Breast Cancer Awareness Month: Research and Activism at WPI

Abstract:
Bulk chemotherapy and drug release strategies for cancer treatment have been associated with lack of specificity and high drug concentrations that often result in toxic side effects. This work presents the results of an experimental study of cancer drugs (prodigiosin or paclitaxel) conjugated to Luteinizing Hormone-Releasing Hormone (LHRH) for the specific targeting and treatment of triple negative breast cancer (TNBC). Injections of LHRH-conjugated drugs (LHRH-prodigiosin or LHRH-paclitaxel) into groups of 4-week-old athymic female nude mice (induced with subcutaneous triple negative xenograft breast tumors) were found to specifically target, eliminate or shrink tumors at early, mid and late stages without any apparent cytotoxicity, as revealed by in vivo toxicity and ex vivo histopathological tests. Our results show that overexpressed LHRH receptors serve as binding sites on the breast cancer cells/tumor and the LHRH-conjugated drugs inhibited the growth of breast cells/tumor in in vitro and in vivo experiments. The inhibitions are attributed to the respective adhesive interactions between LHRH molecular recognition units on the prodigiosin (PGS) and paclitaxel (PTX) drugs and overexpressed LHRH receptors on the breast cancer cells and tumors. The implications of the results are discussed for the development of ligand-conjugated drugs for the specific targeting and treatment of TNBC.

Abstract:
In this paper, we report the development of the nanotube-CTC-chip for isolation of tumor-derived epithelial cells (circulating tumor cells, CTCs) from peripheral blood, with high purity, by exploiting the physical mechanisms of preferential adherence of CTCs on a nanotube surface. The nanotube-CTC-chip is a new 76-element microarray technology that combines carbon nanotube surfaces with microarray batch manufacturing techniques for the capture and isolation of tumor-derived epithelial cells. Using a combination of red blood cell (RBC) lysis and preferential adherence, we demonstrate the capture and enrichment of CTCs with a 5-log reduction of contaminating WBCs. EpCAM negative MDA-MB-231/luciferase-2A-green fluorescent protein (GFP) cells were spiked in the blood of wild mice and enriched using an RBC lysis protocol. The enriched samples were then processed using the nanotube-CTC-chip for preferential CTC adherence on the nanosurface and counting the GFP cells yielded anywhere from 89% to 100% capture from the droplets. Electron microscopy (EM) studies showed focal adhesion with filaments from the cell body to the nanotube surface. We compared the nanotube preferential adherence to collagen adhesion matrix (CAM) scaffolding, reported as a viable strategy for CTC capture in patients. The CAM scaffolding on the device surface yielded 50% adherence with 100% tracking of cancer cells (adhered vs. non-adhered) versus carbon nanotubes with >90% adherence and 100% tracking for the same protocol. The nanotube-CTC-chip successfully captured CTCs in the peripheral blood of breast cancer patients (stage 1–4) with a range of 4–238 CTCs per 8.5 ml blood or 0.5–28 CTCs per ml. CTCs (based on CK8/18, Her2, EGFR) were successfully identified in 7/7 breast cancer patients, and no CTCs were captured in healthy controls (n = 2). CTC enumeration based on multiple markers using the nanotube-CTC-chip enables dynamic views of metastatic progression and could potentially have predictive capabilities for diagnosis and treatment response.

Abstract:
Tamoxifen is a selective estrogen receptor (ER) modulator that is used to treat ER-positive breast cancer, but that at high doses kills both ER-positive and ER-negative breast cancer cells. We recapitulate this off-target effect in Caenorhabditis elegans, which does not have an ER ortholog. We find that different bacteria dramatically modulate tamoxifen toxicity in C. elegans, with a three-order of magnitude difference between animals fed Escherichia coli, Comamonas aquatica, and Bacillus subtilis. Remarkably, host fatty acid (FA) biosynthesis mitigates tamoxifen toxicity, and different bacteria provide the animal with different FAs, resulting in distinct FA profiles. Surprisingly these bacteria modulate tamoxifen toxicity by different death mechanisms, some of which are modulated by FA supplementation and others by antioxidants. Together, this work reveals a complex interplay between microbiota, FA metabolism and tamoxifen toxicity that may provide a blueprint for similar studies in more complex mammals.

Abstract:
Detection of breast cancer in the early stages is associated with higher cure rates and better survival, and also requires fewer intensive treatments. Current breast cancer screening via mammography is unsuitable for use among (younger) women with more dense breasts, and also has limitations in its ability to detect aggressive breast cancers. In this research article, we describe a breast cancer detection test that is based on the detection of ‘circulating tumor cells’ in blood samples. This test can detect breast cancer CTCs with high accuracy across all age groups, hormone receptor subtypes, histological subtypes, and disease grade. In our study, this test detected breast cancer cases and differentiated them from healthy (cancer-free) females as well as those with non-cancerous conditions with high accuracy. This test has negligible risk of false positive findings, as well as high detection rate for early-stage (localized) breast cancer. Clinical adoption of this test can be beneficial in cancer screening as well as in detection of breast cancers in suspected cases.

Abstract:
Inadequate availability of organs and tissues for transplantation is a major problem. While offering an alternative, development of engineered tissue constructs containing a functional vascular network that allows delivery of nutrients and especially O2 remains a challenge. Using native vasculature, especially microvasculature, of a tissue or an organ offers potential to overcome this shortcoming. Decellularized animal organs retain the extracellular matrix (ECM) scaffold and native vascular networks but are challenging to source. A low cost, more readily available, and consistent source of tissues and organs is required. We investigated a plant scaffold model that would mimic input (arterial)-output (venous) flow by grafting two Aptenia cordifolia leaves together with opposite facing petioles. The abaxial epidermis of the top leaf and the adaxial epidermis of the bottom leaf were removed; wounds were soaked for 20 s in 0.1-mg L−1 naphthaleneacetic acid (NAA) and 1-mg L−1 benzylaminopurine (BAP) before horizontally appressing the wounded side of each leaf together. Leaves were decellularized 2 to 4 wk after grafting, lyophilized, and stored at room temperature. Prior to use, grafts were sterilized by ethylene oxide and rehydrated. Structural connections between the leaves were visualized histologically in thin sections using hematoxylin and eosin (H&E) and toluidine blue stains. Ponceau Red dye and red blood cells were perfused into the grafted leaves through one petiole of one leaf after decellularization to observe flow from input petiole into the first and then the second of the grafted leaves prior to exiting via the output petiole. Grafts were recellularized with the green fluorescent protein (GFP) expressing human breast cancer line MDA-MB231 and cell attachment and morphology observed 7 d post cell seeding. Results suggest that these grafted leaves provided an input-output cell-compatible vascularized scaffold as a possible bioscaffold for engineering tissues for transplantation.

Abstract:
Single-cell RNA sequencing (scRNA-seq) is a recent technology that enables fine-grained discovery of cellular subtypes and
specific cell states. Analysis of scRNA-seq data routinely involves machine learning methods, such as feature learning, clustering, and classification, to assist in uncovering novel information from scRNA-seq data. However, current methods are
not well suited to deal with the substantial amount of noise that is created by the experiments or the variation that occurs
due to differences in the cells of the same type. To address this, we developed a new hybrid approach, deep unsupervised
single-cell clustering (DUSC), which integrates feature generation based on a deep learning architecture by using a new
technique to estimate the number of latent features, with a model-based clustering algorithm, to find a compact and informative representation of the single-cell transcriptomic data generating robust clusters. We also include a technique to
estimate an efficient number of latent features in the deep learning model. Our method outperforms both classical and
state-of-the-art feature learning and clustering methods, approaching the accuracy of supervised learning. We applied
DUSC to a single-cell transcriptomics data set obtained from a triple-negative breast cancer tumor to identify potential
cancer subclones accentuated by copy-number variation and investigate the role of clonal heterogeneity. Our method
is freely available to the community and will hopefully facilitate our understanding of the cellular atlas of living organisms
as well as provide the means to improve patient diagnostics and treatment.

Abstract:
The goal of this project was to build policy modules in a synthetic health system to analyze how healthcare policy impacts breast cancer survival rates. To do any inference regarding healthcare policy, researchers need secure and protected health data, which are restricted by privacy laws and interoperability issues. Synthetic health systems generate and help investigate health data without concerns of violating legal restrictions (HIPAA). In this research, we programmed health insurance and loss-of-care modules into a synthetic health system simulator (Synthea) to simulate and analyze the impact of health insurance on breast cancer survival rates. Our goal was for our health insurance and loss-of-care implementations to be realistic and reflective of the real world, in which we were successful.

Abstract:
It has previously been shown that the simultaneous activation of PI3K (phosphatidylinositol 3-kinase) and Ras/MAPK (mitogen-activated protein kinases) pathways facilitate tumor growth despite only inducing cancer cell dormancy individually. Determining the impacts on cellular mechanics each pathway incites alone and in unison is critical to developing non-toxic cancer therapies for triple-negative breast cancers. PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression in healthy MCF-10A human breast epithelial cells activated the PI3K and Ras/MAPK pathways, respectively. Cell stiffness and fluidity were simultaneously measured using atomic force microscopy. Results suggest that PTEN knockout reduced cell stiffness and increased cell fluidity independent of PI3K activation. Effects of activated KRAS overexpression on cell stiffness depends on rigidity of cell culture substrate. Activated KRAS overexpression also counteracts the effects of PTEN knockout.

Abstract:
Recent emphasis has been placed on the role of epigenetic regulators and epigenetic marks as biomarkers for cancer diagnosis and prognosis, and as therapeutic targets for treatment. One such class of regulators is the protein arginine methyltransferase (PRMT) family. The present study examined available curated data regarding the expression and alteration of one of the least studied PRMT family members, PRMT8, in various types of cancer and cancer cell lines. Publicly available cancer data on PRMT8 expression were examined using the Human Protein Atlas and the Kaplan-Meier Plotter, and reverse transcription-polymerase chain reaction was used to screen a selection of human cell lines for variant-specific PRMT8 expression. High levels of PRMT8 expression in breast, ovarian and cervical cancer was observed. Additionally, in patients with breast and ovarian cancer, high PRMT8 expression was correlated with increased patient survival, whereas in gastric cancer, high PRMT8 expression was correlated with decreased patient survival. The present study also investigated the expression of PRMT8 variant 2, a novel transcript variant recently identified in our laboratory, in various cancer cell lines. Variant-specific expression of PRMT8 in numerous distinct cancer cell lines derived from different tissues, including the expression of the novel PRMT8 variant 2 in U87MG glioblastoma cells was demonstrated. The present study proposes the possibility of PRMT8 as a cancer biomarker, based on the high level of PRMT8 expression in various types of cancer, particularly in tissues that would not normally be expected to express PRMT8, and on the correlation of PRMT8 and patient lifespan in several cancer types. Variant-specific expression of PRMT8 in diverse cancer cell lines suggests the possibility of alternate PRMT8 isoforms to have diverse effects on cancer cell phenotypes.

Abstract:
Purpose
Osteoporosis increases the risk of fracture and is often considered a late effect of breast cancer treatment. We examined the prevalence of compromised bone health in a sample of exclusively African-American (AA) breast cancer survivors since bone mineral density (BMD) varies by race/ethnicity in healthy populations.

Methods
Using a case–control design, AA women in a weight loss intervention previously diagnosed and treated for stages I–IIIa breast cancer were matched 1:1 on age, race, sex, and BMI with non-cancer population controls (n = 101 pairs) from National Health and Nutrition Examination Survey (NHANES). Questionnaires and dual-energy x-ray absorptiometry (DXA) scanning were completed, and participants were categorized as having normal bone density, low bone mass, or osteoporosis using the World Health Organization (WHO) definition for femoral neck T-scores.

Results
The majority of these overweight/obese survivors were 6.6 (±4.7) years post-diagnosis, had stage II (n = 46) or stage III (n = 16) disease, and treated with chemotherapy (76 %), radiation (72 %), and/or adjuvant hormone therapies (45 %). Mean femoral neck BMD was significantly lower in cases vs. matched non-cancer population controls (0.85 ± 0.15 vs. 0.91 ± 0.14 g/cm2, respectively; p = 0.007). However, the prevalence of low bone mass and osteoporosis was low and did not significantly differ between groups (n = 101 pairs; p = 0.26), even when restricted to those on adjuvant hormone therapies (n = 45 pairs; p = 0.75). Using conditional logistic regression, controlling for dietary factors and education, the odds of developing compromised bone health in AA breast cancer survivors was insignificant (OR 1.5, 95 % CI 0.52, 5.56).

Conclusions
These null case–control findings challenge the clinical assumption that osteoporosis is highly prevalent among all breast cancer survivors, providing foundational evidence to support differences by race/ethnicity and body weight.

Implications for Cancer Survivors
Routine bone density testing and regular patient–provider dialogue is critical in overweight/obese AA breast cancer survivors to ensure that healthy lifestyle factors (e.g., ideal weight, regular weight-bearing exercises, dietary adequacy of calcium and vitamin D) support optimal skeletal health.

Abstract:
The breast cancer susceptibility gene protein, also known as γ-synuclein, is highly expressed in human breast cancer in a stage-specific manner, with highest expression in late stage cancer. In model systems, γ-synuclein binds phospholipase Cβ2 which is regulated by Gαq to generate intracellular Ca2 + signals. PLCβ2, which is also absent in normal tissue but highly expressed in breast cancer, is additionally regulated by Rac to promote migration pathways. We have found that γ-synuclein binds to the same region of PLCβ2 as Gαq. Using cells that mimic stage 4 breast cancer (MDA MB 231), we show that down-regulation of γ-synuclein reduces the protein level of PLCβ but increases the transcript level over 40 fold. γ-Synuclein down-regulation also promotes the interaction between Gαq and PLCβ resulting in a stronger Ca2 + response to Gαq agonists. The ability of γ-synuclein to interfere with Gαq–PLCβ interactions allows more PLCβ to colocalize with Rac impacting Rac-mediated pathways that may give rise to cancerous phenotypes.