Citations

Background: Discovering risk biomarkers in small benign breast disease (BBD) biopsies is constrained by scarce tissue and microanatomic heterogeneity of terminal duct lobular units (TDLUs). We tested whether tissue-sparing tissue microarray (TMA)–based Digital Spatial Profiling (DSP) can deliver reproducible, biologically coherent protein measurements across benign lobules and breast cancers (BCs), and how well DSP aligns with standard immunoassays. Methods: We performed a pilot using tissues from the Mayo Clinic BBD cohort using TMAs representing four contexts: terminal duct lobular units (TDLUs) from BBD biopsies preceding BC and matched BBD-controls, subsequent BCs, and BC-associated TDLUs. We profiled 79 proteins by DSP (37 retained after QC) and benchmarked against chromogenic IHC and OPAL immunofluorescence. Reproducibility was evaluated using intraclass correlation coefficients (ICCs), cross-platform agreement (weighted kappa), marker correlations, and mixed-effects models with false-discovery-rate (FDR) control. Results: We analyzed 368 BBD-TDLU cores (88 cases; 88 controls), 204 BC cores and 110 BC-associated TDLU cores. ICCs were highest in BC tissues, and lower in BC-associated TDLUs and BBD-TDLUs. Agreement was slight–to-fair in TDLUs but moderate (ER/PR) to substantial (BCL2) in BC. DSP recapitulated expected immunologic correlations (CD45 with T-cell, B-cell, and macrophage markers) and tissue-type gradients (BC > BC-associated TDLUs > BBD-TDLUs). Exploratory case–control differences in BBD-TDLUs did not persist after FDR control. Conclusions: TMA-based DSP is feasible in archival breast tissues and yields biologically coherent, cross-platform-benchmarked profiles that are particularly robust in BC, while conserving scarce TDLUS and clarifying current limits of single-marker risk stratification from benign lobules. These data provide a foundation for refined sampling and expanded panels in future TDLU-focused studies.

Selective inhibitors are essential for targeted therapeutics and for probing enzyme functions in various biological systems. The two main challenges in identifying such protein‐based inhibitors lie in the extensive experimental effort required, including the generation of large libraries, and in tailoring the selectivity of inhibitors to enzymes with homologous structures. To address these challenges, machine learning (ML) is being used to improve protein design by training on targeted libraries and identifying key interface mutations that enhance affinity and specificity. However, such ML‐based methods are limited by inaccurate energy calculations and difficulties in predicting the structural impacts of multiple mutations. Here, we present an ML‐based method that leverages HTS data to streamline the design of selective protease inhibitors. To demonstrate its utility, we applied our new method to find inhibitors of matrix metalloproteinases (MMPs), a family of homologous proteases involved in both physiological and pathological processes. By training ML models on binding data for three MMPs (MMP‐1, MMP‐3, and MMP‐9), we successfully designed a novel N‐TIMP2 variant with a differential specificity profile, namely, high affinity for MMP‐9, moderate affinity for MMP‐3, and low affinity for MMP‐1. Our experimental validation showed that this novel variant exhibited a significant specificity shift and enhanced selectivity compared with wild‐type N‐TIMP2. Through molecular modeling and energy minimization, we obtained structural insights into the variant's enhanced selectivity. Our findings highlight the power of ML‐based methods to reduce experimental workloads, facilitate the rational design of selective inhibitors, and advance the understanding of specific inhibitor–enzyme interactions in homologous enzyme systems.

Selective inhibition of the more than 100 S1 family serine proteases is a long-standing challenge due to their active site similarity. Mesotrypsin, implicated in cancer progression, exemplifies these difficulties; no current inhibitors achieve selectivity over other human trypsins. We found an unexpected autoinhibited conformation of mesotrypsin via x-ray crystallography, revealing a cryptic pocket adjacent to the active site. Using high-throughput virtual screening targeting this cryptic pocket, we identified a conformationally selective small-molecule inhibitor that stabilizes the inactive state of mesotrypsin. This inhibitor demonstrates selectivity for mesotrypsin over other trypsins. Our findings challenge the accepted view of digestive trypsins as constitutively active enzymes lacking potential for allosteric regulation. Furthermore, analyses of other structures suggest that dynamic sampling of closed states with analogous allosteric cryptic pockets appears widespread among S1 serine proteases. These observations point to a potentially generalizable strategy to achieve selective inhibition, offering broad implications for drug development targeting serine proteases in cancer and other diseases.

Abstract Purpose: Black women are disproportionately affected by aggressive breast cancers compared to white women, suggesting underlying biological differences in normal breast tissue. This study aims to identify molecular disparities in normal breast tissue between premenopausal Black and white women that may contribute to increased breast cancer risk. Experimental Procedures: Visium spatial transcriptomic analysis was performed on a pilot cohort of normal breast tissue samples from premenopausal Black and white women. Tissue sections were spatially profiled, and capture spots were classified based on predominant cell types: epithelial, stromal, or endothelial. Differential gene expression analysis between racial groups was conducted for each cell type classification. Multiplex immunofluorescence (mIF) was used to validate selected markers in the pilot cohort. Meta-analysis compared differentially expressed genes with published gene sets associated with breast cancer subtypes. Results: Significant racial differences in gene expression profiles were identified in both epithelial and stromal spots. In epithelial spots, genes upregulated in Black women significantly overlapped with over 20 published gene sets associated with estrogen receptor-negative (ER−) breast cancer, including key genes involved in Wnt signaling inhibition (e.g., WIF1) and basal epithelial markers (e.g., KRT14, KRT15). This suggests alterations in pathways linked to more aggressive breast cancer phenotypes. In stromal spots, differentially expressed genes related to extracellular matrix remodeling and fibroblast activation (e.g., PI16, DPT, EFEMP1) were observed, indicating potential differences in stromal-epithelial interactions. Ongoing deconvolution and spatial analyses are being performed to further elucidate these findings. Preliminary mIF validation confirmed increased expression of PI16 in samples from Black women. Validation of additional markers is underway in a larger cohort of premenopausal Black and white women. Conclusions: Spatial transcriptomic profiling of normal breast tissue reveals distinct molecular differences between Black and white women, particularly in pathways associated with aggressive breast cancer subtypes and stromal interactions. These findings suggest that inherent variations in the normal breast microenvironment may contribute to the increased risk and aggressiveness of breast cancer in Black women. Further validation and analysis may identify potential biomarkers for risk assessment and inform targeted prevention strategies. Citation Format: Camila Pacheco Erak, Nicole Cruz-Reyes, Pria Westerman, Joshua Ogony, Jean R. Clemenceau, Tae Hyun Hwang, Sarah McLaughlin, Derek Radisky. Spatial transcriptomic profiling of normal breast tissue reveals potential mechanisms underlying increased breast cancer risk in Black women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 765.

Abstract Background: Postpartum breast cancer (PPBC) is associated with increased morbidity and mortality among women within 5 to 10 years following childbirth. The postpartum breast undergoes involution, involving tissue remodeling and immune cell infiltration, potentially creating a pro-tumorigenic microenvironment. The molecular mechanisms underlying this elevated risk remain poorly understood. In this study, we compared normal breast tissue samples and human mammary epithelial cells (HMECs) from postpartum women to those from age- and BMI-matched nulliparous women to elucidate potential molecular contributors to PPBC risk. Methods: Transcriptomic profiling was performed on RNA extracted from FFPE normal breast tissues using the NanoString IO 360 and BC 360 platforms to examine gene expression in nulliparous and postpartum women under 45 years of age. HMECs were isolated from organoids derived from breast tissue samples and used for next-generation sequencing and knockdown experiments targeting STAT1 and GBP1. Gene set enrichment analysis (GSEA) was applied to NanoString and RNA-seq datasets to identify differentially enriched genes between postpartum and nulliparous women. Results: Transcriptomic analysis revealed significant upregulation of interferon-stimulated genes, including STAT1 and GBP1, in postpartum breast tissues, consistent with increased immune responses during involution. NanoString data showed that markers of cell proliferation, such as Ki67, STAT1, and GBP1, were significantly higher in samples from parous compared to nulliparous women (p < 0.05). GSEA revealed that cell cycle-promoting genes (GBP1, cyclins, CDKs) were upregulated, while cyclin-dependent kinase inhibitors (CDKNs) were downregulated in postpartum breast tissues. Immunohistochemistry confirmed significantly higher expression of Ki67, STAT1, and GBP1 in postpartum versus nulliparous breast tissues (p < 0.0001). Silencing STAT1 and GBP1 in HMECs resulted in decreased cell proliferation, G1 cell cycle arrest, and increased expression of CDKN1A and CDKN1C. Conclusion: These findings suggest that STAT1 and GBP1 promote proliferation in postpartum breast epithelial cells by downregulating cyclin-dependent kinase inhibitors, potentially contributing to the increased risk of PPBC. Targeting these pathways may offer novel strategies for mitigating postpartum breast cancer risk. Citation Format: Joshua Ogony, Melody Stallings-Mann, Sarah McLaughlin, Mark Sherman, Derek C. Radisky. STAT1 and GBP1 promote proliferation in postpartum breast epithelium by downregulating cyclin-dependent kinase inhibitors: implications for increased postpartum breast cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 187.

Abstract Introduction: Pseudoangiomatous stromal hyperplasia (PASH) is a benign breast lesion characterized by stromal myofibroblast accumulation. While most women with PASH do not develop breast cancer, a subset eventually does, often in the same breast where PASH was originally diagnosed, even after surgical removal of the lesion. This pattern suggests the presence of a field effect, where alterations in the PASH microenvironment extend beyond the lesion itself, potentially influencing neighboring breast tissue. This study aims to identify specific fibroblasts within PASH-containing tissues that may create a microenvironment conducive to cancer initiation. Methods: We conducted a retrospective analysis of formalin-fixed, paraffin-embedded breast tissue samples from women diagnosed with PASH, comparing those who later developed breast cancer (cases) to those who remained cancer-free (controls). Using transcriptional profiling with NanoString IO360/BC360 panels, multiplex immunofluorescence (MxIF), and spatial transcriptomics via NanoString CosMX SMI, we investigated molecular and histopathological differences. We focused on the expression of fibroblast activation markers (ITGB1, PDGFRβ) and their spatial relationships with proliferating epithelial cells, as well as changes in extracellular matrix (ECM) composition and stiffness. Results: Transcriptional profiling revealed significant upregulation of proliferation-associated genes in epithelial cells from cases. MxIF showed higher Ki-67 levels in epithelial cells from cases, with proliferating cells forming clustered regions. Activated fibroblasts expressing ITGB1 and PDGFRβ were markedly increased in cases and were located closer to proliferating epithelial cells. Spatial transcriptomics uncovered gene expression changes in fibroblasts indicative of an activated phenotype, and in epithelial cells, consistent with responses to increased tissue stiffness and mechanotransduction signaling. Polarized light imaging with picrosirius red staining revealed enhanced collagen cross-linking and thick fibril formation in cases, indicating greater ECM stiffness. Conclusions: Our findings suggest that a subset of activated fibroblasts in benign breast tissue creates a pro-tumorigenic microenvironment through ECM remodeling and increased tissue stiffness, promoting epithelial proliferation and potentially driving cancer development. The spatial proximity of these fibroblasts to proliferating epithelial cells underscores their possible role in early carcinogenesis. This study introduces the novel concept of cancer-predisposing fibroblasts in benign breast tissue, providing new insights into microenvironmental changes that precede cancer and offering potential targets for early detection and intervention. Citation Format: Nicole Cruz-Reyes, Pria Westerman, Matilde Rossi, Camila Pacheco Erak, Melody Stallings Mann, Bryan McCauley, Robert Vierkant, Stacey Winham, Mark Sherman, Amy Degnim, Derek Radisky. Activated fibroblasts in benign breast tissue are associated with subsequent breast cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2083.

Abstract Background: Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are the most common lung cancer subtypes. SCC is traditionally resistant to anti-angiogenic therapies, though the mechanisms remain unclear. Nintedanib, a multi-angiokinase receptor inhibitor, is approved for treating ADC in combination with docetaxel, based on selective efficacy in the LUME-1 trial. It is also FDA-approved for idiopathic pulmonary fibrosis due to its antifibrotic properties. Both ADC and SCC feature fibrotic stroma rich in activated tumor-associated fibroblasts (TAFs). Our prior work showed Nintedanib has stronger antifibrotic effects on ADC-TAFs than SCC-TAFs. However, its impact on TAF-driven angiogenesis and the role of TAFs in SCC resistance to multi-angiokinase inhibitors remain unexplored. Methods: The pro-angiogenic activity of TAFs secretome from ADC and SCC patients was assessed. TAFs were stimulated with TGF-β1 to mimic their activated phenotype, then treated with Nintedanib. Conditioned media from treated TAFs was used to assess endothelial cell migration and vascular network formation. Pro-angiogenic factor secretion was analyzed using antibody arrays, and functional roles of selected proteins were validated in vitro and in vivo using genetic approaches. Results: Nintedanib inhibited the pro-angiogenic effects of ADC-TAF secretome but paradoxically enhanced angiogenesis in SCC-TAFs, increasing endothelial cell migration in vitro and in vivo. Notably, Nintedanib selectively upregulated CXCL-1 secretion in SCC-TAFs. Silencing CXCL-1 using siRNA in SCC-TAFs abrogated the Nintedanib-induced angiogenesis. Similar findings with other multi-angiokinase inhibitors suggest aberrant CXCL-1 production in SCC-TAFs may drive universal resistance to these therapies. Conclusion: SCC-TAFs are resistant to the anti-angiogenic effects of Nintedanib, which paradoxically promotes angiogenesis via CXCL-1 upregulation. This study provides the first mechanistic insight into SCC resistance to anti-angiogenic therapies and underscores the critical role of TAFs in modulating therapeutic responses. Targeting CXCL-1 or TAF-mediated pathways may inform alternative anti-angiogenic strategies in SCC, potentially in combination with immune checkpoint inhibitors. Citation Format: Natalia Isabel Diaz Valdivia, Paula Duch, Alejandro Bernardo, Marselina Arshakyan, Noemi Reguart, Derek Radisky, Oriol Casanovas, Jordi Alcaraz. Intrinsic resistance mechanism of squamous cell lung carcinomas to the multiangiokinase receptor inhibitor Nintedanib through CXCL-1 upregulation in tumor associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5554.

Abstract Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the function and activity of matrix metalloproteinases (MMPs), enzymes that are known for their involvement in cancer progression. MMP-9, also known as gelatinase B, plays a crucial role in tumorigenesis of several cancers including triple negative breast cancer (TNBC); tumor cell-produced MMP-9 promotes malignant progression and metastasis of basal-like TNBC. Here, we have used full-length TIMP-1 as a scaffold for engineering inhibitors that bind with higher selectivity to MMP-9, for potential therapeutic applications. We previously developed a yeast surface display library of human TIMP-1, with diversity incorporated at 17 residues within five loops; mutation burden was tuned to 3-5 mutations per clone. Here, we have screened the TIMP-1 library for affinity toward recombinant MMP-9 by using fluorescence-activated cell sorting (FACS). Yeast cells were incubated with an incrementally decreasing concentration of biotinylated MMP-9 (400 nM to 25 nM) in successive rounds of sorting. Samples were analyzed using flow cytometry and highest affinity populations were selected using FACS. Among the TIMP-1 variants with highest enrichment, we identified the TIMP-1-C15 variant that showed 12-fold enhanced binding with improved selectivity toward MMP-9 compared with wild type TIMP-1 (WT TIMP-1). We next expressed and purified soluble recombinant TIMP-1-C15 and conducted inhibition studies using a fluorogenic substrate to evaluate inhibitory potency towards MMP-9 activity. These experiments showed that TIMP-1-C15 inhibits MMP-9 activity with an inhibition constant (Ki) of 0.61 nM, more than 5-fold stronger than WT TIMP-1; these studies also corroborated the improved selectivity towards MMP-9 over other MMPs. Additionally, we further evaluated the functional impact of the TIMP-1-C15 variant on cancer cell invasiveness using TNBC cell lines in cellular invasion assays. The TIMP-1-C15 variant markedly reduced the invasive capabilities of TNBC cells to a greater extent than WT TIMP-1. Currently, we are working to isolate additional TIMP-1 variants from our library that exhibit enhanced selectivity for MMP-9, with MMP-8 included in the sorting process as a competitive target to ensure specificity. Selective inhibition of MMP-9 over MMP-8 is particularly important in breast cancer therapy, as MMP-8 has been shown to have anti-tumorigenic properties, and its inhibition could counteract therapeutic efforts aimed at reducing cancer invasiveness. Our findings suggest that engineered TIMP-1 variants could serve as potent inhibitors of MMP-9, offering a novel approach to reducing the invasiveness of breast cancer cells and potentially limiting the progression of metastatic disease. Citation Format: Alireza Shoari, Alexandra Hockla, Mathew A. Coban, Alexandra M. Dimesa, Derek C. Radisky, Evette S. Radisky. Engineered TIMP-1 for selective MMP-9 inhibition: A novel strategy to suppress breast cancer invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5115.

Abstract Introduction: Fibroadenoma is the most common type of benign breast lesion and is not traditionally considered an indicator of increased breast cancer risk. This study aimed to identify biomarkers within fibroadenoma associated with subsequent breast cancer development using patient samples from the Mayo Clinic Benign Breast Disease (BBD) cohort. Methods: We analyzed formalin-fixed paraffin-embedded (FFPE) breast biopsy samples from patients diagnosed with non-proliferative fibroadenoma who either developed breast cancer later (n=10) or remained cancer-free (n=5). NanoString RNA profiling identified differentially expressed genes in fibroblasts associated with subsequent cancer development. Immunohistochemistry was first done to compare overall expression of PI16 in this cohort. Opal multiplex immunofluorescence staining (mIF) was then performed to evaluate the expression of PI16 in fibroblasts (Vim+PanCK-) and assess immune cell infiltration (CD45+) in different tissue compartments. Fibroblasts were identified by vimentin positivity (Vim+), and epithelial cells were marked by pan-cytokeratin positivity (PanCK+). Results: Compared to patients who remained cancer-free, those who developed breast cancer showed significantly increased expression of PI16 in fibroblasts (PI16+Vim+PanCK-, 3.25-fold increase, p=0.0011). Additionally, the number of immune cells (CD45+) was significantly decreased (0.52-fold, p=0.03) in the fibroadenoma lesions of patients who later developed cancer. Conclusions: Our findings suggest that elevated expression of PI16 in fibroblasts within fibroadenoma may serve as potential biomarkers for predicting breast cancer risk for women with fibroadenoma. PI16 encodes Peptidase Inhibitor 16, which is implicated in modulating fibroblast activity and extracellular matrix remodeling—processes that can contribute to a tumor-supportive microenvironment. Elevated levels of this protein in fibroblasts suggest an altered microenvironment that may foster tumor initiation and progression. The decreased immune cell infiltration further implies an immunosuppressive microenvironment that may facilitate cancer progression. As these markers can be detected in FFPE samples, our results could lead to the development of a readily applicable clinical test to identify fibroadenoma patients at increased risk for breast cancer, enabling earlier intervention and surveillance. Citation Format: Pria Westerman, Nicole Cruz-Reyes, Camila Pacheco Erak, Robert Vierkant, Bryan McCauley, Stacey Winham, Mark Sherman, Amy Degnim, Derek Radisky. Elevated PI16 expression in fibroadenoma fibroblasts is associated with subsequent breast cancer development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2077.