical underpinning as well as ignore important contributions arising from the surface adhesion forces and membrane fluctuations. We develop a protocol comprising a theoretical framework, experimental technique, and statistical approach to accurately quantify the bending and elastic modulus of EVs. The method reveals that membrane fluctuations play a dominant role even for a single EV. The method is then applied to EVs derived from human embryonic kidney cells and their genetically engineered classes altering the tetraspanin expression. The data show a large spread; the area modulus is in the range of 4 to 19 mN/m and the bending modulus is in the range of 15 to 33 kBT, respectively. Surprisingly, data for a single EV, revealed by repeated measurements, also show a spread that is attributed to their compositionally heterogeneous fluid membrane and thermal effects. Our protocol uncovers the influence of membrane protein alterations on the elastic modulus of EVs.

Extracellular vesicles (EVs, ∼ 30 nm−5 μm ) are lipid bilayer-enclosed particles expressingvaluable biological information such as proteins, lipids, and nucleic acids that reflecttheir shedding cell. The discovery of their importance in cell-to-cell communicationsparked a boom in research. Their abundance, ability to freely surpass natural barriersin the body, and reflection to the original cell make them suitable players in fieldssuch as treatment monitoring and targeted drug delivery. By investigating how EVsubpopulations interact with cells, we may also gain further insights into theoreticalquestions such as how cells communicate and how cells respond to external stimuli.Virtually all cells in the body release EVs; each cell may contain multiple origin spots forbiogenesis, and EVs may have different intended purposes that are also reflected in theircomposition. Therefore, EVs are extremely heterogeneous in size, expression level ofbiomolecules, and nanomechanical properties such as elasticity. This heterogeneity andthe small size of the vesicle pose technical challenges for the characterization platformsexisting today. EVs may be studied in bulk with a single output for an entire particleensemble or individually, yielding a characterization of individual EVs in a sample.Bulk methods are often faster, offer higher throughput, and may be the only option foranalyzing some parts, such as RNA. However, for complete characterization, we need toretrieve information on single EVs. This thesis explores techniques to characterize EVson a single vesicle level with three different platforms: a fluorescence microscope, anatomic force microscope, and a combined fluorescence and atomic force microscope.First, a fluorescence microscope is used to study EVs released by cells in a cancercell line model study. The cells are either left untreated or treated with two drugs: onethat the cells should respond to and one that they should be immune to. Five relevantsurface proteins were stained, imaged, and analyzed. The study revealed the possibilityof monitoring drug responses through immunofluorescence. Next, the platform was usedto study lung cancer patients undergoing treatment with EVs retrieved through liquidbiopsy. Each patient generated two sets of EVs: one sample from before treatmentand one sample after treatment, but before the tumor stopped responding to the drug.While the study revealed changes in individual proteins when comparing the two sampleswithin each patient, it was difficult to distinguish a pattern regarding the length oftreatment before drug resistance. It was not until we studied the correlation of proteinsand combined all protein expressions in a sample into a joint probability distributionthat trends became clearer. Longer treatments, for example, were found to have astronger positive correlation among the proteins. This highlights the importance ofincluding sophisticated statistical methods to analyze clinical EV samples on a singleEV level. Next, a theoretical model taking into account the EV’s liquid properties was con-structed. The model agrees with force spectroscopy measurements performed with force microscopy. Three EV samples with different protein expression levels were comparedin terms of elasticity moduli. With the low throughput of EVs in the technique, astatistical framework to compare the distributions of stiffness values was developed. Theframework revealed a large variation in stiffness values extracted from a single vesicle,which is hypothetically attributed to thermal fluctuations and diffusion of membranemolecules.Finally, we combined the fluorescence microscope and atomic force microscope toinvestigate subpopulations and heterogeneity in single EVs with both protein expressionand precise mechanical measurements of size and Young’s modulus. The platformrevealed distinct subpopulations with unique properties in the analyzed parameters. These combined measurements are the first of their kind, and a combined platformcharacterizing EVs in multiple ways may offer great insights into EV biology.

Extracellulära vesiklar (EVs, ~30 nm - 5 µm) är lipiddubbelskiktsinneslutna partiklar som uttrycker värdefull biologisk information som proteiner, lipider och nukleinsyror som reflekterar deras föräldracell.Upptäckten av deras betydelse i cell-till-cellkommunikation utlöste en explosion inom forskning. Deras mängd, förmåga att fritt passera naturliga barriärer i kroppen och reflektion av dess ursprungliga cell gör dem till lämpliga aktörer inom områden som behandlingsövervakning och riktad läkemedelsleverans. Genom att undersöka hur EV-subpopulationer interagerar med celler kan vi också få ytterligare insikter i teoretiska frågor som hur celler kommunicerar och hur celler reagerar på yttre stimuli. Praktiskt taget alla celler i kroppen släpper ut EVs; varje cell kan innehålla flera ursprungsplatser för biogenes, och EVs kan ha olika avsedda syften som också återspeglas i deras sammansättning. Därför är EVs extremt heterogena i storlek, uttrycksnivå av biomolekyler och nanomekaniska egenskaper som elasticitet. Denna heterogenitet och den lilla storleken på vesikeln utgör tekniska utmaningar för de karaktäriseringsplattformar som finns idag. EVs kan studeras i bulk med ett enda resultat för en hel partikelensemble eller individuellt, vilket ger en karakterisering av individuella vesiklar i ett prov. Bulkmetoder är ofta snabbare, erbjuder högre genomströmning och kan ibland vara det enda alternativet för att analysera vissa delar, såsom RNA. Men för en fullständig karaktärisering måste vi hämta information om enstaka vesiklar. Denna avhandling utforskar tekniker för att karakterisera EVs på en enstaka vesikelnivå med tre olika plattformar: ett fluorescensmikroskop, ett atomkraftmikroskop och ett kombinerat fluorescens- och atomkraftmikroskop.

Först används ett fluorescensmikroskop för att studera EVs som frigörs av celler i en modellstudie av cancercellinjer. Cellerna lämnas antingen obehandlade eller behandlas med två läkemedel: ett som cellerna ska svara på och ett som de ska vara immuna mot. Fem relevanta ytproteiner taggades, fotograferades och analyserades.Studien avslöjade möjligheten att bevaka läkemedelssvar genom immunfluorescens. Därefter användes plattformen för att studera lungcancerpatienter som genomgick behandling med EVs hämtade genom flytande biopsi.Varje patient genererade två uppsättningar EVs: ett prov från före behandling och ett prov efter behandling, men innan tumören slutade svara på läkemedlet. Medan studien avslöjade förändringar i individuella proteiner när man jämförde de två proverna inom varje patient, var det svårt att särskilja ett mönster angående behandlingslängden före läkemedelsresistens. Det var inte förrän vi studerade korrelationen mellan proteiner och kombinerade alla proteinuttryck i ett prov till en gemensam sannolikhetsfördelning som trenderna blev tydligare. Längre behandlingar visade sig exempelvis ha en starkare positiv korrelation bland proteinerna. Detta understryker vikten av att inkludera sofistikerade statistiska metoder för att analysera kliniska EV-prover på en enda EV-nivå.

Därefter konstruerades en teoretisk modell som tar hänsyn till vesikelns flytande egenskaper. Modellen överensstämmer med kraftspektroskopimätningar utförda med kraftmikroskopi. Tre EV-prover med olika proteinnivåer jämfördes i termer av elasticitetsmoduler. Med den låga genomströmningen av EVs i plattformen utvecklades ett statistiskt ramverk för att jämföra fördelningarna av styvhetsvärden. Ramverket avslöjade en stor variation i styvhetsvärden extraherade från en enda vesikel, vilket hypotetiskt tillskrivs termiska fluktuationer och diffusion av membranmolekyler.

Slutligen kombinerade vi fluorescensmikroskopet och atomkraftmikroskopet för att undersöka subpopulationer och heterogenitet hos individuella EVs i både proteinuttryck och exakta mekaniska mätningar av storlek och Youngs modul. Plattformen avslöjade distinkta subpopulationer med unika egenskaper i de analyserade parametrarna. Dessa kombinerade mätningar är de första i sitt slag, och en kombinerad plattform som karakteriserar EVs på flera sätt kan ge fantastiska insikter om EV-biologi.

Detection of analytes using streaming current has previously been explored using both experimental approaches and theoretical analyses of such data. However, further developments are needed for establishing a viable microchip that can be exploited to deliver a sensitive, robust, and scalable biosensor device. In this study, we demonstrated the fabrication of such a device on silicon wafer using a scalable silicon microfabrication technology followed by characterization and optimization of this sensor for detection of small extracellular vesicles (sEVs) with sizes in the range of 30 to 200 nm, as determined by nanoparticle tracking analyses. We showed that the sensitivity of the devices, assessed by a common protein-ligand pair and sEVs, significantly outperforms previous approaches using the same principle. Two versions of the microchips, denoted as enclosed and removable-top microchips, were developed and compared, aiming to discern the importance of high-pressure measurement versus easier and better surface preparation capacity. A custom-built chip manifold allowing easy interfacing with standard microfluidic connections was also constructed. By investigating different electrical, fluidic, morphological, and fluorescence measurements, we show that while the enclosed microchip with its robust glass-silicon bonding can withstand higher pressure and thus generate higher streaming current, the removable-top configuration offers several practical benefits, including easy surface preparation, uniform probe conjugation, and improvement in the limit of detection (LoD). We further compared two common surface functionalization strategies and showed that the developed microchip can achieve both high sensitivity for membrane protein profiling and low LoD for detection of sEV detection. At the optimum working condition, we demonstrated that the microchip could detect sEVs reaching an LoD of 10⁴ sEVs/mL (when captured by membrane-sensing peptide (MSP) probes), which is among the lowest in the so far reported microchip-based methods.

Extracellular vesicles (EVs) are nanoparticles encapsulated with a lipid bilayer, and they constitute an excellent source of biomarkers for multiple diseases. However, the heterogeneity in their molecular compositions constitutes a major challenge for their recognition and profiling, thereby limiting their application as an effective biomarker. A single-EV analysis technique is crucial to both the discovery and the detection of EV subpopulations that carry disease-specific signatures. Herein, a plasmonic nanohole array is designed for capturing single EVs and subsequently performing fluorescence detection of their membrane proteins by exploiting plasmonic amplification of the fluorescence signal. Unlike other reported methods, our design relies on an exclusive detection of single EVs captured inside nanoholes, thus allowing us to study only plasmonic effects and avoid other metal-induced phenomena while leveraging on the proximity of emitters to the plasmonic hotspots. The method is optimized through numerical simulations and verified by a combination of atomic force, scanning electron microscopy, and fluorescence microscopy. Fluorescence enhancement is then estimated by measuring the CD9 expression of small EVs derived from the human embryonic kidney (HEK293) cell line and carefully considering the spatial distribution of emission and excitation intensities. Fluorescence intensities of immunostained EVs show a moderate overall enhancement of intensity and follow the intensity trend predicted by simulation for nanohole arrays with different nanohole periods. Moreover, the number of observed EVs in the best-performing nanohole array increases by more than 12 times compared with EVs immobilized on a reference substrate, uncovering a vast amount of weakly fluorescent EVs that would remain undetected with the regular fluorescent method. Our nanohole array provides a basis for a future platform of single-EV analyses, also promising to capture the signature arising from low-expressing proteins.

Precision cancer medicine has changed the treatment landscape of non-small cell lung cancer (NSCLC) as illustrated by the introduction of tyrosine kinase inhibitors (TKIs) towards mutated epidermal growth factor receptor (EGFR). However, as responses to EGFR-TKIs are heterogenous among NSCLC patients, there is a need for ways to early monitor changes in treatment response in a non-invasive way e.g., in patient's blood samples. Recently, extracellular vesicles (EVs) have been identified as a source of tumor biomarkers which could improve on non-invasive liquid biopsy-based diagnosis of cancer. However, the heterogeneity in EVs is high. Putative biomarker candidates may be hidden in the differential expression of membrane proteins in a subset of EVs hard to identify using bulk techniques. Using a fluorescence-based approach, we demonstrate that a single-EV tech-nique can detect alterations in EV surface protein profiles. We analyzed EVs isolated from an EGFR-mutant NSCLC cell line, which is refractory to EGFR-TKIs erlotinib and responsive to osimertinib, before and after treatment with these drugs and after cisplatin chemotherapy. We studied expression level of five proteins; two tetraspanins (CD9, CD81), and three markers of interest in lung cancer (EGFR, programmed death-ligand 1 (PD-L1), human epidermal growth factor receptor 2 (HER2)). The data reveal alterations induced by the osimertinib treatment compared to the other two treatments. These include the growth of the PD-L1/HER2-positive EV population, with the largest increase in vesicles exclusively expressing one of the two proteins. The expression level per EV decreased for these markers. On the other hand, both the TKIs had a similar effect on the EGFR-positive EV population.

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.