Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles.

Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research.

A novel Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis.

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knockdown of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases ATP demand and glycolysis in breast cancer cells, likely by upregulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and the movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

Platelet-derived microvesicles isolated from type-2 diabetes mellitus patients harbour an altered miRNA signature and drive MDA-MB-231 triple-negative breast cancer cell invasion.

The underlying causes of breast cancer are diverse, however, there is a striking association between type 2 diabetes and poor patient outcomes. Platelet activation is a common feature of both type 2 diabetes and breast cancer and has been implicated in tumourigenesis through a multitude of pathways. Here transcriptomic analysis of type 2 diabetes patient-derived platelet microvesicles revealed an altered miRNA signature compared with normoglycaemic control patients. Interestingly, interrogation of these data identifies a shift towards an oncogenic signature in type 2 diabetes-derived platelet microvesicles, with increased levels of miRNAs implicated in breast cancer progression and poor prognosis. Functional studies demonstrate that platelet microvesicles isolated from type 2 diabetes patient blood are internalised by triple-negative breast cancer cells in vitro, and that co-incubation with type 2 diabetes patient-derived platelet microvesicles led to significantly increased expression of epithelial to mesenchymal transition markers and triple-negative breast cancer cell invasion compared with platelet microvesicles from healthy volunteers. Together, these data suggest that circulating PMVs in type 2 diabetes patients may contribute to the progression of triple-negative breast cancer.

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling.

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also show that brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

Exploring the One Health Paradigm in Male Breast Cancer.

How cancer patterns in humans compare to those of other species remains largely unknown and there is an even bigger knowledge gap for rare cancers like male breast cancer. One Health is a convergence of human and animal healthcare that encourages cross-pollination of medical research uniting human and veterinary medicine. Recognising that breast cancer occurs spontaneously in other male species (e.g. primates, canines, felines), and knowing that no laboratory models exist for male breast cancer, which limits our ability to perform functional studies, we explored the feasibility of applying One Health to breast cancer in men by conducting a narrative review of the topic. Spontaneous development of breast cancer was reported in captive male primates and in companion canines and felines. Some parallels in tumour biology of human male breast cancer with canines and primates were found. The age distribution, pattern of biomarker expression and metastasis were similar, with mammary tumours typically detected after two-thirds of average lifespan. However, instances of triple negative and inflammatory breast cancer, which are rarely observed in human male breast cancer, were found in canines and histological classification was inconsistent between species. These disparities need redressing to enable full exploration of the One Health paradigm in rare cancers.

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

Engaging undergraduate medical and dental students with academic medicine: The Aberdeen INSPIRE summer school.

UK medical and dental school curricula limit opportunities for students to gain experience in research. This parallels a decline in the number of clinical academics. To address this at grass roots level, we organised and arranged a residential summer taster week; INSPIRE (Introducing New Skills to Promote Inspirational Research Experience)-Aberdeen). The purpose was to give first and second year medical and dental students who wished to explore a potential clinical academic career a taste of wet laboratory research and to gain experience in basic research skills. Seventeen students from eight different UK medical and dental schools attended this free residential course and were exposed to various laboratory techniques with clinical translation and application in diagnostic and therapeutic medicine. Students were given access to relevant online learning tools of the techniques being used beforehand and seminar style presentations were used to emphasise their clinical application. Students met daily with clinical academics from different specialities to give them a flavour of potential clinical academic career pathways and options. All students felt that the summer school helped them consider academic medicine as a career thus achieving our aim to inspire clinical academics of the future.

Prediction models for hormone receptor status in female breast cancer do not extend to males: further evidence of sex-based disparity in breast cancer.

Breast cancer prognosis and management for both men and women are reliant upon estrogen receptor alpha (ERα) and progesterone receptor (PR) expression to inform therapy. Previous studies have shown that there are sex-specific binding characteristics of ERα and PR in breast cancer and, counterintuitively, ERα expression is more common in male than female breast cancer. We hypothesized that these differences could have morphological manifestations that are undetectable to human observers but could be elucidated computationally. To investigate this, we trained attention-based multiple instance learning prediction models for ERα and PR using H&E-stained images of female breast cancer from the Cancer Genome Atlas (TCGA) (n = 1085) and deployed them on external female (n = 192) and male breast cancer images (n = 245). Both targets were predicted in the internal (AUROC for ERα prediction: 0.86 ± 0.02, p < 0.001; AUROC for PR prediction = 0.76 ± 0.03, p < 0.001) and external female cohorts (AUROC for ERα prediction: 0.78 ± 0.03, p < 0.001; AUROC for PR prediction = 0.80 ± 0.04, p < 0.001) but not the male cohort (AUROC for ERα prediction: 0.66 ± 0.14, p = 0.43; AUROC for PR prediction = 0.63 ± 0.04, p = 0.05). This suggests that subtle morphological differences invisible upon visual inspection may exist between the sexes, supporting previous immunohistochemical, genomic, and transcriptomic analyses.

Trailblazers in cancer research: the next generation - the British Association of Cancer Research early-career conference.

The inaugural 'British Association of Cancer Research (BACR) Early Career Conference, Trailblazers in Cancer Research 2023', was a 2-day meeting held in Manchester, UK. Recognising the disruption caused by the COVID-19 pandemic to early-career researchers (ECRs), the BACR executive committee organised an in-person conference to address the lack of network and training opportunities during this time. The conference brought together PhD students and post-doctoral researchers from across the UK and beyond, who shared their outstanding contributions to cancer research. The meeting incorporated several cutting-edge cancer themes, including 'Cancer Cell Signalling and The Tumour Microenvironment'; 'Emerging Approaches in Cancer Treatment'; 'Cancer Omics and Lifestyle', and 'Nutrition and Cancer'. Alongside showcasing world-class cancer research, the meeting included a career-focused session which allowed industrial and non-academic speakers to provide vital insight into alternative career paths aside from the familiar 'academic' route. Importantly, the conference also introduced delegates to Patient Public Involvement in cancer research, an area of limited experience for many. Overall, the BACR Trailblazers Conference was hugely successful and presented an excellent platform for collaboration and networking among ECRs in cancer research.

Biglycan and reduced glycolysis are associated with breast cancer cell dormancy in the brain.

Exit of quiescent disseminated cancer cells from dormancy is thought to be responsible for metastatic relapse and a better understanding of dormancy could pave the way for novel therapeutic approaches. We used an in vivo model of triple negative breast cancer brain metastasis to identify differences in transcriptional profiles between dormant and proliferating cancer cells in the brain. BGN gene, encoding a small proteoglycan biglycan, was strongly upregulated in dormant cancer cells in vivo. BGN expression was significantly downregulated in patient brain metastases as compared to the matched primary breast tumors and BGN overexpression in cancer cells inhibited their growth in vitro and in vivo. Dormant cancer cells were further characterized by a reduced expression of glycolysis genes in vivo, and inhibition of glycolysis in vitro resulted in a reversible growth arrest reminiscent of dormancy. Our study identified mechanisms that could be targeted to induce/maintain cancer dormancy and thereby prevent metastatic relapse.

Validation of a 3D perfused cell culture platform as a tool for humanised preclinical drug testing in breast cancer using established cell lines and patient-derived tissues.

3D cell culture models of cancer are currently being developed to recapitulate in vivo physiological conditions and to assess therapeutic responses. However, most models failed to incorporate the biochemical and biophysical stimuli from fluid flow. In this study, a three-dimensional scaffold, SeedEZ was applied within the PerfusionPal perfused culture system to investigate how perfusion, and blood-like oxygen delivery influenced breast cancer cell growth and their responses to a commonly used breast cancer drug tamoxifen. Our results showed that breast cancer cells could be maintained over 3 weeks in PerfusionPal with increased cell viability compared to static 3D culture in fully humanised conditions. This platform also supported examining the effect of tamoxifen on breast cancer cell lines and in primary patient-derived breast cancer samples. Future work is warranted to further the adaption for fully humanised assessment of drug effectiveness in a patient personalized approach with the aim to reduce the burden of animal use in cancer research and increase the degree of human pre-clinical data translation to clinic.

Tspan6 stimulates the chemoattractive potential of breast cancer cells for B cells in an EV- and LXR-dependent manner.

The immune microenvironment in breast cancer (BCa) is controlled by a complex network of communication between various cell types. Here, we find that recruitment of B lymphocytes to BCa tissues is controlled via mechanisms associated with cancer cell-derived extracellular vesicles (CCD-EVs). Gene expression profiling identifies the Liver X receptor (LXR)-dependent transcriptional network as a key pathway that controls both CCD-EVs-induced migration of B cells and accumulation of B cells in BCa tissues. The increased accumulation oxysterol ligands for LXR (i.e., 25-hydroxycholesterol and 27-hydroxycholesterol) in CCD-EVs is regulated by the tetraspanin 6 (Tspan6). Tspan6 stimulates the chemoattractive potential of BCa cells for B cells in an EV- and LXR-dependent manner. These results demonstrate that tetraspanins control intercellular trafficking of oxysterols via CCD-EVs. Furthermore, tetraspanin-dependent changes in the oxysterol composition of CCD-EVs and the LXR signaling axis play a key role in specific changes in the tumor immune microenvironment.

Defining genomic, transcriptomic, proteomic, epigenetic, and phenotypic biomarkers with prognostic capability in male breast cancer: a systematic review.

Although similar phenotypically, there is evidence that male and female breast cancer differ in their molecular landscapes. In this systematic review, we consolidated all existing prognostic biomarker data in male breast cancer spanning genetics, transcriptomics, proteomics, and epigenetics, and phenotypic features of prognostic value from articles published over a 29-year period (March 16, 1992, to May 1, 2021). We identified knowledge gaps in the existing literature, discussed limitations of the included studies, and outlined potential approaches for translational biomarker discovery and validation in male breast cancer. We also recognised STC2, DDX3, and DACH1 as underexploited markers of male-specific prognostic value in breast cancer. Finally, beyond describing the cumulative knowledge on the extensively researched markers oestrogen receptor-α, progesterone receptor, HER2, androgen receptor, and BRCA2, we highlighted ATM, CCND1, FGFR2, GATA3, HIF1-α, MDM2, TP53, and c-Myc as well studied predictors of poor survival that also aligned with several hallmarks of cancer.

Integrative, In Silico and Comparative Analysis of Breast Cancer Secretome Highlights Invasive-Ductal-Carcinoma-Grade Progression Biomarkers.

Globally, BC is the most frequently diagnosed cancer in women. The aim of this study was to identify novel secreted biomarkers that may indicate progression to high-grade BC malignancies and therefore predict metastatic potential. A total of 33 studies of breast cancer and 78 of other malignancies were screened via a systematic review for eligibility, yielding 26 datasets, 8 breast cancer secretome datasets, and 18 of other cancers that were included in the comparative secretome analysis. Sequential bioinformatic analysis using online resources enabled the identification of enriched GO_terms, overlapping clusters, and pathway reconstruction. This study identified putative predictors of IDC grade progression and their association with breast cancer patient mortality outcomes, namely, HSPG2, ACTG1, and LAMA5 as biomarkers of in silico pathway prediction, offering a putative approach by which the abovementioned proteins may mediate their effects, enabling disease progression. This study also identified ITGB1, FBN1, and THBS1 as putative pan-cancer detection biomarkers. The present study highlights novel, putative secretome biomarkers that may provide insight into the tumor biology and could inform clinical decision making in the context of IDC management in a non-invasive manner.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Analysis of the Clinical Advancements for BRCA-Related Malignancies Highlights the Lack of Treatment Evidence for BRCA-Positive Male Breast Cancer.

Male breast cancer (MBC) is a rare disease that accounts for less than 1% of all breast cancers and male malignancies. Despite recognised clinico-pathological and molecular differences to female breast cancer (FBC), the clinical management of MBC follows established FBC treatment strategies. Loss of function mutations in the DNA damage response genes BRCA1 and BRCA2, have been strongly implicated in the pathogenesis of MBC. While there have been extensive clinical advancements in other BRCA-related malignancies, including FBC, improvements in MBC remain stagnant. Here we present a review that highlights the lack of treatment evidence for BRCA-related MBC and the required national and global collaborative effort to address this unmet need. In doing so, we summarise the transformative clinical advancements with poly(ADP-ribose) polymerase (PARP) inhibitors in other BRCA-related cancers namely, FBC and prostate cancer.

Raman spectroscopy: current applications in breast cancer diagnosis, challenges and future prospects.

Despite significant improvements in the way breast cancer is managed and treated, it continues to persist as a leading cause of death worldwide. If detected and diagnosed early, when tumours are small and localised, there is a considerably higher chance of survival. However, current methods for detection and diagnosis lack the required sensitivity and specificity for identifying breast cancer at the asymptomatic or very early stages. Thus, there is a need to develop more rapid and reliable methods, capable of detecting disease earlier, for improved disease management and patient outcome. Raman spectroscopy is a non-destructive analytical technique that can rapidly provide highly specific information on the biochemical composition and molecular structure of samples. In cancer, it has the capacity to probe very early biochemical changes that accompany malignant transformation, even prior to the onset of morphological changes, to produce a fingerprint of disease. This review explores the application of Raman spectroscopy in breast cancer, including discussion on its capabilities in analysing both ex-vivo tissue and liquid biopsy samples, and its potential in vivo applications. The review also addresses current challenges and potential future uses of this technology in cancer research and translational clinical application.

Radiotherapy biobanking: current landscape, opportunities, challenges, and future aspirations.

Half of all cancer patients receive radiotherapy, which makes a substantial contribution to their long-term disease control/cure. There are significant inter-patient differences in response, both in terms of efficacy and toxicity (frequently delayed onset) which are difficult to predict. With the introduction of technological improvements (e.g. stereotactic body radiotherapy and proton therapy) and development of combination therapies (e.g. radiotherapy and immune checkpoint inhibition), predictive biomarkers are needed even more. Whilst genomic studies have contributed significantly to predictions of response to anticancer therapy, there is no doubt that more information can be gathered from patient tissue samples. Patients are willing to donate their tissues to biobanks and wish them to be used as widely as possible for high-quality research. We report here a survey of the current practices in the UK from several groups collecting material from patients in radiotherapy trials and have identified barriers to collecting and sharing data and samples. We believe the current situation represents a significant missed opportunity to improve the personalisation of radiotherapy. We propose a greater involvement of patients and/or their advocates, a standardisation of the patient information leaflet, consent form content and data set, with easy linkage to clinical data, which would facilitate widespread sample and data discovery and availability to other researchers. The greater sharing of data and samples, nationally and internationally, would facilitate robust multicentre studies and avoid duplication of effort.

Male breast cancer: an update.

Male breast cancer (MBC) is rare, accounting for less than 1% of all breast cancer but the incidence has increased worldwide. Risk factors include increased longevity, obesity, testicular diseases and tumours, and germline mutations of BRCA2. BRCA2 carriers have 80 times the risk of the general population. Men generally present with breast cancer at an older age compared with women. Histologically, MBC is often of grade 2, hormone receptor positive, HER2 negative, and no special type carcinoma although in situ and invasive papillary carcinomas are common. Reporting and staging are similar to female breast cancer. Metastatic lesions to the male breast do occur and should be differentiated from primary carcinomas. Until recently, MBC was thought to be similar to the usual ER positive post-menopausal female counterpart. However, advances in MBC research and trials have highlighted significant differences between the two. This review provides an up to date overview of the biology, genetics, and histology of MBC with comparison to female breast cancers and differential diagnosis from histological mimics.