Hypoxic Memory Mediates Prolonged Tumor Intrinsic Type I Interferon Suppression to Promote Breast Cancer Progression.

Hypoxia is a common feature of many solid tumors due to aberrant proliferation and angiogenesis that is associated with tumor progression and metastasis. Most of the well-known hypoxia effects are mediated through hypoxia-inducible factors (HIFs). Identification of the long-lasting effects of hypoxia beyond the immediate HIF-induced alterations could provide a better understanding of hypoxia-driven metastasis and potential strategies to circumvent it. Here, we uncovered a hypoxia-induced mechanism that exerts a prolonged effect to promote metastasis. In breast cancer patient-derived circulating tumor cell (CTC) lines and common breast cancer cell lines, hypoxia downregulated tumor intrinsic type I interferon (IFN) signaling and its downstream antigen presentation (AP) machinery in luminal breast cancer cells, via both HIF-dependent and HIF-independent mechanisms. Hypoxia induced durable IFN/AP suppression in certain cell types that was sustained after returning to normoxic conditions, presenting a "hypoxic memory" phenotype. Hypoxic memory of IFN/AP downregulation was established by specific hypoxic priming, and cells with hypoxic memory had an enhanced ability for tumorigenesis and metastasis. Overexpression of IRF3 enhanced IFN signaling and reduced tumor growth in normoxic, but not hypoxic, conditions. The histone deacetylase inhibitor (HDACi) entinostat upregulated IFN targets and erased the hypoxic memory. These results point to a mechanism by which hypoxia facilitates tumor progression through a long-lasting memory that provides advantages for CTCs during the metastatic cascade.

The Space Omics and Medical Atlas (SOMA) and international astronaut biobank.

Spaceflight induces molecular, cellular and physiological shifts in astronauts and poses myriad biomedical challenges to the human body, which are becoming increasingly relevant as more humans venture into space1-6. Yet current frameworks for aerospace medicine are nascent and lag far behind advancements in precision medicine on Earth, underscoring the need for rapid development of space medicine databases, tools and protocols. Here we present the Space Omics and Medical Atlas (SOMA), an integrated data and sample repository for clinical, cellular and multi-omic research profiles from a diverse range of missions, including the NASA Twins Study7, JAXA CFE study8,9, SpaceX Inspiration4 crew10-12, Axiom and Polaris. The SOMA resource represents a more than tenfold increase in publicly available human space omics data, with matched samples available from the Cornell Aerospace Medicine Biobank. The Atlas includes extensive molecular and physiological profiles encompassing genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiome datasets, which reveal some consistent features across missions, including cytokine shifts, telomere elongation and gene expression changes, as well as mission-specific molecular responses and links to orthologous, tissue-specific mouse datasets. Leveraging the datasets, tools and resources in SOMA can help to accelerate precision aerospace medicine, bringing needed health monitoring, risk mitigation and countermeasure data for upcoming lunar, Mars and exploration-class missions.

Collection of biospecimens from the inspiration4 mission establishes the standards for the space omics and medical atlas (SOMA).

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from four crew members longitudinally before (Launch: L-92, L-44, L-3 days), during (Flight Day: FD1, FD2, FD3), and after (Return: R + 1, R + 45, R + 82, R + 194 days) spaceflight, spanning a total of 289 days across 2021-2022. The collection process included venous whole blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies. Venous whole blood was further processed to obtain aliquots of serum, plasma, extracellular vesicles and particles, and peripheral blood mononuclear cells. In total, 2,911 sample aliquots were shipped to our central lab at Weill Cornell Medicine for downstream assays and biobanking. This paper provides an overview of the extensive biospecimen collection and highlights their processing procedures and long-term biobanking techniques, facilitating future molecular tests and evaluations.As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can aid future human spaceflight and space biology experiments.

Single-cell multi-ome and immune profiles of the Inspiration4 crew reveal conserved, cell-type, and sex-specific responses to spaceflight.

Spaceflight induces an immune response in astronauts. To better characterize this effect, we generated single-cell, multi-ome, cell-free RNA (cfRNA), biochemical, and hematology data for the SpaceX Inspiration4 (I4) mission crew. We found that 18 cytokines/chemokines related to inflammation, aging, and muscle homeostasis changed after spaceflight. In I4 single-cell multi-omics data, we identified a "spaceflight signature" of gene expression characterized by enrichment in oxidative phosphorylation, UV response, immune function, and TCF21 pathways. We confirmed the presence of this signature in independent datasets, including the NASA Twins Study, the I4 skin spatial transcriptomics, and 817 NASA GeneLab mouse transcriptomes. Finally, we observed that (1) T cells showed an up-regulation of FOXP3, (2) MHC class I genes exhibited long-term suppression, and (3) infection-related immune pathways were associated with microbiome shifts. In summary, this study reveals conserved and distinct immune disruptions occurring and details a roadmap for potential countermeasures to preserve astronaut health.

Spaceflight induces changes in gene expression profiles linked to insulin and estrogen.

Organismal adaptations to spaceflight have been characterized at the molecular level in model organisms, including Drosophila and C. elegans. Here, we extend molecular work to energy metabolism and sex hormone signaling in mice and humans. We found spaceflight induced changes in insulin and estrogen signaling in rodents and humans. Murine changes were most prominent in the liver, where we observed inhibition of insulin and estrogen receptor signaling with concomitant hepatic insulin resistance and steatosis. Based on the metabolic demand, metabolic pathways mediated by insulin and estrogen vary among muscles, specifically between the soleus and extensor digitorum longus. In humans, spaceflight induced changes in insulin and estrogen related genes and pathways. Pathway analysis demonstrated spaceflight induced changes in insulin resistance, estrogen signaling, stress response, and viral infection. These data strongly suggest the need for further research on the metabolic and reproductive endocrinologic effects of space travel, if we are to become a successful interplanetary species.

Genome and clonal hematopoiesis stability contrasts with immune, cfDNA, mitochondrial, and telomere length changes during short duration spaceflight.

Background: The Inspiration4 (I4) mission, the first all-civilian orbital flight mission, investigated the physiological effects of short-duration spaceflight through a multi-omic approach. Despite advances, there remains much to learn about human adaptation to spaceflight's unique challenges, including microgravity, immune system perturbations, and radiation exposure. Methods: To provide a detailed genetics analysis of the mission, we collected dried blood spots pre-, during, and post-flight for DNA extraction. Telomere length was measured by quantitative PCR, while whole genome and cfDNA sequencing provided insight into genomic stability and immune adaptations. A robust bioinformatic pipeline was used for data analysis, including variant calling to assess mutational burden. Result: Telomere elongation occurred during spaceflight and shortened after return to Earth. Cell-free DNA analysis revealed increased immune cell signatures post-flight. No significant clonal hematopoiesis of indeterminate potential (CHIP) or whole-genome instability was observed. The long-term gene expression changes across immune cells suggested cellular adaptations to the space environment persisting months post-flight. Conclusion: Our findings provide valuable insights into the physiological consequences of short-duration spaceflight, with telomere dynamics and immune cell gene expression adapting to spaceflight and persisting after return to Earth. CHIP sequencing data will serve as a reference point for studying the early development of CHIP in astronauts, an understudied phenomenon as previous studies have focused on career astronauts. This study will serve as a reference point for future commercial and non-commercial spaceflight, low Earth orbit (LEO) missions, and deep-space exploration.

Ectopic Expression of a Truncated Isoform of Hair Keratin 81 in Breast Cancer Alters Biophysical Characteristics to Promote Metastatic Propensity.

Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.

Bone-anchored prostheses for lower limb amputation in a French cohort with 1-15 years of follow-up: implant survival rates, mechanical complications, and reported outcomes.

PURPOSE: To evaluate the implant survival rate, mechanical complications, and reported patient outcomes of bone-anchored prostheses for patients with lower limb amputation in France after 1-15 years of follow-up. METHODS: This retrospective cohort study included patients who underwent surgery at a single center in France between 2007 and 2021. The primary outcomes were the implant survival rate and functional scores assessed by the Questionnaire for Transfemoral Amputees (Q-TFA). Secondary outcomes were adverse events that occurred during follow-up. RESULTS: The cohort consisted of 20 bone-anchored prostheses in 17 patients. The main level of amputation was transfemoral (82%, n = 14). The main reason for amputation was trauma (n = 15). The mean age at amputation was 32 (range 15-54) years, and the mean age at the first stage of osseointegration was 41 (range 21-58) years. The Kaplan-Meier survival curve showed respective survival rates of 90%, 70%, and 60% at 2, 10, and 15 years. All Q-TFA scores were significantly improved at last the follow-up. Eleven patients (65%) experienced mechanical complications. In total, 37 infectious events occurred in 13 patients (76%), mainly comprising stage 1 infections (68%, n = 25). Only two cases of septic loosening occurred (12%), leading to implant removal. CONCLUSION: This is the first French cohort of bone-anchored prostheses and among the series with the longest follow-up periods. The findings indicate that bone-anchored prostheses are safe and reliable for amputee patients who have difficulties with classic prostheses.

Intuitive movement-based prosthesis control enables arm amputees to reach naturally in virtual reality.

Impressive progress is being made in bionic limbs design and control. Yet, controlling the numerous joints of a prosthetic arm necessary to place the hand at a correct position and orientation to grasp objects remains challenging. Here, we designed an intuitive, movement-based prosthesis control that leverages natural arm coordination to predict distal joints missing in people with transhumeral limb loss based on proximal residual limb motion and knowledge of the movement goal. This control was validated on 29 participants, including seven with above-elbow limb loss, who picked and placed bottles in a wide range of locations in virtual reality, with median success rates over 99% and movement times identical to those of natural movements. This control also enabled 15 participants, including three with limb differences, to reach and grasp real objects with a robotic arm operated according to the same principle. Remarkably, this was achieved without any prior training, indicating that this control is intuitive and instantaneously usable. It could be used for phantom limb pain management in virtual reality, or to augment the reaching capabilities of invasive neural interfaces usually more focused on hand and grasp control.

Daily socket comfort in transtibial amputee with a vacuum-assisted suspension system: study protocol of a randomized, multicenter, double-blind multiple N-of-1 trial.

BACKGROUND: The main aim of this paper is to present the feasibility of rigorously designed multiple N-of-1 design in prosthetics research. While research of adequate power and high quality is often lacking in rehabilitation, N-of-1 trials can offer a feasible alternative to randomized controlled group trials, both increasing design power at group level and allowing a rigorous, statistically confirmed evaluation of effectiveness at a single patient level. The paper presents a multiple N-of-1 trial protocol, which aim is to evaluate the effectiveness of Unity, a prosthetic add-on suspension system for amputees, on patient-reported comfort during daily activities (main outcome measure), prosthesis wearing time, perception of limb-prosthesis fitting and stump volume and functional walking parameters. METHODS: Multicenter, randomized, prospective, double-blind multiple N-of-1 trial using an introduction/withdrawal design alternating Unity connected/disconnected phases of randomized length on twenty patients with unilateral transtibial amputation. The primary outcome measure is the Prosthetic Socket Comfort Score (SCS), a validated measure of comfort, administered daily by an phone app designed for the study. Secondary outcomes measures will be collected during the 50 days period of the N-of-1 trial: (1) by the same app, daily for patient-reported limb-prosthesis fitting, stump volume variation, and daily wearing time of the prosthesis; (2) by a pedometer for the number of steps per day; (3) by blind assessors in the rehabilitation center during adjustment visits for functional walking parameter (L-Test, 6-minute walk test), and by the patient for the QUEST, and ABC-S. Effectiveness of the Unity system regarding SCS and daily secondary outcome measures will be tested by randomization test. The secondary outcome measures assessed during visits in the rehabilitation center will be analyzed by Non Overlap of All pairs. An estimate of the effect on the amputee population will be generated by aggregating each individual clinical trial (N-of-1 trial) by Hierarchical Bayesian methods. DISCUSSION: This study protocol was designed to answer the question "which device is best for THIS patient" and to conclude at a group level on the effectiveness of a new devic, using a Multiple N-of-1 trial, which is promising but underused in prosthetics research so far. TRIAL REGISTRATION: N° ID-RCB 2020-A01309-30 Clintrial.gov : NCT04804150 - Retrospectively registered March 20th 2021.

Collection of Biospecimens from the Inspiration4 Mission Establishes the Standards for the Space Omics and Medical Atlas (SOMA).

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from the crew at different stages of the mission, including before (L-92, L-44, L-3 days), during (FD1, FD2, FD3), and after (R+1, R+45, R+82, R+194 days) spaceflight, creating a longitudinal sample set. The collection process included samples such as venous blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies, which were processed to obtain aliquots of serum, plasma, extracellular vesicles, and peripheral blood mononuclear cells. All samples were then processed in clinical and research laboratories for optimal isolation and testing of DNA, RNA, proteins, metabolites, and other biomolecules. This paper describes the complete set of collected biospecimens, their processing steps, and long-term biobanking methods, which enable future molecular assays and testing. As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can also aid future experiments in human spaceflight and space biology.

Insights into brain metastasis: Recent advances in circulating tumor cell research.

BACKGROUND: How tumor cells disseminate to brain and establish brain metastasis remains partly an unsolved problem. This devastating complication of many cancers is initiated by a rare subset of the circulating tumor cells (CTCs) shed into the blood stream. Thus, the profiling of the molecular properties in these brain metastasis-initiating CTCs is essential to uncover the mechanisms underlying brain metastasis. RECENT FINDINGS: Important efforts to improve the enrichment and detection of CTCs enabled the detailed molecular and functional analysis of CTCs that drive brain metastasis. In this review, we highlight key findings on existing preclinical studies that provide insights toward a comprehensive picture of brain metastasis-precursors in CTCs and the potential clinical implications. CONCLUSION: A deeper understanding of the brain metastasis precursors should help to stratify high-risk patients and improve preventive therapeutic strategies. Although all these preclinical evidences have yet to be translated into patients, they provide considerable hope to benefit patients with brain metastases in the future.

Author Correction: PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood-brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. SIGNIFICANCE: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo-cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1.

PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells.

Circulating tumor cells (CTCs) shed from solid tumors can serve as a minimally invasive liquid biopsy for monitoring disease progression. Because CTCs are rare and heterogeneous, their biological properties need to be investigated at the single cell level, which requires efficient ways to isolate and analyze live single CTCs. Current methods for CTC isolation and identification are either performed on fixed and stained cells or need multiple procedures to isolate pure live CTCs. Here, we used the AccuCyte-RareCyte system to develop a Protocol for Integrated Capture and Retrieval of Ultra-pure single live CTCs using Negative and positive selection (PIC&RUN). The positive selection module of PIC&RUN identifies CTCs based on detection of cancer surface markers and exclusion of immune markers. Combined with a two-step cell picking protocol to retrieve ultrapure single CTCs, the positive selection module is compatible for downstream single cell transcriptomic analysis. The negative selection module of PIC&RUN identifies CTCs based on a live cell dye and the absence of immune markers, allowing retrieval of viable CTCs that are suitable for ex vivo culture. This new assay combines the CTC capture and retrieval in one integrated platform, providing a valuable tool for downstream live CTC analyses.

Quality of life assessment following amputation for septic shock: a long-term descriptive survey after symmetric peripheral gangrene.

PURPOSE: To assess health-related quality of life (HRQOL) following rehabilitation of amputees suffering symmetric peripheral gangrene (SPG) after septic shock. MATERIAL AND METHODS: A retrospective cohort study was conducted in nine French specialized rehabilitation centers. Thirty-two ICU adult patients hospitalized between 2005 and 2015 for septic shock who additionally presented with SPG resulting in at least two major amputations were enrolled. HRQOL was assessed by EQ-5D-3 L questionnaire. RESULTS: All patients (mean ICU length of stay 39 ±â€¯22d, SAPS II 58 ±â€¯18) had both lower limbs amputated and 84% were quadruple amputees. HRQOL, assessed 4.8 ±â€¯2.8 years after amputation, was inferior to the French reference. However, patients' self-rated health status was similar to the reference at the time of HRQOL assessment. The main factor of impaired HRQOL was intense phantom pain, not the mobility or self-care dimensions of EQ-5D. All patients except one preferred to be treated again for SPG despite disability. CONCLUSION: ICU survivors referred to rehabilitation centers after SPG-related amputations had impaired HRQOL. At the time of HRQOL assessment, they considered themselves in good health and preferred to be treated again despite disability. Appraisal of long-term functional outcome should not be used to guide end-of-life decision-making in this situation.

DDB2 (damaged-DNA binding 2) protein: a new modulator of nanomechanical properties and cell adhesion of breast cancer cells.

DDB2, known for its role in DNA repair, was recently shown to reduce mammary tumor invasiveness by inducing the transcription of IκBα, an inhibitor of NF-κB activity. Since cellular adhesion is a key event during the epithelial to mesenchymal transition (EMT) leading to the invasive capacities of breast tumor cells, the aim of this study was to investigate the role of DDB2 in this process. Thus, using low and high DDB2-expressing MDA-MB231 and MCF7 cells, respectively, in which DDB2 expression was modulated experimentally, we showed that DDB2 overexpression was associated with a decrease of adhesion abilities on glass and plastic areas of breast cancer cells. Then, we investigated cell nanomechanical properties by atomic force microscopy (AFM). Our results revealed significant changes in the Young's Modulus value and the adhesion force in MDA-MB231 and MCF7 cells, whether DDB2 was expressed or not. The cell stiffness decrease observed in MDA-MB231 and MCF7 expressing DDB2 was correlated with a loss of the cortical actin-cytoskeleton staining. To understand how DDB2 regulates these processes, an adhesion-related gene PCR-Array was performed. Several adhesion-related genes were differentially expressed according to DDB2 expression, indicating that important changes are occurring at the molecular level. Thus, this work demonstrates that AFM technology is an important tool to follow cellular changes during tumorigenesis. Moreover, our data revealed that DDB2 is involved in early events occurring during metastatic progression of breast cancer cells and will contribute to define this protein as a new marker of metastatic progression in this type of cancer.

Manganese superoxide dismutase in breast cancer: from molecular mechanisms of gene regulation to biological and clinical significance.

Breast cancer is one of the most common malignancies of all cancers in women worldwide. Many difficulties reside in the prediction of tumor metastatic progression because of the lack of sufficiently reliable predictive biological markers, and this is a permanent preoccupation for clinicians. Manganese superoxide dismutase (MnSOD) may represent a rational candidate as a predictive biomarker of breast tumor metastatic progression, because its gene expression is profoundly altered between early and advanced breast cancer, in contrast to expression in the normal mammary gland. In this review, we report the characterization of some gene polymorphisms and molecular mechanisms of SOD2 gene regulation, which allows a better understanding of how MnSOD is decreased in early breast cancer and increased in advanced breast cancer. Several studies display the biological significance of MnSOD level in proliferation as well as in invasive and angiogenic abilities of breast tumor cells by controlling superoxide anion radical (O2(•-)) and hydrogen peroxide (H2O2). Particularly, they report how these reactive oxygen species may activate some signaling pathways involved in breast tumor growth. Emerging understanding of these findings provides an interesting framework for guiding translational research and suggests a way to define precisely the clinical interest of MnSOD as a prognostic and/or predicting marker in breast cancer, by associating with some regulators involved in SOD2 gene regulation and other well-known biomarkers, in addition to the typical clinical parameters.

DDB2: a novel regulator of NF-κB and breast tumor invasion.

The DNA repair protein damaged DNA-binding 2 (DDB2) has been implicated in promoting cell-cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are noninvasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-κB and the expression of its target matrix metalloprotease 9 (MMP9). Mechanistic investigations indicated that DDB2 decreased NF-κB activity by upregulating expression of IκBα by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IκBα gene expression restored NF-κB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest in DDB2 as a prognostic marker or therapeutic target in this setting.