Integrating automated liquid handling in the separation workflow of extracellular vesicles enhances specificity and reproducibility.

BACKGROUND: Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. RESULTS: Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine. CONCLUSIONS: In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.

Interaction between prostate cancer cells and prostate fibroblasts promotes accumulation and proteolytic processing of basement membrane proteins.

BACKGROUND: Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. METHODS: We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. RESULTS: In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, ß2, and ß3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. CONCLUSIONS: Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.

Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms.

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1ß1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.

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.

Platelet response to a small molecule inhibitor of α2ß1 integrin is associated with ITGA2 C807T dimorphism.

High expression of the collagen receptor, α2ß1 integrin, on platelets of ITGA2 807T-allele carriers has been identified as a risk factor for thromboembolic conditions, and α2ß1 inhibitors are considered to be potential therapeutic agents. In 59 genotyped individuals, we measured α2 expression levels on platelets and analyzed platelet adhesion to collagen under flow conditions. A sulfonamide-type small-molecule inhibitor of α2ß1 integrin decreased average platelet adhesion in individuals with the C/T807T genotype but not in those harboring C807C. Thus, genotype can be used to select a human subpopulation that has the highest probability of showing a positive response to α2ß1 inhibitors.

Embigin deficiency leads to delayed embryonic lung development and high neonatal mortality in mice.

Embigin (Gp70), a receptor for fibronectin and an ancillary protein for monocarboxylate transporters, is known to regulate stem cell niches in sebaceous gland and bone marrow. Here, we show that embigin expression is at high level during early mouse embryogenesis and that embigin is essential for lung development. Markedly increased neonatal mortality of Emb-/- mice can be explained by the compromised lung maturation: in Emb-/- mice (E17.5) the number and the size of the small airways and distal airspace are significantly smaller, there are fewer ATI and ATII cells, and the alkaline phosphatase activity in amniotic fluid is lower. Emb-/- lungs show less peripheral branching already at E12.5, and embigin is highly expressed in lung primordium. Thus, embigin function is essential at early pseudoglandular stage or even earlier. Furthermore, our RNA-seq analysis and Ki67 staining results support the idea that the development of Emb-/- lungs is rather delayed than defected.

Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets.

Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.

Inhibition of TGF-ß signaling, invasion, and growth of cutaneous squamous cell carcinoma by PLX8394.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. The prognosis of patients with metastatic cSCC is poor emphasizing the need for new therapies. We have previously reported that the activation of Ras/MEK/ERK1/2 and transforming growth factor ß (TGF-ß)/Smad2 signaling in transformed keratinocytes and cSCC cells leads to increased accumulation of laminin-332 and accelerated invasion. Here, we show that the next-generation B-Raf inhibitor PLX8394 blocks TGF-ß signaling in ras-transformed metastatic epidermal keratinocytes (RT3 cells) harboring wild-type B-Raf and hyperactive Ras. PLX8394 decreased phosphorylation of TGF-ß receptor II and Smad2, as well as p38 activity, MMP-1 and MMP-13 synthesis, and laminin-332 accumulation. PLX8394 significantly inhibited the growth of human cSCC tumors and in vivo collagen degradation in xenograft model. In conclusion, our data indicate that PLX8394 inhibits several serine-threonine kinases in malignantly transformed human keratinocytes and cSCC cells and inhibits cSCC invasion and tumor growth in vitro and in vivo. We identify PLX8394 as a potential therapeutic compound for advanced human cSCC.

Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics.

The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT; six preservation and five non-preservation) and three blood processing intervals (BPI; 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies.

Inflammation-related citrullination of matrisome proteins in human cancer.

Introduction: Protein 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. Methods: To 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 discussion: Our 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.

Detection of melanoma-derived cancer-testis antigen CT16 in patient sera by a novel immunoassay.

Cancer-testis antigens (CTAs) are expressed mainly in various cancer tissues and in testis or placenta. Because of their restricted expression pattern, the CTAs can be potentially used for vaccine development and diagnostic applications. CTA CT16 has been found to be expressed in lung and renal cancers as well as in melanomas. Detection of CT16 protein directly from patient serum could facilitate monitoring of tumor growth and response to therapy in CT16-positive patients. A highly sensitive time-resolved fluorescence-based immunoassay measuring CTA CT16 in serum was developed. Generally, CTAs have not been measured directly from body fluids. CT16 level was detectable in 14 of 23 (61%) patients with metastatic melanoma, whereas none of the nine healthy volunteers collected by us had measurable CT16 level. For an unknown reason, 1 of 20 commercial control serum samples gave a positive result. The Wilcoxon-Mann-Whitney exact test showed statistically significant difference when patients with metastatic melanoma were compared to our control group (p = 0.006) or to the commercial set (p < 0.001). Four melanoma patients had exceptionally high serum CT16 level. CT16 did not correlate either with S100B, a recognized marker of progressing melanoma, or with unspecific serum marker lactate dehydrogenase. Elevation of CT16 titers preceded or followed the clinical diagnosis of disease progression in four patients with metastatic melanoma. As a conclusion, our results show that CT16 protein can be measured directly from patient serum, and the developed assay has a potential for clinical use.

Recombinant extracellular vesicles as biological reference material for method development, data normalization and assessment of (pre-)analytical variables.

The diagnostic and therapeutic use of extracellular vesicles (EV) is under intense investigation and may lead to societal benefits. Reference materials are an invaluable resource for developing, improving and assessing the performance of regulated EV applications and for quantitative and objective data interpretation. We have engineered recombinant EV (rEV) as a biological reference material. rEV have similar biochemical and biophysical characteristics to sample EV and function as an internal quantitative and qualitative control throughout analysis. Spiking rEV in bodily fluids prior to EV analysis maps technical variability of EV applications and promotes intra- and inter-laboratory studies. This protocol, which is an Extension to our previously published protocol (Tulkens et al., 2020), describes the production, separation and quality assurance of rEV, their dilution and addition to bodily fluids, and the detection steps based on complementary fluorescence, nucleic acid and protein measurements. We demonstrate the use of rEV for method development, data normalization and assessment of pre-analytical variables. The protocol can be adopted by researchers with standard laboratory and basic EV separation/characterization experience and requires ~4-5 d.

H-Ras activation and fibroblast-induced TGF-ß signaling promote laminin-332 accumulation and invasion in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-ß (TGF-ß) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-ß upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-ß signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-ß signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-ß-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion.

Feasibility of Mechanical Extrusion to Coat Nanoparticles with Extracellular Vesicle Membranes.

Biomimetic functionalization to confer stealth and targeting properties to nanoparticles is a field of intense study. Extracellular vesicles (EV), sub-micron delivery vehicles for intercellular communication, have unique characteristics for drug delivery. We investigated the top-down functionalization of gold nanoparticles with extracellular vesicle membranes, including both lipids and associated membrane proteins, through mechanical extrusion. EV surface-exposed membrane proteins were confirmed to help avoid unwanted elimination by macrophages, while improving autologous uptake. EV membrane morphology, protein composition and orientation were found to be unaffected by mechanical extrusion. We implemented complementary EV characterization methods, including transmission- and immune-electron microscopy, and nanoparticle tracking analysis, to verify membrane coating, size and zeta potential of the EV membrane-cloaked nanoparticles. While successful EV membrane coating of the gold nanoparticles resulted in lower macrophage uptake, low yield was found to be a significant downside of the extrusion approach. Our data incentivize more research to leverage EV membrane biomimicking as a unique drug delivery approach in the near future.

Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine.

Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers.

Role of prolyl hydroxylation in the molecular interactions of collagens.

Co- and post-translational hydroxylation of proline residues is critical for the stability of the triple helical collagen structure. In this review, we summarise the biology of collagen prolyl 4-hydroxylases and collagen prolyl 3-hydroxylases, the enzymes responsible for proline hydroxylation. Furthermore, we describe the potential roles of hydroxyproline residues in the complex interplay between collagens and other proteins, especially integrin and discoidin domain receptor type cell adhesion receptors. Qualitative and quantitative regulation of collagen hydroxylation may have remarkable effects on the properties of the extracellular matrix and consequently on the cell behaviour.

FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-ß-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and ß-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.

Integrin α2ß1 decelerates proliferation, but promotes survival and invasion of prostate cancer cells.

High expression level of integrin α2ß1 is a hallmark of prostate cancer stem cell like cells. The role of this collagen receptor is controversial since it is down regulated in poorly differentiated carcinomas, but concomitantly proposed to promote metastasis. Here, we show that docetaxel resistant DU145 prostate cancer cells express high levels of α2ß1 and that α2ß1High subpopulation of DU145 cells proliferates slower than the cells representing α2ß1Low subpopulation. To further study this initial observation we used Crispr/Cas9 technology to create an α2ß1 negative DU145 cell line. Furthermore, we performed rescue experiment by transfecting α2 knockout cells with vector carrying α2 cDNA or with an empty vector for appropriate control. When these two cell lines were compared, α2ß1 positive cells proliferated slower, were more resistant to docetaxel and also migrated more effectively on collagen and invaded faster through matrigel or collagen. Integrin α2ß1 was demonstrated to be a positive regulator of p38 MAPK phosphorylation and a selective p38 inhibitor (SB203580) promoted proliferation and inhibited invasion. Effects of α2ß1 integrin on the global gene expression pattern of DU145 cells in spheroid cultures were studied by RNA sequencing. Integrin α2ß1 was shown to regulate several cancer progression related genes, most notably matrix metalloproteinase-1 (MMP-1), a recognized invasion promoting protein. To conclude, the fact that α2ß1 decelerates cell proliferation may explain the dominance of α2ß1 negative/low cells in primary sites of poorly differentiated carcinomas, while the critical role of α2ß1 integrin in invasion stresses the importance of this adhesion receptor in cancer dissemination.