Multiparametric profiling of HER2-enriched extracellular vesicles in breast cancer using Single Extracellular VEsicle Nanoscopy.

BACKGROUND: Patients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy - such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids. RESULTS: Using Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls. CONCLUSIONS: SEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.

Disparities in the use of screening breast magnetic resonance imaging persist in Louisiana after the Affordable Care Act: A question of access, policy, institutional support, or something else?

BACKGROUND: Louisiana continues to have one of the highest breast cancer mortality rates in the nation, and Black women are disproportionally affected. Louisiana has made advances in improving access to breast cancer screening through the expansion of Medicaid. There remains, however, broad underuse of advanced imaging technology such as screening breast magnetic resonance imaging (MRI), particularly for Black women. METHODS: Breast MRI has been proven to be very sensitive for the early detection of breast cancer in women at high risk. MRI is more sensitive than mammography for aggressive, invasive breast cancer types, which disproportionally affect Black women. Here the authors identify potential barriers to breast MRI screening in Black women, propose strategies to address disparities in access, and advocate for specific recommendations for change. RESULTS: Cost was identified as one of the greatest barriers to screening breast MRI. The authors propose implementation of cost-saving, abbreviated protocols to address cost along with lobbying for further expansion of the Affordable Care Act (ACA) to include coverage for screening breast MRI. In addition, addressing gaps in communication and knowledge and facilitating providers' ability to readily identify women who might benefit from MRI could be particularly impactful for high-risk Black women in Louisiana communities. CONCLUSIONS: Since the adoption of the ACA in Louisiana, Black women have continued to have disproportionally high breast cancer mortality rates. This persistent disparity provides evidence that additional change is needed. This change should include exploring innovative ways to make advanced imaging technology such as breast MRI more accessible and expanding research to specifically address community and culturally specific barriers.

Arginine regulates HSPA5/BiP translation through ribosome pausing in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a high mortality rate due to a lack of therapeutic targets. Many TNBC cells are reliant on extracellular arginine for survival and express high levels of binding immunoglobin protein (BiP), a marker of metastasis and endoplasmic reticulum (ER) stress response. METHODS: In this study, the effect of arginine shortage on BiP expression in the TNBC cell line MDA-MB-231 was evaluated. Two stable cell lines were generated in MDA-MB-231 cells: the first expressed wild-type BiP, and the second expressed a mutated BiP free of the two arginine pause-site codons, CCU and CGU, termed G-BiP. RESULTS: The results showed that arginine shortage induced a non-canonical ER stress response by inhibiting BiP translation via ribosome pausing. Overexpression of G-BiP in MDA-MB-231 cells promoted cell resistance to arginine shortage compared to cells overexpressing wild-type BiP. Additionally, limiting arginine led to decreased levels of the spliced XBP1 in the G-BiP overexpressing cells, potentially contributing to their improved survival compared to the parental WT BiP overexpressing cells. CONCLUSION: In conclusion, these findings suggest that the downregulation of BiP disrupts proteostasis during arginine shortage-induced non-canonical ER stress and plays a key role in cell growth inhibition, indicating BiP as a target of codon-specific ribosome pausing upon arginine shortage.

Molecular features of androgen-receptor low, estrogen receptor-negative breast cancers in the Carolina breast cancer study.

PURPOSE: Androgen receptor (AR) expression is absent in 40-90% of estrogen receptor (ER)-negative breast cancers. The prognostic value of AR in ER-negative patients and therapeutic targets for patients absent in AR remains poorly explored. METHODS: We used an RNA-based multigene classifier to identify AR-low and AR-high ER-negative participants in the Carolina Breast Cancer Study (CBCS; N = 669) and The Cancer Genome Atlas (TCGA; N = 237). We compared AR-defined subgroups by demographics, tumor characteristics, and established molecular signatures [PAM50 risk of recurrence (ROR), homologous recombination deficiency (HRD), and immune response]. RESULTS: AR-low tumors were more prevalent among younger (RFD = + 10%, 95% CI = 4% to 16%) participants in CBCS and were associated with HER2 negativity (RFD = - 35%, 95% CI = - 44% to - 26%), higher grade (RFD = + 17%, 95% CI = 8% to 26%), and higher risk of recurrence scores (RFD = + 22%, 95% CI = 16.1% to 28%), with similar results in TCGA. The AR-low subgroup was strongly associated with HRD in CBCS (RFD = + 33.3%, 95% CI = 23.8% to 43.2%) and TCGA (RFD = + 41.5%, 95% CI = 34.0% to 48.6%). In CBCS, AR-low tumors had high adaptive immune marker expression. CONCLUSION: Multigene, RNA-based low AR expression is associated with aggressive disease characteristics as well as DNA repair defects and immune phenotypes, suggesting plausible precision therapies for AR-low, ER-negative patients.

Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression.

A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.

Harmonization of Busulfan Plasma Exposure Unit (BPEU): A Community-Initiated Consensus Statement.

Busulfan therapeutic drug monitoring (TDM) is often used to achieve target plasma exposures. Variability in busulfan plasma exposure units (BPEU) is a potential source for misinterpretation of publications and protocols and is a barrier to data capture by hematopoietic cell transplantation (HCT) registry databases. We sought to harmonize to a single BPEU for international use. Using Delphi consensus methodology, iterative surveys were sent to an increasing number of relevant clinical stakeholders. In survey 1, 14 stakeholders were asked to identify ideal properties of a BPEU. In survey 2, 52 stakeholders were asked (1) to evaluate BPEU candidates according to ideal BPEU properties established by survey 1 and local position statements for TDM and (2) to identify potential facilitators and barriers to adoption of the harmonized BPEU. The most frequently used BPEU identified, in descending order, were area under the curve (AUC) in µM × min, AUC in mg × h/L, concentration at steady state (Css) in ng/mL, AUC in µM × h, and AUC in µg × h/L. All respondents conceptually agreed on the ideal properties of a BPEU and to adopt a harmonized BPEU. Respondents were equally divided between selecting AUC in µM × min versus mg × h/L for harmonization. AUC in mg × h/L was finally selected as the harmonized BPEU, because it satisfied most of the survey-determined ideal properties for the harmonized BPEU and is read easily understood in the clinical practice environment. Furthermore, 10 major professional societies have endorsed AUC in mg × h/L as the harmonized unit for reporting to HCT registry databases and for use in future protocols and publications.

Obesity and Triple-Negative Breast Cancer: Disparities, Controversies, and Biology.

Once considered a problem of Western nations, obesity (body mass index ≥30 kg/m2) has rapidly increased since the 1970s to become a major threat to world health. Since 1970, the face of obesity has changed from a disease of affluence and abundance to a disease of poverty. During the last 10 years, studies have mechanistically linked obesity and an obese tumor microenvironment with signaling pathways that predict aggressive breast cancer biology. For example, in the United States, African American women are more likely than non-Hispanic European American women to be obese and to be diagnosed with triple-negative breast cancer (TNBC). In 2008, the Carolina Breast Study found that obesity (increased waist/hip ratio) was linked to an increased incidence of TNBC in premenopausal and postmenopausal African American women. Subsequently, several groups have investigated the potential link between obesity and TNBC in African American women. To date, the data are complex and sometimes contradictory. We review epidemiologic studies that investigated the potential association among obesity, metabolic syndrome, and TNBC in African American women and mechanistic studies that link insulin signaling to the obese breast microenvironment, tissue inflammation, and aggressive TNBC biology.

Förster resonance energy transfer to impart signal-on and -off capabilities in a single microRNA biosensor.

Many studies have found that over- or under-expression of biomolecules called microRNAs (miRNA) regulates several diseases. Biosensors are in need to visually identify the relative expression level of miRNA to determine the direction these miRNA change in cells and tissues. Our established reporter+probe miRNA biosensor design requires that miRNA outcompete and displace the reporter from the probe. Once displaced, the reporter folds into a hairpin structure to force together a pair of Förster Resonance Energy Transfer (FRET) dyes. The donor and acceptor signal changes can be used to indicate the over-/under-expression of miRNA. The bright signal from the donor will indicate miRNA under-expression; the bright acceptor signal will indicate miRNA over-expression. Since close proximity of the dyes to each other and nucleic acids often quench fluorescence, polyethylene glycol spacers were added in-between the dyes and nucleic acids. We compared reporter designs with and without spacers to investigate the effects on the following analytical metrics: (1) extent of signal change, (2) limits of detection and quantitation, and (3) sensitivity. Systematic errors and amount of reporter+probe biosensor formed were evaluated for one of the biosensors. Cy3|Cy5 and 6-carboxyfluorescein (6-FAM)|ATTO 633 dye pairs on reporters containing spacers showed an increase in the acceptor signal change by ∼190 and ∼484%, respectively, compared to no spacers. Transduction mechanisms that enhance and quench the signal both showed LODs that ranged from 3-17 nanomolar (nM) with 100 nM of the biosensor.

Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance.

Trastuzumab therapy in HER2+ breast cancer patients has mixed success owing to acquired resistance to therapy. A detailed understanding of downstream molecular cascades resulting from trastuzumab resistance is yet to emerge. In this study, we investigate the cellular mechanisms underlying acquired resistance using trastuzumab-sensitive and -resistant cancer cells (BT474 and BT474R) treated with endogenous ligands EGF and HRG across time. We probe early receptor organization through microscopy and signaling events through multiomics measurements and assess the bioenergetic state through mitochondrial measurements. Integrative analyses of our measurements reveal significant alterations in EGF-treated BT474 HER2 membrane dynamics and robust downstream activation of PI3K/AKT/mTORC1 signaling. EGF-treated BT474R shows a sustained interferon-independent activation of the IRF1/STAT1 cascade, potentially contributing to trastuzumab resistance. Both cell lines exhibit temporally divergent metabolic demands and HIF1A-mediated stress responses. BT474R demonstrates inherently increased mitochondrial activity. HRG treatment in BT474R leads to a pronounced reduction in AR expression, affecting downstream lipid metabolism with implications for treatment response. Our results provide novel insights into mechanistic changes underlying ligand treatment in BT474 and BT474R and emphasize the pivotal role of endogenous ligands. These results can serve as a framework for furthering the understanding of trastuzumab resistance, with therapeutic implications for women with acquired resistance.

Insulin Resistance in Women Correlates with Chromatin Histone Lysine Acetylation, Inflammatory Signaling, and Accelerated Aging.

BACKGROUND: Epigenetic changes link medical, social, and environmental factors with cardiovascular and kidney disease and, more recently, with cancer. The mechanistic link between metabolic health and epigenetic changes is only starting to be investigated. In our in vitro and in vivo studies, we performed a broad analysis of the link between hyperinsulinemia and chromatin acetylation; our top "hit" was chromatin opening at H3K9ac. METHODS: Building on our published preclinical studies, here, we performed a detailed analysis of the link between insulin resistance, chromatin acetylation, and inflammation using an initial test set of 28 women and validation sets of 245, 22, and 53 women. RESULTS: ChIP-seq identified chromatin acetylation and opening at the genes coding for TNFα and IL6 in insulin-resistant women. Pathway analysis identified inflammatory response genes, NFκB/TNFα-signaling, reactome cytokine signaling, innate immunity, and senescence. Consistent with this finding, flow cytometry identified increased senescent circulating peripheral T-cells. DNA methylation analysis identified evidence of accelerated aging in insulin-resistant vs. metabolically healthy women. CONCLUSIONS: This study shows that insulin-resistant women have increased chromatin acetylation/opening, inflammation, and, perhaps, accelerated aging. Given the role that inflammation plays in cancer initiation and progression, these studies provide a potential mechanistic link between insulin resistance and cancer.

Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men.

Lung cancer is the third most common cancer with Black/AA men showing higher risk and poorer outcomes than NHW men. Lung cancer disparities are multifactorial, driven by tobacco exposure, inequities in care access, upstream health determinants, and molecular determinants including biological and genetic factors. Elevated expressions of protein arginine methyltransferases (PRMTs) correlating with poorer prognosis have been observed in many cancers. Most importantly, our study shows that PRMT6 displays higher expression in lung cancer tissues of Black/AA men compared to NHW men. In this study, we investigated the underlying mechanism of PRMT6 and its cooperation with PRMT1 to form a heteromer as a driver of lung cancer. Disrupting PRMT1/PRMT6 heteromer by a competitive peptide reduced proliferation in non-small cell lung cancer cell lines and patient-derived organoids, therefore, giving rise to a more strategic approach in the treatment of Black/AA men with lung cancer and to eliminate cancer health disparities.

Does the Vaginal Microbiome Operate Differently by Race to Influence Risk of Precervical Cancer?

Background: The vaginal microbiome (VMB) plays an important role in the persistence of human papillomavirus (HPV) infection and differs by race and among women with cervical intraepithelial neoplasia (CIN). Materials and Methods: We explored these relationships using 16S rRNA VMB taxonomic profiles of 3050 predominantly Black women. VMB profiles were assigned to three subgroups based on taxonomic markers indicative of vaginal wellness: optimal (Lactobacillus crispatus, L. gasseri, and L. jensenii), moderate (L. iners), and suboptimal (Gardnerella vaginalis, Atopobium vaginae, Ca. Lachnocurva vaginae, and others). Multivariable Firth logistic regression models were adjusted for age, smoking, VMB, HPV, and pregnancy status. Results: VMB prevalence by subgroup was 18%, 30%, and 51% for the optimal, moderate, and suboptimal groups, respectively. In fully adjusted models, the risk of CIN grade 3 (CIN3) among non-Latina (nL) Blacks was twice that of nL Whites (odds ratio [OR] = 2.0, 95% confidence interval [CI]: 1.1, 3.9, p = 0.02). The VMB modified this association (p = 0.04) such that the risk of CIN3 was significantly higher for nL Blacks than for nL Whites only among women with optimal VMBs (OR = 7.8, 95% CI: 1.7, 74.5, p = 0.007). Within racial groups, the risk of CIN3 was only elevated among nL White women with suboptimal VMBs (OR = 6.0, 95% CI: 1.3, 56.9, p = 0.02) compared with their racial counterparts with optimal VMBs. Conclusions: Our findings suggest that race is a modifier of the VMB in HPV carcinogenesis. An optimal VMB does not appear to be protective for nL Black women compared with nL White women.

Pilot and feasibility study: prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways.

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

Racial Disparity in Quadruple Negative Breast Cancer: Aggressive Biology and Potential Therapeutic Targeting and Prevention.

Black/African-American (AA) women, relative to their White/European-American (EA) counterparts, experience disproportionately high breast cancer mortality. Central to this survival disparity, Black/AA women have an unequal burden of aggressive breast cancer subtypes, such as triple-negative breast cancer (ER/PR-, HER2-wild type; TNBC). While TNBC has been well characterized, recent studies have identified a highly aggressive androgen receptor (AR)-negative subtype of TNBC, quadruple-negative breast cancer (ER/PR-, HER2-wildtype, AR-; QNBC). Similar to TNBC, QNBC disproportionately impacts Black/AA women and likely plays an important role in the breast cancer survival disparities experienced by Black/AA women. Here, we discuss the racial disparities of QNBC and molecular signaling pathways that may contribute to the aggressive biology of QNBC in Black/AA women. Our immediate goal is to spotlight potential prevention and therapeutic targets for Black/AA QNBC; ultimately our goal is to provide greater insight into reducing the breast cancer survival burden experienced by Black/AA women.