NMR-Based Stable Isotope Tracing of Cancer Metabolism.

NMR is widely used for metabolite profiling (metabolomics, metabonomics) particularly of various readily obtainable biofluids such as plasma and urine. It is especially valuable for stable isotope tracer studies to track metabolic pathways under control or perturbed conditions in a wide range of cell models as well as animal models and human subjects. NMR has unique properties for utilizing stable isotopes to edit or simplify otherwise complex spectra acquired in vitro and in vivo, while quantifying the level of enrichment at specific atomic positions in various metabolites (i.e., isotopomer distribution analysis).In this protocol, we give an overview with specific protocols for NMR-based stable isotope-resolved metabolomics, or SIRM, with a workflow from administration of isotope-enriched precursors, via sample preparation through to NMR data collection and reduction. We focus on indirect detection of common NMR-active stable isotopes including 13C, 15N, 31P, and 2H, using a variety of 1H-based two-dimensional experiments. We also include the application and analyses of multiplex tracer experiments.

How I treat Blastic Plasmacytoid Dendritic Cell Neoplasm.

Historically, treatment options for blastic plasmacytoid dendritic cell neoplasm (BPDCN) were limited to conventional chemotherapy, adopted from regimens used to treat acute myeloid or acute lymphoblastic leukemias, or lymphomas. Nowadays, a novel therapy targeting CD123 is available to treat BPDCN. Yet, regardless of treatment choice, achieving a first complete remission (CR1) represents the main goal of therapy, because it represents the best opportunity to prolong survival in BPDCN, if offered an allogeneic hematopoietic cell transplant (allo-HCT) as consolidative therapy. Although no specific conditioning regimen is considered standard-of-care in allo-HCT eligible patients, recent data from two large registries reported a survival advantage when offering total body irradiation-based myeloablative conditioning (MAC) regimens. Unfortunately, applicability of MAC regimens is not feasible in older/unfit patients, which represents a considerable proportion of patients presenting worldwide. In such cases, reduced intensity conditioning regimens represent the next best option. Auto-HCT could be considered in older/unfit patients who did not have BM involvement at initial presentation and at time of the procedure, albeit data supporting this option is less abundant. Future research is needed to decipher the interplay between clinical, genetic, and molecular features of the disease to personalize treatment accordingly, by enhancing efficacy and avoiding unnecessary toxicities.

Reduced Sense of Smell in Patients with Severe Chronic Rhinosinusitis and its Implications for Diagnosis and Management: A Narrative Review.

Reduced sense of smell is a common symptom in patients with chronic rhinosinusitis (CRS). Although it is often under-diagnosed by healthcare providers, reduced sense of smell can have a substantial negative impact on patient's quality of life as measured by health-related quality of life (HRQoL) assessments and patient-reported outcomes. This narrative review describes current smell loss diagnosis and management guidelines in CRS, and the relationship between smell loss and CRS. Reduced sense of smell can be an indication of CRS disease severity in patients with (CRSwNP) and without nasal polyps (CRSsNP), and recovery of smell can be an indicator of successful CRS treatment. The current first-line therapeutic options for smell loss are intranasal corticosteroids and nasal irrigation, and second-line therapeutic options include systemic steroids and surgery. Shared decision-making between patient, caregiver, and healthcare provider is important when choosing the most appropriate CRS treatment option. Emerging biologic therapies that target type 2 inflammation signaling pathways, such as dupilumab, omalizumab, and mepolizumab, have been shown to improve smell and taste in randomized controlled trials of patients with CRSwNP.A graphical abstract and video abstract are available with this article.

Chronic rhinosinusitis (CRS) is an inflammatory condition often associated with a loss of smell and taste. Patients with CRS and a loss of smell often rate their quality of life as poor and are more likely to also suffer from depression and anxiety than patients without smell loss. Patients with severe smell loss are also more likely to have increased severity of CRS disease by other measures. Standard treatments for smell loss include topical steroids, corticosteroids absorbed into the whole body system (systemic), and/or sinonasal surgery, but the effects may not last, and patients may experience side effects when they use repeated short bursts or long-term treatment with systemic corticosteroids. A newer treatment option for CRS is biologic therapy, which targets the immunologic pathways associated with inflammation. Biologic therapies have been shown to be effective in the treatment of CRS with nasal polyps including improvement in sense of smell. Here, we review the most common diagnostic tests and treatment options for CRS-associated smell loss and show how severity of smell loss is linked to severity of CRS. Supplementary file1 (MP4 60193 kb).

Lipid profiling of RON and DEK-dependent signaling in breast cancer guides discovery of gene networks predictive of poor outcomes.

Recurrent and metastatic breast cancer is frequently treatment resistant. A wealth of evidence suggests that reprogrammed lipid metabolism supports cancer recurrence. Overexpression of the RON and DEK oncoproteins in breast cancer is associated with poor outcome. Both proteins promote cancer metastasis in laboratory models, but their influence on lipid metabolite levels remain unknown. To measure RON- and DEK-dependent steady-state lipid metabolite levels, a nuclear magnetic resonance (NMR)-based approach was utilized. The observed differences identified a lipid metabolism-related gene expression signature that is prognostic of overall survival (OS), distant metastasis-free survival (DMFS), post-progression survival (PPS), and recurrence-free survival (RFS) in patients with breast cancer. RON loss led to decreased cholesterol and sphingomyelin levels, whereas DEK loss increased total fatty acid levels and decreased free glycerol levels. Lipid-related genes were then queried to define a signature that predicts poor outcomes for patients with breast cancer patients. Taken together, RON and DEK differentially regulate lipid metabolism in a manner that predicts and may promote breast cancer metastasis and recurrence.

ATM phosphorylation of CD98HC increases antiporter membrane localization and prevents chronic toxic glutamate accumulation in Ataxia telangiectasia.

Ataxia telangiectasia (A-T) is a rare genetic disorder characterized by neurological defects, immunodeficiency, cancer predisposition, radiosensitivity, decreased blood vessel integrity, and diabetes. ATM, the protein mutated in A-T, responds to DNA damage and oxidative stress, but its functional relationship to the progressive clinical manifestation of A-T is not understood. CD98HC chaperones cystine/glutamate (xc -) and cationic/neutral amino acid (y+L) antiporters to the cell membrane, and CD98HC phosphorylation by ATM accelerates membrane localization to acutely increase amino acid transport. Loss of ATM impacts tissues reliant on SLC family antiporters relevant to A-T phenotypes, such as endothelial cells (telangiectasia) and pancreatic α-cells (fatty liver and diabetes) with toxic glutamate accumulation. Bypassing the antiporters restores intracellular metabolic balance both in ATM-deficient cells and mouse models. These findings provide new insight into the long-known benefits of N-acetyl cysteine to A-T cells beyond oxidative stress through removing excess glutamate by production of glutathione.

Association Between Smell Loss, Disease Burden, and Dupilumab Efficacy in Chronic Rhinosinusitis with Nasal Polyps.

OBJECTIVE: To evaluate the association between smell loss and other aspects of disease, and evaluate dupilumab efficacy in patients with severe chronic rhinosinusitis with nasal polyps (CRSwNP) and moderate or severe smell loss. METHODS: This post-hoc analysis of the SINUS-24/52 studies (NCT02912468/NCT02898454) analyzed nasal polyp score (NPS, 0-8), nasal congestion/obstruction (NC, 0-3), Lund-Mackay CT-scan score (LMK-CT, 0-24), rhinosinusitis severity visual analog scale (RS-VAS, 0-10), and 22-item Sinonasal Outcome Test (SNOT-22, 0-110) according to baseline monthly average patient-reported loss of smell scores (LoS, 0-3) of >1 to 2 (moderate) or >2 to 3 (severe) in patients randomized to dupilumab 300 mg or placebo every 2 weeks. RESULTS: Of 724 patients randomized, baseline LoS was severe in 601 (83%) and moderate in 106 (15%). At baseline, severe versus moderate LoS was associated with 1-point greater severity of NC (odds ratio [OR] 6.01 [95% confidence interval, (CI) 3.95, 9.15]), 5-point greater severity of LMK-CT (OR 2.19 [1.69, 2.85]), and 8.9-point greater severity of SNOT-22 (OR 1.35 [1.20, 1.49]). At Week 24, least squares mean differences (95% CI) dupilumab versus placebo in change from baseline were: NPS -1.90 (-2.56, -1.25) and -1.95 (-2.20, -1.70) in the moderate and severe baseline LoS subgroups, respectively; NC -.35 (-.64, -.06) and -1.00 (-1.13, -.87); LMK-CT -6.30 (-7.88, -4.72) and -6.22 (-6.82, -5.63); RS-VAS -1.18 (-2.20, -.16) and -3.47 (-3.90, -3.03); and SNOT-22 -7.52 (-14.55, -.48) and -21.72 (-24.63, -18.82); all nominal P < .05 versus placebo. Improvements with dupilumab in NC, RS-VAS, and SNOT-22 were statistically greater in patients with severe versus moderate baseline LoS. CONCLUSION: Significant smell impairment in severe CRSwNP is associated with significant disease (NC, RS-VAS, LMK), health-related quality of life impairment (SNOT-22), asthma, and non-steroidal anti-inflammatory drug-exacerbated respiratory disease. Dupilumab significantly improved NPS, NC, LMK-CT, RS-VAS, and SNOT-22 in subjects with moderate and severe baseline smell loss.

Challenges of Spatially Resolved Metabolism in Cancer Research.

Stable isotope-resolved metabolomics comprises a critical set of technologies that can be applied to a wide variety of systems, from isolated cells to whole organisms, to define metabolic pathway usage and responses to perturbations such as drugs or mutations, as well as providing the basis for flux analysis. As the diversity of stable isotope-enriched compounds is very high, and with newer approaches to multiplexing, the coverage of metabolism is now very extensive. However, as the complexity of the model increases, including more kinds of interacting cell types and interorgan communication, the analytical complexity also increases. Further, as studies move further into spatially resolved biology, new technical problems have to be overcome owing to the small number of analytes present in the confines of a single cell or cell compartment. Here, we review the overall goals and solutions made possible by stable isotope tracing and their applications to models of increasing complexity. Finally, we discuss progress and outstanding difficulties in high-resolution spatially resolved tracer-based metabolic studies.

Molecular patterns and mechanisms of tumorigenesis in HPV-associated and HPV-independent sinonasal squamous cell carcinoma.

Mechanisms of tumorigenesis in sinonasal squamous cell carcinoma (SNSCC) remain poorly described due to its rare nature. A subset of SNSCC are associated with the human papillomavirus (HPV); however, it is unknown whether HPV is a driver of HPV-associated SNSCC tumorigenesis or merely a neutral bystander. We hypothesized that performing the first large high-throughput sequencing study of SNSCC would reveal molecular mechanisms of tumorigenesis driving HPV-associated and HPV-independent SNSCC and identify targetable pathways. High-throughput sequencing was performed on 64 patients with HPV-associated and HPV-independent sinonasal carcinomas. Mutation annotation, viral integration, copy number, and pathway-based analyses were performed. Analysis of HPV-associated SNSCC revealed similar mutational patterns observed in HPV-associated cervical and head and neck squamous cell carcinoma, including lack of TP53 mutations and the presence of known hotspot mutations in PI3K and FGFR3. Further similarities included enrichment of APOBEC mutational signature, viral integration at known hotspot locations, and frequent mutations in epigenetic regulators. HPV-associated SNSCC-specific recurrent mutations were also identified including KMT2C , UBXN11 , AP3S1 , MT-ND4 , and MT-ND5 . Mutations in KMT2D and FGFR3 were associated with decreased overall survival. We developed the first known HPV-associated SNSCC cell line and combinatorial small molecule inhibition of YAP/TAZ and PI3K pathways synergistically inhibited tumor cell clonogenicity. In conclusion, HPV-associated SNSCC and HPV-independent SNSCC are driven by molecularly distinct mechanisms of tumorigenesis. Combinatorial blockade of YAP/TAZ and vertical inhibition of the PI3K pathway may be useful in targeting HPV-associated SNSCC whereas targeting MYC and horizontal inhibition of RAS/PI3K pathways for HPV-independent SNSCC. One Sentence Summary: This study solidifies HPV as a driver of HPV-associated SNSCC tumorigenesis, identifies molecular mechanisms distinguishing HPV-associated and HPV-independent SNSCC, and elucidates YAP/TAZ and PI3K blockade as key targets for HPV-associated SNSCC.

Metabolic Reprogramming in Human Cancer Patients and Patient-Derived Models.

Stable isotope-resolved metabolomics delineates reprogrammed intersecting metabolic networks in human cancers. Knowledge gained from in vivo patient studies provides the "benchmark" for cancer models to recapitulate. It is particularly difficult to model patients' tumor microenvironment (TME) with its complex cell-cell/cell-matrix interactions, which shapes metabolic reprogramming crucial to cancer development/drug resistance. Patient-derived organotypic tissue cultures (PD-OTCs) represent a unique model that retains an individual patient's TME. PD-OTCs of non-small-cell lung cancer better recapitulated the in vivo metabolic reprogramming of patient tumors than the patient-derived tumor xenograft (PDTX), while enabling interrogation of immunometabolic response to modulators and TME-dependent resistance development. Patient-derived organoids (PDOs) are also good models for reconstituting TME-dependent metabolic reprogramming and for evaluating therapeutic responses. Single-cell based 'omics on combinations of PD-OTC and PDO models will afford an unprecedented understanding on TME dependence of human cancer metabolic reprogramming, which should translate into the identification of novel metabolic targets for regulating TME interactions and drug resistance.

The role of revision sinus surgery in the initiation of dupilumab therapy: A real-world study of molecular and cellular features.

KEY POINTS: A persistent type 2 endotype signature exists in recalcitrant chronic rhinosinusitis with nasal polyps mucosa on dupilumab. Revision sinus surgery immediately prior to dupilumab reduces long-term interleukin (IL)-4/IL-13 tissue mRNA. Pre-dupilumab revision surgery is associated with reduced tissue eosinophils and GATA-3+ cells.

Panniculus Retractor Use for Visualization of Fetal Anatomy: A Randomized Controlled Trial.

OBJECTIVE: To evaluate whether use of a panniculus retractor device for pregnant patients with body mass index (BMI) 40 or higher and a panniculus improves the completion rate of the fetal anatomic examination. METHODS: This was a randomized trial in which eligible patients with BMI 40 or higher and a panniculus were randomized to undergo their detailed fetal anatomic examination with a panniculus retractor device in place compared with usual care. The primary outcome was the completion rate of 16 prespecified views from the anatomic examination. Secondary outcomes included completion rate of all 64 views from our institution's detailed anatomic examination, duration of examination, major fetal anomaly detection rate, depth from the skin to amniotic cavity before and after retractor placement, patient and ultrasonographer satisfaction, and prespecified adverse events. We assumed a baseline completion rate of 23% for the primary outcome and targeted a twofold improvement with 80% power and two-sided α of 0.05, which resulted in a sample size of 132 participants. The goal enrollment was increased to 150 participants to account for potential dropout. Statistical tests included the Student's t test, χ 2 , and relative risks (RRs) as appropriate. RESULTS: From March to July of 2023, 150 participants completed the study: 74 in the retractor group and 76 in the usual care group. Baseline characteristics were similar between groups except for panniculus grade. The completion rate of 16 prespecified views was 25.7% (19/74) in the retractor group and 31.6% (24/76) in the control group (RR 0.81, 95% CI, 0.49-1.35). There were no significant differences between groups for any of the secondary outcomes. Patient satisfaction and ultrasonographer satisfaction were similar between groups. CONCLUSION: Use of a panniculus retractor device during the fetal anatomic examination for patients with BMI 40 or higher and a panniculus was well tolerated by patients and ultrasonographers but did not improve the completion rate of 16 prespecified fetal anatomic views. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov , NCT05764408.

Stable isotope-resolved metabolomics analyses of metabolic phenotypes reveal variable glutamine metabolism in different patient-derived models of non-small cell lung cancer from a single patient.

INTRODUCTION: Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors. OBJECTIVES: We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC1) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses. METHODS: To address the issue of fidelity, we have carried out a detailed Stable Isotope-Resolved Metabolomics study of freshly resected tissue slices, mouse patient derived xenografts (PDXs), and cells derived from a single patient using both 13C6-glucose and 13C5,15N2-glutamine tracers. RESULTS: Although we found similar glucose metabolism in the three models, glutamine utilization was markedly higher in the isolated cell culture and in cell culture-derived xenografts compared with the primary cancer tissue or direct tissue xenografts (PDX). CONCLUSIONS: This suggests that caution is needed in interpreting cancer biochemistry using patient-derived cancer cells in vitro or in xenografts, even at very early passage, and that direct analysis of patient derived tissue slices provides the optimal model for ex vivo metabolomics. Further research is needed to determine the generality of these observations.

Targetable leukaemia dependency on noncanonical PI3Kγ signalling.

Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.

Leukemia circulation kinetics revealed through blood exchange method.

Leukemias and their bone marrow microenvironments undergo dynamic changes over the course of disease. However, little is known about the circulation kinetics of leukemia cells, nor the impact of specific factors on the clearance of circulating leukemia cells (CLCs) from the blood. To gain a basic understanding of CLC dynamics over the course of disease progression and therapeutic response, we apply a blood exchange method to mouse models of acute leukemia. We find that CLCs circulate in the blood for 1-2 orders of magnitude longer than solid tumor circulating tumor cells. We further observe that: (i) leukemia presence in the marrow can limit the clearance of CLCs in a model of acute lymphocytic leukemia (ALL), and (ii) CLCs in a model of relapsed acute myeloid leukemia (AML) can clear faster than their untreated counterparts. Our approach can also directly quantify the impact of microenvironmental factors on CLC clearance properties. For example, data from two leukemia models suggest that E-selectin, a vascular adhesion molecule, alters CLC clearance. Our research highlights that clearance rates of CLCs can vary in response to tumor and treatment status and provides a strategy for identifying basic processes and factors that govern the kinetics of circulating cells.

Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing.

BACKGROUND: Mammalian cells both import exogenous fatty acids and synthesize them de novo. Palmitate, the end product of fatty acid synthase (FASN) is a substrate for stearoyl-CoA desaturases (Δ-9 desaturases) that introduce a single double bond into fatty acyl-CoA substrates such as palmitoyl-CoA and stearoyl-CoA. This process is particularly upregulated in lipogenic tissues and cancer cells. Tracer methodology is needed to determine uptake versus de novo synthesis of lipids and subsequent chain elongation and desaturation. Here we describe an NMR method to determine the uptake of 13C-palmitate from the medium into HCT116 human colorectal cancer cells, and the subsequent desaturation and incorporation into complex lipids. RESULTS: Exogenous 13C16-palmitate was absorbed from the medium by HCT116 cells and incorporated primarily into complex glycerol lipids. Desaturase activity was determined from the quantification of double bonds in acyl chains, which was greatly reduced by ablation of the major desaturase SCD1. SIGNIFICANCE: The NMR approach requires minimal sample preparation, is non-destructive, and provides direct information about the level of saturation and incorporation of fatty acids into complex lipids.

Organ involvement in adults with BPDCN is associated with sun exposure history, TET2 and RAS mutations, and survival.

ABSTRACT: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) can involve skin, bone marrow (BM), central nervous system (CNS), and non-CNS extramedullary sites. Preclinical models demonstrated clonal advantage of TET2-mutated plasmacytoid dendritic cells exposed to UV radiation. However, whether sun exposure, disease characteristics, and patient survival are clinically related is unclear. We classified organ involvement in 66 patients at diagnosis as skin only (n = 19), systemic plus skin (n = 33), or systemic only (n = 14). BM involvement was absent, microscopic (<5%), or overt (≥5%). UV exposure was based on clinical and demographic data. Patients with skin only BPDCN were more frequently aged ≥75 years (47% vs 19%; P = .032) and had lower rates of complex karyotype (0 vs 32%, P = .022) and mutated NRAS (0 vs 29%, P = .044). Conversely, those without skin involvement had lower UV exposure (23% vs 59%, P = .03) and fewer TET2 mutations (33% vs 72%, P = .051). The median overall survival (OS) was 23.5, 20.4, and 17.5 months for skin only, systemic plus skin, and systemic only, respectively. Patients with no BM involvement had better OS vs overt involvement (median OS, 27.3 vs 15.0 months; P = .033) and comparable with microscopic involvement (27.3 vs 23.5 months; P = .6). Overt BM involvement remained significant for OS when adjusted for baseline characteristics and treatment received. In summary, BPDCN clinical characteristics are associated with disease genetics and survival, which together may impact prognosis and indicate informative disease subtypes for future research.

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via ß2-adrenergic receptor (ß2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using ß2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

Does Playing Tennis with a Low-Compression Ball Effect Psychophysiological Responses and Match Characteristics in Recreational Adult Players?

This study aimed to compare the effects of playing tennis using low-compression balls (Lc-Balls) and standard balls (St-Balls) on psychophysiological responses and match characteristics among recreational adult tennis players. Participants (N = 24; age: 20.5 ± 1.3 years) were randomly matched to play two singles matches over three sets: one match was played with a Lc-Ball and one match was played with a St-Ball, resulting in twenty-four matches. Heart-rate responses and match characteristics were assessed during each match. Post-match measures included retrospective assessments of perceived exertion, ratings of enjoyment towards physical activity, and ratings of mental effort and mood. Results showed higher psychophysiological responses and more intensive play during the game when playing with the Lc-Ball (p ≤ 0.05, d values ranging from 0.24 to 1.93 [small to very large effect]). Further, playing with a Lc-Ball related to reporting a lower rating of perceived exertion (p = 0.00, d = 0.90 [moderate effect]) and greater physical enjoyment (p = 0.00, d = 1.73 [large effect]). Playing with the St-Ball was associated with higher unpleasant mood responses including depression, tension, anger, and fatigue. In conclusion, the results suggest that using the Lc-Ball may lead to better match performance with higher enjoyment in the tennis match-play in recreational adult tennis players.