Single-cell and spatial profiling identify three response trajectories to pembrolizumab and radiation therapy in triple negative breast cancer.

Strategies are needed to better identify patients that will benefit from immunotherapy alone or who may require additional therapies like chemotherapy or radiotherapy to overcome resistance. Here we employ single-cell transcriptomics and spatial proteomics to profile triple negative breast cancer biopsies taken at baseline, after one cycle of pembrolizumab, and after a second cycle of pembrolizumab given with radiotherapy. Non-responders lack immune infiltrate before and after therapy and exhibit minimal therapy-induced immune changes. Responding tumors form two groups that are distinguishable by a classifier prior to therapy, with one showing high major histocompatibility complex expression, evidence of tertiary lymphoid structures, and displaying anti-tumor immunity before treatment. The other responder group resembles non-responders at baseline and mounts a maximal immune response, characterized by cytotoxic T cell and antigen presenting myeloid cell interactions, only after combination therapy, which is mirrored in a murine model of triple negative breast cancer.

HEG1 Protects Against Atherosclerosis by Regulating Stable Flow-Induced KLF2/4 Expression in Endothelial Cells.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow-induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA-mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1iECKO) mice. To determine the role of HEG1 in atherosclerosis, HEG1iECKO and littermate-control mice were injected with an adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow-induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow-induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

Characterization of CSF inflammatory markers after hemorrhagic stroke and their relationship to disease severity.

Background: The inflammatory response within the central nervous system is a key driver of secondary brain injury after hemorrhagic stroke, both in patients with intracerebral hemorrhage (ICH) and aneurysmal subarachnoid hemorrhage (aSAH). In this study, we aimed to characterize inflammatory molecules in the blood and cerebrospinal fluid (CSF) of patients within 72 hours of hemorrhage to understand how such molecules vary across disease types and disease severity. Methods: Biological samples were collected from patients admitted to a single-center Neurosciences Intensive Care Unit with a diagnosis of ICH or aSAH between 2014 and 2022. Control CSF samples were collected from patients undergoing CSF diversion for normal pressure hydrocephalus. A panel of immune molecules in the plasma and CSF samples was analyzed using Cytometric Bead Array assays. Clinical variables, including demographics, disease severity, and intensive care unit length of stay were collected. Results: Plasma and/or CSF samples were collected from 260 patients (188 ICH patients, 54 aSAH patients, 18 controls). C-C motif chemokine ligand-2 (CCL2), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF), were detectable in the CSF within the first 3 days after hemorrhage, and all were elevated compared to plasma. Compared with controls, CCL2, IL-6, IL-8, G-CSF, and VEGF were elevated in the CSF of both ICH and aSAH patients (p<0.01 for all comparisons). VEGF was increased in ICH patients compared to aSAH patients (p<0.01). CCL2, G-CSF, and VEGF in the CSF were associated with more severe disease in aSAH patients only. Conclusions: Within 3 days of hemorrhagic stroke, proinflammatory molecules can be detected in the CSF at higher concentrations than in the plasma. Early concentrations of some pro-inflammatory molecules may be associated with markers of disease severity.

Using Adversarial Images to Assess the Robustness of Deep Learning Models Trained on Diagnostic Images in Oncology.

PURPOSE: Deep learning (DL) models have rapidly become a popular and cost-effective tool for image classification within oncology. A major limitation of DL models is their vulnerability to adversarial images, manipulated input images designed to cause misclassifications by DL models. The purpose of the study is to investigate the robustness of DL models trained on diagnostic images using adversarial images and explore the utility of an iterative adversarial training approach to improve the robustness of DL models against adversarial images. METHODS: We examined the impact of adversarial images on the classification accuracies of DL models trained to classify cancerous lesions across three common oncologic imaging modalities. The computed tomography (CT) model was trained to classify malignant lung nodules. The mammogram model was trained to classify malignant breast lesions. The magnetic resonance imaging (MRI) model was trained to classify brain metastases. RESULTS: Oncologic images showed instability to small pixel-level changes. A pixel-level perturbation of 0.004 (for pixels normalized to the range between 0 and 1) resulted in most oncologic images to be misclassified (CT 25.6%, mammogram 23.9%, and MRI 6.4% accuracy). Adversarial training improved the stability and robustness of DL models trained on oncologic images compared with naive models ([CT 67.7% v 26.9%], mammogram [63.4% vs 27.7%], and MRI [87.2% vs 24.3%]). CONCLUSION: DL models naively trained on oncologic images exhibited dramatic instability to small pixel-level changes resulting in substantial decreases in accuracy. Adversarial training techniques improved the stability and robustness of DL models to such pixel-level changes. Before clinical implementation, adversarial training should be considered to proposed DL models to improve overall performance and safety.

Commensal bacteria and fungi differentially regulate tumor responses to radiation therapy.

Studies suggest that the efficacy of cancer chemotherapy and immunotherapy is influenced by intestinal bacteria. However, the influence of the microbiome on radiation therapy is not as well understood, and the microbiome comprises more than bacteria. Here, we find that intestinal fungi regulate antitumor immune responses following radiation in mouse models of breast cancer and melanoma and that fungi and bacteria have opposite influences on these responses. Antibiotic-mediated depletion or gnotobiotic exclusion of fungi enhances responsiveness to radiation, whereas antibiotic-mediated depletion of bacteria reduces responsiveness and is associated with overgrowth of commensal fungi. Further, elevated intratumoral expression of Dectin-1, a primary innate sensor of fungi, is negatively associated with survival in patients with breast cancer and is required for the effects of commensal fungi in mouse models of radiation therapy.

Functionally distinct tendons have different biomechanical, biochemical and histological responses to in vitro unloading.

Tendons with different in vivo functions are known to have different baseline biomechanics, biochemistry and ultrastructure, and these can be affected by changes in loading. However it is not know whether different tendon types respond in the same, or different ways, to changes in loading. This study performed in vitro un-loading (stress deprivation) in culture on ovine medial extensor tendons (MET, a positional tendon), and superficial and deep digital flexor tendons (SDFTs and DDFTs, with energy-storing and intermediate functions respectively), for 21 days (n = 14 each). Tensile strength and elastic modulus were then measured, followed by biochemical assays for sulphated glycosaminoglycan (sGAG) and hydroxyproline content. Histological inspection for cell morphology, cell density and collagen alignment was also performed. The positional tendon (MET) had a significant reduction (∼50%) in modulus and strength (P < 0.001) after in vitro stress-deprivation, however there were no significant effects on the energy-storing tendons (SDFT and DDFT). In contrast, sGAG was not affected in the MET, but was reduced in the SDFT and DDFT (P < 0.001). All tendons lost compactness and collagen organisation, and had reduced cell density, but these were more rapid in the MET than the SDFT and DDFT. These results suggest that different tendon types respond to identical stimuli in different ways, thus; (i) the results from an experiment in one tendon type may not be as applicable to other tendon types as previously thought, (ii) positional tendons may be particularly vulnerable to clinical stress-deprivation, and (iii) graft tendon source may affect the biological response to loading in ligament and tendon reconstruction.

Current and future molecular diagnostics of gastric cancer.

Introduction: Gastric cancer (GC) is the fifth most common cancer and confers the second-highest mortality among other cancers. Improving the survival rates of GC patients requires prompt and accurate diagnosis and effective treatment which is often preceded by the poorly understood pathogenic mechanisms. Area covered: This literature review aims to summarize current understanding of genetic and molecular alterations that promote carcinogenesis including (1) activation of oncogenes, (2) overexpression of growth factors, receptors and matrix metalloproteinases, (3) inactivation of tumor suppressor genes, DNA repair genes, and cell adhesion molecules and (4) alterations of cell-cycle regulators that regulate biological characteristics of cancer cells. Moreover, the significance of molecular biomarkers such as micro-RNAs (miRNAs) and long non-coding RNAs (lncRNAs) and advanced molecular techniques including droplet digital polymerase chain reaction (ddPCR), quantitative PCR (qPCR) and next-generation sequencing (NGS) are also discussed. Expert opinion: A GC-specific panel of biomarkers based on the NGS or ddPCR has the potential for diagnosis, prognosis, and monitoring treatment response in GC patients. Despite the requirements for validation in larger population in clinical studies, race-specific differences in the gene panel have also to be examined by performing the clinical trials in subjects with different races.

Clinical value of transoral robotic surgery: Nationwide results from the first 5 years of adoption.

OBJECTIVES: To compare long-term oncologic outcomes and adjuvant therapies for patients treated with transoral robotic surgery (TORS), nonrobotic surgery, or transoral laser microsurgery (TLM). STUDY DESIGN: A retrospective analysis of the National Cancer Database (2010-2014). METHODS: Patients with clinical tumor (T)1 and T2 oropharyngeal squamous cell carcinomas (OPSCC) were classified into those receiving TORS versus nonrobotic surgery versus TLM. Univariate and multivariate survival analyses were conducted with chi-square tests; Kaplan-Meier log-rank test; and Cox multivariate, logistic regression, and multinomial regression modeling. RESULTS: We identified 2,224 OPSCC TORS patients; 6,697 nonrobotic surgery patients; and 333 TLM patients. The majority of patients were white males with a mean age of approximately 59 years. No significant difference was noted between the cohorts in tumor stage; however, TORS patients were more likely to have nodal (N)1 to N3 disease than nonrobotic surgery and TLM patients, respectively (69.8% vs. 62.0% vs. 59.7%, P < 0.001). TORS was associated with a lower likelihood of positive margins when compared to nonrobotic surgery, although not TLM (nonrobotic surgery: hazard ratio [HR] 1.51, P < 0.001, TLM: HR 1.13, P = 0.582). TORS was associated with lower likelihood of postsurgical chemoradiotherapy (TLM: HR 2.07, P < 0.001, nonrobotic surgery: 1.65, P < 0.001) but not adjuvant radiotherapy alone (TLM: HR 1.06, P = 0.569, nonrobotic surgery: 0.96, P = 0.655). On multivariate Cox analysis of overall survival, the use of TORS was not associated with increased survival (TLM: HR 1.31, P = 0.233, nonrobotic surgery: HR 1.12, P < 0.303). CONCLUSION: The advantages of TORS for early-stage OPSCC may be a lower likelihood of postsurgical positive margins and subsequent need for adjuvant chemoradiotherapy. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1844-1855, 2019.

Cellular, matrix, and mechano-biological differences in load-bearing versus positional tendons throughout development and aging: a narrative review.

PURPOSE: Summarise available evidence comparing the cellular, biochemical, structural and biomechanical properties, and the changes that occur in these parameters in response to stimuli, in differentially loaded tendons across different stages of life. METHODS: The PubMed database was searched for literature pertaining to differences between tendons using the term "tendon" or "tendinopathy", plus one or more of the following descriptors: "loading", "positional", "weight- or load-bearing", and "energy-storing". The abstracts were reviewed and relevant full-length articles retrieved and used to assemble a narrative review. RESULTS: The incidence and prevalence of tendon disorders ("tendinopathies") is increasing in Western societies, with limited evidence that currently available treatments have any significant long-term effect on the disease course. A key emerging hypothesis is that disease in different tendons and even different regions within a tendon may be distinct. The available literature indicates that there are phenotypic differences, not only in the constitutive compositional and material properties but also in resident cells of positional compared with load-bearing tendons. Evident during early tendon growth, such differences have become well established by adulthood. CONCLUSIONS: The pheno-endotype of tendinopathy may be distinct between load-bearing tendons compared to positional tendons, which has translational implications with regard to preventing and managing tendinopathy. Better understanding of the molecular, cellular, and biomechanical pathophysiology underlying disease phenotypes, will allow more targeted/personalised treatment and therefore improve outcomes.

Interval increase in the prevalence of symptomatic cholelithiasis-associated non-alcoholic fatty liver disease over a ten-year period in an Asian population.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is frequently associated with cholelithiasis. The prevalence of NAFLD in Asia has been on the rise, but the magnitude of this increase had not been studied previously. METHODS: A retrospective cohort study was conducted on consecutive patients who underwent laparoscopic or open cholecystectomy from November 2001 to November 2004 (Cohort 1) and from November 2011 to November 2014 (Cohort 2) at Singapore General Hospital. Preoperative diagnostic scans (e.g. ultrasonography, computed tomography and magnetic resonance imaging) and clinical data were reviewed for the presence of fatty liver. Statistical analysis was performed. RESULTS: In Cohorts 1 and 2, 127 patients and 99 patients were operated on, respectively. Cohort 2 had significantly higher proportions of patients with NAFLD (56.6% vs. 40.2%; p < 0.015) and hyperlipidaemia (45.5% vs. 18.9%; p < 0.001) as compared to Cohort 1. Binary logistic regression analysis showed that hypertension (odds ratio [OR] 2.558; p < 0.004) and Indian ethnicity (OR 5.448; p < 0.043) were significantly associated with NAFLD. CONCLUSION: Similar to other international studies, we found a significant increase in the prevalence of patients with NAFLD presenting symptomatically for cholecystectomy over an interval of ten years in Singapore. Hypertension and Indian ethnicity were significantly associated with NAFLD in both time periods. This trend supports the need for concerted public health intervention to stem the increasing incidence of NAFLD and prevent its progression to more advanced liver disease.

Chondroitin sulphate glycosaminoglycans contribute to widespread inferior biomechanics in tendon after focal injury.

Both mechanical and structural properties of tendon change after injury however the causal relationship between these properties is presently unclear. This study aimed to determine the extent of biomechanical change in post-injury tendon pathology and whether the sulphated glycosaminoglycans (glycosaminoglycans) present are a causal factor in these changes. Equine superficial digital flexor tendons (SDF tendons) were surgically-injured in vivo (n=6 injured, n=6 control). Six weeks later they were harvested and regionally dissected into twelve regions around the lesion (equal medial/lateral, proximal/distal). Glycosaminoglycans were removed by enzymatic (chondroitinase) treatment. Elastic modulus (modulus) and ultimate tensile strength (UTS) were measured under uniaxial tension to failure, and tendon glycosaminoglycan content was measured by spectrophotometry. Compared to healthy tendons, pathology induced by the injury decreased modulus (-38%; 95%CI -49% to -28%; P<0.001) and UTS (-38%; 95%CI -48% to -28%; P<0.001) and increased glycosaminoglycan content (+52%; 95%CI 39% - 64%; P<0.001) throughout the tendon. Chondroitinase-mediated glycosaminoglycan removal (50%; 95%CI 21-79%; P<0.001) in surgically-injured pathological tendons caused a significant increase in modulus (5.6MPa/µg removed; 95%CI 0.31-11; P=0.038) and UTS (1.0MPa per µg removed; 95%CI 0.043-2; P=0.041). These results demonstrate that the chondroitin/dermatan sulphate glycosaminoglycans that accumulate in pathological tendon post-injury are partly responsible for the altered biomechanical properties.