A living biobank of ovarian cancer ex vivo models reveals profound mitotic heterogeneity.

High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a "living biobank" of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making.

Desmoid fibromatosis through the patients' eyes: time to change the focus and organisation of care?

PURPOSE: Desmoid fibromatosis (DF) is a rare, unpredictable disease with no established, evidence-based treatments. Individual management is based on consensus algorithms. This study aimed to examine the specific health-related quality of life challenges faced by DF patients, current experiences and expectations of care. METHODS: Twenty-seven DF patients were purposively sampled from The Royal Marsden Hospital. Two focus groups and 13 interviews (males 12, females 15; mean age at study 39.5 years) explored health-related quality of life issues and experiences of healthcare. Thematic content was analysed. RESULTS: Discussions revealed four key themes (diagnostic pathway; treatment pathway; living with DF; supportive care). Diagnostic delay resulted from lack of recognition by patients and healthcare professionals. Some patients received an initial diagnosis of cancer, causing significant distress. Treatment decisions were challenging, and patients experienced uncertainty among clinicians about optimal therapies. Side-effects of treatment were severe, including fatigue, nausea, anorexia, low libido and depression. Pain was the most debilitating symptom and dependency on painkillers was a significant concern. Functional limitation and restricted mobility frequently affected daily activities. Patients experienced difficulty accomplishing their role in society; relationship problems, caring for children, employment and financial difficulties. Social isolation and lack of understanding were common. The psychological impact of this "life-changing and life-long" condition was profound. All patients requested knowledgeable healthcare professionals, more information, continuity of care and peer support. CONCLUSIONS: DF patients face complex physical, psychological and practical challenges. Comprehensive care services are needed. Increasing awareness may help to improve diagnostic pathways and overall patient experience.

Altered lower extremity movement variability in female soccer players during side-step cutting after anterior cruciate ligament reconstruction.

BACKGROUND: Anterior cruciate ligament (ACL) reconstruction (ACLR) is common after an ACL tear and is thought to restore functional stability to the knee. A recent investigation demonstrated that individuals who have undergone ACLR exhibited increased lower extremity coupling variability during gait, suggestive of altered dynamic stability. However, little is known about whether they exhibit alterations in lower extremity variability during dynamic sport-specific tasks. PURPOSE: To determine if female soccer players who have had an ACLR demonstrate differences in lower extremity coupling variability as compared with athletes with no history of knee injury during a side-step cutting maneuver. STUDY DESIGN: Controlled laboratory study. METHODS: Ten female soccer players who had undergone ACLR served as the experimental group, and 10 female soccer players with no history of knee ligament injury composed the control group (CON). Three-dimensional kinematics and ground-reaction forces were collected while each participant performed a side-step cutting maneuver. Based on known ACL loading patterns, 7 lower extremity intralimb couplings were created. With use of a vector-coding technique, the coordination variability was calculated for each coupling. Independent t tests were used to determine group differences in variability for each coupling (P ≤ .05). RESULTS: Individuals who had undergone ACLR exhibited increased lower extremity variability during side-step cutting as compared with control subjects in the following couplings: hip rotation/knee abduction-adduction (27.2° ± 11.5° [ACLR] vs 19.7° ± 6.8° [CON]; P = .04), hip flexion-extension/knee abduction-adduction (26.0° ± 13.3° [ACLR] vs 18.6° ± 5.3° [CON]; P = .05), knee abduction-adduction/knee flexion-extension (13.5° ± 5.7° [ACLR] vs 7.3° ± 2.7° [CON]; P < .01), and knee abduction-adduction/knee rotation (26.4° ± 10.8° [ACLR] vs 19.3° ± 4.5° [CON]; P = .03). In addition, there was a trend toward increased variability in the hip rotation/ankle inversion-eversion coupling (22.9° ± 9.3° [ACLR] vs 18.0° ± 6.7° [CON]; P = .09) and knee abduction-adduction/ankle inversion-eversion coupling (25.9° ± 10.0° [ACLR] vs 20.2° ± 9.7° [CON]; P = .10). CONCLUSION: Female soccer players who have undergone ACLR and returned to sports participation exhibit altered lower extremity coupling variability during side-step cutting. CLINICAL RELEVANCE: While individuals who have had an ACLR exhibit mechanical knee stability before returning to sports, the observed increased movement variability during side-step cutting is likely reflective of altered neuromuscular control and may contribute to the known increased risk for ACL reinjury and knee osteoarthritis after return to sports participation. Improving the understanding of altered lower extremity coupling variability after ACLR will aid in the development of more effective rehabilitation programs.

Early molecular and functional changes in colonic epithelium that precede increased gut permeability during colitis development in mdr1a(-/-) mice.

BACKGROUND: The early molecular changes preceding the onset of mucosal inflammation in colitis and their temporal relationship with gut permeability remain poorly defined. This study investigated functional and transcriptomic changes in mdr1a(-/-) mice lacking the intestinal transporter P-glycoprotein, which develop colitis spontaneously when exposed to normal enteric flora. METHODS: Mdr1a(-/-) mice were housed in specific pathogen-free conditions to slow colitis development and compared to congenic controls. Mucosal permeability and cytokine secretion were analyzed in ex vivo colon. Gene expression in colonic mucosal and epithelial preparations was analyzed by microarray and qPCR. Colonocyte responsiveness to bacterial antigens was measured in short-term culture. RESULTS: Colon from 4-5-week-old, disease-free mdr1a(-/-) mice was histologically normal with no evidence of increased permeability compared to controls. However, these tissues display a distinctive pattern of gene expression involving significant changes in a small number of genes. The majority of upregulated genes were associated with bacterial recognition and the ubiquitin-proteasome system and were gamma-interferon (IFN-gamma) responsive. Expression of the antiinflammatory factor pancreatitis-associated protein (PAP) and the related gene RegIIIgamma were markedly reduced. Colonocytes from 4-5-week mdr1a(-/-) exhibit similar transcriptomic changes, accompanied by higher basal chemokine secretion and increased responsiveness to LPS. Significant increases in colonic permeability were associated with older (12-16-week) mdr1a(-/-) mice displaying molecular and functional evidence of active inflammation. CONCLUSIONS: These studies show that early epithelial changes associated with altered responsiveness to bacteria precede increased permeability and mucosal inflammation in this model of colitis, highlighting the importance of P-glycoprotein in regulating interactions with the commensal microflora.

The ETS domain transcription factor Elk-1 regulates the expression of its partner protein, SRF.

The ternary complex factors (TCF) are a subfamily of ETS domain transcription factors that bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). Here, we have identified the SRF gene as a target for the TCFs, thereby providing a positive feedback loop whereby TCF activation leads to the enhancement of the expression of its partner protein SRF. The binding of the TCF Elk-1 to the SRF promoter and subsequent regulation of SRF expression occurs in a ternary complex-dependent manner. Our data therefore reveal that SRF is an important target for the ERK and Rho signaling pathways that converge on a ternary TCF-SRF complex at the SRE on the SRF promoter.

Ternary complex factor-serum response factor complex-regulated gene activity is required for cellular proliferation and inhibition of apoptotic cell death.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.