Multi-variate coding for possession: methodology and preliminary results.

In this work we are presenting a database structure to encode the phenomenon of differential possession across languages, considering noun possession classes and possessive constructions as independent but linked. We show how this structure can be used to study different dimensions of possession: semantics, noun valence, and possessive constructions. We present preliminary survey results from a global sample of 120 languages and show that there is a universal semantic core in both inalienable and non-possessible noun classes. Inalienables are centered on body parts and kinship. Non-possessibles are centered on animals, humans, and natural elements.

Intramuscular vs Intravenous SARS-CoV-2 Neutralizing Antibody Sotrovimab for Treatment of COVID-19 (COMET-TAIL): A Randomized Noninferiority Clinical Trial.

Background: Convenient administration of coronavirus disease 2019 (COVID-19) treatment in community settings is desirable. Sotrovimab is a pan-sarbecovirus dual-action monoclonal antibody formulated for intravenous (IV) or intramuscular (IM) administration for early treatment of mild/moderate COVID-19. Method: This multicenter phase 3 study based on a randomized open-label design tested the noninferiority of IM to IV administration according to an absolute noninferiority margin of 3.5%. From June to August 2021, patients aged ≥12 years with COVID-19, who were neither hospitalized nor receiving supplemental oxygen but were at high risk for progression, were randomized 1:1:1 to receive sotrovimab as a single 500-mg IV infusion or a 500- or 250-mg IM injection. The primary composite endpoint was progression to (1) all-cause hospitalization for >24 hours for acute management of illness or (2) all-cause death through day 29. Results: Sotrovimab 500 mg IM was noninferior to 500 mg IV: 10 (2.7%) of 376 participants vs 5 (1.3%) of 378 met the primary endpoint, respectively (absolute adjusted risk difference, 1.06%; 95% CI, -1.15% to 3.26%). The 95% CI upper limit was lower than the prespecified noninferiority margin of 3.5%. The 250-mg IM group was discontinued early because of the greater proportion of hospitalizations vs the 500-mg groups. Serious adverse events occurred in <1% to 2% of participants across groups. Four participants experienced serious disease-related events and died (500 mg IM, 2/393, <1%; 250 mg IM, 2/195, 1%). Conclusions: Sotrovimab 500-mg IM injection was well tolerated and noninferior to IV administration. IM administration could expand outpatient treatment access for COVID-19. Clinical Trials Registration: ClinicalTrials.gov: NCT04913675.

Multimodal imaging demonstrates enhanced tumor exposure of PEGylated FUD peptide in breast cancer.

In breast cancer, the extracellular matrix (ECM) undergoes remodeling and changes the tumor microenvironment to support tumor progression and metastasis. Fibronectin (FN) assembly is an important step in the regulation of the tumor microenvironment since the FN matrix precedes the deposition of various other ECM proteins, controls immune cell infiltration, and serves as a reservoir for cytokines and growth factors. Therefore, FN is an attractive target for breast cancer therapy and imaging. Functional Upstream Domain (FUD) is a 6-kDa peptide targeting the N-terminal 70-kDa domain of FN, which is critical for fibrillogenesis. FUD has previously been shown to function as an anti-fibrotic peptide both in vitro and in vivo. In this work, we conjugated the FUD peptide with 20-kDa of PEG (PEG-FUD) and demonstrated its improved tumor exposure compared to non-PEGylated FUD in a murine breast cancer model via multiple imaging modalities. Importantly, PEG-FUD peptide retained a nanomolar binding affinity for FN and maintained in vitro plasma stability for up to 48 h. Cy5-labeled PEG-FUD bound to exogenous or endogenous FN assembled by fibroblasts. The in vivo fluorescence imaging with Cy5-labeled FUD and FUD conjugates demonstrated that PEGylation of the FUD peptide enhanced blood exposure after subcutaneous (SC) injection and significantly increased accumulation of FUD peptide in 4T1 mammary tumors. Intravital microscopy confirmed that Cy5-labeled PEG-FUD deposited mostly in the extravascular region of the tumor microenvironment after SC administration. Lastly, positron emission tomography/computed tomography imaging showed that 64Cu-labeled PEG-FUD preferentially accumulated in the 4T1 tumors with improved tumor uptake compared to 64Cu-labeled FUD (48 h: 1.35 ± 0.05 vs. 0.59 ± 0.03 %IA/g, P < 0.001) when injected intravenously (IV). The results indicate that PEG-FUD targets 4T1 breast cancer with enhanced tumor retention compared to non-PEGylated FUD, and biodistribution profiles of PEG-FUD after SC and IV injection may guide the optimization of PEG-FUD as a therapeutic and/or imaging agent for use in vivo.

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant.

Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with extremely high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content in exosomes, but the current methods require laborious and time-consuming multistep sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, to simplify exosomal lysis, effectively extract proteins, and expedite digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3466 proteins and quantified 2288 proteins using a reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. Here, 3166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision that this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.

Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers.

It is well established that collagen alignment in the breast tumor microenvironment provides biophysical cues to drive disease progression. Numerous mechanistic studies have demonstrated that tumor cell behavior is driven by the architecture and stiffness of the collagen matrix. However, the mechanical properties within a 3D collagen microenvironment, particularly at the scale of the cell, remain poorly defined. To investigate cell-scale mechanical cues with respect to local collagen architecture, we employed a combination of intravital imaging of the mammary tumor microenvironment and a 3D collagen gel system with both acellular pNIPAAm microspheres and MDA-MB-231 breast carcinoma cells. Within the in vivo tumor microenvironment, the displacement of collagen fiber was identified in response to tumor cells migrating through the stromal matrix. To further investigate cell-scale stiffness in aligned fiber architectures and the propagation of cell-induced fiber deformations, precise control of collagen architecture was coupled with innovative methodology to measure mechanical properties of the collagen fiber network. This method revealed up to a 35-fold difference in directional cell-scale stiffness resulting from contraction against aligned fibers. Furthermore, the local anisotropy of the matrix dramatically altered the rate at which contractility-induced fiber displacements decayed over distance. Together, our results reveal mechanical properties in aligned matrices that provide dramatically different cues to the cell in perpendicular directions. These findings are supported by the mechanosensing behavior of tumor cells and have important implications for cell-cell communication within the tissue microenvironment. STATEMENT OF SIGNIFICANCE: It is widely appreciated that the architecture of the extracellular matrix impacts cellular behavior in normal and disease states. Numerous studies have determined the fundamental role of collagen matrix architecture on cellular mechanosensing, but effectively quantifying anisotropic mechanical properties of the collagen matrix at the cell-scale remains challenging. Here, we developed innovative methodology to discover that collagen alignment results in a 35-fold difference in cell-scale stiffness and alters contractile force transmission through the fiber network. Furthermore, we identified bias in cell response along the axis of alignment, where local stiffness is highest. Overall, our results define cell-scale stiffness and fiber deformations due to collagen architecture that may instruct cell communication within a broad range of tissue microenvironments.

Hyperpolarized 13C Magnetic Resonance Spectroscopic Imaging of Pyruvate Metabolism in Murine Breast Cancer Models of Different Metastatic Potential.

This study uses dynamic hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (MRSI) to estimate differences in glycolytic metabolism between highly metastatic (4T1, n = 7) and metastatically dormant (4T07, n = 7) murine breast cancer models. The apparent conversion rate of pyruvate-to-lactate (kPL) and lactate-to-pyruvate area-under-the-curve ratio (AUCL/P) were estimated from the metabolite images and compared with biochemical metabolic measures and immunohistochemistry (IHC). A non-significant trend of increasing kPL (p = 0.17) and AUCL/P (p = 0.11) from 4T07 to 4T1 tumors was observed. No significant differences in tumor IHC lactate dehydrogenase-A (LDHA), monocarboxylate transporter-1 (MCT1), cluster of differentiation 31 (CD31), and hypoxia inducible factor-α (HIF-1α), tumor lactate-dehydrogenase (LDH) activity, or blood lactate or glucose levels were found between the two tumor lines. However, AUCL/P was significantly correlated with tumor LDH activity (ρspearman = 0.621, p = 0.027) and blood glucose levels (ρspearman = -0.474, p = 0.042). kPL displayed a similar, non-significant trend for LDH activity (ρspearman = 0.480, p = 0.114) and blood glucose levels (ρspearman = -0.414, p = 0.088). Neither kPL nor AUCL/P were significantly correlated with blood lactate levels or tumor LDHA or MCT1. The significant positive correlation between AUCL/P and tumor LDH activity indicates the potential of AUCL/P as a biomarker of glycolytic metabolism in breast cancer models. However, the lack of a significant difference between in vivo tumor metabolism for the two models suggest similar pyruvate-to-lactate conversion despite differing metastatic potential.

Rejewski & Enigma.

Alan Turing and Bletchley Park are rightly recognized for their work on breaking the Enigma code. However, this was built on a foundation of work during the 1930s by the Polish cryptographer, Marian Rejewski. Often working alone, and with limited resources, he found ways to break early Enigma code. This article attempts to highlight the man and his invaluable contribution.

Photocleavable Surfactant-Enabled Extracellular Matrix Proteomics.

The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains challenging owing to the extremely low solubility of the ECM. Herein, enabled by effective solubilization of ECM proteins using our recently developed photocleavable surfactant, Azo, we have developed a streamlined ECM proteomic strategy that allows fast tissue decellularization, efficient extraction and enrichment of ECM proteins, and rapid digestion prior to reversed-phase liquid chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM proteins from mouse mammary tumors have been identified using 1D and 2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229 (from 2DLC-MS/MS) post-translational modifications of ECM proteins, including glycosylation, phosphorylation, and hydroxylation, were identified and localized. This Azo-enabled ECM proteomics strategy will streamline the analysis of ECM proteins and promote the study of ECM biology.

A live cell reporter of exosome secretion and uptake reveals pathfinding behavior of migrating cells.

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.

Fibrillar Collagen Quantification With Curvelet Transform Based Computational Methods.

Quantification of fibrillar collagen organization has given new insight into the possible role of collagen topology in many diseases and has also identified candidate image-based bio-markers in breast cancer and pancreatic cancer. We have been developing collagen quantification tools based on the curvelet transform (CT) algorithm and have demonstrated this to be a powerful multiscale image representation method due to its unique features in collagen image denoising and fiber edge enhancement. In this paper, we present our CT-based collagen quantification software platform with a focus on new features and also giving a detailed description of curvelet-based fiber representation. These new features include C++-based code optimization for fast individual fiber tracking, Java-based synthetic fiber generator module for method validation, automatic tumor boundary generation for fiber relative quantification, parallel computing for large-scale batch mode processing, region-of-interest analysis for user-specified quantification, and pre- and post-processing modules for individual fiber visualization. We present a validation of the tracking of individual fibers and fiber orientations by using synthesized fibers generated by the synthetic fiber generator. In addition, we provide a comparison of the fiber orientation calculation on pancreatic tissue images between our tool and three other quantitative approaches. Lastly, we demonstrate the use of our software tool for the automatic tumor boundary creation and the relative alignment quantification of collagen fibers in human breast cancer pathology images, as well as the alignment quantification of in vivo mouse xenograft breast cancer images.

MRI of the joint and evaluation of the granulocyte-macrophage colony-stimulating factor-CCL17 axis in patients with rheumatoid arthritis receiving otilimab: a phase 2a randomised mechanistic study.

BACKGROUND: Otilimab is a human monoclonal antibody that inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF), a driver in many immune-mediated inflammatory conditions. We evaluated the effect of otilimab on the GM-CSF-chemokine (C-C motif) ligand 17 (CCL17) axis and synovitis in patients with rheumatoid arthritis. METHODS: This phase 2a, randomised, double-blind, multicentre, placebo-controlled, parallel-group study was done at nine sites across the USA, Poland, and Germany. Patients aged 18 years or older with rheumatoid arthritis per American College of Rheumatology-European League Against Rheumatism 2010 criteria and receiving stable methotrexate were randomly assigned (3:1) by an interactive response technology system to either subcutaneous otilimab 180 mg or placebo once weekly for 5 weeks, then every other week until week 10 (within a 12-week treatment period), followed by a 10-week safety follow-up. Randomisation was stratified by early rheumatoid arthritis (≤2 years since diagnosis) and established rheumatoid arthritis (>2 years since diagnosis). Patients and study personnel (except for an unblinded coordinator or nurse who prepared and administered the study drug) were blinded to treatment assignment; the syringe was shielded during administration. Patients were enrolled by study investigators and allocated to a treatment by central randomisation on the basis of a schedule generated by the sponsor. The primary endpoint was change over time (assessed at baseline and weeks 1, 2, 4, 6, 8, 12, and 22 of follow-up) in 112 biomarkers, including target engagement biomarkers and those that may be indicative of rheumatoid arthritis disease activity and response to otilimab. Secondary endpoints were change from baseline in synovitis, osteitis and erosion assessed by rheumatoid arthritis MRI scoring system (RAMRIS) and rheumatoid arthritis MRI quantitative score (RAMRIQ), and safety evaluation. The primary, secondary, and safety endpoints were assessed in the intention-to-treat population. Biomarker and MRI endpoints were analysed for differences between treatment groups using a repeated measures model. This study is registered with ClinicalTrials.gov, NCT02799472. FINDINGS: Between Aug 9, 2016, and Oct 30, 2017, 39 patients were randomly assigned and included in the analysis (otilimab n=28; placebo n=11). In the otilimab group, mean serum concentrations of GM-CSF-otilimab complex peaked at week 4 (138·4 ng/L, 95% CI 90·0-212·9) but decreased from week 6-12. CCL17 concentrations decreased from baseline to week 1, remained stable to week 8, and returned to baseline at week 12; least-squares mean ratio to baseline was 0·65 (95% CI 0·49-0·86; coefficient of variation 13·60) at week 2, 0·68 (0·53-0·88; 12·51) at week 4, 0·78 (0·60-1·00; 12·48) at week 6, and 0·68 (0·54-0·85; 11·21) at week 8. No meaningful change in CCL17 concentrations was observed with placebo. In the otilimab group, the least-squares mean ratio to baseline in MMP-degraded type I collagen was 0·86-0·91 over weeks 1-8, returning to baseline at week 12; concentrations remained above baseline at all timepoints in the placebo group. There were no observable differences between otilimab and placebo for all other biomarkers. At week 12, least-squares mean change in RAMRIS synovitis score from baseline was -1·3 (standard error [SE] 0·6) in the otilimab group and 0·8 (1·2) with placebo; RAMRIQ synovitis score showed a least-squares mean change from baseline of -1417·0 µl (671·5) in the otilimab group and -912·3 µl (1405·8) with placebo. Compared with placebo, otilimab did not show significant reductions from baseline to week 12 in RAMRIS synovitis, osteitis and bone erosion, or in RAMRIQ synovitis and erosion damage. Adverse events were reported in 11 (39%) of 28 otilimab-treated and four (36%) of 11 placebo-treated patients, most commonly cough in the otilimab group (2 [7%] of 28; not reported in placebo group), and pain in extremity (four [36%] of 11) and rheumatoid arthritis (two [18%] of 11) in the placebo group (not reported in otilimab group). There were no serious adverse events or deaths. INTERPRETATION: Serum concentrations of GM-CSF-otilimab complex indicated that target engagement was achieved with initial weekly dosing, but not sustained with every other week dosing. CCL17 might be a pharmacodynamic biomarker for otilimab activity in future studies. Otilimab was well tolerated and, despite suboptimal exposure, showed some evidence for improved synovitis over 12 weeks in patients with active rheumatoid arthritis. FUNDING: GlaxoSmithKline.

Efficacy, patient-reported outcomes, and safety of the anti-granulocyte macrophage colony-stimulating factor antibody otilimab (GSK3196165) in patients with rheumatoid arthritis: a randomised, phase 2b, dose-ranging study.

BACKGROUND: The human monoclonal antibody otilimab inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF), a key driver in immune-mediated inflammatory conditions. We aimed to evaluate the efficacy, safety, and key patient-reported outcomes related to pain in patients with active rheumatoid arthritis receiving otilimab. METHODS: This phase 2b, dose-ranging, multicentre, placebo-controlled study was done at 64 sites across 14 countries. Patients aged 18 years or older with rheumatoid arthritis who were receiving stable methotrexate were randomly assigned (1:1:1:1:1:1) to subcutaneous placebo or otilimab 22·5 mg, 45 mg, 90 mg, 135 mg, or 180 mg, plus methotrexate, once weekly for 5 weeks, then every other week until week 50. The randomisation schedule was generated by the sponsor, and patients were assigned to treatment by interactive response technology. Randomisation was blocked (block size of six) but was not stratified. Investigators, patients, and the sponsor were blinded to treatment. An unblinded administrator prepared and administered the study drug. The primary endpoint was the proportion of patients who achieved disease activity score for 28 joints with C-reactive protein (DAS28-CRP) <2·6 at week 24. Patients who were not in the otilimab 180 mg group, without a good or moderate European League Against Rheumatism response (week 12) or with DAS28-CRP >3·2 (week 24) escaped to otilimab 180 mg. Patients who escaped were treated as non-responders in their original assigned group. Safety endpoints were incidence of adverse events and serious adverse events, infections, and pulmonary events. Efficacy and safety outcomes were assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02504671. FINDINGS: Between July 23, 2015, and Dec 29, 2017, 222 patients were randomly assigned (37 to each group). 86 (49%) of 175 escaped to otilimab 180 mg at week 12 and 57 (69%) of 83 at week 24. At week 24, the proportion of patients with DAS28-CRP <2·6 was two (5%) of 37 in the otilimab 22·5 mg group, six (16%) of 37 in the 45 mg group, seven (19%) of 37 in the 90 mg group, five (14%) of 37 in the 135 mg group, five (14%) of 37 in the 180 mg, and one (3%) of 37 in the placebo group. The largest difference was achieved with otilimab 90 mg (16·2%; odds ratio [OR] 8·39, 95% CI 0·98-72·14; p=0·053). Adverse events were reported pre-escape in 19-24 (51-65%) patients and post escape in 10-17 (40-61%) patients across otilimab dose groups and in 18 (49%) of 37 and 22 (67%) of 33 in the placebo group. The most common adverse event was nasopharyngitis: 3-9 (8-24%) in otilimab groups and one (3%) in the placebo group pre-escape and 1-3 (4-10%) in otilimab groups and seven (21%) in the placebo group post escape. Pre-escape serious adverse events were foot fracture (otilimab 45 mg); arthralgia, myocardial infarction, dizziness (otilimab 90 mg); oesophageal spasm, acute pyelonephritis (otilimab 22·5 mg), and uterine leiomyoma (otilimab 135 mg). Post-escape serious adverse events were ankle fracture (placebo) and rheumatoid arthritis (otilimab 135 mg). There were no deaths or pulmonary events of clinical concern, and rates of serious infection were low. INTERPRETATION: Otilimab plus methotrexate was well tolerated and, despite not achieving the primary endpoint of DAS28-CRP remission, there were improvements compared with placebo in disease activity scores. Of note, patients reported significant improvement in pain and physical function, supporting further clinical development of otilimab in rheumatoid arthritis. FUNDING: GlaxoSmithKline.

Target engagement and cellular fate of otelixizumab: a repeat dose escalation study of an anti-CD3ε mAb in new-onset type 1 diabetes mellitus patients.

AIMS: This paper describes the pharmacological findings from a study where otelixizumab, an anti-CD3ɛ mAb, was dosed in new onset Type 1 diabetes mellitus (NOT1DM) patients. This is the first time that the full dose-response of an anti-CD3ɛ mAb has been investigated in the clinic. The data have been validated using a previously developed pharmacokinetic/pharmacodynamic (PK/PD) model of otelixizumab to simulate the interplay between drug administration, CD3ɛ target engagement and downmodulation. METHODS: Patients were randomized to control or active treatment with otelixizumab (1:4), administered via infusion over 6 days, in a dose-ascending study consisted of three cohorts (n = 10 per cohort) at doses of 9, 18 or 27 mg respectively. The study allowed quantification of otelixizumab PK, CD3ɛ target engagement and its pharmacodynamic effect (CD3ε/TCR modulation on circulating T lymphocytes). RESULTS: Otelixizumab concentrations increased and averaged to 364.09 (54.3), 1625.55 (72.5) and 2781.35 (28.0) ng ml-1 (Geom.mean, %CV) at the 9, 18 and 27 mg dose respectively. CD3ɛ target engagement was found to be rapid (within the first 30 min), leading to a receptor occupancy of ~60% within 6 h of dosing in all three doses. A dose-response relationship was observed with the two highest doses achieving a ~90% target engagement and consequential CD3ɛ/TCR downmodulation by Day 6. CONCLUSIONS: Data from this study revealed maximum target engagement and CD3ɛ/TCR modulation is achieved at doses of 18, 27 mg of otelixizumab. These findings can be useful in guiding dose selection in clinical trials with anti-CD3ɛ mAbs.

Mechano-Signal Transduction in Mesenchymal Stem Cells Induces Prosaposin Secretion to Drive the Proliferation of Breast Cancer Cells.

In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here, we show that ECM stiffness leads to mechano-signal transduction in MSC, which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of α-smooth muscle actin, a marker of CAF, compared with MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin that promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells. Cancer Res; 77(22); 6179-89. ©2017 AACR.

Low risk of concussions in top-level karate competition.

BACKGROUND: Although it is well known that injuries occur in combat sports, the true incidence of concussions is not clearly defined in the literature for karate competition. AIM: To determine the incidence of concussions in top-level (World Karate Federation World Championships) karate competition. METHODS: Injuries that took place in 4 consecutive World Karate Championships (from 2008 to 2014) were prospectively registered. A total of 4625 fights (2916 in the male category and 1709 in the female category) were scrutinised, and concussions were identified and analysed separately for frequency (rate per fight) and injury risk. RESULTS: A total of 4 concussions were diagnosed by the attending physicians after carrying out athlete examinations. Globally, there was 1 concussion in every 1156 fights, or 0.43/1000 athlete-exposures (AE). In male athletes, the rate of concussion was 1/5832 min of fighting, and in female athletes, it was 1/6836 min. OR for concussion in women is 0.57 (95% CI 0.06 to 5.47; z=0.489; p=0.6249) and risk ratio for concussions in men is RR 1.478 (95% CI 0.271 to 8.072), p=0.528, representing a higher risk of definite concussions in men than in women, but not statistically significant. There is not a significantly higher risk of concussions in team competition (no weight limit) when compared with individual competition (held with strict weight limits for each category). CONCLUSION: The risk of concussions in top-level karate competition is low, with a tendency for an increased risk for men and for competition without weight limits, but not statistically significant with respect to women or individual competition.

In Vivo Visualization of Stromal Macrophages via label-free FLIM-based metabolite imaging.

Macrophage infiltration and recruitment in breast tumors has been correlated with poor prognosis in breast cancer patients and has been linked to tumor cell dissemination. Much of our understanding comes from animal models in which macrophages are labeled by expression of an extrinsic fluorophore. However, conventional extrinsic fluorescence labeling approaches are not readily applied to human tissue and clinical use. We report a novel strategy that exploits endogenous fluorescence from the metabolic co-factors NADH and FAD with quantitation from Fluorescence Lifetime Imaging Microscopy (FLIM) as a means to non-invasively identify tumor-associated macrophages in the intact mammary tumor microenvironment. Macrophages were FAD(HI) and demonstrated a glycolytic-like NADH-FLIM signature that was readily separated from the intrinsic fluorescence signature of tumor cells. This non-invasive quantitative technique provides a unique ability to discern specific cell types based upon their metabolic signatures without the use of exogenous fluorescent labels. Not only does this provide high resolution temporal and spatial views of macrophages in live animal breast cancer models, this approach can be extended to other animal disease models where macrophages are implicated and has potential for clinical applications.

Neutrophils drive accelerated tumor progression in the collagen-dense mammary tumor microenvironment.

BACKGROUND: High mammographic density has been correlated with a 4-fold to 6-fold increased risk of developing breast cancer, and is associated with increased stromal deposition of extracellular matrix proteins, including collagen I. The molecular and cellular mechanisms responsible for high breast tissue density are not completely understood. METHODS: We previously described accelerated tumor formation and metastases in a transgenic mouse model of collagen-dense mammary tumors (type I collagen-α1 (Col1α1)(tm1Jae) and mouse mammary tumor virus - polyoma virus middle T antigen (MMTV-PyVT)) compared to wild-type mice. Using ELISA cytokine arrays and multi-color flow cytometry analysis, we studied cytokine signals and the non-malignant, immune cells in the collagen-dense tumor microenvironment that may promote accelerated tumor progression and metastasis. RESULTS: Collagen-dense tumors did not show any alteration in immune cell populations at late stages. The cytokine signals in the mammary tumor microenvironment were clearly different between wild-type and collagen-dense tumors. Cytokines associated with neutrophil signaling, such as granulocyte monocyte-colony stimulated factor (GM-CSF), were increased in collagen-dense tumors. Depleting neutrophils with anti-Ly6G (1A8) significantly reduced the number of tumors, and blocked metastasis in over 80 % of mice with collagen-dense tumors, but did not impact tumor growth or metastasis in wild-type mice. CONCLUSION: Our study suggests that tumor progression in a collagen-dense microenvironment is mechanistically different, with pro-tumor neutrophils, compared to a non-dense microenvironment.

COX-2 modulates mammary tumor progression in response to collagen density.

BACKGROUND: High breast density is linked to an increased risk of breast cancer, and correlates with changes in collagen. In a mouse model of mammary carcinoma in the context of increased collagen deposition, the MMTV-PyMT/Col1a1 (tm1jae) , there is accelerated mammary tumor formation and progression. Previous gene expression analysis suggests that increased collagen density elevates expression of PTGS2 (prostaglandin-endoperoxide synthase 2), the gene for cyclooxygenase-2 (COX-2). METHODS: To understand the role of COX-2 in tumor progression within a collagen-dense microenvironment, we treated MMTV-PyMT or MMTV-PyMT/Col1a1 (tm1jae) tumors prior to and after tumor formation. Animals received treatment with celecoxib, a specific COX-2 inhibitor, or placebo. Mammary tumors were examined for COX-2, inflammatory and stromal cell components, and collagen deposition through immunohistochemical analysis, immunofluorescence, multiplex cytokine ELISA and tissue imaging techniques. RESULTS: PyMT/Col1a1 (tm1jae) tumors were larger, more proliferative, and expressed higher levels of COX-2 and PGE2 than PyMT tumors in wild type (WT) mice. Treatment with celecoxib significantly decreased the induced tumor size and metastasis of the PyMT/Col1a1 tumors, such that their size was not different from the smaller PyMT tumors. Celecoxib had minimal effect on the PyMT tumors. Celecoxib decreased expression levels of COX-2, PGE2, and Ki-67. Several cytokines were over-expressed in PyMT/Col1a1 compared to PyMT, and celecoxib treatment prevented their over-expression. Furthermore, macrophage and neutrophil recruitment were enhanced in PyMT/Col1a1 tumors, and this effect was inhibited by celecoxib. Notably, COX-2 inhibition reduced overall collagen deposition. Finally, when celecoxib was used prior to tumor formation, PyMT/Col1a1 tumors were fewer and smaller than in untreated animals. CONCLUSION: These findings suggest that COX-2 has a direct role in modulating tumor progression in tumors arising within collagen-dense microenvironments, and suggest that COX-2 may be an effective therapeutic target for women with dense breast tissue and early-stage breast cancer.

Low Risk of Injuries in Young Adolescents Participating in Top-Level Karate Competition.

BACKGROUND: Prospective studies on injuries in martial arts competition are scarce, especially those involving young practitioners, but the upsurge of children and adolescents taking part in organized training and competition in these sports requires clarification of the injury risk that they represent for youths. HYPOTHESIS: Top-level karate competition for young adolescents (cadets, or 14- to 15-year-olds) has a low injury rate and can be safely promoted. STUDY DESIGN: Descriptive epidemiological study. METHODS: Prospective recording of the injuries resulting from all bouts in 3 consecutive World Karate Championships (2009, 2011, and 2013) for cadets was performed. Data were collected prospectively in situ with checklists that described competitor sex, bout category, and weight as well as injured area, diagnosis, mechanism of injury, severity, and treatment. RESULTS: A total of 1020 bouts were reviewed, 671 in the male category and 349 in the female category. A total of 61 injuries were recorded. Of those, only 3 were time-loss injuries. During the 2009 and 2011 championships, there was 1 injury per 25.6 fights, while during the 2013 championship the number of injuries increased, with 1 injury per 10 fights (P = .003). There was no statistical difference in the total injury rate between the male and female categories (P = .71), with an odds ratio of 1.16 (95% CI, 0.52-2.55). CONCLUSION: The injury rate for cadet top-level karate competition found in this prospective study is much lower than the rates previously published for karate or other martial arts competitions, but there seems to be a marked increase as more championships are held, which is a matter of concern.