Measuring FRET in flow cytometry and microscopy

This unit presents protocols describing the measurement of protein associations usingFRET by flow and image cytometry. The theoretical background of FRET is describedin detail inUNIT 1.12, and will not be discussed here. FRET is ideal for the investigation ofprotein associations, but can also be used for the sorting of cells in which interaction ofone protein with another is detected by FRET (Sz¨oll´o´si et al., 1998; M´atyus et al., 2001;Nagy et al., 2005; van Wageningen et al., 2006). The proteins under investigation canbe labeled by fluorescent antibodies or fluorescent protein (FP) variants. The protocolsdescribed are applicable to both situations, except where indicated. Four protocols will bepresented. Basic Protocol 1 describes flow cytometric FRET based on the measurementof donor quenching. This method provides a FRET value on a population basis. BasicProtocol 2 covers flow cytometric FRET based on the measurement of fluorescenceintensities in the donor, FRET, and acceptor channels, providing cell-by-cell FRETvalues. Alternate Protocol 1 is based on cell-by-cell correction for autofluorescence andrequires the measurement of four fluorescence intensities. The algorithm described canbe applied for image cytometric FRET as well. Alternate Protocol 2 is a procedurefor application of the FRET protocol to microscopy. Basic Protocol 3 describes imagecytometric FRET resolved by donor photobleaching. Consult Table 12.8.1 for applicablecombinations of donor and acceptor dye pairs

Two-sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy

The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, beta1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of beta1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of beta1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-by-pixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between beta1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between beta1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of beta1-integrin-ErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with beta1-integrins

Trastuzumab derived HER2-specific CARs for the treatment of trastuzumab-resistant breast cancer: CAR T cells penetrate and eradicate tumors that are not accessible to antibodies

HER2-targeted monoclonal antibodies improve the outcome for advanced breast cancer patients; however, resistance to therapy is still frequent. Epitope masking and steric hindrance to antibody binding through matrix components are thought to be the major mechanism. We asked whether tumors resistant to trastuzumab can still be eliminated by CAR T cells redirected by the same antibody domain. While saturating doses of trastuzumab in the presence of CD16.176V.NK-92 effector cells and trastuzumab derived CAR T cells equally well recognized and killed HER2-positive tumor cells in a monolayer, only CAR T cells penetrated into the core region of tumor spheroids and exhibited cytotoxic activity in vitro, whereas antibodies failed. In NSG mice treatment with trastuzumab and CD16.176V.NK-92 cells only transiently retarded tumor growth but did not induce regression of clinically trastuzumab-resistant breast cancer xenografts. In contrast, one dose of HER2-specific CAR T cells eradicated established tumors resulting in long-term survival. Data indicate that CAR T cells can successfully combat antibody resistant tumors by targeting the same epitope suggesting that CAR T cells can penetrate the tumor matrix which is a barrier for antibodies

Különböző eredetű malignus agydaganatok invazivitásának panelszerű vizsgálata

Az agydaganatok kezelésének hatékonyságát környezeti infi ltrációs képességük jelentősen befolyásolja. A várható túlélés az intrakraniális tumorok eltávolíthatóságától nagymértékben függ. A glioblasztómák kifejezett en magas kiújulási arányában döntő szerepe van a környezeti infi ltrációnak. Ezzel szemben a hasonlóan anaplasztikus bronchogén adenokarcinóma agyi átt étei sokkal kevésbé szűrik be az agyállományt, így radikális eltávolításuk általában rutinszerűen kivitelezhető. A glioblasztómák kiugróan magas infi ltrációs képessége molekuláris hátt erének pontosabb megismeréséhez az invázióban résztvevő molekulák mRNS-expressziójának meghatározásával kerülhetünk közelebb. E molekulák együtt es, panelszerű vizsgálatát végeztük el két eltérő inváziós képességgel rendelkező tumorban. Idegsebészeti műtétt el eltávolított öt glioblasztóma és öt adenokarcinóma átt éti tumormintán 29 molekula mRNS-expresszióját határoztuk meg kvantitatív reverz transzkriptáz polimeráz láncreakcióval (QRT-PCR), majd 9 célmolekulára vonatkozóan immunhisztokémiai vizsgálatok történtek. Célmolekuláink az epidermális növekedési faktor receptorok (EGFR), az integrin-receptorcsalád és legfontosabb ligandjaik: a kollagének, fi bronektin és lamininek köréből kerültek ki. Hat molekula esetében észleltünk szignifi káns mRNS-expresszióbeli különbséget a glioblasztóma és az átt éti daganat között . Immunhisztokémiai vizsgálatokkal négy esetben találtunk proteinszinten verifi kálható eltéréseket. A glioblasztómák agyi átt éti daganatot jelentősen meghaladó inváziós tulajdonságáért felelős molekulák panelszerű vizsgálatával egyszerűen és gyorsan meghatározhatók a két daganatt ípusra jellemző fő különbségek: eredményeink a gliómák invazivitásában már ismert ErbB1 mellett az alfa7 integrin szerepére is felhívják a fi gyelmet