Secretomics reveals hormone-therapy of breast cancer may induce survival by facilitating hypercoagulation and immunomodulation in vitro.

Tumour cell haematogenous dissemination is predicated on molecular changes that enhance their capacity for invasion and preparation of the pre-metastatic niche. It is increasingly evident that platelets play an essential role in this transformation. The systemic nature of signalling molecules and extravascular factors that participate in mediating platelet-tumour cell interactions led to the development of an in vitro co-culture using whole blood and breast tumour cells, allowing us to decipher the impact of hormone-therapy on tumour cells and associated changes in the plasma proteome. Using mass spectrometry, we determined dysregulation of proteins associated with maintaining an invasive tumour phenotype. Tumour changes in genes associated with EMT and survival were documented. This is postulated to be induced via tumour cell interactions with the coagulatory and immune systems. Results highlight tumour cell adaptability to both treatment and blood resulting in a pro-tumorigenic response and a hypercoagulatory state. We illustrate that the breast cancer cell secretome can be altered by hormone-therapy, subject to the tumour subphenotype and linked to platelet activation. More sophisticated co-culture systems are required to recapitulate these interactions to better understand tumorigenesis. Moreover, deeper plasma profiling, using abundant protein depleted and/or vesicle enriched strategies, will likely reveal additional secretory proteins related to tumour cell-platelet interactions.

FGFR2 genetic variants in women with breast cancer.

Black African populations are more genetically diverse than others, but genetic variants have been studied primarily in European populations. The present study examined the association of four single nucleotide polymorphisms (SNPs) of the fibroblast growth factor receptor 2, associated with breast cancer in non­African populations, with breast cancer in Black, southern African women. Genomic DNA was extracted from whole blood samples of 1,001 patients with breast cancer and 1,006 controls (without breast cancer), and the rs2981582, rs35054928, rs2981578, and rs11200014 polymorphisms were analyzed using allele­specific Kompetitive allele­specific PCR™, and the χ2 or Fisher's exact tests were used to compare the genotype frequencies. There was no association between those SNPs and breast cancer in the studied cohort, although an association was identified between the C/C homozygote genotype for rs2981578 and invasive lobular carcinoma. These results show that genetic biomarkers of breast cancer risk in European populations are not necessarily associated with risk in sub­Saharan African populations. African populations are more heterogenous than other populations, and the information from this population can help focus genetic risks of cancer in this understudied population.

Breast Tumor Cells Evade the Cytotoxic Action of Anastrozole, Aspirin, and Clopidogrel Cocktail.

Globally, breast cancer is among the most frequently diagnosed and common cause of death among women. Aromatase inhibitors, such as anastrozole, are one of the first-line therapies used in the treatment of breast cancer in postmenopausal women; however, thromboembolic complications are common. Thus, this study investigated the combined effects of anastrozole and antiplatelet therapies, aspirin and clopidogrel, on breast cancer cytotoxicity and survival in vitro. Breast cancer cell lines (MCF-7 and T47D) were treated with varying Cmax concentrations of anastrozole and/or antiplatelet therapies for 24 h. A wound-healing scratch assay was used to measure migration and the WST-1 assay for cellular proliferation. An autophagy/cytotoxicity dual staining kit was used to assay cell death and survival. Changes in cell morphology were assessed using scanning electron microscopy. Data were analyzed with Statistica software. Our findings showed that sub-phenotypic differences exist between the luminal-A breast cancer cell lines, with T47D cells being more aggressive than MCF-7 cells. Cellular proliferation and migration responded in a dose-dependent manner for the different treatment groups. Notably, anastrozole combined with aspirin and clopidogrel mediated higher levels of cell survival than each agent individually, with autophagy levels being significantly increased in comparison to that induced with antiplatelet therapy alone.

Discordance between PAM50 intrinsic subtyping and immunohistochemistry in South African women with breast cancer.

PURPOSE: Breast cancer is a heterogeneous disease with different gene expression profiles, treatment options and outcomes. In South Africa, tumors are classified using immunohistochemistry. In high-income countries multiparameter genomic assays are being utilized with implications for tumor classification and treatment. METHODS: In a cohort of 378 breast cancer patients from the SABCHO study, we investigated the concordance between tumor samples classified by IHC and the PAM50 gene assay. RESULTS: IHC classified patients as ER-positive (77.5%), PR-positive (70.6%), and HER2-positive (32.3%). These results, together with Ki67, were used as surrogates for intrinsic subtyping, and showed 6.9% IHC-A-clinical, 72.7% IHC-B-clinical, 5.3% IHC-HER2-clinical and 15.1% triple negative cancer (TNC). Typing using the PAM50 gave 19.3% luminal-A, 32.5% luminal-B, 23.5% HER2-enriched and 24.6% basal-like. The basal-like and TNC had the highest concordance, while the luminal-A and IHC-A group had the lowest concordance. By altering the cutoff for Ki67, and realigning the HER2/ER/PR-positive patients to IHC-HER2, we improved concordance with the intrinsic subtypes. CONCLUSION: We suggest that the Ki67 be changed to a cutoff of 20-25% in our population to better reflect the luminal subtype classifications. This change would inform treatment options for breast cancer patients in settings where genomic assays are unaffordable.

Targeting Platelet Activation Pathways to Limit Tumour Progression: Current State of Affairs.

The association between cancer and a hypercoagulatory environment is well described. Thrombotic complications serve not only as a major mortality risk but the underlying molecular structure and function play significant roles in enhancing tumour progression, which is defined as the tumour's capacity to survive, invade and metastasise, amongst other hallmarks of the disease. The use of anticoagulant or antiplatelet drugs in cardiovascular disease lessens thrombotic effects, but the consequences on tumour progression require interrogation. Therefore, this review considered developments in the management of platelet activation pathways (thromboxane, ADP and thrombin), focusing on the use of Aspirin, Clopidogrel and Atopaxar, and their potential impacts on tumour progression. Published data suggested a cautionary tale in ensuring we adequately investigate not only drug-drug interactions but also those unforeseen reciprocal interactions between drugs and their targets within the tumour microenvironment that may act as selective pressures, enhancing tumour survival and progression.

Differential Expression of Platelet Activation Markers, CD62P and CD63, after Exposure to Breast Cancer Cells Treated with Kigelia Africana, Ximenia Caffra and Mimusops Zeyheri Seed Oils In Vitro.

Cancer patients, including breast cancer patients, live in a hypercoagulable state. Chemo- and hormone- therapy used in the treatment of breast cancer increases the risk of thrombosis. Due to differences in health care services between developed and developing countries, the survival rate of women with breast cancer in developing countries is low. Consequently, ethnomedicines are used and their efficacy as potential alternatives are being scientifically explored. The seed oils of Kigelia africana, Ximenia caffra and Mimusops zeyheri have anti-proliferative effects on hormone-dependent (MCF-7) and cytotoxic effects on hormone-independent (MDA-MB-231) breast cancer cells. In this study, we determined if these seed oils reduce the thrombogenic ability of breast cancer cells by measuring the platelet surface expression of the activation-specific antigens CD62P and CD63. MDA-MB-231 and MCF-7 cells were pretreated with the seed oils before being exposed to whole blood of human female volunteers. An increase in CD62P and CD63 expression following whole blood exposure to untreated breast cancer cells was observed. Treated MDA-MB-231 cells reduced CD62P and CD63 expression while treated MCF-7 cells increased CD62P and decreased CD63 expression. Kigelia africana, Ximenia caffra and Mimusops zeyheri seed oils are able to reduce the thrombogenic ability of MDA-MB-231 breast cancer cells.

Antiplatelet Therapy Combined with Anastrozole Induces Features of Partial EMT in Breast Cancer Cells and Fails to Mitigate Breast-Cancer Induced Hypercoagulation.

Thromboembolic complications are a leading cause of morbidity and mortality in cancer patients. Cancer patients often present with an increased risk for thrombosis including hypercoagulation, so the application of antiplatelet strategies to oncology warrants further investigation. This study investigated the effects of anastrozole and antiplatelet therapy (aspirin/clopidogrel cocktail or atopaxar) treatment on the tumour responses of luminal phenotype breast cancer cells and induced hypercoagulation. Ethical clearance was obtained (M150263). Blood was co-cultured with breast cancer cell lines (MCF7 and T47D) pre-treated with anastrozole and/or antiplatelet drugs for 24 h. Hypercoagulation was indicated by thrombin production and platelet activation (morphological and molecular). Gene expression associated with the epithelial-to-mesenchymal transition (EMT) was assessed in breast cancer cells, and secreted cytokines associated with tumour progression were evaluated. Data were analysed with the PAST3 software. Our findings showed that antiplatelet therapies (aspirin/clopidogrel cocktail and atopaxar) combined with anastrozole failed to prevent hypercoagulation and induced evidence of a partial EMT. Differences in tumour responses that modulate tumour aggression were noted between breast cancer cell lines, and this may be an important consideration in the clinical management of subphenotypes of luminal phenotype breast cancer. Further investigation is needed before this treatment modality (combined hormone and antiplatelet therapy) can be considered for managing tumour associated-thromboembolic disorder.

Breast Cancer Cells Induce a Pro-inflammatory Response to Mitigate Immune Mediation in a 3D Culture Model.

BACKGROUND: Growth factors and cytokines mediate complex interactions between cells within the breast tumour microenvironment. In advanced cancer, an excess of regulatory T (TREG) lymphocytes and lack of natural killer (NK) cells in tumour-infiltrating lymphocyte populations may reflect a shift to pro-tumorigenic adaptive immune mechanisms. To facilitate targeted assessment of the interactions between tumour and immune cells ex vivo, three-dimensional (3D) culture systems are able to better recapitulate the in vivo microenvironment, recreating the anatomy of tumours. MATERIALS AND METHODS: We used 3D breast tumour models to determine morphological alterations, and the levels of secreted transforming growth factor-ß (TGFß) and induced cytokines. 3D luminal phenotype models and basal phenotype models were generated by culturing NK cells and CD4+CD25+ TREG cells with MCF-7 cells and MDA-MB-231 cells respectively, in growth factor-reduced Matrigel. TGFß was qualitatively assessed by immunolocalisation and cytokine data from culture supernatant was acquired with a multiplex cytokine assay. Traditional statistical analysis and principal component analysis were employed to unravel the cytokine response. RESULTS AND CONCLUSION: We identified that an interleukin-6 (IL6)-chemokine axis associated with TGFß is primarily responsible for differences detected between breast cancer models, with luminal and basal phenotype tumours responding differentially to immune mediation. Identified cytokines are implicated in facilitating tumour cell subversion of immune cell function to promote an invasive phenotype. Moreover, the disruption of the extracellular matrix and failure to form well-differentiated tumour masses/networks is indicative of enhanced malignancy. Tumour cells are implicated in promoting a pro-inflammatory microenvironment to attenuate NK cell function and in inducing a pro-tumorigenic profile that is facilitated by TREG lymphocytes.

Analysis of Immune-Tumor Cell Interactions Using a 3D Co-culture Model.

Three-dimensional (3D) cultures are better able to reflect the tumor microenvironment than two-dimensional (2D) monolayer cultures by facilitating cell-cell interactions in the appropriate spatial dimensions. Here I describe the isolation and co-culture of immune cells with tumor cell lines in a three-dimensional system facilitated by a basement membrane extract. This allows for further downstream applications to analyze interactions between these cell types.

A case of benign osteogenic tumour in Homo naledi: Evidence for peripheral osteoma in the U.W. 101-1142 mandible.

The reported incidence of neoplasia in the extinct hominin record is rare. We describe here the first palaeopathological analysis of an osteogenic lesion in the extinct hominin Homo naledi from Dinaledi Cave (Rising Star), South Africa. The lesion presented as an irregular bony growth, found on the right lingual surface of the body of the adult mandible U.W. 101-1142. The growth was macroscopically evaluated and internally imaged using micro-focus x-ray computed tomography (µCT). A detailed description and differential diagnosis were undertaken using gross and micromorphology, and we conclude that the most probable diagnosis is peripheral osteoma - a benign osteogenic neoplasia. These tumours are cryptic in clinical expression, though they may present localised discomfort and swelling. It has been suggested that muscle traction may play a role in the development and expression of these tumours. The impact of this lesion on the individual affected is unknown. This study adds to the growing corpus of palaeopathological data from the South African fossil record, which suggests that the incidence of neoplastic disease in deep prehistory was more prevalent than traditionally accepted. The study also highlights the utility of micro-computed tomography in assisting accurate diagnoses of ancient pathologies.

Ultrastructural investigation of the time-dependent relationship between breast cancer cells and thrombosis induction.

Thromboembolic complications are a common cause of death in breast cancer patients. The in vivo relationship between coagulation factors and circulating tumours is a multifaceted one, with tumour cells implicated in thrombocytosis and platelets associated with coagulation-mediated metastasis. Platelets and fibrin networks are known to be morphologically altered in patients with cancer, and their relationship with breast cancer cells themselves may increase thrombosis susceptibility. This was investigated in vitro, assessing such morphological alterations through the establishment of a MCF-7 breast cancer cell co-culture system with blood plasma. Co-culture duration ranged from zero to thirty minutes, with five-minute intervals, in order to assess the time-dependent ultrastructural conformations of platelet and fibrin networks, using scanning electron microscopy. It was found that enhanced coagulability was related to co-culture duration. Changes in platelet and fibrin network morphology from normal were visible as early as five minutes in co-culture with MCF-7 cells. With longer co-culture duration thrombosis-linked variation in structural configuration was intensified, including advanced platelet aggregation and adherence characteristics, compression of fibrin networks into plaques of increased density, and upsurge of microparticulate extrusion implicated in amplifying procoagulant events during the metastatic process. These results confirm that cancer cells are stimulators of coagulation even in an in vitro system and breast cancer patients may become increasingly susceptible to thrombotic-related consequences with increased exposure to tumour cells, especially during metastasis.

Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer.

Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell-cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.

Anastrozole and RU486: Effects on estrogen receptor α and Mucin 1 expression and correlation in the MCF-7 breast cancer cell line.

Anastrozole and RU486 are shown to reduce hormone-responsive breast cancer progression when used as adjuvant treatments to surgical intervention, however, a high incidence of cancer recurrence remains. Estrogen receptor alpha (ERα) and Mucin 1 (MUC1), a glycoprotein, are both implicated in breast cancer progression. We assessed whether Anastrozole and RU486 treatment affects the expression of, and relationship between, ERα and MUC1 in the ERα(+) MUC1(+) MCF-7 breast cancer cell line. MCF-7 cells, treated with physiological concentrations of either Anastrozole or RU486 for 18 h or 72 h, were subjected to immunolocalization of both markers. CellProfiler software was used to quantify intensity for statistical analyses. ERα expression increased at both time periods following treatment. MUC1 expression increased with RU486-treatment at both times, whereas Anastrozole induced increased MUC1 expression at 72 h only. The biomarkers demonstrated increased point association at 72 h within treatment groups despite MUC1 diverging from correlation with ERα. We propose that tumor progression is independent of MUC1 and ERα correlation. These preliminary results indicate that withdrawal of adjuvant treatment may result in residual cell populations expressing increased ERα and MUC1. This phenotype may allow enhanced estrogenic and metastatic capacity influencing cancer recurrence, a hypothesis we are investigating further.

Signals from pancreatic mesoderm influence the expression of a pancreatic phenotype in hepatic stem-like cell line PHeSC-A2 in vitro: a preliminary study.

The ex vivo generation of pancreatic cells from adult hepatic stem cells for subsequent transplantation has been proposed as a novel treatment for Diabetes mellitus. The pancreas and liver, closely related developmentally, may retain a shared (hepatopancreatic) stem cell whose plasticity could be exploited to differentiate into either lineage, dependent on environmental signals. This novel study investigated whether signals from pancreatic mesoderm could induce the differentiation of adult hepatic stem cell-like cells into pancreatic endocrine cells in vitro. A porcine hepatic stem-like cell line, designated PHeSC-A2, was co-cultured with quail pancreatic mesoderm in a Growth Factor Reduced Matrigel-Ham's F12.ITS culture system. Immunocytochemical studies revealed insulin- and glucagon-producing cells. Assessment of nuclear morphology indicated that these endocrine cells were PHeSC-A2-derived. It is thus proposed that the PHeSC-A2 cell line has a higher level of plasticity than previously indicated. These preliminary results and assessment of published data have led to the following postulations: (a) permissive signaling from pancreatic mesoderm suffices to induce hepatic stem cells to assume a pancreatic lineage, (b) the pancreatic phenotype assumed by hepatic stem cells is a default state, (c) the differentiation capacity embodied by these cells indicates the existence of a hepatopancreatic stem cell lineage.