The recognition of nitric oxide and nitrogen oxides as key elements in human biology has driven the development of one of the most extensive areas of research in recent decades, consistently uncovering new insights into therapy and disease. These molecules are central to redox biology, with their inorganic chemistry playing a significant role in many biological systems. This review explores how fundamental chemical properties, kinetics, and thermodynamics influence important biological outcomes, including cancer, covering both normal physiology and disease states. The distinct chemical traits of these compounds allow for precise regulation of biological processes. Emerging concepts in cancer and other diseases will be discussed to highlight innovative treatments for advanced-stage conditions.

Tumor NOS2 and COX2 expression induced by IFNg and TNFa

Nitric oxide synthase 2 (NOS2) and cyclooxygenase 2 (COX2) tumor expression present significant obstacles for effective treatment of aggressive tumors including ER-negative breast cancer. Spatial analysis of NOS2, COX2 and CD8 expression in patient tumors has identified mechanisms of treatment inhibition that were further elucidated using the 4T1 mouse model of triple negative breast cancer and live cell culture studies. NOS2 and COX2 activate each other via a feed-forward paracrine mechanism. While NOS2 promotes cancer stemness and the formation of metastatic niches, COX2 mediates CD8+ T cell suppression. Quantitative spatial analysis has revealed NOS2/COX2 roles during the temporal progression from immune hot in low COX2 expressing tumors to three immune cold stages in the tumor microenvironment of high COX2 expressing tumors. Type 1: immune cold with stroma-restricted CD8+ T cell secretion of interferon gamma, which activates COX2 expression at the tumor margin as well as NOS2 expression at the tumor periphery. Type 2: developing immune desserts lack stroma-restricted CD8+ T cells with tumor NOS2 and COX2 restricted to the tumor periphery. Type 3: mature immune desserts exhibit abated NOS2, COX2 and CD8+ T cells, and induction of B7H4 and cancer-associated fibroblasts driven by significant tumor hypoxia and necrosis. These three types of immune desserts can coexist with each other and with immune hot regions in the same tumor. The coordinated interplay of NOS2 and COX2 indicates that targeting both these enzymes provides an effective treatment strategy that is supported by ongoing clinical trials demonstrating improved clinical outcomes in patients who have otherwise exhausted treatment options.

Supplementary Table I summarizes pathological features of tumor immune microenvironment where NOS2+ inflamed regions are significantly higher in tumors from Deceased patients.

Nitric oxide synthase 2 (NOS2) and cyclooxygenase 2 (COX2) lie at a critical intersection between inflammation, metabolism, and oncogenic signaling, where they cooperatively promote and establish a Nitric Oxide (NO)-driven Warburg phenotype in advanced cancers. Early work in macrophages established NOS2-derived NO as both a signaling molecule and metabolic stressor that inhibits oxidative phosphorylation (OXPHOS) by targeting iron-sulfur enzymes and respiratory complexes, forcing neighboring cells to rewire metabolism. In human tumors, sustained NOS2 expression in cancer cells and tumor-associated macrophages (TAMs) enforces a Warburg-like state characterized by high glycolytic flux, glutamine dependence, and enhanced NADPH production, supporting proliferation, biosynthesis, and resistance to oxidative stress. At nitrosative-signaling concentrations (≈100-500 nM), NO breaks carbon entry into the TCA cycle at aconitase and pyruvate dehydrogenase, progressively disables dehydrogenase complexes containing dihydrolipoamide dehydrogenase (DLD) and electron-transport complexes (ETCs), and activates hypoxia-inducible factor 1-alpha (HIF-1), phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), extracellular signal-regulated kinase (ERK)/pyruvate kinase M2 (PKM2)/c-Myc signaling axis, nuclear factor erythroid 2-related factor 2 (Nrf2), and transforming growth factor Beta (TGF-β)/SMAD pathways. These biochemical and signaling effects convert transient glycolytic adaptation into chemically enforced dependency, further stabilized by metabolite-driven inhibition of ten-eleven translocation (TET) and Jumonji demethylases, creating an "epigenetic lock" that maintains oncogenic transcriptional programs. NOS2 and COX2 form a reciprocal feed-forward circuit in which NO, prostaglandin E2 (PGE2), interleukin (IL)-6, and IL-8 reinforce one another, driving tumor-promoting inflammation, immunosuppression, angiogenesis, and metastasis while depleting nutrients and acidifying the tumor interstitial fluid. Spatially, NOS2/COX2 niches at the tumor-stroma interface and within immune deserts generate gradients of NO, PGE2, oxygen, and metabolites that partition tumors into microdomains with distinct metabolic states, immune composition, and therapeutic vulnerabilities. Integrating these insights with Hanahan's updated hallmarks of cancer, we propose that NOS2-derived NO functions as a node synchronizing deregulated energetics, inflammation, immune evasion, plasticity, and therapy resistance within the tumor microenvironment (TME). Targeting the NOS2-COX2 axis and its downstream NO-iron-epigenetic circuitry may therefore disrupt multiple hallmarks and reveal combinatorial strategies to exploit NO-induced metabolic liabilities in cancer.

Tumor NOS2 and COX2 expression induced by IFNg and TNFa

Tumor immunosuppression affects survival and treatment efficacy. Tumor NOS2/COX2 coexpression strongly predicts poor outcome in estrogen receptor-negative (ER-) breast cancer by promoting metastasis, drug resistance, cancer stemness, and immune suppression. Herein, a spatially distinct NOS2/COX2 and CD3+CD8+PD1-T effector (TEff) cell landscape correlated with poor survival in ER-tumors. NOS2 was primarily expressed at the tumor margin, whereas COX2 together with B7H4 was associated with immune desert regions lacking TEff cells, where a higher ratio of tumor NOS2 or COX2 to TEff cells predicted poor survival. Also, programmed cell death ligand 1/programmed cell death 1, regulatory T cells (TRegs), and IDO1 were primarily associated with stroma-restricted TEff cells. Regardless of the survival outcome, CD4+ T cells and macrophages were primarily in stromal lymphoid aggregates. Finally, in a 4T1 model, COX2 inhibition led to increased CD8+ TEff/CD4+ TReg ratio and CD8+ TEff infiltration while Nos2 deficiency had no significant effect, thus reinforcing our observations that COX2 is an essential component of immunosuppression through CD8+ TEff cell exclusion from the tumor. Our study indicates that tumor NOS2/COX2 expression plays a central role in tumor immune evasion, suggesting that strategies combining clinically available NOS2/COX2 inhibitors with immune therapy could provide effective options for the treatment of aggressive and drug-resistant ER-breast tumors.

Supplementary Fig. 4. Supplementary Fig. 4. Spatial UMAP analysis of CD8+/-NOS2+/-COX2+/- phenotypes in Deceased vs Alive patient tumors. Single cell neighborhood profile summary.

Supplementary Table II summarizes �8+ T cells in designated regions of the tumors.

The Journal retracts the article titled “Nitric Oxide Donor DETA/NO Inhibits the Growth of Endometrial Cancer Cells by Upregulating the Expression of RASSF1 and CDKN1A” [...]

Spatial analysis of tumor NOS2/COX2 expression with respect to survival. Spatial landscape of NOS2 (red) and COX2 (green) with DAPI (white), the tumor marker CKSOX10 (blue) for (A) deceased and (B) alive patients at 5-year survival. C, Quantification of NOS2/COX2 tumor expression at the single cell level, with each dot representing a tumor sample (also shown in Supplementary Fig. S2). D, Heat density maps of tumor NOS2/COX2 expression in deceased and alive patient tumors. E, Significant Pearson’s correlation between tumor NOS2 and COX2 expression R2 = 0.8481 and P < 0.0001 in tumors from deceased patients but not alive patient samples (F). Mann–Whitney *, P < 0.05; **, P ≤ 0.01.

Nitric oxide (NO) and reactive nitrogen species (RNS) exert profound biological impacts dictated by their chemistry. Understanding their spatial distribution is essential for deciphering their roles in diverse biological processes. This review establishes a framework for the chemical biology of NO and RNS, exploring their dynamic reactions within the context of cancer. Concentration-dependent signaling reveals distinctive processes in cancer, with three levels of NO influencing oncogenic properties. In this context, NO plays a crucial role in cancer cell proliferation, metastasis, chemotherapy resistance, and immune suppression. Increased NOS2 expression correlates with poor survival across different tumors, including breast cancer. Additionally, NOS2 can crosstalk with the proinflammatory enzyme cyclooxygenase-2 (COX-2) to promote cancer progression. NOS2 and COX-2 co-expression establishes a positive feed-forward loop, driving immunosuppression and metastasis in estrogen receptor-negative (ER-) breast cancer. Spatial evaluation of NOS2 and COX-2 reveals orthogonal expression, suggesting the unique roles of these niches in the tumor microenvironment (TME). NOS2 and COX2 niche formation requires IFN-γ and cytokine-releasing cells. These niches contribute to poor clinical outcomes, emphasizing their role in cancer progression. Strategies to target these markers include direct inhibition, involving pan-inhibitors and selective inhibitors, as well as indirect approaches targeting their induction or downstream effectors. Compounds from cruciferous vegetables are potential candidates for NOS2 and COX-2 inhibition offering therapeutic applications. Thus, understanding the chemical biology of NO and RNS, their spatial distribution, and their implications in cancer progression provides valuable insights for developing targeted therapies and preventive strategies.

Spatial landscape of CD44v6 and EpCAM expression relative to tumor NOS2/COX2 expression and survival. A, Enriched regions showing spatially distinct CD44v6 and EpCAM expression. B, Quantification of CD44v6 and EpCAM as well as ratios of NOS2 or COX2 to CD44v6 or EpCAM, respectively, in tumors from deceased vs. alive patients. Significance was determined using the Mann–Whitney test, in which *, P < 0.05; **, P = 0.007. C, Lymphoid aggregate and EpCAM+ tumor cell boarder. Pearson’s correlation coefficient shows significant associations between T effector cells or IFNγ and EpCAM expression in tumors from deceased vs. alive patients. D, Spatial landscape of CD44v6 and EpCAM expression in annotated tumor, lymphoid aggregate, invasive tumor/stroma, and immune desert regions. These regions show EpCAM+ and CD44v6+ tumor cells that have invaded the stroma.

S-UMAP analysis for tumor NOS2/COX2 and CD8 expression. Density heat maps show spatially distinct tumor NOS2, COX2, and CD8 clustering in tumors from (A) deceased and (B) alive patients. Spatial distribution plots based upon positive pixels for CD8 (magenta), NOS2 (red; also shown in Fig. 1D), and COX2 (green; also shown in Fig. 1D) expression are shown. C, Masks show distinct neighborhood cluster prevalence in deceased (red) or alive (blue) patient tumors. D, Ratio of phenotype density profiles (i.e., neighborhood cluster density profiles) in the D3/alive as well as the alive/D3 demonstrates the predictive value of CD8−NOS2+COX2+ (yellow diamonds) and CD8+NOS2−COX2− (purple triangle) phenotypes due to their vast differences in deceased vs. alive patient tumors. E, Spatial dot plot and nearest-neighbor analysis at 50 μm distances of the defined phenotypes with a predictive value. F, mIF of CD8 (magenta) NOS2 (red) and COX2 (green) expression in areas of interest (red square boxes) in the spatial dot plot shown in E. F, (Top) entire tumor image; F, (middle) a satellite or tumor budding metastatic niche (yellow box) expressing elevated tumor NOS2; (bottom) immune desert region. G, Enhanced magnification of these areas of interest (F, yellow and green boxes) shows a CD8−NOS2+COX2+ metastatic niche (top image; also shown in Fig. 1A) and CD8−NOS2−COX2+ immune desert (bottom image).

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. The binding and dissociation of ligands tunes the inherent conformational flexibility of these important drug targets towards distinct functional states. To trigger such protein-ligand interaction dynamics within the human adenosine A 2A receptor, we designed seven photochemical affinity switches derived from the anti-Parkinson’s drug istradefylline. In a rational approach based on UV/Vis spectroscopy, time-resolved absorption spectroscopy, differential scanning fluorimetry and cryo-crystallography, we identified compounds suitable for time-resolved serial crystallography. Our analysis of millisecond-scale dynamics revealed how trans-cis isomerization shifts selected istradefylline derivatives within the binding pocket. Depending on the chemical nature of the ligand, this disrupts interactions between extracellular loops 2 and 3, acting as a lid on the binding pocket, followed by large-scale receptor rearrangements upon ligand dissociation. This innovative approach provides insights into GPCR dynamics at the atomic level, offering potential for developing novel pharmaceutics.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. The binding and dissociation of ligands tunes the inherent conformational flexibility of these important drug targets towards distinct functional states. Here we show how to trigger and resolve protein-ligand interaction dynamics within the human adenosine A2A receptor. For this, we designed seven photochemical affinity switches derived from the anti-Parkinson's drug istradefylline. In a rational approach based on UV/Vis spectroscopy, time-resolved absorption spectroscopy, differential scanning fluorimetry and cryo-crystallography, we identified compounds suitable for time-resolved serial crystallography. Our analysis of millisecond-scale dynamics revealed how trans-to-cis isomerization shifts selected istradefylline derivatives within the binding pocket. Depending on the chemical nature of the ligand, interactions between extracellular loops 2 and 3, acting as a lid on the binding pocket, are disrupted and rearrangement of the orthosteric binding pocket is invoked upon ligand dissociation. This innovative approach provides insights into GPCR dynamics at the atomic level, offering potential for developing novel pharmaceuticals.

Estrogen receptor-negative breast cancer is an aggressive subtype with limited therapeutic options. Elevated nitric oxide synthase 2 (NOS2) and COX2 mediate immunosuppression and poor survival in these tumors. Therefore, the influence of tumor NOS2/COX2 on immune architecture was examined in 16 African American and five Caucasian estrogen receptor-negative tumors. Elevated tumor NOS2/COX2 limited CD8+ T-cell infiltration at 5-year survival. Distinct CD8+/-NOS2+/-COX2+/- phenotypes defining metastatic and cancer stem cell niches and immune desert regions were identified. These results were supported by an unbiased, unsupervised nonlinear dimensionality reduction technique, Uniform Manifold Approximation and Projection for Dimension Reduction, incorporating spatial relations between cells and validated in a separate gene expression cohort using NOS2/CD8 and COX2/CD8 ratios. Additionally, elongated tumor cells were specifically in CD8(-)NOS2(+)COX2(+) regions, suggesting metastatic hot spots. This work demonstrates predictive power of spatial analyses of CD8/NOS2/COX2 architecture and supports the use of clinically available NOS2/COX2 inhibitors for improved survival in patients with these aggressive tumors. Significance: This work identifies CD8(-)NOS2(+)COX2(+) and CD8(-)NOS2(-)COX2(+) unique cellular neighborhoods that drive the tumor immune spatial architecture of CD8(+ )T cells predictive of clinical outcome and can be targeted with clinically available NOS inhibitors and NSAIDs.

Acinetobacter baumannii, a commonly multidrug-resistant Gram-negative bacterium responsible for large numbers of bloodstream and lung infections worldwide, is increasingly difficult to treat and constitutes a growing threat to human health. Structurally novel antibacterial chemical matter that can evade existing resistance mechanisms is essential for addressing this critical medical need. Herein, we describe our efforts to inhibit the essential A. baumannii lipooligosaccharide (LOS) ATP-binding cassette (ABC) transporter MsbA. An unexpected impurity from a phenotypic screening was optimized as a series of dimeric compounds, culminating with 1 (cerastecin D), which exhibited antibacterial activity in the presence of human serum and a pharmacokinetic profile sufficient to achieve efficacy against A. baumannii in murine septicemia and lung infection models.

Defined CD8 NOS2 and COX2 spatial landscape. Five basic regions showing (A) margin or stroma-restricted lymphoid aggregates (orange circles) in which a gap of 50 μm (double orange arrow) between CD3+ T-cell aggregates and the CKSOX10+ tumor edge (green circle) was observed. Blue and dashed magenta circles identify immune desert regions lacking CD8+ T cells. B, Tumor fragmentation or satellite region. C, Tumor edge with proximal stroma regions. D, Significant differences in % cell composition is shown for CD8+ T cells as well as (E) NOS2+ and (F) COX2+ tumor cells. G, Graphic summary of tumor NOS2/COX2 landscape and CD8+ T-cell regional distributions with respect to the tumor–stroma interface. H, Spatial architecture of predictive phenotypes in tumors from deceased vs. alive patients. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.