Biology subjects

Macdonald, J. K.

Publications and source records attributed to Macdonald, J. K..

4 recordsLinked to original sources

bioRxiv2026

Multimodal Imaging of the Cellular and Extracellular Microenvironment on the Same Formalin-Fixed Paraffin-Embedded Tissue Section

Same-tissue section multimodal imaging is a powerful strategy that spatially profiles tissue histology, cell populations and molecular composition while maximizing tissue economy, preserving spatial molecular relationships, and increasing co-registration capacity. However, performing multiple modalities on the same tissue section can destroy or chemically alter the tissue, compromising downstream data. Here, we systematically assess integration of picrosirius red staining, hematoxylin and eosin staining, and multiplexed immunofluorescence into N-glycan and extracellular matrix peptide matrix-assisted laser/desorption ionization imaging mass spectrometry (IMS) workflows. We evaluate alterations in tissue morphology, stain efficiency, IMS feature intensity as well as IMS feature localization after upstream modality integration. We propose an optimized multimodal sequence that maximizes data quality and follows a very specific order of: autofluorescence microscopy, multiplexed immunofluorescence, picrosirius red staining, N-glycan IMS, hematoxylin and eosin staining, and extracellular matrix peptide IMS. Overall, this work develops an optimized multimodal workflow that comprehensively images tissue morphology, collagen fibers, and cell populations at single-cell resolution as well as multiplexed N-glycan composition and multiplexed extracellular matrix peptides with post-translational modification status from a single tissue section.

biochemistry↗

bioRxiv2026

Extracellular Matrix Proteomic Signatures Associate with Disease-Free Survival in Later Events of Ductal Carcinoma In Situ or Invasive Breast Cancer

BackgroundDuctal carcinoma in situ (DCIS) is a noninvasive breast lesion with variable risk of progression to invasive breast cancer (IBC). Current transcription and cell marker investigations suggest ECM decreases in later events but are limited in details of ECM proteomic composition, including post-translational modifications. We investigated whether the extracellular matrix (ECM) proteome alters with later breast events of DCIS or IBC. MethodsECM-targeted mass spectrometry imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to ten tissue microarrays from the Resource of Archival Human Breast Tissue cohort (RAHBT). Primary DCIS specimens (n=136) were analyzed in relation to later events of DCIS (n=40) or IBC(n=30), with a mean follow-up of 192.1 months 95% CI [179.1,205.1]. Statistical modeling, survival analyses, and exploratory machine learning approaches were used to identify ECM peptide signatures associated with later events. ResultsDistinct ECM peptide profiles were associated with later events of DCIS or IBC. Fifteen peptides derived from fibrillar collagens (COL1A1, COL1A2, COL3A1) and elastin, showed significantly reduced abundance in patients who developed IBC. Lower expression of specific collagen peptides associated with overall 19.9% 95% CI [17.92, 21.81] decreased disease-free survival for IBC. Lower expression of these peptides was significantly associated with reduced disease-free survival (age-adjusted hazard ratio [HR] = 2.45, 95% CI: 2.33-2.57; P < 0.05). Patient-matched samples of primary DCIS, later DCIS, and later invasive breast cancer further demonstrated reduction in ECM peptide detection. Exploratory predictive modeling from patient-matched samples achieved high performance (AUROC >0.98, accuracy >93%) in distinguishing primary from later events. Following prior work in the RAHBT cohort, reduction of certain collagen peptides was also observed in primary DCIS samples from higher risk patient groups. ConclusionsECM proteomic remodeling, particularly decreases of specific collagen domains, is strongly associated with later events of DCIS and IBC. These findings highlight ECM proteome as a critical regulator of breast cancer emergence with potential as a prognosticator of risk stratification to guide clinical management of DCIS.

pathology↗

bioRxiv2025

Spatial Proteomics of the Normal Breast Collagen Stroma: Links to BI-RADS Categories and Body Mass Index

Collagen breast stroma is the basis of increased breast density and a well-established breast cancer risk factor, yet proteomic regulation of normal breast stroma remains poorly defined. This study reports spatial regulation of the collagen proteome in normal breast tissue sections annotated by clinical characteristics. Normal breast samples from the Susan G. Komen tissue bank included data on genetic ancestry (n=40 total; n=20 African ancestry; n=20 European ancestry), body-mass-index (BMI), age, and mammogram density by the Breast Imaging Reporting and Data System (BI-RADS). Multiplexed cell marker staining showed CD44 and COL1A1 markers modulated with BMI. Collagen fiber widths by second harmonic generation (SHG) showed potential contrasts in BMI categories by genetic ancestry. Targeted extracellular matrix proteomics mass spectrometry imaging showed collagen alpha-1(I) chain domain proteome was spatially heterogenous across the normal breast microenvironment with site specific post-translational modification of proline hydroxylation. Signatures computationally extracted from breast stroma reported that 47 collagen peptides distinguished BI-RADS categories (area under the receiver operating curve>0.7; p-value>0.05). Proteomic alterations were found between overweight to obese categories with strong positive associations to BMI by multivariate analysis. This study provides the first spatial analysis of the collagen proteome in normal breast within contexts of cellular markers and clinical characteristics.

cancer biology↗

bioRxiv2024

Flagellar motility and the mucus environment influence aggregation mediated antibiotic tolerance of Pseudomonas aeruginosa in chronic lung infection

Pseudomonas aeruginosa frequently causes chronic lung infection in individuals with muco-obstructive airway diseases (MADs). Chronic P. aeruginosa infections are difficult to treat, primarily owing to antibiotic treatment failure, which is often observed in the absence of antimicrobial resistance. In MADs, P. aeruginosa forms biofilm-like aggregates within the luminal mucus. While the contribution of mucin hyperconcentration towards antibiotic tolerance has been described, the mechanism for mucin driven antibiotic tolerance and the influence of aggregates have not been fully elucidated. In this study, we investigated the contribution of flagellar motility towards aggregate formation as it relates to the diseased mucus environment. We found that loss of flagellar motility resulted in increased P. aeruginosa aggregation and tolerance to multiple classes of antibiotics. Further, we observed differential roles in antimicrobial tolerance of the motAB and motCD stators, which power the flagellum. Additionally, we found that control of fliC expression was important for aggregate formation and antibiotic tolerance as a strain constitutively expressing fliC was unable to form aggregates and was highly susceptible to treatment. Lastly, we demonstrate that neutrophil elastase, an abundant immune mediator and biomarker of chronic lung infection, promotes aggregation and antibiotic tolerance by impairing flagellar motility. Collectively, these results highlight the key role of flagellar motility in aggregate formation and antibiotic tolerance and deepens our understanding of how the MADs lung environment promotes antibiotic tolerance of P. aeruginosa. IMPORTANCEAntibiotic recalcitrance of chronic Pseudomonas aeruginosa infections in muco-obstructive airway diseases is a primary driver of mortality. Mechanisms that drive antibiotic tolerance are poorly understood. We investigated motility phenotypes related to P. aeruginosa adaptation and antibiotic tolerance in the diseased mucus environment. Loss of flagellar motility drives antibiotic tolerance by promoting aggregate formation. Regulation of flagellar motility appears to be a key step in aggregate formation as the inability to turn off flagellin expression resulted in poor aggregate formation and increased antibiotic susceptibility. These results deepen our understanding of the formation of antibiotic tolerant aggregates within the MADs airway and opens novel avenues and targets for treatment of chronic P. aeruginosa infections.

microbiology↗