Abstract COX-2 is an inducible enzyme key to the production of inflammatory prostaglandins. COX-2 also has tumor intrinsic oncogenic activity in mouse models of breast cancer. Previously, we reported that increased expression of Cys-526-nitrosylated COX-2 (SNO-COX-2), but not non-nitrosylated COX-2, associated with progression of early-stage human breast cancer to invasive ductal carcinoma. Here, we used a 3D culture model of early-stage human breast cancer (MCF10DCIS cells) to investigate the relationship between SNO-COX-2 expression and mesenchymal/invasive tumor cell morphology. We find that SNO-COX-2, but not non-nitrosylated COX-2, closely associated with mesenchymal phenotypes induced by fibrillar type I collagen. Interestingly, invasive phenotypes did not associate with induction of classic epithelial-to-mesenchymal transition (EMT) markers including SNAIL, CDH2 (N-cadherin), and VIM (vimentin). By contrast TGFβ-1 strongly induced EMT-related transcripts, but not SNO-COX-2 protein expression nor mesenchymal phenotypes. These observations suggest that in MCF10DCIS cells, SNO-COX-2 associates with mesenchymal phenotypes more strongly than non-nitrosylated COX-2 protein or expression of classic EMT transcripts. Supporting these observations in vivo, a heterogeneous mouse breast tumor model (D2A1 cell injection) demonstrates that invasive mesenchymal tumor regions also have increased SNO-COX-2 expression compared to epithelial tumor regions. Further, using a microenvironment microarray to test MCF10DCIS.com cells 300 distinct tumor microenvironment conditions we find SNO-COX-2 protein expression is driven by inflammation, wound resolution, and cancer-associated factors. Standouts include TNC, SPP1, decorin, fibrillar type I and III collagens, INF-γ, and IL-4/13, with evidence for specific extracellular matrix-ligand interactions driving both high and low SNO-COX-2 expression. In sum, in MCF10DCIS cells, expression of SNO-COX-2 is highly microenvironment-dependent and strongly associated with invasive/mesenchymal growth, indicating potential for SNO-COX-2 as a biomarker to assess risk of early-stage breast cancer progression. Citation Format: Reuben J. Hoffmann, AeSoon Bensen, Mark Dane, Jane Arterberry, Rebecca Smith, James Korkola, Pepper Schedin. S-Nitrosylated COX-2 is a microenvironment-regulated breast cancer biomarker of mesenchymal phenotypes [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 6155.

Platinum-based chemotherapy is commonly used for non-small cell lung cancer (NSCLC) and high-grade serous ovarian cancer (HGSOC) treatments, yet clinical outcomes remain poor. Cellular senescence and its associated secretory phenotype (SASP) can have multiple tumor-promoting activities, but both are largely unexplored in these cancers. In this study, using xenograft, orthotopic and KrasG12V-driven murine NSCLC models, we demonstrate that cisplatin-induced senescence strongly promotes malignant phenotypes and tumor progression, which is stimulated by aging. Mechanistically, we found that a transforming growth factor-beta (TGFβ)-enriched SASP drives pro-proliferative effects through TGFBR1 and AKT/mTOR. TGFBR1 inhibition with galunisertib or senolytic treatment reduces tumor progression driven by cisplatin-induced senescence, and concomitant use of TGFBR1 inhibitors with platinum-based chemotherapy reduces tumor burden and improves survival. Finally, we validate the translational relevance of tumor-promoting TGFβ-enriched SASP using clinical NSCLC and HGSOC samples from patients who received neoadjuvant platinum-based chemotherapy. Together, our findings identify a potential cancer therapy resistance mechanism and provide preclinical proof of concept for future trials.

COX-2, an inducible enzyme key to production of inflammatory prostaglandins, has tumor cell-intrinsic oncogenic activity. Previously, we reported Cys-526-nitrosylated COX-2 (SNO-COX-2) associates with breast cancer progression and poor-prognostic young onset breast cancer. Here, using a 3D culture model of early-stage human breast cancer (MCF10DCIS cells), we report SNO-COX-2, but not non-nitrosylated COX-2, closely associated with mesenchymal cell phenotypes induced by fibrillar Col1. Inhibition of nitric oxide synthase (NOS) activity did not reduce SNO-COX-2 levels, suggesting alternative nitrosylation mechanisms. In 3D MCF10DCIS culture, mesenchymal phenotypes and SNO-COX-2 protein induced by Col1 did not associate with transcription of classic epithelial-to-mesenchymal transition (EMT) markers nor common cancer signaling pathways. Conversely, TGFβ-1 strongly induced EMT- and cancer signaling-related transcripts but was insufficient to increase SNO-COX-2 protein or mesenchymal phenotypes. These data suggest the mesenchymal phenotype and SNO-COX-2 expression in MCF10DCIS are driven by a non-transcriptional mechanism dependent on Col1. We tested 300 additional microenvironmental conditions and find SNO-COX-2 expression is driven by inflammatory, wound-resolving, and cancer-associated TME factors, including TNC, SPP1, decorin, Col1, Col3, INF-γ, and IL-4/13, with specific extracellular matrix-ligand combinations driving both high and low SNO-COX-2 expression. In sum, these observations show that in MCF10DCIS cells, SNO-COX-2 associates with mesenchymal phenotypes more strongly than non-nitrosylated COX-2; expression of classic EMT transcripts is neither sufficient nor necessary for acquisition of mesenchymal phenotypes; and expression of SNO-COX-2 is highly microenvironment-dependent. Future studies evaluating SNO-COX-2 as a biomarker for early-stage breast cancer with increased risk for progression, and its regulation, are warranted.

Metastatic solid tumors persist by evolving therapeutic resistance through complex, heterogeneous adaptive strategies that challenge standard precision medicine approaches. Current clinical decision making relies on bulk biomarkers, failing to resolve the spatial architecture and cellular contexts in which resistance mechanisms emerge. We present a patient-centric spatial framework, profiling 345,207 cells from four metastatic breast cancer patients across ten biopsies spanning personalized treatment courses of up to 3.5 years. By integrating probabilistic topic modeling with spatial deep learning, we observe fundamental principles of metastatic survival: pathway independence, microenvironment remodeling, and compensatory signaling. While these principles are universal, the underlying mechanisms are distinct: pathway independence manifested variously as the extinction of luminal identity, constitutive ESR1 activation, or spatial partitioning into drug-refractory invasive nests. Immune sanctuary was achieved through either genetic evasion mechanisms or physical exclusion via expanded fibroblast barriers. Compensatory transcriptional programs were engaged through rewired ligand-receptor networks and alternative survival pathway activation. These findings establish spatial profiling as a means to identify which mechanisms underlie each resistance principle in individual patients, enabling rational design of multi-axis combination therapies and earlier therapeutic decisions.

Senescence and the senescence associated secretory phenotype (SASP) are implicated in promoting early tumorigenesis but due to the complexity of SASP it has been difficult to identify the responsible factors. We used canonical SASP factors on our microenvironment microarray (MEMA) platform to systematically identify SASP-associated drivers of tumorigenesis in breast and lung cancer cells. We found multiple SASP factors enhanced the proliferation and overall cell numbers for both lung and breast cells grown on the MEMA, and that there was significant overlap in SASP-associated growth-promoting factors between the two different cell types. We validated the ability of several factors, including IL-6, TGF-β and EGF, to drive growth in in vitro assays. Interestingly, these factors were effective in driving growth and survival in cells that were altered (either immortalized or fully transformed) but not in normal cells and impacted breast cells differently depending on the age of the patient. RNAseq identified upregulation of wound-healing and stem-cell programs in SASP factor-treated cells. Many of these same SASP factors were present in conditioned media collected from senescent cells, which enhanced the growth of both lung and breast cancer cells, and inhibitors of the specific SASP factors partially reduced growth. Similarly, targeted inhibition of EGF partially reduced lung tumour growth in xenografts when senescent but not normal fibroblasts were co-implanted. Our findings have identified core SASP drivers of tumorigenesis and suggest that effective tumorigenesis driven by SASP is multifactorial and requires alterations in the target cells to achieve maximal response.

Paclitaxel is a standard of care neoadjuvant therapy for patients with triple negative breast cancer (TNBC); however, it shows limited benefit for locally advanced or metastatic disease. Here we used a coordinated experimental-computational approach to explore the influence of paclitaxel on the cellular and molecular responses of TNBC cells. We found that escalating doses of paclitaxel resulted in multinucleation, promotion of senescence, and initiation of DNA damage induced apoptosis. Single-cell RNA sequencing (scRNA-seq) of TNBC cells after paclitaxel treatment revealed upregulation of innate immune programs canonically associated with interferon response and downregulation of cell cycle progression programs. Systematic exploration of transcriptional responses to paclitaxel and cancer-associated microenvironmental factors revealed common gene programs induced by paclitaxel, IFNB, and IFNG. Transcription factor (TF) enrichment analysis identified 13 TFs that were both enriched based on activity of downstream targets and also significantly upregulated after paclitaxel treatment. Functional assessment with siRNA knockdown confirmed that the TFs FOSL1, NFE2L2 and ELF3 mediate cellular proliferation and also regulate nuclear structure. We further explored the influence of these TFs on paclitaxel-induced cell cycle behavior via live cell imaging, which revealed altered progression rates through G1, S/G2 and M phases. We found that ELF3 knockdown synergized with paclitaxel treatment to lock cells in a G1 state and prevent cell cycle progression. Analysis of publicly available breast cancer patient data showed that high ELF3 expression was associated with poor prognosis and enrichment in programs associated with cell cycle progression. Together these analyses disentangle the diverse aspects of paclitaxel response and identify ELF3 upregulation as a putative biomarker of paclitaxel resistance in TNBC.

Triple-negative breast cancer (TNBC) shows considerable intratumoral heterogeneity, which contributes to therapeutic resistance. Recent studies show that targeted therapeutics can steer TNBC toward homogeneous, drug-resistant states, but little is understood about how the microenvironment modulates these responses. We report studies to determine how components of the microenvironment impact response to trametinib and cellular heterogeneity. We find that multiple microenvironmental factors, including HGF and neuregulin 1, can drive therapeutic resistance and that treatment with hepatocyte growth factor (HGF) inhibitors restores trametinib sensitivity. Interestingly, treatment with these ligands reverses trametinib-induced homogeneity, restoring heterogeneity to levels comparable to baseline both in vitro and in vivo. Analysis of patient data demonstrates that TNBC with high HGF expression levels has a poor outcome and increased expression of basal and mesenchymal state markers. Our data suggest that common growth factors drive therapeutic resistance and maintain tumor heterogeneity in TNBC, and that co-targeting these factors may improve therapeutic response.

COX-2 is an inducible enzyme key to the production of inflammatory prostaglandins. COX-2 also has tumor intrinsic oncogenic activity in mouse models of breast cancer. Previously, we reported increased expression of Cys-526-nitrosylated COX-2 (SNO-COX-2), but not non-nitrosylated COX-2, with progression of early-stage human breast cancer to invasive ductal carcinoma. Here, we used a 3D culture model of early-stage human breast cancer (MCF10DCIS cells) to investigate the relationship between SNO-COX-2 expression and mesenchymal/invasive tumor cell morphology. We find that SNO-COX-2, but not non-nitrosylated COX-2, closely associated with mesenchymal phenotypes induced by fibrillar type I collagen. Interestingly, invasive phenotypes did not associate with induction of the classic epithelial-to-mesenchymal transition (EMT) markers SNAIL , CDH2 (N-cadherin), and VIM (vimentin). By contrast TGFβ-1 strongly induced EMT-related transcripts, but not SNO-COX-2 protein expression or mesenchymal phenotypes. These observations suggest that in MCF10DCIS cells, SNO-COX-2 associates with mesenchymal phenotypes more strongly than non-nitrosylated COX-2 protein, or expression of classic EMT transcripts. In a mouse model with breast tumor heterogeneity, mesenchymal tumor regions also have increased SNO-COX-2 expression. Testing 300 distinct tumor microenvironment conditions, we find SNO-COX-2 protein expression is driven by inflammation, wound resolution, and cancer-associated factors, especially TNC, SPP1, decorin, fibrillar type I and III collagens, INF-γ, and IL-4/13, with evidence for specific extracellular matrix-ligand interactions driving both high and low SNO-COX-2 expression. In sum, in MCF10DCIS cells, expression of SNO-COX-2 is highly microenvironment-dependent and strongly associated with invasive/mesenchymal growth, indicating potential for SNO-COX-2 as a biomarker to assess risk of early-stage breast cancer progression.

Microenvironment signals are potent determinants of cell fate and arbiters of tissue homeostasis, however understanding how different microenvironment factors coordinately regulate cellular phenotype has been experimentally challenging. Here we used a high-throughput microenvironment microarray comprised of 2640 unique pairwise signals to identify factors that support proliferation and maintenance of primary human mammary luminal epithelial cells. Multiple microenvironment factors that modulated luminal cell number were identified, including: HGF, NRG1, BMP2, CXCL1, TGFB1, FGF2, PDGFB, RANKL, WNT3A, SPP1, HA, VTN, and OMD. All of these factors were previously shown to modulate luminal cell numbers in painstaking mouse genetics experiments, or were shown to have a role in breast cancer, demonstrating the relevance and power of our high-dimensional approach to dissect key microenvironmental signals. RNA-sequencing of primary epithelial and stromal cell lineages identified the cell types that express these signals and the cognate receptors in vivo. Cell-based functional studies confirmed which effects from microenvironment factors were reproducible and robust to individual variation. Hepatocyte growth factor (HGF) was the factor most robust to individual variation and drove expansion of luminal cells via cKit+ progenitor cells, which expressed abundant MET receptor. Luminal cells from women who are genetically high risk for breast cancer had significantly more MET receptor and may explain the characteristic expansion of the luminal lineage in those women. In ensemble, our approach provides proof of principle that microenvironment signals that control specific cellular states can be dissected with high-dimensional cell-based approaches.

Observed (experimentally measured) 1D growth patterns

Simulation shown in Fig. 4D

Platinum-based chemotherapy is commonly used for non-small cell lung cancer (NSCLC) treatment, yet clinical outcomes remain poor. Cellular senescence and its associated secretory phenotype (SASP) can have multiple tumour-promoting activities, although these are largely unexplored in lung cancer. Here we show that cisplatin-derived SASP enhances the malignant phenotype of lung cancer cells. Using xenograft, orthotopic and Kras G12V -driven murine NSCLC models, we demonstrate that cisplatin-induced senescent cells strongly promote tumour progression. Mechanistically, we find that a TGF-β-enriched SASP drives pro-proliferative effects through TGFβR1 and Akt/mTOR pathway activation. We validate the translational relevance of chemotherapy-induced SASP using clinical NSCLC samples from patients who received neoadjuvant platinum-based chemotherapy. Importantly, TGFβR1 inhibition with galunisertib or senolytic treatment significantly reduces tumour promotion driven by cisplatin-induced senescence. Finally, we demonstrate, using distinct murine NSCLC models, that addition of TGFBR1 inhibitors to platinum-based chemotherapy reduces tumour burden and improves survival, providing pre-clinical proof-of-concept for future trial designs.

Simulation shown in Fig. 4F

Astrocytes and brain endothelial cells are components of the neurovascular unit that comprises the blood-brain barrier (BBB) and their dysfunction contributes to pathogenesis in Huntington's disease (HD). Defining the contribution of these cells to disease can inform cell-type-specific effects and uncover new disease-modifying therapeutic targets. These cells express integrin (ITG) adhesion receptors that anchor the cells to the extracellular matrix (ECM) to maintain the integrity of the BBB. We used HD patient-derived induced pluripotent stem cell (iPSC) modeling to study the ECM-ITG interface in astrocytes and brain microvascular endothelial cells and found ECM-ITG dysregulation in human iPSC-derived cells that may contribute to the dysfunction of the BBB in HD. This disruption has functional consequences since reducing ITG expression in glia in an HD Drosophila model suppressed disease-associated CNS dysfunction. Since ITGs can be targeted therapeutically and manipulating ITG signaling prevents neurodegeneration in other diseases, defining the role of ITGs in HD may provide a novel strategy of intervention to slow CNS pathophysiology to treat HD.

Background Platinum-based chemotherapy is commonly used for the treatment of non-small cell lung cancer (NSCLC), yet clinical outcomes and survival rates remain very poor and continues to be a cancer of unmet need. Recent evidence points to cellular senescence, a response to oncogenic- and therapy-induced genotoxic stress, and its associated proinflammatroy secretory phenotype (SASP) as an emerging hallmark of cancer. Hence, targeting senescence and its associated tumor-promoting activities is emerging as a novel and promising therapeutic strategy but largely unexplored in lung cancer. Experimental Procedures Our study includes a variety of methodologies, including: - Functional in vitro analyses: proliferation, colony formation, tumor spheres, migration assays. High throughput unbiased analyses: RNAseq, proteomics and microenvironment microarrays (MEMA). - In vivo models of lung cancer: xenografts, orthotopic and genetically engineered mouse models. Longitudinal tumor burden by IVIS and microCT and mouse survival. - Clinical samples: Histological and in silico analyses. Results Here we show that cisplatin-derived SASP enhances the malignant phenotype of lung cancer cells. Using xenograft, orthotopic and KrasG12V-driven murine NSCLC models, we demonstrate that cisplatin-induced senescent cells strongly promote tumor progression. Mechanistically, we find that a TGF-β-enriched SASP drives pro-proliferative effects through TGFβR1 and Akt/mTOR pathway activation. We validate the translational relevance of chemotherapy-induced SASP using clinical NSCLC samples from a trial with patients who received neoadjuvant platinum-based chemotherapy. Importantly, TGFβR1 inhibition with galunisertib or senolytic treatment significantly reduces tumor promotion driven by cisplatin-induced senescence. Finally, we demonstrate, using distinct murine NSCLC models, that addition of TGFBR1 inhibitors to platinum-based chemotherapy reduces tumor burden and improves survival, providing pre-clinical proof-of-concept for future trial designs on combination therapies. Conclusions and Impact We regard this work as a major conceptual dissection of tumor-promoting activities of therapy-induced senescence, and a preclinical advance in the management of lung cancer with potential wide therapeutic applications in precision medicine. Of note, we expect our findings to have implications for multiple cancer types, including ovarian, breast, mesothelioma, oesophageal, head and neck, bladder and brain cancers, where platinum-based therapies remain important standard-of-care treatments. Citation Format: Estela González-Gualda, David Macias, Samir Morsli, José Ezequiel Martín, Hui-Ling Ou, Mary Denholm, Ioana Olan, Reuben Hoffmann, Mark Dane, Dimitris Veroutis, Guillermo Medrano, Francisca Mulero, Carla P. Martins, Mariano Barbacid, Vassilis Gorgoulis, James E. Korkola, Doris M. Rassl, Gary J. Doherty, Robert C. Rintoul, Masashi Narita, Daniel Muñoz-Espín. A tumor-promoting senescent secretome triggered by platinum chemotherapy exploits a targetable TGFβR1/Akt-mTOR axis in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5504.

Simulation shown in Fig. 4E

Supplementary Methods, Figures 1-6 and Tables 1-3.

Simulated 1D growth patterns

Identifying effective therapeutic treatment strategies is a major challenge to improving outcomes for patients with breast cancer. To gain a comprehensive understanding of how clinically relevant anti-cancer agents modulate cell cycle progression, here we use genetically engineered breast cancer cell lines to track drug-induced changes in cell number and cell cycle phase to reveal drug-specific cell cycle effects that vary across time. We use a linear chain trick (LCT) computational model, which faithfully captures drug-induced dynamic responses, correctly infers drug effects, and reproduces influences on specific cell cycle phases. We use the LCT model to predict the effects of unseen drug combinations and confirm these in independent validation experiments. Our integrated experimental and modeling approach opens avenues to assess drug responses, predict effective drug combinations, and identify optimal drug sequencing strategies.

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.