The induction of a mesenchymal phenotype by platelet cloaking of cancer cells is a universal phenomenon.

Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

Investigating the clearance of VWF A-domains using site-directed PEGylation and novel N-linked glycosylation.

BACKGROUND: Previous studies have demonstrated that the A1A2A3 domains of von Willebrand factor (VWF) play a key role in regulating macrophage-mediated clearance in vivo. In particular, the A1-domain has been shown to modulate interaction with macrophage low-density lipoprotein receptor-related protein-1 (LRP1) clearance receptor. Furthermore, N-linked glycans within the A2-domain have been shown to protect VWF against premature LRP1-mediated clearance. Importantly, however, the specific regions within A1A2A3 that enable macrophage binding have not been defined. OBJECTIVE AND METHODS: To address this, we utilized site-directed PEGylation and introduced novel targeted N-linked glycosylation within A1A2A3-VWF and subsequently examined VWF clearance. RESULTS: Conjugation with a 40-kDa polyethylene glycol (PEG) moiety significantly extended the half-life of A1A2A3-VWF in VWF-/- mice in a site-specific manner. For example, PEGylation at specific sites within the A1-domain (S1286) and A3-domain (V1803, S1807) attenuated VWF clearance in vivo, compared to wild-type A1A2A3-VWF. Furthermore, PEGylation at these specific sites ablated binding to differentiated THP-1 macrophages and LRP1 cluster II and cluster IV in-vitro. Conversely, PEGylation at other positions (Q1353-A1-domain and M1545-A2-domain) had limited effects on VWF clearance or binding to LRP1.Novel N-linked glycan chains were introduced at N1803 and N1807 in the A3-domain. In contrast to PEGylation at these sites, no significant extension in half-life was observed with these N-glycan variants. CONCLUSIONS: These novel data demonstrate that site specific PEGylation but not site specific N-glycosylation modifies LRP1-dependent uptake of the A1A2A3-VWF by macrophages. This suggests that PEGylation, within the A1- and A3-domains in particular, may be used to attenuate LRP1-mediated clearance of VWF.

Aspirin and P2Y12 inhibition attenuate platelet-induced ovarian cancer cell invasion.

BACKGROUND: Platelet-cancer cell interactions play a key role in successful haematogenous metastasis. Disseminated malignancy is the leading cause of death among ovarian cancer patients. It is unknown why different ovarian cancers have different metastatic phenotypes. To investigate if platelet-cancer cell interactions play a role, we characterized the response of ovarian cancer cell lines to platelets both functionally and at a molecular level. METHODS: Cell lines 59 M and SK-OV-3 were used as in vitro model systems of metastatic ovarian cancer. Platelet cloaking of each cell line was quantified by flow cytometry. Matrigel invasion chamber assays were used to assess the invasive capacity of the cell lines. The induction of an EMT was assessed by morphology analysis and by gene expression analysis of a panel of 11 EMT markers using TaqMan RT-PCR. RESULTS: SK-OV-3 cells adhered to and activated more platelets than 59 M cells (p = 0.0333). Platelets significantly promoted the ability of only SK-OV-3 cells to invade (p ≤ 0.0001). Morphology and transcritpome analysis indicated that platelets induce an epithelial-to-mesenchymal transition phenotype in both cells lines, with a more exaggerated response in SK-OV-3 cells. Next, we investigated if antiplatelet agents could abrogate the platelet-induced aggressive phenotype in SK-OV-3 cells. Both aspirin (p ≤ 0.05) and 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (P2Y12 inhibitor; p ≤ 0.01) significantly decreased their invasion capacity, and effectively reverted invasion to levels comparable to SK-OV-3 cells alone. CONCLUSION: While there is increasing evidence for the cancer-protective effect of aspirin, this study suggests P2Y12 inhibition may also play a role. Understanding these complex interactions between platelets and cancer cells could ultimately allow the establishment of therapies tailored to inhibiting metastasis, thus significantly reducing cancer morbidity.

Living in shear: platelets protect cancer cells from shear induced damage.

Pharmacologically and genetically induced thrombocytopenia is associated with decreased metastasis, highlighting the importance of platelets in the bloodborne dissemination of cancer cells. It is frequently suggested that platelets support metastasis, in part, by protecting cancer cells from shear stress, a biomechanical force generated by blood flow. However, there is currently no evidence to support this hypothesis. To address this, we investigated the effect of shear stress on A2780 ovarian cancer cells in the presence and absence of platelets. Using a cone and plate viscometer, suspensions of A2780 cells with and without platelets were exposed to shear rates representing venous (200 s(-1)) and arterial (1,500 s(-1)) blood flow. Lactate dehydrogenase (LDH) release was used to quantify shear induced membrane damage. Both venous and arterial shear rates induced the release of LDH from A2780 cells, demonstrating their susceptibility to shear forces. In contrast, platelets released minimal levels of LDH in response to similar conditions. In the presence of platelets, there was a significant decrease in LDH release by A2780 cells under shear conditions, suggesting that platelets can confer protection against shear induced damage. The disruption of platelet-cancer cell interactions could increase the shear stress induced destruction of cancer cells in vivo.

Response of extraintestinal pathogenic Escherichia coli to human serum reveals a protective role for Rcs-regulated exopolysaccharide colanic acid.

Extraintestinal Escherichia coli (ExPEC) organisms are the leading cause of Gram-negative bacterial bloodstream infections. These bacteria adapt to survival in the bloodstream through expression of factors involved in scavenging of nutrients and resisting the killing activity of serum. In this study, the transcriptional response of a prototypic ExPEC strain (CFT073) to human serum was investigated. Resistance of CFT073 to the bactericidal properties of serum involved increased expression of envelope stress regulators, including CpxR, σE, and RcsB. Many of the upregulated genes induced by active serum were regulated by the Rcs two-component system. This system is triggered by envelope stress such as changes to cell wall integrity. RcsB-mediated serum resistance was conferred through induction of the exopolysaccharide colanic acid. Production of this exopolysaccharide may be protective while cell wall damage caused by serum components is repaired.

Increased platelet reactivity in patients with late-stage metastatic cancer.

Platelet hyperreactivity is associated with an increased risk of thrombosis. Cancer patients are at an increased risk of thrombosis, a risk that increases with disease progression. While cancer patients show evidence of platelet activation in vivo, few studies have extensively assessed whether these patients display platelet hyperreactivity. We hypothesized that patients with metastatic cancer would display platelet hyperreactivity, reflecting their associated high risk of thrombosis. In a cohort of patients with metastatic cancer (n = 13), we assessed platelet function using well-established assays of platelet reactivity (agonist-induced platelet aggregation, spontaneous platelet aggregation, and agonist-induced P-selectin expression). In comparison with healthy controls (n = 10), patients with metastatic cancer displayed global platelet hyperreactivity. Agonist-induced platelet aggregation responses to ADP (adenosine diphosphate), epinephrine, collagen, arachidonic acid, and PAR-1 (protease-activated receptor-1) activating peptide, as well as spontaneous platelet aggregation, were significantly increased in patients with metastatic cancer. Furthermore, agonist-induced platelet P-selectin expression was also significantly increased within the patient cohort. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis.

Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae.

A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.

Major differences exist in frequencies of virulence factors and multidrug resistance between community and nosocomial Escherichia coli bloodstream isolates.

Escherichia coli is a major cause of bloodstream infections and death due to sepsis. It is the most frequent Gram-negative bacterial pathogen recovered from cultures of blood from both community-acquired and nosocomial cases. We set out to determine the relationships between E. coli virulence factors (VFs), phylogenetic groups, and antibiotic resistance and whether bacteremia cases had a community, health care-associated. or nosocomial origin. Isolates from consecutive episodes of E. coli bacteremia in 303 patients presenting to a university hospital were screened for their VFs, phylogenetic group, and antibiotic resistance. The majority of VFs present in the collection were equally distributed between antibiotic-susceptible and multiple-drug-resistant (MDR) isolates, but the overall VF score was higher for isolates of community and health care-associated origin than those of nosocomial origin (P = 0.0002 and P = 0.0172, respectively); the papA, papG allele II, hlyA, and hek VFs were more prevalent in this cohort. Most isolates belonged to phylogenetic group B2, which harbored a greater proportion of antibiotic-susceptible isolates than MDR isolates (P = 0.04). The community, health care-associated, or nosocomial origin of E. coli bacteremia determines the virulence capacity of an isolate better than the phylogenetic group does. This study provides new insights into the relationships between the pathogenesis and epidemiology of E. coli bacteremia.