Identification of natural, bioactive compounds has been of interest to the biomedical field, in particular for the treatment of cancer. Flavonoids are a family of compounds, present across many plant species, known for their polyphenolic structure that results in cellular bioactivity. The flavonoid, sakuranetin, is produced due to external stressors, through the naringenin biosynthetic pathway. Binding affinity and docking simulations confirmed sakuranetin binding to the estrogen receptor alpha (ERα) pocket in a manner similar to, but with weaker affinity than 17β-estradiol (E2). Due to sakuranetin having chemical similarities to estrogen, this study evaluated the potential of sakuranetin to act as an endocrine modulator in estrogen receptor-positive (ERα+) breast cancer cell lines. In the ERα + cell lines, MCF-7 and T-47D, sakuranetin treatment induced dose-dependent estrogenic activity (≥ 10 μM). Sakuranetin significantly increased colony formation and cell proliferation in the MCF-7 cell line. Breast cancer cell lines with constitutively active ER through an inserted mutation in the ERα (Y537S) demonstrated no significant changes in estrogenic activity or cellular proliferation following sakuranetin treatment, except at an elevated dose (50 μM). Finaly, sakuranetin treatment enhanced genes associated with ER signaling and significantly increased ERα-mediated gene (PGR and CXCL12) expression. These results support the role of sakuranetin as a natural estrogenic compound.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of cancer with poor clinical outcomes. There is a critical need to identify novel, druggable targets for TNBC to improve therapy response and patient outcomes. Due to their roles in critical processes driving cancer progression, kinases have been a major focus of drug discovery efforts. The role of extracellular signal-regulated kinase 5 (ERK5) in mediating TNBC extracellular matrix (ECM) has previously been described in 2D culture and in vivo. Here, we characterized the impact of ERK5 on breast cancer biology in 2D culture, 3D spheroids, and our 3D breast adipose-macrophysiological system (BA-MaPS). Methods: We assessed migration changes in MDA-MB-231 parental and ERK5-knockout (ERK5-ko) cells cultured in the three in vitro models using transwell, scratch, and spheroid pseudo-migration assays. Differential gene expression among these cell lines in the three platforms was assessed by RNA sequencing and pathway analysis. Stromal remodeling of adipocytes and matrix was evaluated by H&E and Masson's Trichrome. Results: Across the in vitro models, ERK5 deletion impaired TNBC cell migration. ERK5-mediated transcriptomic changes included genes associated with epithelial-to-mesenchymal transition (EMT) and migration, with further analysis showing significant alterations in core and associated matrisome. Histological staining corroborated the downregulation of collagen with ERK5 depletion in the BA-MaPS. The NFκB pathway was significantly upregulated only in the ERK5-ko 2D-cultured cells, not in 3D spheroids nor the BA-MaPS model. Conclusions: These results indicate a link between ERK5 and TNBC progression through regulation of TME remodeling, EMT, and cell motility. Differences in 2D culture, 3D spheroid, and BA-MaPS underscore the importance of using physiologically relevant models in breast cancer research.

Glioblastoma (GBM) is the most aggressive and prevalent primary brain tumor in adults, characterized by rapid growth, diffuse infiltration, and a dismal prognosis. Despite advances in conventional therapies, the median survival remains approximately one year, emphasizing the urgent need for novel therapeutic strategies. GD2, a disialoganglioside overexpressed in several malignancies, has been implicated in tumorigenesis and metastasis and has been identified as a cancer stem cell marker. While previous reports have identified high levels of GD2 expression in gliomas compared to normal brain tissue, its role in GBM stemness remains controversial. In this study, we revisited prior findings refuting GD2 ' s involvement in GBM stemness by replicating key tumorigenesis experiments and further explored its impact on stemness properties such as migration and metabolic plasticity. Additionally, a phytochemical screen was used to identify natural compounds as potential inhibitors targeting GD2-mediated tumorigenesis. Our findings aim to clarify GD2 ' s role in GBM and provide insights into novel therapeutic interventions.

Current 3D tumor models for aggressive breast cancers inadequately recapitulate the native tumor microenvironment (TME), leading to poor translational potential. There is a critical need for models capable of mimicking the unique biochemical signals present in the TME. To address this gap, breast tissue and a patient-derived xenograft tumor were decellularized and processed to produce breast tissue- and tumor-specific decellularized extracellular matrices (dECM). Histology confirmed complete cellular removal while maintaining the ECM. Further, DNA content was significantly reduced while ECM composition (POSTN, COLI, FN1) was retained. Breast dECM was incorporated (0, 5, 10, 20, and 50 µg/mL) with triple-negative breast cancer cell lines to generate spheroids. Imaging and histology demonstrated that cells in low dECM (5 and 10 µg/mL) formed compact singular spheres, while higher dECM concentrations (20 and 50 µg/mL) resulted in cells concentrated on the outer edge of the sphere and irregular sphere circularity. RNA-sequencing of MDA-MB-231 dECM spheres demonstrated that gene changes were mediated by both the inclusion of dECM and its composition. High-density tumor dECM upregulated genes associated with metastasis, while high-density breast dECM enhanced tumor suppressors and anti-metastasis genes. These findings indicate that dECM provides physiological cues in 3D tumor models by incorporating TME.

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype accounting for 10-15% of all breast cancer cases. It is further characterized by high resistance to chemotherapy and low survival rates. Lipid composition and patient demographics, such as obesity, have been associated with TNBC, and obesity related fatty acids in the tumor microenvironment (TME) can drive metabolic inflammation and promote TNBC progression. Despite this, the unique impact of the TNBC subtype on lipid composition within the TME, and its subsequent role as an energy source for TNBC cell growth and disease progression, remains poorly understood. Therefore, deciphering the lipid heterogeneity in the TME of TNBC will provide novel insights into TNBC progression and ultimately unveil novel therapeutic targets. Prior work by our collaborative group has demonstrated a heterogeneous distribution of lipids across TNBC tumors compared to matched normal tissue. Further, there was an increase in distinct lipid species, including linoleic acid (LA) and oleic acid (OA). We hypothesized that the distinct TNBC lipid composition enriched in LA and OA fuels TNBC progression. To understand the impact of TNBC-driven TME remodeling in the context of lipid composition, we developed a model of TNBC enriched for LA and OA through the co-culture of differentiated breast adipose-derived ASCs (BrASC) and TNBC. We demonstrated that an adipocyte-TNBC co-culture model can be successfully constructed by treating BrASC with fatty acids distinct to TNBC during differentiation to adipogenic lineage; fatty acid production and uptake was confirmed with quantitative BODIPY fluorescence microscopy. TNBC cell viability was confirmed with quantitative fluorescence microscopy, and up to 200µM of lipid can be added to the system while retaining cell viability. Our results demonstrated that BrASC incorporated exogenous lipids during adipogenic differentiation, and the co-culture of these BrASC with TNBC provided lipid transfer. Overall, our results suggest that an in vitro model can be developed to interrogate the impact of heterogeneous lipids on the TNBC-TME. Future studies will include evaluating the impact of an altered lipid profile in BrASC on TNBC progression. In conclusion, the long-term goal of this research is to develop an in vitro tool to study the impact of the unique lipid TME on TNBC, thus improving upon current pre-clinical models for TNBC. Citation Format: Khudeja Salim, Elnaz Sheikh, Jorge A. Belgodere, Bridgette M. Collins Burow, Van H. Barnes, Manas R. Gartia, Matthew E. Burow, Elizabeth C. Martin. Development of breast adipose-cancer co-culture model to decipher the impact of lipid heterogeneity on triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB241.

Abstract Metaplastic breast cancer accounts for >1% of all breast cancers and is marked by an aggressive phenotype with poor patient survival. Individuals diagnosed with metaplastic breast cancer have higher rates of recurrence, metastasis, and limited therapeutics options. Further the five-year survival rate of metaplastic breast cancer is 55%. Metaplastic breast cancer is characterized by unique histological features and recent evidence suggests that metaplastic breast tumors have extensive extracellular matrix (ECM) remodeling, including altered protein expression and increased ECM stiffness. Due to the scarcity of this tumor type, accurate modeling of the metaplastic ECM would provide enhanced in vitro testing and ultimately guide the discovery of novel targeted treatments for this rare disease. Here we demonstrate changes in the metaplastic TNBC tumor microenvironment and preliminary modeling of the metaplastic matrix in vitro. Specifically, SEM imaging revealed enhanced pore size and stiffness in the metaplastic tumor compared to matched distal breast adipose tissue. Further, metaplastic TNBC had significant enrichment for ECM proteins, notably glycoproteins (MFAP2, POSTN, FN1), compared to distal adipose. The enhanced expression of the glycoprotein MFAP2 in primary metaplastic and non-metaplastic TNBC breast cancer cell lines demonstrated enrichment of genes associated with the biological process: epithelial-to-mesenchymal transition. In depth analysis of genes elevated with MFAP2 expression in metaplastic TNBC demonstrated elevated expression of genes associated with a cancer stem like phenotype and ECM remodeling. Overall, our results establish an extracellular signature and onco-architecture for the metaplastic triple-negative tumor type. Citation Format: Elizabeth Martin, Katherine Hebert, Mackenzie Hawes, Thomas Cheng, Delia Carlino, Matthew E. Burow, Bridgette M. Collins-Burow, Jorge Belgodere. Modeling the metaplastic triple negative breast cancer matrix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4926.

Alterations in the tumor extracellular environment and matrix stiffness promote tumor progression. Furthermore, correlational studies have identified enrichment of extracellular matrix (ECM) proteins (glycoproteins, collagens) in breast tumors. Despite these findings, there has yet to be an interdisciplinary analysis of both ECM composition and structural architecture in rare breast tumors, such as metaplastic breast cancer. Here, we explored changes in ECM protein expression and architecture in a triple-negative breast cancer (TNBC) metaplastic tumor through SEM, proteomics, and RNA sequencing. SEM revealed that the tumor pore size was larger compared to the control adipose tissue. Oscillating rheometry demonstrated increased ECM stiffness in the tumor compared to the control adipose breast adipose. Proteomic analysis of the metaplastic TNBC tumor showed significant enrichment for ECM proteins, notably glycoproteins compared to the control adipose. Interestingly, these samples showed no observed changes in expression for major fibrillar collagens COL1A1 and COL1A2, and a reduced expression of COL3A1. To determine the impact of less characterized ECMs in metaplastic TNBC, we overexpressed MFAP2 in primary metaplastic breast cancer cells and performed RNA sequencing. MFAP2 overexpression was associated with upregulation of epithelial-to-mesenchymal transition-related genes. Overall, our results establish an extracellular signature and onco-architecture for the metaplastic triple-negative tumor type.

Triple-negative breast cancer (TNBC) is a highly invasive breast cancer subtype that is challenging to treat due to inherent heterogeneity and absence of estrogen, progesterone, and human epidermal growth factor 2 receptors. Kinase signaling networks drive cancer growth and development, and kinase inhibitors are promising anti-cancer strategies in diverse cancer subtypes. Kinase inhibitor screens are an efficient, valuable means of identifying compounds that suppress cancer cell growth in vitro, facilitating the identification of kinase vulnerabilities to target therapeutically. The Kinase Chemogenomic Set is a well-annotated library of 187 kinase inhibitor compounds that indexes 215 kinases of the 518 in the known human kinome representing various kinase networks and signaling pathways, several of which are understudied. Our screen revealed 14 kinase inhibitor compounds effectively inhibited TNBC cell growth and proliferation. Upon further testing, three compounds, THZ531, THZ1, and PFE-PKIS 29, had the most significant and consistent effects across a range of TNBC cell lines. These cyclin-dependent kinase (CDK)12/CDK13, CDK7, and phosphoinositide 3-kinase inhibitors, respectively, decreased metabolic activity in TNBC cell lines and promote a gene expression profile consistent with the reversal of the epithelial-to-mesenchymal transition, indicating these kinase networks potentially mediate metastatic behavior. These data identified novel kinase targets and kinase signaling pathways that drive metastasis in TNBC.

Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer, with high resistance to chemotherapy and low survival rates, and accounts for 10-15% of all breast cancer cases. TNBC incidence has been associated with obesity and fatty acids in the tumor microenvironment (TME) derived from lipids can induce obesity-driven metabolic inflammation and affect TNBC pathology. However, the unique impact of the TNBC subtype on lipid composition in the TME, and its subsequent role as an energy source for cancer cell metabolism and progression is unknown. Understanding lipid heterogeneity in the TME will provide novel insights to target TNBC. It is, therefore, crucial to understand the impact of TNBC-driven TME remolding in the context of lipid composition. Our study aims to decipher the role of lipid composition in the progression of TNBC. Spatial lipid profiling of hormone receptor-positive (HR+) and TNBC primary tumors was performed and compared to matched normal breast adipose tissue. Raman mapping of lipids showed a heterogeneous distribution of lipids across the TNBC tissues, including five omega-3 fatty acids, two omega-6 fatty acids, and one unsaturated fatty acid were upregulated in TNBC tumors compared to normal-matched adipose. To determine the unique impact of lipids elevated in TNBC, we next demonstrated that the uptake of lipid compounds (linoleic acid, docosahexaenoic acid, and oleate acid) by TNBC cells increased cell proliferation after 72 hours of treatment. Overall, our results suggest that lipid composition plays a key role in breast TME and may represent a novel therapeutic strategy to target TNBC by alternate sensitivity to therapeutic modalities including apoptosis and ferroptosis. Citation Format: Khudeja Salim, Elnaz Sheikh, Bridgette M. Collins-Burow, Van T. Hoang, Elizabeth C. Martin, Matthew E. Burow, Manas R. Gartia. Impact of lipid composition on triple-negative breast cancer progression and survival [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-06-09.

Kudzu's invasive nature has contributed to its classification as a weed, as it frequently outcompetes native plant species, leading to extensive overgrowth. Efforts to control kudzu have proven challenging, with moderate success using physical or biological agents. In this study, we evaluated the effects of two such control agents, ultraviolet C radiation and Myrothecium verrucaria, to significantly increase the production of tuberosin, a phytoalexin isoflavone. Our findings demonstrate that estrogenic activity of tuberosin is cell-type-dependent, displaying antagonist or competitive inhibition when combined with 17-β-estradiol in the estrogen receptor (ER) positive cell lines MCF-7 and T-47D, while showing dose-dependent agonist activity in HEK293 cells transfected to express both ER receptors (α and β). Tuberosin was shown to modulate ER pathways, alter ER-mediated gene expression, and increase cell proliferation in a dose-dependent manner while maintaining expression of the ERα protein. Binding affinity and docking simulations confirmed tuberosin binding to the ERα pocket in a similar but weaker manner compared to synthetic estrogen. Tuberosin-treated endothelial cells suppressed vascular network assembly and maturation without affecting the cellular proliferative capacity. The presented studies leverage current kudzu management methods to naturally produce tuberosin, examine cell-type-specific effects, and support further investigation as an antiestrogen for breast cancer treatment.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of hormone receptors and human epidermal growth factor receptor 2. This subtype is difficult to treat due to lack of known protein targets, is often refractory to chemotherapy, and has a high rate of recurrence. Stromal components, like collagens in the extracellular matrix (ECM) in the TNBC tumor microenvironment (TME) can contribute to treatment resistance by altering the immune microenvironment. Identifying the drivers of immunosuppressive ECM will yield novel predictive biomarkers in TNBC and guide effective treatment options. In the TME, different stromal cells secrete different proteins and breast adipose-derived stromal cells (BrASCs) produce different collagens than differentiated breast adipocytes. Thus adipogenesis in the TNBC TME can alter the ECM significantly. Furthermore, adipogenesis in TNBC is associated with increased pro-tumorigenic M2 macrophages. To determine the interplay between TNBC and components of the TME on immune recruitment, we performed spatial profiling of primary patient TNBC tumors with a specific focus on the transcriptome of tumor regions with macrophage infiltration compared to tumor regions without macrophage infiltration. Results demonstrated that in M2 macrophage infiltrated areas of TNBC tumor, expression of FN1 and COL5A1 was decreased in both the tumor and the stromal compartments. In contrast regions without macrophage infiltration had enriched FN1 and COL5A1. To determine if TME stroma composition modulated the collagen type and thus macrophage phenotype, we next developed a 3D tumor spheroid model using either BrASCs or BrASCs differentiated to an adipocyte lineage. Model analysis revealed a phenotypic change in differentiated and non-differentiated 3D BrASC spheroids. Post induction of differentiation with supplemented media, spheroids were measured for circularity, referenced to a perfect circle value of 1; induced spheroids retained compact circularity ∼0.8, while non-induced controls were eccentric and irregularly shaped ∼0.5. Spheroids in both conditions were viable on day 7, as assessed by staining with calcein AM and ethidium homodimer-1. Next spheroids were evaluated for adipogenesis and collagen production. Once validated, this model will be used as a platform to understand the impact TNBC ECM on immune regulation. Citation Format: Nicole Cullen, Khudeja Salim, Megan C. Benz, Jovanny Zabaleta, Bruce A. Bunnell, Van T. Hoang, Matthew E. Burow, Elizabeth C. Martin, Bridgette M. Collins-Burow. Use of breast adipose-derived stromal cells in a 3D spheroid model to recapitulate the triple-negative breast cancer tumor microenvironment [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-06-05.

Objectives/Goals: The never in mitosis kinase (NEK) family regulates vital processes, namely cell cycle progression, but their potential as therapeutic targets in TNBC has not been fully explored. Our studies aim to develop a toolkit to investigate the functional roles of NEKs in pathologies including carcinogenesis. Methods/Study Population: To assess differential NEK expression in normal and tumor tissues and correlation of gene expression with patient survival, we used Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier Plotter (KMPlot) pan-cancer analysis, respectively. Basal NEK protein levels were determined by immunoblot across a panel of cell lines, including breast cancer, osteosarcoma, hepatocellular carcinoma, and non-cancerous cells, to identify appropriate systems for evaluation of NEK function. Doxycycline-inducible cell lines were generated by transduction with lentiviral stocks of NEK shRNA and overexpression constructs and antibiotic selection. Expression was analyzed by qPCR and immunoblot. Results/Anticipated Results: Expression of NEK2, 4, 5, 6, 8, and 11 was higher in breast tumors compared to normal tissue by GEPIA analysis. Further examination using KMPlot showed a correlation between elevated NEK6 expression and decreased overall survival in patients with aggressive cancers. As an initial proof-of-concept study, we analyzed NEK6 protein expression in breast cancer cells. Levels of NEK6 were elevated in TNBC cells (MDA-MB-231) compared to hormone receptor positive (HR+) breast cancer cells (MCF7). Using complementary approaches to investigate the functional role of NEK6 in breast cancer, we depleted NEK6 expression using shRNAs in TNBC cells and expressed NEK6 in HR+ cells Discussion/Significance of Impact: Because kinase dysregulation promotes oncogenesis and metastasis, targeting kinases is a key strategy in therapeutic development. A NEK-specific molecular toolkit allows researchers to elucidate NEK functions and contributions to carcinogenesis, promoting advancement of novel therapies.

The Never-in-Mitosis A-Related Kinase (NEK) family is an important, yet largely understudied, family of protein kinases involved in the regulation of a variety of critical cellular processes. Consequently, dysregulation of NEK function has been linked to the etiology and progression of several disorders, including cancer, ciliopathies, neurodegenerative disorders, inflammatory disorders, and other pervasive diseases. In this review, we have summarized recent findings to provide an overview of the NEK family and their diverse functions within various cellular contexts. In parallel, we have highlighted the emerging roles of NEK family members in human health, identifying potential therapeutic targets within the NEK family and exploring their potential for future clinical applications. Finally, we have addressed ongoing challenges and emerging research directions in this rapidly evolving field, aiming to pave the way for future discoveries and innovations.

Naringenin (Nar) is a citrus fruit-derived phytoestrogen, a group of dietary compounds produced by a wide variety of plants. Due to structural similarity to 17‐β‐estradiol (E2), phytoestrogens can bind to estrogen receptors (ERs) to exert context-dependent estrogenic and/or anti-estrogenic effects. As such, there are potential health benefits and risks associated with phytoestrogen exposure. To investigate the effects of Nar in breast cancer, based on its activity as a phytoestrogen, we treated MCF-7 ER-positive (ER+) breast cancer cells with Nar (10 µM) and examined transcriptomic changes in MCF-7 cells induced by Nar treatment. Pathway analysis included in our dataset shows upregulation of genes associated with estrogen signaling and epithelial to mesenchymal transition in breast cancer cells treated with naringenin.

Hormone receptor positive (HR+) breast cancer is the predominant molecular subtype in postmenopausal women. Although menopause decreases the concentration of estrogen in the body, HR+ breast tumors are sustained by local production of estrogens in the surrounding adipose tissue. The aged breast is characterized by an expansion of white adipose tissue, a phenomenon with unknown implications for breast cancer progression and treatment. Current clinical trials focused on HR+ breast cancer treatment fail to accurately represent aged women, with the average age of enrollment 7.76 years younger than the average age at diagnosis. It is important to understand how age-related changes to this environment impact disease progression, as breast adipose tissue both surrounds and bidirectionally communicates with cancer cells. Considering that age is not accurately represented in clinical trials, there remains a critical need for clinically relevant models of aged HR+ tumors and breast adipose in vitro. This project set out to characterize how age-based differences in the microenvironment alter HR+ tumor growth and disease progression, using a combination of in vivo analyses of age and in vitro conditioned media studies. Our results show that tumors from aged mice have a higher growth rate and overall total tumor volume at endpoint compared to young counterparts, indicating age as a driver in vivo tumorigenesis. In vitro proteomic analysis demonstrated that HR+ cancer cells exposed to conditioned media from aged breast adipose derived stem cells (brASC) have elevated expression of markers associated with adhesion, EMT, and angiogenesis at the mRNA and protein levels. Cytokine arrays of aged tumors show decreased expression of CXCL12/SDF1 and increased expression of SERPINE1, supporting possible changes in adhesion, EMT, and extracellular matrix with age. These findings suggest that age-educated components of the tumor microenvironment alter signaling within the tumor and promote tumorigenesis. Citation Format: Mackenzie Hawes, Megan C. Benz, Sophie R. Dietrich, Jack D. North, Bruce A. Bunnell, Bridgette M. Collins-Burow, Van T. Hoang, Elizabeth C. Martin, Matthew E. Burow. Age Conditions the Tumor Microenvironment of Hormone Receptor Positive Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-06-03.

The novel (nua) kinases 1 and 2 are two of 12 AMP-activated protein-related kinases whose signaling pathways are involved in cancer progression, as well as neurologic, fibrotic, and inflammatory diseases. Currently, there are 80 Food and Drug Administration-approved kinase inhibitors which target roughly 24 of the 500+ known human kinases, leaving most kinases underexplored, including NUAK1 and NUAK2. Thus, there is a critical need for selective inhibition of NUAK1 and NUAK2 signaling. Here, we review the protein structure, known upstream regulators and downstream targets, and expression profiles of NUAK1 and NUAK2 in cancerous compared to noncancerous tissue. We also delineate the biological roles and signaling pathways of the NUAK kinases in a range of malignancies, focusing on cancer but also covering noncancerous physiology, and the therapeutic potential of NUAK kinase inhibition. We summarize the known small-molecule NUAK kinase inhibitors in preclinical models and one inhibitor in clinical trials. This review highlights the signaling mechanisms and therapeutic value of targeting NUAK kinase signaling pathways with specific, small-molecule NUAK inhibitors.

Introduction: Hormone receptor positive (HR+) breast cancer accounts for nearly 60% of all breast cancer cases, with roughly 40% of patients experiencing resistance to endocrine treatment. There are known mechanisms of HR heterogeneity in breast cancer cells such as estrogen receptor-a gene (ER) gene mutations, co-activators, enhanced Ki-67 expression and growth factor activated pathways. Despite this, we do not have adequate predictive markers for response to therapy. Currently we have an incomplete understanding of how heterogeneity in the tumor microenvironment (TME) impacts the ER response in breast cancer. It is well established that the TME can modulate cellular proliferation, survival, and resistance to therapy in breast cancer. There is currently a gap in the ability of models to accurately mimic the TME in vitro, thus limiting our understanding of drug resistance and development of novel therapeutics. Here, we have modeled the pre-menopause, obese HR+ breast TME through the incorporation of six pre-menopause, obese human breast tissue (HBT) donors into a novel ex vivo breast tumor model to investigate the influence of the TME on HR+ breast cancer response to endocrine treatment. Methods: Ex-vivo breast tumors (EVBTs) were created by sandwiching. and then anchoring, healthy human breast tissue seeded with breast cancer cells between two confluent monolayers of breast adipose-derived stromal cells (brASCs). Breast adipose tissue from six different young (<50 years of age), obese (BMI≥28) donors was used to construct the EVBTs which were then seeded with the same representative HR+ breast cancer cell line and treated with vehicle, E2 100ρM, or Fulvestrant 1μM for 48 hours in median environment depleted of exogenous hormones and growth factors. RNA sequencing was used to demonstrate that the breast cancer cell line signatures was retained in the EVBT system and demonstrated ER response through evaluation of ERα and the ER responsive genes (PGR). Additional analysis done with qRT-PCR demonstrated relative gene expression changes in the microenvironment in response to endocrine treatment. To determine the source of transcriptional changes, individual cell populations were evaluated for ER and ECM genes using qRT-PCR following treatment with endocrine therapy. Results: Results demonstrated the retention of the breast cancer cell line transcriptome in the ex vivo breast tumor for up to 14 days in vitro. In addition, some donors displayed decreased PGR gene expression after treatment with ICI, elevated ERα gene expression, and increased COL6A6 expression. compared to breast tissue not seeded with HR+ breast cancer. Post-endocrine treatment qRT-PCR results showed variable ECM-gene expression changes between the breast tissue donors. To determine the source of the ECM changes, HR+ breast cancer cells and brASCs were evaluated post-endocrine treatment. Results demonstrated that expression of COL6A6 increased in breast ASCs cultured alone following treatment with Fulvestrant. No significant changes were noted in HR breast cancer cells cultured alone after endocrine treatment. Conclusion: Given the impact of the TME in breast cancer pathology and therapeutic resistance, it is critical for the contributions of the TME to be included in pre-clinical models of breast cancer. The findings from this study are ongoing, however, data suggest ECM remodeling occurs in response to endocrine therapy which is influenced by the TME. Further studies are needed to better define the interaction of ECM with HR+ breast cancer as well as the impact of endocrine therapies on ECM and TME remodeling. Citation Format: Megan Benz, Jack D. North, Mackenzie L. Hawes, Jack R. Elliott, Delia A. Carlino, Katherine L. Hebert, Van T. Hoang, Bridgette M. Collins-Burow, Frank H. Lau, Matthew E. Burow, Elizabeth C. Martin. Utilization of a novel breast microphysiological system to investigate the influence of tumor microenvironment on hormone receptor positive breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-04-23.