Inflammation-related citrullination of matrisome proteins in human cancer

IntroductionProtein arginine deiminases (PADs) are intracellular enzymes that may, especially in pathological conditions, also citrullinate extracellular substrates, including matrisome proteins such as structural proteins in extracellular matrix (ECM). PADs are abundantly expressed in human cancer cells. Citrullination of matrisome proteins has been reported in colon cancer but the phenomenon has never been systematically studied.MethodsTo gain a broader view of citrullination of matrisome proteins in cancer, we analyzed cancer proteomics data sets in 3 public databases for citrullinated matrisome proteins. In addition, we used three-dimensional cell cocultures of fibroblasts and cancer cells and analyzed citrullination of ECM.Results and discussionOur new analysis indicate that citrullination of ECM occurs in human cancer, and there is a significant variation between tumors. Most frequently citrullinated proteins included fibrinogen and fibronectin, which are typically citrullinated in rheumatoid inflammation. We also detected correlation between immune cell marker proteins, matrix metalloproteinases and ECM citrullination, which suggests that in cancer, citrullination of matrisome proteins is predominantly an inflammation-related phenomenon. This was further supported by our analysis of three-dimensional spheroid co-cultures of nine human cancer cell lines and fibroblasts by mass spectrometry, which gave no evidence that cancer cells or fibroblasts could citrullinate matrisome proteins in tumor stroma. It also appears that in the spheroid cultures, matrisome proteins are protected from citrullination.</p

The composition of prostate core matrisome in vivo and in vitro unveiled by mass spectrometric analysis

Background: The composition and organization of extracellular matrix (ECM) are important regulators of cell behavior. In particular in the prostate, this central role of the ECM is further stressed by the fact that several potential markers of prostate stem cells are matrix receptors. Methods: We established 12 fibroblastic cell lines from cancerous and non‐cancerous areas of six prostates and allowed the cells to produce ECM under cell culture conditions. We also performed a proteome wide analysis of the ECM components by mass spectrometry. To study the in vitro activation of fibroblastic cells we compared the differences between the ECM produced in cell culture by six non‐cancerous‐tissue‐derived fibroblasts and the in vivo matrisome from the corresponding non‐cancerous tissue of prostate. Results: Our results suggest that at tissue level the ECM is mainly produced by fibroblastic cells and that it contains standard collagen I fibrils and fibril‐associated proteins. Beaded‐filament forming collagen VI is also abundant and basement membranes potentially contain five laminin subtypes and collagens XV and XVIII. As the main finding, we also detected differences when in vivo and in vitro matrisomes were compared. Only 65 out of 206 proteins were found to be common for both in vivo and in vitro samples. Majority of the 55 proteins, which were solely detected in in vivo samples, were considered to be plasma derived. Eighty‐six proteins were solely found from in vitro fibroblast‐derived ECM, and most of them were related to matrix remodeling or growth factor action, proposing that the activation of fibroblasts in cell culture may remarkably modify their gene expression profile. Finally, in comparison to traditional 2D in vitro cell culture, the ECM composition of 3D spheroid culture was analyzed. The matrisome in spheroid culture did not resemble the in vivo ECM more closely than in monolayer culture. Conclusions: Artificial activation of ECM remodeling seems to be a distinctive feature in in vitro models. In conclusion the constitution of ECM produced by prostate derived fibroblasts in vitro is similar, but not identical to the prostate ECM in vivo as shown here by mass spectrometric analysis. </p

EphB2 Promotes Progression of Cutaneous Squamous Cell Carcinoma

Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo. Tumor cell–specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability, migration of tumor cells, and invasion of tumor cells. Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13. Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer

MISpheroID: a knowledgebase and transparency tool for minimum information in spheroid identity

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility. © 2021, The Author(s)

Säätiövalvonta hyvässä hallinnossa

Säätiölain 14:1 § velvoittaa viranomaisen valvomaan säätiöiden toimintaa. Lain nojalla viranomaisen eli Patentti- ja rekisterihallituksen tehtävänä on valvoa, että säätiön toiminnassa noudatetaan säätiölakia ja säätiön sääntöjen määräyksiä. Vuosikymmenet, oikeastaan koko vanhan säätiölain voimassaoloajan, säätiövalvonta perustui lähinnä ainoastaan mekaaniseen tarkastukseen. Suurimpana syynä syvällisten valvontatoimenpiteiden puuttumiselle oli viranomaisen resurssipula. Tilanne on kohentunut, kun vuonna 2014 tuli voimaan laki säätiöiden valvontamaksusta, jonka avulla on saatu lisäresursseja valvontatyöhön. Vuoden 2015 loppupuolella voimaan tullut uusi säätiölaki on myös merkittävästi mahdollistanut tehokkaamman valvonnan. Samalla on myös haettu uutta rajanvetoa viranomaisen valvontatyöhön. Tutkielmassa tarkastelen, kuinka säätiövalvonta on kehittynyt ja toiminut vanhan sekä voimassa olevan säätiölain aikana. Näkökulmana on hyvän hallinnon toteutuminen viranomaisen säätiövalvontatehtävässä. Säätiöiden valvonta toteutuu sisäisenä valvontana säätiön toimielinten toimesta. Tässä tutkielmassa keskitytään säätiöiden ulkoiseen valvontaan, viranomaisvalvontaan. Säätiöiden tilintarkastuksella on olennainen merkitys säätiöiden viranomaisvalvonnan toteuttamiseen. Asianmukainen tilintarkastuskaan ei kuitenkaan välttämättä tuo julki mahdollisia väärinkäytöksiä säätiössä, jolloin ne jäävät tulematta ilmi huolellisesta tilintarkastuksesta ja viranomaisvalvonnasta huolimatta. Tutkielmassa tuodaan esiin, että myös muilla tavoin voidaan tuoda julki väärinkäytösepäilyjä, jolloin viranomainen voi niiden johdosta ryhtyä tarvittaviin valvontatoimiin. Tutkimusote on lähtökohtaisesti lainopillinen eli asiaa pyritään systematisoimaan ja tulkitsemaan säätiö- ja hallintolain näkökulmasta. Lähdeaineistona on voimassa oleva sekä sitä edeltänyt lainsäädäntö ja niihin liittyvä valmisteluaineisto. Lähteenä on myös käytetty oikeuskirjallisuutta sekä otantaa valvontakäytännöistä. Valvontatapauksia koskevaa käytäntöä tarkastelemalla pyritään tunnistamaan ja konkretisoimaan säätiöiden toiminnassa tapahtuneita väärinkäytöksiä sekä niihin liittyviä valvonnallisia ongelmia ja valvontaa koskevia soveltamiskysymyksiä. Säätiöiden viranomaisvalvontaa on käsitelty julkisuudessa lähinnä väärinkäytösten ilmettyä. Selvitän muutamien valvontatapausten avulla, miten viranomainen on valvontatehtäväänsä toteuttanut. Nähdäkseni säätiövalvonta toteutuu voimassa olevien lakien avulla varsin hyvin hyvän hallinnon valossa, mutta hallinnon tulevaisuuden kannalta haluan kiinnittää huomion siihen, kuinka valvontatehtävässä tarvittava osaaminen ja asiantuntemus ei saisi siirtyä valtionhallinnon ulkopuolelle. Mielestäni valtionhallinnon omaa asiantuntemusta pitäisi tulevaisuudessa kehittää ja parantaa. Pitäisi pystyä säilyttämään monipuolinen osaaminen siellä, missä valvontavelvoite on

Inhibition of cholesterol synthesis disrupts integrin internalization and EV1 infection.

<p>A) Thin layer chromatography analysis of sterols in SAOS-α2β1 cells. B) Representative pictures of internalization of α2 integrin after ketoconazole, 5% LPDS or 10% FCS treatments. Internalized integrin is seen as green labeling and surface-bound integrin as red or yellow dye. The ratio of voxels between surface and internalized integrin was quantified with internalization algorithm embedded in BioImageXD software. Higher ratio means higher amount of integrin in plasma membrane. Results are averages from together 33 cells from 3 independent tests+SE. C) Electron microscopic example images of integrin structures after antibody clustering in 5% LPDS+ketoconazole cells at 0.5 and 3.5 h time points. D) Proportional change of EV1 infectivity in cells treated with 10% FCS DMEM, 5% LDPS DMEM or 5% LPDS DMEM with ketoconazole. Results are averages of four independent tests (+SE). **P<0.05, ***P<0.001. E) EV1 infectivity in cells treated with 10% FCS DMEM, 5% LDPS DMEM or 5% LPDS+mβCD-cholesterol. Results are averages of three independent tests (+SE), together over 750 cells were counted. F) The effect of U18666A (3 µg/ml) on EV1 infection percentage. EV1 infectivity was calculated together from 750 cells from three individual tests (+SE). Cholesterol labeling with filipin and lysosomal labeling with Lamp-1 was performed to confirm the efficacy of the drug. Bars 10 µm.</p

Collagen uptake is disturbed by ketoconazole treatment.

<p>A) Percentage of cells showing internalized collagen vesicles vs. cell surface-enriched collagen was counted from 77 to 80 cells cultivated in 10% DMEM or 5% LPDS DMEM with ketoconazole, respectively. Typical images used in calculations are shown. Bars 10 µm. B) Ratio of cell surface-enriched vs. internalized collagen was calculated from confocal sections from 20 cells cultivated for 6 h in 10% DMEM, 5% LPDS or 5% LPDS with ketoconazole. Surface-labeled collagen is seen as green or yellow and internalized collagen as red staining. Representative images are shown. Higher ratio means higher collagen label at plasma membrane. Results are mean values from together 20 cells of 2 independent tests (+ SE). Bars 10 µm.</p

Cholesterol sequestering drugs inhibit α2β1 integrin internalization and EV1 infection.

<p>A) Differential staining of the internalized (green) and surface-bound (red or yellow) α2β1 integrin 2 h after internalization. Nystatin (50 µg/ml), filipin (1 µg/ml) or equal amount of DMSO (control) were used. Bars, 10 µm. The ratio of surface versus internalized α2β1 integrin was determined using the internalization algorithm in BioImageXD software and the higher ratio means lower amount of internalization. Results are expressed as mean values measured from 30 cells from 3 independent experiments ± standard error (SE). ***P<0.001. B) The effect of nystatin (50 µg/ml) or filipin (1 µg/ml) on EV1 infectivity was determined. Results are expressed as mean values from three independent experiments ± SE (more than 400 cells counted). ***P<0.001. The EV1 capsid proteins were visualized after 7 h p.i. Bars, 10 µm.</p

The effects of cholesterol aggregating drugs on EV1 uncoating.

<p>A) EV1 infectivity of control cells and cells treated with filipin and nystatin after 5 min p.i. (P<0.001, binomial <i>t</i>-test). Confocal images show labeled EV1 capsid proteins at 7 h p.i. Bars, 10 µm. B) EV1 infection percentage of cells treated with nystatin or filipin that were added at different time points p.i. The results are mean values of 3 independent experiments ± SE (more than 700 cells counted). C) Infection percentage of neutral-red labeled EV1 (NR-EV1) with light treatments in different times p.i. The control cells were not exposed to light reaction. Results are averages of 2 independent tests (+ SE) and over 800 cells were counted at the minimum. D) Sucrose gradient sedimentation assay of uncoating with [³⁵S]methionine-labeled EV1 at 4 h p.i. RNA containing virus sediments at 160S, whereas the 80S represents empty capsids from which the viral RNA genome is released. E-F) As in D except filipin or nystatin was added to the cells 15 min p.i. G) Cointernalized Fluospheres and clustered α2 integrin colocalize in endosomes. Total intensity of FluoSpheres in endosomes was analysed from confocal sections by using the intensity algorithm in BioImageXD. Together 20 cells from 2 separate tests were analysed (+ SE). *P<0.01. Bars, 10 µm.</p

Colocalization of α2 integrin with collagen.

<p>Representative images of cells showing α2 integrin (red) and collagen (green) labeling, 2 or 6 h after plating cells on soluble collagen with or without ketoconazole. Bars 10 µm.</p