Using a pan-cancer atlas to investigate tumour associated macrophages as regulators of immunotherapy response.

The paradigm for macrophage characterization has evolved from the simple M1/M2 dichotomy to a more complex model that encompasses the broad spectrum of macrophage phenotypic diversity, due to differences in ontogeny and/or local stimuli. We currently lack an in-depth pan-cancer single cell RNA-seq (scRNAseq) atlas of tumour-associated macrophages (TAMs) that fully captures this complexity. In addition, an increased understanding of macrophage diversity could help to explain the variable responses of cancer patients to immunotherapy. Our atlas includes well established macrophage subsets as well as a number of additional ones. We associate macrophage composition with tumour phenotype and show macrophage subsets can vary between primary and metastatic tumours growing in sites like the liver. We also examine macrophage-T cell functional cross talk and identify two subsets of TAMs associated with T cell activation. Analysis of TAM signatures in a large cohort of immune checkpoint inhibitor-treated patients (CPI1000 + ) identify multiple TAM subsets associated with response, including the presence of a subset of TAMs that upregulate collagen-related genes. Finally, we demonstrate the utility of our data as a resource and reference atlas for mapping of novel macrophage datasets using projection. Overall, these advances represent an important step in both macrophage classification and overcoming resistance to immunotherapies in cancer.

Analysis of SARS-CoV-2 antibody seroprevalence in Northern Ireland during 2020-2021.

Background: With the spread of SARS-CoV-2 impacting upon public health directly and socioeconomically, further information was required to inform policy decisions designed to limit virus spread during the pandemic. This study sought to contribute to serosurveillance work within Northern Ireland to track SARS-CoV-2 progression and guide health strategy. Methods: Sera/plasma samples from clinical biochemistry laboratories were analysed for anti-SARS-CoV-2 antibodies. Samples were assessed using an Elecsys anti-SARS-CoV-2 or anti-SARS-CoV-2 S ECLIA (Roche) on an automated cobas e 801 analyser. Samples were also assessed via an anti-SARS-CoV-2 ELISA (Euroimmun). A subset of samples assessed via the Elecsys anti-SARS-CoV-2 ECLIA were subsequently analysed in an ACE2 pseudoneutralisation assay using a V-PLEX SARS-CoV-2 Panel 7 for IgG and ACE2 (Meso Scale Diagnostics). Results: Across three testing rounds (June-July 2020, November-December 2020 and June-July 2021 (rounds 1-3 respectively)), 4844 residual sera/plasma specimens were assayed for anti-SARS-CoV-2 antibodies. Seropositivity rates increased across the study, peaking at 11.6 % (95 % CI 10.4 %-13.0 %) during round 3. Varying trends in SARS-CoV-2 seropositivity were noted based on demographic factors. For instance, highest rates of seropositivity shifted from older to younger demographics across the study period. In round 3, Alpha (B.1.1.7) variant neutralising antibodies were most frequently detected across age groups, with median concentration of anti-spike protein antibodies elevated in 50-69 year olds and anti-S1 RBD antibodies elevated in 70+ year olds, relative to other age groups. Conclusions: With seropositivity rates of <15 % across the assessment period, it can be concluded that the significant proportion of the Northern Ireland population had not yet naturally contracted the virus by mid-2021.

Changes in the immune landscape of TNBC after neoadjuvant chemotherapy: correlation with relapse.

Introduction: Patients with high-risk, triple negative breast cancer (TNBC) often receive neoadjuvant chemotherapy (NAC) alone or with immunotherapy. Various single-cell and spatially resolved techniques have demonstrated heterogeneity in the phenotype and distribution of macrophages and T cells in this form of breast cancer. Furthermore, recent studies in mice have implicated immune cells in perivascular (PV) areas of tumors in the regulation of metastasis and anti-tumor immunity. However, little is known of how the latter change during NAC in human TNBC or their impact on subsequent relapse, or the likely efficacy of immunotherapy given with or after NAC. Methods: We have used multiplex immunofluorescence and AI-based image analysis to compare the immune landscape in untreated and NAC-treated human TNBCs. We quantified changes in the phenotype, distribution and intercellular contacts of subsets of tumor-associated macrophages (TAMs), CD4+ and CD8+ T cells, and regulatory T cells (Tregs) in PV and non-PV various areas of the stroma and tumor cell islands. These were compared in tumors from patients who had either developed metastases or were disease-free (DF) after a three-year follow up period. Results: In tumors from patients who remained DF after NAC, there was a marked increase in stromal CD163+ TAMs, especially those expressing the negative checkpoint regulator, T-cell immunoglobulin and mucin domain 3 (TIM-3). Whereas CD4+ T cells preferentially located to PV areas in the stroma of both untreated and NAC-treated tumors, specific subsets of TAMs and Tregs only did so only after NAC. Distinct subsets of CD4+ and CD8+ T cells formed PV clusters with CD163+ TAMs and Tregs. These were retained after NAC. Discussion: Quantification of stromal TIM-3+CD163+ TAMs in tumor residues after NAC may represent a new way of identifying patients at high risk of relapse. PV clustering of immune cells is highly likely to regulate the activation and function of T cells, and thus the efficacy of T cell-based immunotherapies administered with or after NAC.

Evaluating The Precocial-altricial Axis of Motor Skill at Birth in A Preterm Pig Model.

The pace of locomotor development is a critical component of lifetime evolutionary fitness. Developmental researchers often divide species into two broad categories based on functional competence at birth: precocial infants who can independently stand and locomote soon after birth versus altricial infants who are either incapable of independent movement or can only do so in a rudimentary manner. However, investigating the lower level neuromotor and biomechanical traits that account for perinatal variation in motor development is complicated by the lack of experimental control inherent to all comparative analyses. Precocial and altricial animals often differ along a host of dimensions that can obfuscate the specific factors controlling motor development per se. Here, we propose an alternative approach of examining locomotor development in a nominally precocial species-the domestic pig (Sus scrofa)-in which gestation length has been experimentally manipulated, thereby creating "functionally altricial" cohorts for comparison. We have used standard biomechanical testing to evaluate balance and locomotor performance in preterm pigs born at 94% full-term gestation (N = 29 individuals) and compared these data to a similar dataset on age-matched full-term piglets (N = 15 individuals). Static balance tests showed that preterm pigs were characterized by increased postural sway, particularly in the fore-aft (anteroposterior) direction. Locomotor analyses showed that preterm piglets tended to take shorter, more frequent strides, use higher duty factors, and preferentially choose gait patterns that ensured they were supported by at least three limbs during most of the stride cycle, though differences between preterm and full-term animals were often modulated by variation in locomotor speed. Morphometric analysis showed no differences in relative extensor muscle mass between preterm and full-term animals, suggesting that neurological immaturity might be more determinant of preterm piglet motor dysfunctions than musculoskeletal immaturity per se (though much work remains to be done to fully document the neuromotor phenotype of the preterm infant pig model). In many ways, the postural and locomotor deficits shown by the preterm piglets paralleled the locomotor phenotype of altricial mammals. Overall, our study demonstrates the utility of a "within-species" design for studying the biomechanical correlates and neuromotor basis of evolutionary variation in motor skill at birth.

Technical note on the exploration of COVID-19 in autopsy material.

Interrogation of immune response in autopsy material from patients with SARS-CoV-2 is potentially significant. We aim to describe a validated protocol for the exploration of the molecular physiopathology of SARS-CoV-2 pulmonary disease using multiplex immunofluorescence (mIF).The application of validated assays for the detection of SARS-CoV-2 in tissues, originally developed in our laboratory in the context of oncology, was used to map the topography and complexity of the adaptive immune response at protein and mRNA levels.SARS-CoV-2 is detectable in situ by protein or mRNA, with a sensitivity that could be in part related to disease stage. In formalin-fixed, paraffin-embedded pneumonia material, multiplex immunofluorescent panels are robust, reliable and quantifiable and can detect topographic variations in inflammation related to pathological processes.Clinical autopsies have relevance in understanding diseases of unknown/complex pathophysiology. In particular, autopsy materials are suitable for the detection of SARS-CoV-2 and for the topographic description of the complex tissue-based immune response using mIF.

Targeting circulating monocytes with CCL2-loaded liposomes armed with an oncolytic adenovirus.

Oncolytic viruses (OVs) selectively replicate in and destroy cancer cells resulting in anti-tumor immunity. However, clinical use remains a challenge because of virus clearance upon intravenous delivery. OV packaging using a nanomedicine approach could overcome this. Here we encapsulate an oncolytic adenovirus (Ad[I/PPT-E1A]) into CCL2-coated liposomes in order to exploit recruitment of CCR2-expressing circulating monocytes into tumors. We demonstrate successful encapsulation of Ad[I/PPT-E1A] into CCL2-coated liposomes that were preferentially taken up by CCR2-expressing monocytes. No complex-related toxicities were observed following incubation with prostate tumor cells and the encapsulation did not affect virus oncolytic activity in vitro. Furthermore, intravenous administration of our nanomedicine resulted in a significant reduction in tumor size and pulmonary metastasis in prostate cancer-bearing mice whereby a 1000-fold less virus was needed compared to Ad[I/PPT-E1A] alone. Taken together our data provide an opportunity to target OVs via circulation to inaccessible tumors using liposome-assisted drug delivery.

Macrophage Regulation of the Development of Castration-Resistant Prostate Cancer.

Androgen deprivation therapy (ADT) is the front-line treatment for early and metastatic prostate cancer, and the development of tumor resistance to it has major clinical consequences. Cancer cells start to proliferate and tumors begin to regrow, requiring the administration of more generic anticancer treatments like surgery, radiotherapy, and/or chemotherapy. Tumor-associated macrophages are known to drive tumor resistance to a number of anti-cancer therapies. El-Kenawi and colleagues now demonstrate a novel mechanism underpinning their ability to do so in prostate tumors during ADT. This involves the accumulation of cholesterol by macrophages in tumors and its transfer to cancer cells, where it acts as a precursor for androgen biosynthesis and results in the activation of androgen receptors.See related article by El-Kenawi and colleagues, p. 5477.

The Potential of Digital Image Analysis to Determine Tumor Cell Content in Biobanked Formalin-Fixed, Paraffin-Embedded Tissue Samples.

Introduction: Best practices dictate that biobanks ensure accurate determination of tumor content before supplying formalin-fixed, paraffin-embedded (FFPE) tissue samples to researchers for nucleic acid extraction and downstream molecular testing. It is advisable that trained and competent individuals, who understand the requirements of the downstream molecular tests, perform the microscopic morphological examination. However, the special skills, time, and costs associated with these assessments can be prohibitive, especially in large case cohorts requiring extensive pathological review. Determination of tumor content reliably by digital image analysis (DIA) could represent a significant advantage if validated, utilized, and deployed by biobanks. Materials and Methods: Whole slide digital scanned images of colorectal, lung, and breast cancer specimens were created. The scanned images were imported into the DIA software QuPath and digital annotations were completed by biobank technicians, under the direction of trained histopathology senior scientists. Automated cell detection was conducted and tumor epithelial cells were classified and quantified. Results: DIA scores were highly concordant with the manual assessment for 376 of 435 samples (86%). A detailed review of discordant cases indicated digital scores had a higher accuracy than the manual estimation. Conclusion: Automated digital quantification has the potential to replace visual estimations with reduced subjectivity and increased reliability compared with manual tumor estimations. We recommend the use of DIA by biobanks involved in provision of FFPE tissue samples, especially in large research studies requiring high volumes of cases to be analyzed.

Economic Threshold Analysis of Supplementing Dairy Cow Diets with Betaine and Fat during a Heat Challenge: A Pre- and Post-Experimental Comparison.

Ex ante economic analysis can be used to establish the production threshold for a proposed experimental diet to be as profitable as the control treatment. This study reports (1) a pre-experimental economic analysis to estimate the milk production thresholds for an experiment where dietary supplements were fed to dairy cows experiencing a heat challenge, and (2) comparison of these thresholds to the milk production results of the subsequent animal experiment. The pre-experimental thresholds equated to a 1% increase in milk production for the betaine supplement, 9% increase for the fat supplement, and 11% increase for fat and betaine in combination, to achieve the same contribution to farm profit as the control diet. For the post-experimental comparison, previously modelled climate predictions were used to extrapolate the milk production results from the animal experiment over the annual hot-weather period for the dairying region in northern Victoria, Australia. Supplementing diets with fat or betaine had the potential to produce enough extra milk to exceed the production thresholds, making either supplement a profitable alternative to feeding the control diet during the hot-weather period. Feeding fat and betaine in combination failed to result in the extra milk required to justify the additional cost when compared to the control diet.

Macrophages Mediate the Antitumor Effects of the Oncolytic Virus HSV1716 in Mammary Tumors.

Oncolytic viruses (OV) have been shown to activate the antitumor functions of specific immune cells like T cells. Here, we show OV can also reprogram tumor-associated macrophage (TAM) to a less immunosuppressive phenotype. Syngeneic, immunocompetent mouse models of primary breast cancer were established using PyMT-TS1, 4T1, and E0771 cell lines, and a metastatic model of breast cancer was established using the 4T1 cell line. Tumor growth and overall survival was assessed following intravenous administration of the OV, HSV1716 (a modified herpes simplex virus). Infiltration and function of various immune effector cells was assessed by NanoString, flow cytometry of dispersed tumors, and immunofluorescence analysis of tumor sections. HSV1716 administration led to marked tumor shrinkage in primary mammary tumors and a decrease in metastases. This was associated with a significant increase in the recruitment/activation of cytotoxic T cells, a reduction in the presence of regulatory T cells and the reprograming of TAMs towards a pro-inflammatory, less immunosuppressive phenotype. These findings were supported by in vitro data demonstrating that human monocyte-derived macrophages host HSV1716 replication, and that this led to immunogenic macrophage lysis. These events were dependent on macrophage expression of proliferating cell nuclear antigen (PCNA). Finally, the antitumor effect of OV was markedly diminished when TAMs were depleted using clodronate liposomes. Together, our results show that TAMs play an essential role in support of the tumoricidal effect of the OV, HSV1716-they both host viral replication via a novel, PCNA-dependent mechanism and are reprogramed to express a less immunosuppressive phenotype.

Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/ß-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1α, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

Preoperative Nutrition Status and Postoperative Outcomes in Patients Undergoing Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) is a complex surgery to treat peritoneal surface malignancy (PSM). PSM and gastrointestinal (GI) resection from CRS can lead to significant GI symptoms and malnutrition. There is limited research into the nutrition status of this patient group and the impact of malnutrition on morbidity. OBJECTIVE: This study aims to determine if preoperative malnutrition, assessed using the Subjective Global Assessment (SGA), is associated with postoperative morbidity and increased length of stay (LOS) in patients undergoing CRS/HIPEC for PSM. METHODS: This study prospectively assessed the nutritional status of patients undergoing CRS/HIPEC using a validated nutrition assessment tool. Preoperative clinical symptoms, Peritoneal Cancer Index (PCI), intraoperative blood transfusions, operative time, GI resections, postoperative morbidity, and LOS, as well as pre- and postoperative nutritional interventions, were recorded. The impact of preoperative nutritional status was assessed in relation to postoperative complications and hospital LOS. RESULTS: The study included 102 participants; 34 patients (33%) were classified as malnourished (SGA = B or C). Preoperative weight loss (15% vs. 74%; p ≤ 0.001) and the presence of clinical symptoms (18% vs. 47%; p = 0.002) were significantly higher in malnourished patients. While PCI, intraoperative blood transfusions, and GI resections were independent predictors of morbidity, malnutrition was significantly associated with infectious complications and LOS. For each grade of worsening malnutrition, LOS increased by an average of 7.65 days. CONCLUSIONS: Preoperative malnutrition is prevalent in patients undergoing CRS/HIPEC and postoperative morbidity is common. Malnutrition is linked to LOS and plays a role in postoperative outcomes such as infection. Clear pre- and postoperative nutrition pathways are needed to optimize nutrition support and postoperative recovery.

Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models.

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.

Diverse Functions of Macrophages in Different Tumor Microenvironments.

Tumor-associated macrophages are a major constituent of malignant tumors and are known to stimulate key steps in tumor progression. In our review in this journal in 2006, we postulated that functionally distinct subsets of these cells exist in different areas within solid tumors. Here, we review the many experimental and clinical studies conducted since then to investigate the function(s), regulation, and clinical significance of macrophages in these sites. The latter include three sites of cancer cell invasion, tumor nests, the tumor stroma, and areas close to, or distant from, the tumor vasculature. A more complete understanding of macrophage diversity in tumors could lead to the development of more selective therapies to restore the formidable, anticancer functions of these cells. Cancer Res; 78(19); 5492-503. ©2018 AACR.

Perivascular macrophages in health and disease.

Macrophages are a heterogeneous group of cells that are capable of carrying out distinct functions in different tissues, as well as in different locations within a given tissue. Some of these tissue macrophages lie on, or close to, the outer (abluminal) surface of blood vessels and perform several crucial activities at this interface between the tissue and the blood. In steady-state tissues, these perivascular macrophages maintain tight junctions between endothelial cells and limit vessel permeability, phagocytose potential pathogens before they enter tissues from the blood and restrict inappropriate inflammation. They also have a multifaceted role in diseases such as cancer, Alzheimer disease, multiple sclerosis and type 1 diabetes. Here, we examine the important functions of perivascular macrophages in various adult tissues and describe how these functions are perturbed in a broad array of pathological conditions.

RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples.

Immunohistochemistry remains the overwhelming technique of choice for test biomarker evaluation in both clinical or research settings when using formalin-fixed, paraffin embedded tissue sections. However, validations can be complex with significant issues about specificity, sensitivity and reproducibility. The vast array of commercially available antibodies from many vendors may also lead to non-standard approaches which are difficult to cross-reference. In contrast mRNA detection, by in situ hybridization (ISH) with sequence specific probes, offers a realistic alternative, with less validation steps and more stringent and reproducible assessment criteria. In the present study mRNA ISH was evaluated in prospectively and retrospectively collected FFPE samples within a cancer biobank setting. Three positive control probes, POLR2A, PPIB and UBC were applied to FFPE sections from a range of tumour types in FFPE whole-face (prospective collection) or TMA (retrospective collection) formats and evaluated semi-quantitatively and by image analysis. Results indicate that mRNA can be robustly evaluated by ISH in prospectively and retrospectively collected tissue samples. Furthermore, for 2 important test biomarkers, PD-L1 and c-MET, we show that mRNA ISH is a technology that can be applied with confidence in the majority of tissue samples because there are quantifiable levels of control probes indicating overall mRNA integrity.

Enhancing utility and understanding of evidence based practice through undergraduate nurse education.

BACKGROUND: The concept of evidence-based practice is globally relevant in current healthcare climates. However, students and teachers struggle with integrating evidence based practice effectively into a curriculum. This has implications for nurse education and in particular the way in which research is presented and delivered to students. A new undergraduate Evidence Based Practice module (Evidence Based Nursing 1) was developed in a large University within the United Kingdom. It commenced in October 2014 running in year one of a 3 year undergraduate nursing programme. This study sought to formally evaluate attitudes and beliefs, knowledge level and utilization of evidence based practice though using two validated questionnaires: Evidence Based Practice Beliefs Scale© and Evidence Based Practice Implementation Scale©. METHOD: This was a pilot study using quantitative pre and post-test design. Anonymised data was collected from Year 1 undergraduate student nurses in the September 2014 intake (n = 311) at two time points. Time 1: pre-module in September 2014; and Time 2: post -module in August 2015. All data was collected via Survey Monkey. RESULTS: Results demonstrate that the educational initiative positively impacted on both the beliefs and implementation of evidence based practice. Analysis highlighted statistically significant changes (p < 0.05) in both the Evidence Based Practice Beliefs Scale (7/16 categories) and the Evidence Based Practice Implementation Scale (13 / 18 categories). CONCLUSIONS: The significance of integrating evidence based practice into undergraduate nurse education curriculum cannot be underestimated if evidence based practice and its positive impact of patient care are to be appreciated in healthcare settings internationally.

Building a 'Repository of Science': The importance of integrating biobanks within molecular pathology programmes.

Repositories containing high quality human biospecimens linked with robust and relevant clinical and pathological information are required for the discovery and validation of biomarkers for disease diagnosis, progression and response to treatment. Current molecular based discovery projects using either low or high throughput technologies rely heavily on ready access to such sample collections. It is imperative that modern biobanks align with molecular diagnostic pathology practices not only to provide the type of samples needed for discovery projects but also to ensure requirements for ongoing sample collections and the future needs of researchers are adequately addressed. Biobanks within comprehensive molecular pathology programmes are perfectly positioned to offer more than just tumour derived biospecimens; for example, they have the ability to facilitate researchers gaining access to sample metadata such as digitised scans of tissue samples annotated prior to macrodissection for molecular diagnostics or pseudoanonymised clinical outcome data or research results retrieved from other users utilising the same or overlapping cohorts of samples. Furthermore, biobanks can work with molecular diagnostic laboratories to develop standardised methodologies for the acquisition and storage of samples required for new approaches to research such as 'liquid biopsies' which will ultimately feed into the test validations required in large prospective clinical studies in order to implement liquid biopsy approaches for routine clinical practice. We draw on our experience in Northern Ireland to discuss how this harmonised approach of biobanks working synergistically with molecular pathology programmes is a key for the future success of precision medicine.