Spatial N-glycomics of the normal breast microenvironment reveals fucosylated and high-mannose N-glycan signatures related to BI-RADS density and ancestry.

Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.

Mass Spectrometry Imaging of Fibroblasts: Promise and Challenge.

INTRODUCTION: Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast signaling contributes to many disease states and extracellular matrix secreted by fibroblasts has been used to stratify patient by outcome, recurrence, and therapeutic resistance. Recent advances in imaging mass spectrometry allow access to single cell fibroblasts and their ECM niche within clinically relevant tissue samples. AREAS COVERED: We review biological and technical challenges as well as new solutions to proteomic access of fibroblast expression within the complex tissue microenvironment. Review topics cover conventional proteomic methods for single fibroblast analysis and current approaches to accessing single fibroblast proteomes by imaging mass spectrometry approaches. Strategies to target and evaluate the single cell stroma proteome on the basis of cell signaling are presented. EXPERT OPINION: The promise of defining proteomic signatures from fibroblasts and their extracellular matrix niches is the discovery of new disease markers and the ability to refine therapeutic treatments. Several imaging mass spectrometry approaches exist to define the fibroblast in the setting of pathological changes from clinically acquired samples. Continued technology advances are needed to access and understand the stromal proteome and apply testing to the clinic.

Metabolic Links to Socioeconomic Stresses Uniquely Affecting Ancestry in Normal Breast Tissue at Risk for Breast Cancer.

A primary difference between black women (BW) and white women (WW) diagnosed with breast cancer is aggressiveness of the tumor. Black women have higher mortalities with similar incidence of breast cancer compared to other race/ethnicities, and they are diagnosed at a younger age with more advanced tumors with double the rate of lethal, triple negative breast cancers. One hypothesis is that chronic social and economic stressors result in ancestry-dependent molecular responses that create a tumor permissive tissue microenvironment in normal breast tissue. Altered regulation of N-glycosylation of proteins, a glucose metabolism-linked post-translational modification attached to an asparagine (N) residue, has been associated with two strong independent risk factors for breast cancer: increased breast density and body mass index (BMI). Interestingly, high body mass index (BMI) levels have been reported to associate with increases of cancer-associated N-glycan signatures. In this study, we used matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) to investigate molecular pattern changes of N-glycosylation in ancestry defined normal breast tissue from BW and WW with significant 5-year risk of breast cancer by Gail score. N-glycosylation was tested against social stressors including marital status, single, education, economic status (income), personal reproductive history, the risk factors BMI and age. Normal breast tissue microarrays from the Susan G. Komen tissue bank (BW=43; WW= 43) were used to evaluate glycosylation against socioeconomic stress and risk factors. One specific N-glycan (2158 m/z) appeared dependent on ancestry with high sensitivity and specificity (AUC 0.77, Brown/Wilson p-value<0.0001). Application of a linear regression model with ancestry as group variable and socioeconomic covariates as predictors identified a specific N-glycan signature associated with different socioeconomic stresses. For WW, household income was strongly associated to certain N-glycans, while for BW, marital status (married and single) was strongly associated with the same N-glycan signature. Current work focuses on understanding if combined N-glycan biosignatures can further help understand normal breast tissue at risk. This study lays the foundation for understanding the complexities linking socioeconomic stresses and molecular factors to their role in ancestry dependent breast cancer risk.

Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry.

Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.