Frailty Reduces Vaccine Effectiveness Against SARS-CoV-2 Infection: A Test-Negative Case Control Study Using National VA Data.

OBJECTIVES: To assess the variation of vaccine effectiveness against SARS-CoV-2 infection during the Delta wave according to frailty status among U.S. veterans. DESIGN: Test-negative case-control study of SARS-CoV-2 mRNA vaccine effectiveness. SETTING: Veterans Health Administration (VHA) medical centers. PARTICIPANTS: Veterans 19 years and older who had at least one COVID-19/Flu like symptoms and received a SARS-CoV-2 PCR or antigen test at VHA medical centers between July 25 to September 30, 2021. INTERVENTION: mRNA vaccination. MEASUREMENTS: New SARS-CoV-2 infection. Vaccine effectiveness was defined as 1-odds of vaccination in cases/odds of vaccination in controls, where cases were patients who had a COVID-19 test and tested positive for SARS-CoV-2, and controls were those who tested negative. Frailty was measured using the VA frailty index, categorized as robust (0-<0.1), pre-frail (≥0.1-<0.21) and frail (≥0.21). RESULTS: A total of 58,604 patients (age:58.9±17.0, median:61, IQR:45-72; 87.5%men; 68.1%white; 1.3%African American, 8.3%Hispanic) were included in the study. Of these, 27,733 (47.3%) were robust, 16,276 (27.8%) were prefrail, and 14,595 (24.9%) were frail. mRNA vaccine effectiveness against the Delta variant symptomatic infection was lower in patients with frailty, 62.8 %(95%CI:59.8-65.7), versus prefrail 73.9%(95%CI:72.0-75.7), and robust, 77.0 %(95%CI:75.7-78.3). CONCLUSIONS: This test-negative case control study showed that mRNA vaccine effectiveness against infection declined in veterans with frailty. Frailty status is a factor to consider when designing, developing, and evaluating COVID-19 vaccines.

Temporal profiling of the breast tumour microenvironment reveals collagen XII as a driver of metastasis.

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.

Activation of the viral sensor oligoadenylate synthetase 2 (Oas2) prevents pregnancy-driven mammary cancer metastases.

BACKGROUND: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice. METHODS: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry. RESULTS: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present. CONCLUSIONS: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy.

ALTEN: A High-Fidelity Primary Tissue-Engineering Platform to Assess Cellular Responses Ex Vivo.

To fully investigate cellular responses to stimuli and perturbations within tissues, it is essential to replicate the complex molecular interactions within the local microenvironment of cellular niches. Here, the authors introduce Alginate-based tissue engineering (ALTEN), a biomimetic tissue platform that allows ex vivo analysis of explanted tissue biopsies. This method preserves the original characteristics of the source tissue's cellular milieu, allowing multiple and diverse cell types to be maintained over an extended period of time. As a result, ALTEN enables rapid and faithful characterization of perturbations across specific cell types within a tissue. Importantly, using single-cell genomics, this approach provides integrated cellular responses at the resolution of individual cells. ALTEN is a powerful tool for the analysis of cellular responses upon exposure to cytotoxic agents and immunomodulators. Additionally, ALTEN's scalability using automated microfluidic devices for tissue encapsulation and subsequent transport, to enable centralized high-throughput analysis of samples gathered by large-scale multicenter studies, is shown.

Advancements in 3D Cell Culture Systems for Personalizing Anti-Cancer Therapies.

Over 90% of potential anti-cancer drug candidates results in translational failures in clinical trials. The main reason for this failure can be attributed to the non-accurate pre-clinical models that are being currently used for drug development and in personalised therapies. To ensure that the assessment of drug efficacy and their mechanism of action have clinical translatability, the complexity of the tumor microenvironment needs to be properly modelled. 3D culture models are emerging as a powerful research tool that recapitulates in vivo characteristics. Technological advancements in this field show promising application in improving drug discovery, pre-clinical validation, and precision medicine. In this review, we discuss the significance of the tumor microenvironment and its impact on therapy success, the current developments of 3D culture, and the opportunities that advancements that in vitro technologies can provide to improve cancer therapeutics.

Mammary Tumor Organoid Culture in Non-Adhesive Alginate for Luminal Mechanics and High-Throughput Drug Screening.

Mammary tumor organoids have become a promising in vitro model for drug screening and personalized medicine. However, the dependency on the basement membrane extract (BME) as the growth matrices limits their comprehensive application. In this work, mouse mammary tumor organoids are established by encapsulating tumor pieces in non-adhesive alginate. High-throughput generation of organoids in alginate microbeads is achieved utilizing microfluidic droplet technology. Tumor pieces within the alginate microbeads developed both luminal- and solid-like structures and displayed a high similarity to the original fresh tumor in cellular phenotypes and lineages. The mechanical forces of the luminal organoids in the alginate capsules are analyzed with the theory of the thick-wall pressure vessel (TWPV) model. The luminal pressure of the organoids increase with the lumen growth and can reach 2 kPa after two weeks' culture. Finally, the mammary tumor organoids are treated with doxorubicin and latrunculin A to evaluate their application as a drug screening platform. It is found that the drug response is related to the luminal size and pressures of organoids. This high-throughput culture for mammary tumor organoids may present a promising tool for preclinical drug target validation and personalized medicine.

Tumor dissociation of highly viable cell suspensions for single-cell omic analyses in mouse models of breast cancer.

Cell preparation with a high rate of viable cells is required to obtain reliable single-cell transcriptomic and epigenomic data. This protocol describes a technique for digestion and single-cell isolation from mouse mammary tumors to achieve ∼90% of viable cells, which can be subsequently processed in a diverse array of high-throughput single-cell "omic platforms," both in an unbiased manner or after selection of a specific cell population. For complete details on the use and execution of this protocol, please refer to Valdes-Mora et al. (2021).

Impact of Social Frailty on Relocation of Older Adults.

BACKGROUND: The relationship between frailty and variables such as housing are the least included in models of frailty and research on frailty or social frailty and relocation is negligible. The decision to relocate is complex and demanding for older adults with a loss of independence but little is known about what makes older adults relocate to congregated housing designated for older adults, let alone in combination with social frailty, and how they navigate this transition. OBJECTIVES: This mixed method descriptive study aims to understand the influence of social frailty for a population of French-speaking semi-independent older adults relocating to a housing continuum community. DESIGN: Semi-structured individual interviews including sociodemographic data and the PRISMA-7 Frailty Scale were conducted with recently relocated older adults. SETTING: A newly opened French-speaking housing continuum community in Eastern Canada that offers luxury apartments for independent older adults, two assisted living facilities for semi-independent older adults along with a long-term care facility. PARTICIPANTS: Twenty-nine older adults with a mean age of 85 years, mostly female, married or widowed and highly educated. MEASUREMENTS: Content analysis of the transcribed recorded interviews and descriptive statistical analyses to examine relationships between the frailty PRISMA-7 scale, answers to additional questions and the sociodemographic data. RESULTS: There was not a significant difference in the scores for socialization before and after relocation nor between prior help and current help; however, there was a significant negative correlation between help and socialization before and after relocation. Three main themes included: imposed influences, push and pull factors and post relocation. CONCLUSIONS: The results indicate that several social factors contributed to relocation and that participants were experiencing social frailty. Participants were at the crossover point of being vulnerable to experiencing additional deficits which would potentially have led to higher frailty had they not relocated.

Generation and analysis of 3D cell culture models for drug discovery.

Successful preclinical drug testing relies in part on data generated using in vitro cell culture models that recapitulate the structure and function of tumours and other tissues in vivo. The growing evidence that 3D cell models can more accurately predict the efficacy of drug responses compared to traditionally utilised 2D cell culture systems has led to continuous scientific and technological advances that enable better physiologically representative in vitro modelling of in vivo tissues. This review will provide an overview of the utility of current 3D cell models from a drug screening perspective and explore the future of 3D cell models for drug discovery applications.

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer.

BACKGROUND: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. METHODS: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. RESULTS: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. CONCLUSIONS: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.

Myeloid-Derived Suppressor Cells as a Therapeutic Target for Cancer.

The emergence of immunotherapy has been an astounding breakthrough in cancer treatments. In particular, immune checkpoint inhibitors, targeting PD-1 and CTLA-4, have shown remarkable therapeutic outcomes. However, response rates from immunotherapy have been reported to be varied, with some having pronounced success and others with minimal to no clinical benefit. An important aspect associated with this discrepancy in patient response is the immune-suppressive effects elicited by the tumour microenvironment (TME). Immune suppression plays a pivotal role in regulating cancer progression, metastasis, and reducing immunotherapy success. Most notably, myeloid-derived suppressor cells (MDSC), a heterogeneous population of immature myeloid cells, have potent mechanisms to inhibit T-cell and NK-cell activity to promote tumour growth, development of the pre-metastatic niche, and contribute to resistance to immunotherapy. Accumulating research indicates that MDSC can be a therapeutic target to alleviate their pro-tumourigenic functions and immunosuppressive activities to bolster the efficacy of checkpoint inhibitors. In this review, we provide an overview of the general immunotherapeutic approaches and discuss the characterisation, expansion, and activities of MDSCs with the current treatments used to target them either as a single therapeutic target or synergistically in combination with immunotherapy.

ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance.

MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.

The Impact of Prior Season Vaccination on Subsequent Influenza Vaccine Effectiveness to Prevent Influenza-related Hospitalizations Over 4 Influenza Seasons in Canada.

BACKGROUND: Recent studies have demonstrated the possibility of negative associations between prior influenza vaccines and subsequent influenza vaccine effectiveness (VE), depending on season and strain. We investigated this association over 4 consecutive influenza seasons (2011-2012 through 2014-2015) in Canada. METHODS: Using a matched test-negative design, laboratory-confirmed influenza cases and matched test-negative controls admitted to hospitals were enrolled. Patients were stratified into 4 groups according to influenza vaccine history (not vaccinated current and prior season [referent], vaccinated prior season only, vaccinated current season only, and vaccinated both current and prior season). Conditional logistic regression was used to estimate VE; prior vaccine impact was assessed each season for overall effect and effect stratified by age (<65 years, ≥65 years) and type/subtype (A/H1N1, A/H3N2, influenza B). RESULTS: Overall, mainly nonsignificant associations were observed. Trends of nonsignificant decreased VE among patients repeatedly vaccinated in both prior and current season relative to the current season only were observed in the A/H3N2-dominant seasons of 2012-2013 and 2014-2015. Conversely, in 2011-2012, during which B viruses circulated, and in 2013-2014, when A/H1N1 circulated, being vaccinated in both seasons tended to result in a high VE in the current season against the dominant circulating subtype. CONCLUSIONS: Prior vaccine impact on subsequent VE among Canadian inpatients was mainly nonsignificant. Even in circumstances where we observed a trend of negative impact, being repeatedly vaccinated was still more effective than not receiving the current season's vaccine. These findings favor continuation of annual influenza vaccination recommendations, particularly in older adults. CLINICAL TRIALS REGISTRATION: NCT01517191.

Single-Cell Transcriptomics in Cancer Immunobiology: The Future of Precision Oncology.

Cancer is a heterogeneous and complex disease. Tumors are formed by cancer cells and a myriad of non-cancerous cell types that together with the extracellular matrix form the tumor microenvironment. These cancer-associated cells and components contribute to shape the progression of cancer and are deeply involved in patient outcome. The immune system is an essential part of the tumor microenvironment, and induction of cancer immunotolerance is a necessary step involved in tumor formation and growth. Immune mechanisms are intimately associated with cancer progression, invasion, and metastasis; as well as to tumor dormancy and modulation of sensitivity to drug therapy. Transcriptome analyses have been extensively used to understand the heterogeneity of tumors, classifying tumors into molecular subtypes and establishing signatures that predict response to therapy and patient outcomes. However, the classification of the tumor cell diversity and specially the identification of rare populations has been limited in these transcriptomic analyses of bulk tumor cell populations. Massively-parallel single-cell RNAseq analysis has emerged as a powerful method to unravel heterogeneity and to study rare cell populations in cancer, through unsupervised sampling and modeling of transcriptional states in single cells. In this context, the study of the role of the immune system in cancer would benefit from single cell approaches, as it will enable the characterization and/or discovery of the cell types and pathways involved in cancer immunotolerance otherwise missed in bulk transcriptomic information. Thus, the analysis of gene expression patterns at single cell resolution holds the potential to provide key information to develop precise and personalized cancer treatment including immunotherapy. This review is focused on the latest single-cell RNAseq methodologies able to agnostically study thousands of tumor cells as well as targeted single-cell RNAseq to study rare populations within tumors. In particular, we will discuss methods to study the immune system in cancer. We will also discuss the current challenges to the study of cancer at the single cell level and the potential solutions to the current approaches.

Social and societal implications of frailty, including impact on Canadian healthcare systems.

Frailty has many social and societal implications. Social circumstances are key both as contributors to frail older adults' health outcomes and as practical facilitators or barriers to intervention and supports. Frailty also has important societal implications for health systems and social care policy. In this discussion paper, we use a social ecology framework to consider the social and societal implications and impact of frailty at each level, from the individual, through relationships with family and friend caregivers, institutions, health systems, neighborhoods and communities, to society at large. We conclude by arguing that attention to these issues at a policy level is critical. We identify three target actions: 1) Social dimensions of frailty should be systematically considered when frailty is assessed. 2) Action is needed at the level of policies and programs to improve support for caregivers. 3) Policy review across all portfolios will benefit from a social frailty lens.

Static droplet array for culturing single live adherent cells in an isolated chemical microenvironment.

We present here a new method to easily and reliably generate an array of hundreds of dispersed nanoliter-volume semi-droplets for single-cells culture and analysis. The liquid segmentation step occurs directly in indexed traps by a tweezer-like mechanism and is stabilized by spatial confinement. Unlike common droplet-based techniques, the semi-droplet wets its surrounding trap walls thus supporting the culturing of both adherent and non-adherent cells. To eliminate cross-droplet cell migration and chemical cross-talk each semi-droplet is separated from a nearby trap by an ∼80 pL air plug. The overall setup and injection procedure takes less than 10 minutes, is insensitive to fabrication defects and supports cell recovery for downstream analysis. The method offers a new approach to easily capture, image and culture single cells in a chemically isolated microenvironment as a preliminary step towards high-throughput single-cell assays.

MASTL overexpression promotes chromosome instability and metastasis in breast cancer.

MASTL kinase is essential for correct progression through mitosis, with loss of MASTL causing chromosome segregation errors, mitotic collapse and failure of cytokinesis. However, in cancer MASTL is most commonly amplified and overexpressed. This correlates with increased chromosome instability in breast cancer and poor patient survival in breast, ovarian and lung cancer. Global phosphoproteomic analysis of immortalised breast MCF10A cells engineered to overexpressed MASTL revealed disruption to desmosomes, actin cytoskeleton, PI3K/AKT/mTOR and p38 stress kinase signalling pathways. Notably, these pathways were also disrupted in patient samples that overexpress MASTL. In MCF10A cells, these alterations corresponded with a loss of contact inhibition and partial epithelial-mesenchymal transition, which disrupted migration and allowed cells to proliferate uncontrollably in 3D culture. Furthermore, MASTL overexpression increased aberrant mitotic divisions resulting in increased micronuclei formation. Mathematical modelling indicated that this delay was due to continued inhibition of PP2A-B55, which delayed timely mitotic exit. This corresponded with an increase in DNA damage and delayed transit through interphase. There were no significant alterations to replication kinetics upon MASTL overexpression, however, inhibition of p38 kinase rescued the interphase delay, suggesting the delay was a G2 DNA damage checkpoint response. Importantly, knockdown of MASTL, reduced cell proliferation, prevented invasion and metastasis of MDA-MB-231 breast cancer cells both in vitro and in vivo, indicating the potential of future therapies that target MASTL. Taken together, these results suggest that MASTL overexpression contributes to chromosome instability and metastasis, thereby decreasing breast cancer patient survival.

Andy's Algorithms: new automated digital image analysis pipelines for FIJI.

Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy's Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3'-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays. Andy's Algorithms incorporates a step-by-step tutorial and optimization pipeline to make batch image analysis simple for the untrained user and adaptable across laboratories. Andy's algorithms provide a simpler, faster, standardized work flow compared to existing programs, while offering equivalent performance and additional features, in a free to use open-source application of FIJI. Andy's Algorithms are available at GitHub, publicly accessed at https://github.com/andlaw1841/Andy-s-Algorithm .