Hypofractionated Radiotherapy-Related Lymphopenia Is Associated With Worse Survival in Unresectable Intrahepatic Cholangiocarcinoma.

OBJECTIVE: The aim of this study was to evaluate the incidence of radiotherapy (RT)-related lymphopenia, its predictors, and association with survival in unresectable intrahepatic cholangiocarcinoma (ICC) treated with hypofractionated-RT (HF-RT). METHODS: Retrospective analysis of 96 patients with unresectable ICC who underwent HF-RT (median 58.05 Gy in 15 fractions) between 2009 and 2022 was performed. Absolute lymphocyte count (ALC) nadir within 12 weeks of RT was analyzed. Primary variable of interest was severe lymphopenia, defined as Grade 3+ (ALC <0.5 k/µL) per CTCAE v5.0. Primary outcome of interest was overall survival (OS) from RT. RESULTS: Median follow-up was 16 months. Fifty-two percent of patients had chemotherapy pre-RT, 23% during RT, and 40% post-RT. Pre-RT, median ALC was 1.1 k/µL and 5% had severe lymphopenia. Post-RT, 68% developed RT-related severe lymphopenia. Patients who developed severe lymphopenia had a significantly lower pre-RT ALC (median 1.1 vs. 1.5 k/µL, P =0.01) and larger target tumor volume (median 125 vs. 62 cm 3 , P =0.02). In our multivariable Cox model, severe lymphopenia was associated with a 1.7-fold increased risk of death ( P =0.04); 1-year OS rates were 63% vs 77% ( P =0.03). Receipt of photon versus proton-based RT (OR=3.50, P =0.02), higher mean liver dose (OR=1.19, P <0.01), and longer RT duration (OR=1.49, P =0.02) predicted severe lymphopenia. CONCLUSIONS: HF-RT-related lymphopenia is an independent prognostic factor for survival in patients with unresectable ICC. Patients with lower baseline ALC and larger tumor volume may be at increased risk, and use of proton therapy, minimizing mean liver dose, and avoiding treatment breaks may reduce RT-related lymphopenia.

High-resolution CTCF footprinting reveals impact of chromatin state on cohesin extrusion dynamics.

DNA looping is vital for establishing many enhancer-promoter interactions. While CTCF is known to anchor many cohesin-mediated loops, the looped chromatin fiber appears to predominantly exist in a poorly characterized actively extruding state. To better characterize extruding chromatin loop structures, we used CTCF MNase HiChIP data to determine both CTCF binding at high resolution and 3D contact information. Here we present FactorFinder, a tool that identifies CTCF binding sites at near base-pair resolution. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF) state is rare genome-wide with locus-specific variation from ~1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics, and find that active enhancers and RNA Pol II impede cohesin extrusion, facilitating an enrichment of enhancer-promoter contacts in the partially extruded loop state. We propose a model of topological regulation whereby the transient, partially extruded states play active roles in transcription.

Integrative PTEN Enhancer Discovery Reveals a New Model of Enhancer Organization.

Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.

Small-molecule targeted therapies induce dependence on DNA double-strand break repair in residual tumor cells.

Residual cancer cells that survive drug treatments with targeted therapies act as a reservoir from which eventual resistant disease emerges. Although there is great interest in therapeutically targeting residual cells, efforts are hampered by our limited knowledge of the vulnerabilities existing in this cell state. Here, we report that diverse oncogene-targeted therapies, including inhibitors of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), KRAS, and BRAF, induce DNA double-strand breaks and, consequently, ataxia-telangiectasia mutated (ATM)-dependent DNA repair in oncogene-matched residual tumor cells. This DNA damage response, observed in cell lines, mouse xenograft models, and human patients, is driven by a pathway involving the activation of caspases 3 and 7 and the downstream caspase-activated deoxyribonuclease (CAD). CAD is, in turn, activated through caspase-mediated degradation of its endogenous inhibitor, ICAD. In models of EGFR mutant non-small cell lung cancer (NSCLC), tumor cells that survive treatment with small-molecule EGFR-targeted therapies are thus synthetically dependent on ATM, and combined treatment with an ATM kinase inhibitor eradicates these cells in vivo. This led to more penetrant and durable responses in EGFR mutant NSCLC mouse xenograft models, including those derived from both established cell lines and patient tumors. Last, we found that rare patients with EGFR mutant NSCLC harboring co-occurring, loss-of-function mutations in ATM exhibit extended progression-free survival on first generation EGFR inhibitor therapy relative to patients with EGFR mutant NSCLC lacking deleterious ATM mutations. Together, these findings establish a rationale for the mechanism-based integration of ATM inhibitors alongside existing targeted therapies.

Circulating Tumor DNA Predicts Pathologic and Clinical Outcomes Following Neoadjuvant Chemoradiation and Surgery for Patients With Locally Advanced Rectal Cancer.

PURPOSE: This study was designed to assess the ability of perioperative circulating tumor DNA (ctDNA) to predict surgical outcome and recurrence following neoadjuvant chemoradiation for locally advanced rectal cancer (LARC). MATERIALS AND METHODS: Twenty-nine patients with newly diagnosed LARC treated between January 2014 and February 2018 were enrolled. Patients received long-course neoadjuvant chemoradiation prior to surgery. Plasma ctDNA was collected at baseline, preoperatively, and postoperatively. Next-generation sequencing was used to identify mutations in the primary tumor, and mutation-specific droplet digital polymerase chain reaction was used to assess mutation fraction in ctDNA. RESULTS: The median age was 54 years. The overall margin-negative, node-negative resection rate was 73% and was significantly higher among patients with undetectable preoperative ctDNA (n = 17, 88%) versus patients with detectable preoperative ctDNA (n = 9, 44%; P = .028). Undetectable ctDNA was also associated with more favorable neoadjuvant rectal scores (univariate linear regression, P = .029). Recurrence-free survival (RFS) was calculated for the subset (n = 19) who both underwent surgery and had postoperative ctDNA available. At a median follow-up of 20 months, patients with detectable postoperative ctDNA experienced poorer RFS (hazard ratio, 11.56; P = .007). All patients (4 of 4) with detectable postoperative ctDNA recurred (positive predictive value = 100%), whereas only 2 of 15 patients with undetectable ctDNA recurred (negative predictive value = 87%). CONCLUSION: Among patients treated with neoadjuvant chemoradiation for LARC, patients with undetectable preoperative ctDNA were more likely to have a favorable surgical outcome as measured by the rate of margin-negative, node-negative resections and neoadjuvant rectal score. Furthermore, we have confirmed prior reports indicating that detectable postoperative ctDNA is associated with worse RFS. Future prospective study is needed to assess the potential for ctDNA to assist with personalizing treatment for LARC.

Results and Molecular Correlates from a Pilot Study of Neoadjuvant Induction FOLFIRINOX Followed by Chemoradiation and Surgery for Gastroesophageal Adenocarcinomas.

PURPOSE: We performed a NCI-sponsored, prospective study of neoadjuvant FOLFIRINOX followed by chemoradiation with carboplatin/paclitaxel followed by surgery in patients with locally advanced gastric or gastroesophageal cancer. PATIENTS AND METHODS: The primary objective was to determine completion rate of neoadjuvant FOLFIRINOX × 8 followed by chemoradiation. Secondary endpoints were toxicity and pathologic complete response (pCR) rate. Exploratory analysis was performed of circulating tumor DNA (ctDNA) to treatment response. RESULTS: From October 2017 to June 2018, 25 patients were enrolled. All patients started FOLFIRINOX, 92% completed all eight planned cycles, and 88% completed chemoradiation. Twenty (80%) patients underwent surgical resection, and 7 had a pCR (35% in resected cohort, 28% intention to treat). Tumor-specific mutations were identified in 21 (84%) patients, of whom 4 and 17 patients had undetectable and detectable ctDNA at baseline, respectively. Presence of detectable post-chemoradiation ctDNA (P = 0.004) and/or postoperative ctDNA (P = 0.045) were associated with disease recurrence. CONCLUSIONS: Here we show neoadjuvant FOLFIRINOX followed by chemoradiation for locally advanced gastroesophageal cancer is feasible and yields a high rate of pCR. ctDNA appears to be a promising predictor of postoperative recurrence.See related commentary by Catenacci, p. 6281.

Chemoradiation-Related Lymphopenia and Its Association with Survival in Patients with Squamous Cell Carcinoma of the Anal Canal.

BACKGROUND: Although treatment-related lymphopenia (TRL) is common and associated with poorer survival in multiple solid malignancies, few data exist for anal cancer. We evaluated TRL and its association with survival in patients with anal cancer treated with chemoradiation (CRT). MATERIALS AND METHODS: A retrospective analysis of 140 patients with nonmetastatic anal squamous cell carcinoma (SCC) treated with definitive CRT was performed. Total lymphocyte counts (TLC) at baseline and monthly intervals up to 12 months after initiating CRT were analyzed. Multivariable Cox regression analysis was performed to evaluate the association between overall survival (OS) and TRL, dichotomized by grade (G)4 TRL (<0.2k/µL) 2 months after initiating CRT. Kaplan-Meier and log-rank tests were used to compare OS between patients with versus without G4 TRL. RESULTS: Median time of follow-up was 55 months. Prior to CRT, 95% of patients had a normal TLC (>1k/µL). Two months after initiating CRT, there was a median of 71% reduction in TLC from baseline and 84% of patients had TRL: 11% G1, 31% G2, 34% G3, and 8% G4. On multivariable Cox model, G4 TRL at two months was associated with a 3.7-fold increased risk of death. On log-rank test, the 5-year OS rate was 32% in the cohort with G4 TRL versus 86% in the cohort without G4 TRL. CONCLUSION: TRL is common and may be another prognostic marker of OS in anal cancer patients treated with CRT. The association between TRL and OS suggests an important role of the host immunity in anal cancer outcomes. IMPLICATIONS FOR PRACTICE: This is the first detailed report demonstrating that standard chemoradiation (CRT) commonly results in treatment-related lymphopenia (TRL), which may be associated with a poorer overall survival (OS) in patients with anal squamous cell carcinoma. The association between TRL and worse OS observed in this study supports the importance of host immunity in survival among patients with anal cancer. These findings encourage larger, prospective studies to further investigate TRL, its predictors, and its relationship with survival outcomes. Furthermore, the results of this study support ongoing efforts of clinical trials to investigate the potential role of immunotherapy in anal cancer.

Single-cell lineage analysis reveals genetic and epigenetic interplay in glioblastoma drug resistance.

BACKGROUND: Tumors can evolve and adapt to therapeutic pressure by acquiring genetic and epigenetic alterations that may be transient or stable. A precise understanding of how such events contribute to intratumoral heterogeneity, dynamic subpopulations, and overall tumor fitness will require experimental approaches to prospectively label, track, and characterize resistant or otherwise adaptive populations at the single-cell level. In glioblastoma, poor efficacy of receptor tyrosine kinase (RTK) therapies has been alternatively ascribed to genetic heterogeneity or to epigenetic transitions that circumvent signaling blockade. RESULTS: We combine cell lineage barcoding and single-cell transcriptomics to trace the emergence of drug resistance in stem-like glioblastoma cells treated with RTK inhibitors. Whereas a broad variety of barcoded lineages adopt a Notch-dependent persister phenotype that sustains them through early drug exposure, rare subclones acquire genetic changes that enable their rapid outgrowth over time. Single-cell analyses reveal that these genetic subclones gain copy number amplifications of the insulin receptor substrate-1 and substrate-2 (IRS1 or IRS2) loci, which activate insulin and AKT signaling programs. Persister-like cells and genomic amplifications of IRS2 and other loci are evident in primary glioblastomas and may underlie the inefficacy of targeted therapies in this disease. CONCLUSIONS: A method for combined lineage tracing and scRNA-seq reveals the interplay between complementary genetic and epigenetic mechanisms of resistance in a heterogeneous glioblastoma tumor model.

Dosimetric Analysis and Normal-Tissue Complication Probability Modeling of Child-Pugh Score and Albumin-Bilirubin Grade Increase After Hepatic Irradiation.

PURPOSE: This study aimed to develop robust normal-tissue complication probability (NTCP) models for patients with hepatocellular carcinoma treated with radiation therapy (RT) using Child-Pugh (CP) score and albumin-bilirubin (ALBI) grade increase as endpoints for hepatic toxicity. METHODS AND MATERIALS: Data from 108 patients with hepatocellular carcinoma treated with RT between 2008 and 2017 were evaluated, of which 47 patients (44%) were treated with proton RT. Of these patients, 29 received stereotactic body RT and 79 moderately hypofractionated RT to median physical tumor doses of 43 Gy in 5 fractions and 59 Gy in 15 fractions, respectively. A generalized Lyman-Kutcher-Berman (LKB) model was used to model the NTCP using 2 clinical endpoints, both evaluated at 3 months after RT: CP score increase of ≥2 and ALBI grade increase of ≥1 from the pre-RT baseline. Confidence intervals on LKB fit parameters were determined using bootstrap resampling. RESULTS: Compared with previous NTCP models, this study found a stronger correlation between normal liver volume receiving low doses of radiation (5-10 Gy) and a CP score or ALBI grade increase. A CP score increase exhibited a stronger correlation to normal liver volumes irradiated than an ALBI grade increase. LKB models for CP increase found values for the volume-effect parameter of a = 0.06 for all patients, and a = 0.02/0.09 when fit to photon/proton patients separately. Subset analyses for patients with superior initial liver functions showed consistent dose-volume effects (a = 0.1) and consistent dose-response relationships. CONCLUSIONS: This study presents an update of liver NTCP models in the era of modern RT techniques using relevant endpoints of hepatic toxicity, CP score and ALBI grade increase. The results show a stronger influence of low-dose bath on hepatic toxicity than those found in previous studies, indicating that RT techniques that minimize the low-dose bath may be beneficial for patients.

Hypofractionated Radiation Therapy for Unresectable/Locally Recurrent Intrahepatic Cholangiocarcinoma.

OBJECTIVE: The aim of this study was to evaluate outcomes for patients with unresectable intrahepatic cholangiocarcinoma (ICC) treated with hypofractionated proton or photon radiation therapy (HF-RT). METHODS: We retrospectively identified 66 patients with ICC who were treated with HF-RT from 2008 to 2018. Median age at RT was 76 years (range 30-92), including 27 patients (41%) aged ≥ 80 years. Median RT dose was 58.05 Gy (range 37.5-67.5), all delivered in 15 daily fractions. Thirty-two patients received proton RT and 34 patients received photon RT. RESULTS: Median follow-up times from diagnosis and RT start were 21 months and 14 months, respectively. In total, five patients (7.6%) developed local failure. The 2-year outcomes were 84% local control (LC) and 58% OS. Among the 51 patients treated with definitive intent, the 2-year LC rate was 93% and the OS rate was 62%. On multivariate analysis for LC, older age was associated with a lower risk of local failure [hazard ratio (HR) 0.91; p = 0.02], while prior surgery (HR 16.5; p = 0.04) and macrovascular invasion (HR 123.93; p = 0.02) were independently associated with an increased risk of local failure. On multivariate analysis for OS, female sex (HR 0.33; p = 0.001) and prior chemotherapy (HR 0.38; p = 0.003) remained significantly associated with OS. On multivariate analysis for OS, compared with photon RT, there was a trend towards improved survival with proton RT (HR 0.50; p = 0.05). The rate of overall grade 3 + toxicity was 11%. One patient developed radiation-induced liver disease and was treated with corticosteroids. CONCLUSIONS: HF-RT yields high rates of local control and is an effective modality to optimize biliary control for unresectable/locally recurrent ICC.

Pretreatment plasma HGF as potential biomarker for susceptibility to radiation-induced liver dysfunction after radiotherapy.

Radiotherapy shows excellent local control in liver cancers but carries the risk of radiation-induced liver dysfunction and liver failure. We conducted a study of plasma hepatocyte growth factor (HGF) in a clinical trial of proton radiotherapy in patients with unresectable liver cancers (NCT00976898), and in an observational study for liver cancer patients undergoing surgical treatments. Liver dysfunction within 3 months after radiotherapy-a Childs-Turcotte-Pugh (CTP) score increase of 1 point or more-occurred in 9/34 (26%) of patients. Patients with no increase in CTP score had lower pretreatment plasma HGF level (p = 0.015). Both the increase in CTP score (p = 0.034) and the pretreatment plasma HGF (p = 0.017) were associated with OS. Plasma HGF was significantly associated with presence of cirrhosis (p = 0.0027) and with Model for End-stage Liver Disease (MELD) score (p < 0.0001), but not with OS in surgical liver cancer patients. Pretreatment plasma HGF is a candidate biomarker for patient selection for radiotherapy.

Liver reirradiation for patients with hepatocellular carcinoma and liver metastasis.

PURPOSE: This study aimed to assess the safety and efficacy of administering liver reirradiation to patients with primary liver tumors or liver metastasis. METHODS AND MATERIALS: A total of 49 patients (with 64 individual tumors) who received liver reirradiation at our institution between June 2008 and December 2016 were identified for retrospective review. Patients were treated to the same, different, or a combination of previously treated liver tumors for recurrent primary (53%) or metastatic (47%) disease using photons or protons. Clinical and treatment-related factors were compiled and patients were monitored for toxicity and evidence of classic or nonclassic radiation-induced liver disease. Survival was estimated with the Kaplan-Meier method and cumulative incidence of local failure (LF) was used to estimate LF using the Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: The median age at the time of reirradiation was 72 years and the median interval between radiation courses was 9 months. At a median follow-up of 10.5 months, 36 patients (73%) had died, 9 patients (18%) were alive, and 4 patients (8%) were lost to follow-up. The median survival for the cohort was 14 months. The overall 1-year estimate of LF was 46.4%. The 1-year estimates of LF for liver metastases and hepatocellular carcinoma were 61.0% and 32.5%, respectively. The average prescription dose was similar between the reirradiation and initial courses (equivalent dose in 2 Gy fractions EQD2: 65.0 vs 64.3 Gyα/ß = 10, respectively) but the average dose to the untreated liver was lower at the time of reirradiation (EQD2: 10.5 vs 13.9 Gyα/ß = 3, respectively, P = .01). Among patients with hepatocellular carcinoma, the average normal liver dose was significantly larger for patients who exhibited a worsening of Child-Pugh score after reirradiation compared with those who did not (1210 cGy vs 759 cGy, P = .04). With regard to toxicity, 85.7% of patients experienced grade 1 to 2 toxicity, 4.1% developed grade 3, and only 2 patients (4.1%) met the criteria for radiation-induced liver disease after reirradiation. CONCLUSIONS: Liver reirradiation may be an effective and safe option for select patients; however, further prospective study is necessary to establish treatment guidelines and recommended dosing.

Differential Association Between Circulating Lymphocyte Populations With Outcome After Radiation Therapy in Subtypes of Liver Cancer.

PURPOSE: Irradiation may have significant immunomodulatory effects that impact tumor response and could potentiate immunotherapeutic approaches. The purposes of this study were to prospectively investigate circulating lymphoid cell population fractions during hypofractionated proton therapy (HPT) in blood samples of liver cancer patients and to explore their association with survival. METHODS AND MATERIALS: We collected serial blood samples before treatment and at days 8 and 15 of HPT from 43 patients with liver cancer-22 with hepatocellular carcinoma (HCC) and 21 with intrahepatic cholangiocarcinoma (ICC)-enrolled in a phase 2 clinical trial. All patients received 15 fractions of proton therapy to a median dose of 58 Gy (relative biological effectiveness). We used flow cytometry to measure the changes in the fractions of total CD3+, CD4+, and CD8+ T cells; CD4+ CD25+ T cells; CD4+ CD127+ T cells; CD3+ CD8+ CD25+ activated cytotoxic T lymphocytes (CTLs); and CD3- CD56+ natural killer cells. RESULTS: With a median follow-up period of 42 months, median overall survival (OS) in the study cohort was 30.6 months for HCC and 14.5 months for ICC patients. Longer OS was significantly correlated with greater CD4+ CD25+ T-cell (P = .003) and CD4+ CD127+ T-cell (P = .01) fractions at baseline only in ICC patients. In HCC patients, the fraction of activated CTLs mid treatment (at day 8) was significantly associated with OS (P = .007). These findings suggest a differential relevance of immunomodulation by HPT in these liver cancers. CONCLUSIONS: Antitumor immunity may depend on maintenance of a sufficiently high number of activated CTLs during HPT in HCC patients and CD4+ CD25+ T cells and CD4+ CD127+ T cells prior to treatment in ICC patients. These results could guide the design of future studies to determine the optimal treatment schedules when combining irradiation with specific immunotherapy approaches.

Looking in the miR-ror: TGF-ß-mediated activation of NF-κB in glioma.

The explosive growth in our understanding of the molecular underpinnings of glioblastomas has served as an instructive paradigm for other cancers. However, the exact nature by which many of the pathogenic drivers connect is less well known, and elucidation of relationships between critical genetic and signaling alterations may inform the development of therapeutic approaches to the disease. In this issue, Song et al. identify miR-182 induction as a mechanism by which TGF-ß stimulation aberrantly activates NF-κB signaling in glioblastoma cells, clarifying a critical point of cross-talk between molecular signaling pathways. Their findings provide a greater understanding of the complex interplay between signaling pathways in cancer that may ultimately prove useful in the development of synergistic targeting approaches.

Glioma stem cell proliferation and tumor growth are promoted by nitric oxide synthase-2.

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Nonreceptor tyrosine kinase BMX maintains self-renewal and tumorigenic potential of glioblastoma stem cells by activating STAT3.

Glioblastomas display cellular hierarchies containing tumor-propagating glioblastoma stem cells (GSCs). STAT3 is a critical signaling node in GSC maintenance but molecular mechanisms underlying STAT3 activation in GSCs are poorly defined. Here we demonstrate that the bone marrow X-linked (BMX) nonreceptor tyrosine kinase activates STAT3 signaling to maintain self-renewal and tumorigenic potential of GSCs. BMX is differentially expressed in GSCs relative to nonstem cancer cells and neural progenitors. BMX knockdown potently inhibited STAT3 activation, expression of GSC transcription factors, and growth of GSC-derived intracranial tumors. Constitutively active STAT3 rescued the effects of BMX downregulation, supporting that BMX signals through STAT3 in GSCs. These data demonstrate that BMX represents a GSC therapeutic target and reinforces the importance of STAT3 signaling in stem-like cancer phenotypes.

Bacterial flavohemoglobin: a molecular tool to probe mammalian nitric oxide biology.

A wide range of mammalian signaling and stress pathways are mediated by nitric oxide (NO), which is synthesized in vivo by the nitric oxide synthase (NOS) family of enzymes. Experimental manipulations of NO are frequently achieved by either inhibition or activation of endogenous NOS or via providing exogenous NO sources. On the contrary, many microbes consume NO via flavohemoglobin (FlavoHb), a highly efficient NO-dioxygenase that protects from nitrosative stress. Here we report a novel resource for studying NO in mammalian cells by heterologously expressing Escherichia coli FlavoHb within a lentiviral delivery system. This technique boosts endogenous cellular consumption of NO, thus providing a simple and efficacious approach to studying mammalian NO biology that can be employed as both a primary experimental and confirmatory tool.