Examining the Effects of Altitude on Workload Demands in Professional Basketball Players during the Preseason Phase.

Basketball involves frequent high-intensity movements requiring optimal aerobic power. Altitude training can enhance physiological adaptations, but research examining its effects in basketball is limited. This study aimed to characterize the internal/external workload of professional basketball players during preseason and evaluate the effects of altitude and playing position. Twelve top-tier professional male basketball players (Liga Endesa, ACB; guards: n = 3, forwards: n = 5, and centers: n = 4) participated in a crossover study design composed of two training camps with nine sessions over 6 days under two different conditions: high altitude (2320 m) and sea level (10 m). Internal loads (heart rate, %HRMAX) and external loads (total distances covered across speed thresholds, accelerations/decelerations, impacts, and jumps) were quantified via wearable tracking and heart rate telemetry. Repeated-measures MANOVA tested the altitude x playing position effects. Altitude increased the total distance (+10%), lower-speed running distances (+10-39%), accelerations/decelerations (+25-30%), average heart rate (+6%), time in higher-intensity HR zones (+23-63%), and jumps (+13%) across all positions (p < 0.05). Positional differences existed, with guards accruing more high-speed running and centers exhibiting greater cardiovascular demands (p < 0.05). In conclusion, a 6-day altitude block effectively overloads training, providing a stimulus to enhance fitness capacities when structured appropriately. Monitoring workloads and individualizing training by playing position are important when implementing altitude training, given the varied responses.

Discovery of a selective inhibitor of doublecortin like kinase 1.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

Negative-Binomial and quasi-poisson regressions between COVID-19, mobility and environment in São Paulo, Brazil.

Brazil, the country most impacted by the coronavirus disease 2019 (COVID-19) on the southern hemisphere, use intensive care admissions per day, mobility and other indices to monitor quarantines and prevent the transmissions of SARS-CoV-2. In this study we quantified the associations between residential mobility index (RMI), air pollution, meteorology, and daily cases and deaths of COVID-19 in São Paulo, Brazil. We applied a semiparametric generalized additive model (GAM) to estimate: 1) the association between RMI and COVID-19, accounting for ambient particulate matter (PM2.5), ozone (O3), relative humidity, temperature and delayed exposure between 4 and 21 days, and 2) the association between COVID-19 and exposure to for ambient particulate matter (PM2.5), ozone (O3), accounting for relative humidity, temperature and mobility. We found that an RMI of 45.28% results in 1212 cases (95% CI: 1189 to 1235) and 44 deaths (95% CI: 40 to 47). Increasing the isolation from 45.28% to 50% would avoid 438 cases and 21 deaths. Also, we found that an increment of 10 µg⋅m-³ of PM2.5 results in a risk of 1.140 (95% CI: 1.021 to 1.274) for cases and 1.086 (95% CI: 1.008 to 1.170) for deaths, while O3 produces a relative risk of 1.075 (95% CI: 1.006 to 1.150) for cases and 1.063 (95% CI: 1.006 to 1.124) for deaths, respectively. We compared our results with observations and literature review, finding well agreement. Policymakers can use such mobility indices as tools to control social distance activities. Spatial distancing is an important factor to control COVID-19, however, measuring face-mask usage would enhance the understanding the pandemic dynamic. Small increments of air pollution result in an increased number of COVID-19 cases and deaths.

Complement anaphylatoxins C3a and C5a: Emerging roles in cancer progression and treatment.

Recent insights into the role of complement anaphylatoxins C3a and C5a in cancer provide new opportunities for the development of innovative biomarkers and therapeutic strategies. These two complement activation products can maintain chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and increase the motility and metastatic potential of cancer cells. Still, the diverse heterogeneity of responses mediated by these peptides poses a challenge both to our understanding of the role played by these molecules in cancer progression and to the development of effective treatments. This review attempts to summarize the evidence surrounding the involvement of anaphylatoxins in the biological contexts associated with tumor progression. We also describe the recent developments that support the inhibition of anaphylatoxins, or their cognate receptors C3aR and C5aR1, as a treatment option for maximizing the clinical efficacy of current immunotherapies that target the PD-1/PD-L1 immune checkpoint.

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. METHODS: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Follow-up RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. RESULTS: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. CONCLUSIONS: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. LAY SUMMARY: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.

Evolution of Vehicle Emission Factors in a Megacity Affected by Extensive Biofuel Use: Results of Tunnel Measurements in São Paulo, Brazil.

Since 2001, four emission measurement campaigns have been conducted in multiple traffic tunnels in the megacity of São Paulo, Brazil, an area with a fleet of more than 7 million vehicles running on fuels with high biofuel contents: gasoline + ethanol for light-duty vehicles (LDVs) and diesel + biodiesel for heavy-duty vehicles (HDVs). Emission factors for LDVs and HDVs were calculated using a carbon balance method, the pollutants considered including nitrogen oxides (NOx), carbon monoxide (CO), and sulfur dioxide, as well as carbon dioxide and ethanol. From 2001 to 2018, fleet-average emission factors for LDVs and HDVs, respectively, were found to decrease by 4.9 and 5.1% per year for CO and by 5.5 and 4.2% per year for NOx. These reductions demonstrate that regulations for vehicle emissions adopted in Brazil in the last 30 years improved air quality in the megacity of São Paulo significantly, albeit with a clear delay. These findings, especially those for CO, indicate that official emission inventories underestimate vehicle emissions. Here, we demonstrated that the adoption of emission factors calculated under real-world conditions can dramatically improve air quality modeling in the region.

Spread of SARS-CoV-2 through Latin America and the Caribbean region: A look from its economic conditions, climate and air pollution indicators.

We have evaluated the spread of SARS-CoV-2 through Latin America and the Caribbean (LAC) region by means of a correlation between climate and air pollution indicators, namely, average temperature, minimum temperature, maximum temperature, rainfall, average relative humidity, wind speed, and air pollution indicators PM10, PM2.5, and NO2 with the COVID-19 daily new cases and deaths. The study focuses in the following LAC cities: Mexico City (Mexico), Santo Domingo (Dominican Republic), San Juan (Puerto Rico), Bogotá (Colombia), Guayaquil (Ecuador), Manaus (Brazil), Lima (Perú), Santiago (Chile), São Paulo (Brazil) and Buenos Aires (Argentina). The results show that average temperature, minimum temperature, and air quality were significantly associated with the spread of COVID-19 in LAC. Additionally, humidity, wind speed and rainfall showed a significant relationship with daily cases, total cases and mortality for various cities. Income inequality and poverty levels were also considered as a variable for qualitative analysis. Our findings suggest that and income inequality and poverty levels in the cities analyzed were related to the spread of COVID-19 positive and negative, respectively. These results might help decision-makers to design future strategies to tackle the spread of COVID-19 in LAC and around the world.

Monitoring the External and Internal Load Under 2 Teaching Methodologies.

González-Espinosa, S, Antúnez, A, Feu, S, and Ibáñez, SJ. Monitoring the external and internal load under 2 teaching methodologies. J Strength Cond Res 34(10): 2920-2928, 2020-The purpose of this study was to analyze and compare the physical demands produced by the implementation of 2 sport teaching methods during a class and their relation with the learning achieved by the students. Two intervention programs were used to teach the sport of basketball, one based on the direct instruction (DI) method and the other on the tactical-game approach (TGA). The intervention programs were administered to 4 groups of students, 2 for each methodology, with 88 students participating in the study. A total of 10 practical sessions were given, as well as a pre-test and post-test. The practical sessions and the tests were recorded with an inertial system for monitoring and recording physical activity and movement in real time. Differences were found between the methodologies in the teaching sessions in the variables m·min, acc·min, PL·min, and HRmax (p < 0.000). In the assessment tests, there were also differences between both methodologies in the walk and sprint variables (p < 0.005). The results obtained from the analysis of the sessions using each methodology show that the TGA method obtained better results in the variables of external and internal loads than the DI methodology. The TGA methodology permitted a greater development of the students' physical fitness. Moreover, performance in play was not associated with the achievement of higher values in the results of external and internal load in the tests. The students trained with the TGA methodology recorded better performance indicators in the game. These results lead us to recommend physical education teachers to use a student-centered approach in their lessons because it improved both the students' physical fitness and their sports performance.

Blockade of the Complement C5a/C5aR1 Axis Impairs Lung Cancer Bone Metastasis by CXCL16-mediated Effects.

RATIONALE: C5aR1 (CD88), a receptor for complement anaphylatoxin C5a, is a potent immune mediator. Its impact on malignant growth and dissemination of non-small cell lung cancer cells is poorly understood. OBJECTIVES: To investigate the contribution of the C5a/C5aR1 axis to the malignant phenotype of non-small cell lung cancer cells, particularly in skeletal colonization, a preferential lung metastasis site. METHODS: Association between C5aR1 expression and clinical outcome was assessed in silico and validated by immunohistochemistry. Functional significance was evaluated by lentiviral gene silencing and ligand l-aptamer inhibition in in vivo models of lung cancer bone metastasis. In vitro functional assays for signaling, migration, invasion, metalloprotease activity, and osteoclastogenesis were also performed. MEASUREMENTS AND MAIN RESULTS: High levels of C5aR1 in human lung tumors were significantly associated with shorter recurrence-free survival, overall survival, and bone metastasis. Silencing of C5aR1 in lung cancer cells led to a substantial reduction in skeletal metastatic burden and osteolysis in in vivo models. Furthermore, metalloproteolytic, migratory, and invasive tumor cell activities were modulated in vitro by C5aR1 stimulation or gene silencing. l-Aptamer blockade or C5aR1 silencing significantly reduced the osseous metastatic activity of lung cancer cells in vivo. This effect was associated with decreased osteoclastogenic activity in vitro and was rescued by the exogenous addition of the chemokine CXCL16. CONCLUSIONS: Disruption of C5aR1 signaling in lung cancer cells abrogates their tumor-associated osteoclastogenic activity, impairing osseous colonization. This study unveils the role played by the C5a/C5aR1 axis in lung cancer dissemination and supports its potential use as a novel therapeutic target.

Bursting Tumor Bubbles to Improve CAR T-cell Therapy.

Chimeric antigen receptor (CAR) T cells have had dramatic success in B-cell malignancies, but this efficacy has not yet translated to more common solid tumors. In this issue of Cancer Research, Zhong and colleagues demonstrated that tumor-derived small extracellular vesicles (sEV) contain CAR target antigens like mesothelin, enabling them to preferentially interact with and suppress the activity of CAR T cells in vivo. PD-L1 in tumor-derived sEVs increased upon CAR T-cell infusion and induced PD-L1-dependent suppression of CAR T cells that could be completely reversed by PD-L1 blockade. Strategies to inhibit sEV secretion, via genetic manipulation of tumor cells or pharmacologic inhibition, significantly improved CAR T-cell accumulation, function, and antitumor activity in vivo, suggesting that therapeutic targeting of sEV secretion could be a promising new approach to improving the efficacy of CAR T-cell therapy. See related article by Zhong et al., p. 2790.

Gene knock-outs in human CD34+ hematopoietic stem and progenitor cells and in the human immune system of mice.

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate "humanized mice". To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and transplant these modified cells in immunodeficient mice with high engraftment levels and multilineage hematopoietic differentiation. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.

Game Space and Game Situation as Mediators of the External Load in the Tasks of School Handball.

The teacher's didactic intervention also requires knowledge and control of learning tasks' workloads. The objectives of the study were as follows: (i) to quantify the subjective external load-eTL of tasks framed in didactic units designed by in-service teachers; and (ii) to analyze the differences in the subjective eTL according to the game situation and the game space. A total of 306 learning tasks designed by seven in-service teachers (five men and two women), with more than 10 years of teaching practice, were analyzed. These tasks were coded through the Integral System for Training Task Analysis (SIATE, acronym in Spanish). The interobserver reliability of the coded variables obtained a considerable concordance (MKfree > 0.70). The results indicated that there were significant differences in the subjective eTL according to the game situation and game space. The situations of small-sided games in numerical equality or inequality and full games, in medium spaces or large spaces, presented a higher subjective eTL and therefore the highest physiological and motor demands on students. The inclusion of attacking or defending players and an adequate selection of the game space indicated the importance of planning and organizing learning tasks.

Design and validation of a tunable inertial microfluidic system for the efficient enrichment of circulating tumor cells in blood.

The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial-based separation is a promising high-throughput, marker-free sorting strategy for the enrichment and isolation of CTCs. Here, we present and validate a double spiral microfluidic device that efficiently isolates CTCs with a fine-tunable cut-off value of 9 µm and a separation range of 2 µm. We designed the device based on computer simulations that introduce a novel, customized inertial force term, and provide practical fabrication guidelines. We validated the device using calibration beads, which allowed us to refine the simulations and redesign the device. Then we validated the redesigned device using blood samples and a murine model of metastatic breast cancer. Finally, as a proof of principle, we tested the device using peripheral blood from a patient with hepatocellular carcinoma, isolating more than 17 CTCs/ml, with purity/removal values of 96.03% and 99.99% of white blood cell and red blood cells, respectively. These results confirm highly efficient CTC isolation with a stringent cut-off value and better separation results than the state of the art.

Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

Tumor ENPP1 (CD203a)/Haptoglobin Axis Exploits Myeloid-Derived Suppressor Cells to Promote Post-Radiotherapy Local Recurrence in Breast Cancer.

ABSTRACT: Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse. SIGNIFICANCE: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.

Learning Basketball Using Direct Instruction and Tactical Game Approach Methodologies.

This study was to analyze and compare the learning acquired by the students in the sport of basketball in two different methodologies. The sample was composed of 40 students divided into two groups. The intervention programs had previously been validated. A descriptive analysis of the learning indicators, a t-test for independent samples to identify the differences between the methods, and a t-test for related samples to analyze the differences in each group were performed. There are differences between the performance profiles of students in the Direct Instruction in Basketball program and those in the Tactical Game in Basketball program in nine variables. Significant differences are found in the situations of dribbling, shooting, passing and movement, spacing, off-ball defense, and help and in the performance indicator for decision making, execution, and total, which are favorable to the Tactical Game in Basketball program. The students of the Direct Instruction in Basketball program only improved in three variables after the program, while the Tactical Game in Basketball students improved in thirteen variables. It is recommended that the teachers at the schools use the Tactical Game in Basketball methodology for their basketball teaching lessons, because student learning is better than in the Direct Instruction in Basketball program.

Simulation of O3 and NOx in São Paulo street urban canyons with VEIN (v0.2.2) and MUNICH (v1.0).

We evaluate the performance of the Model of Urban Network of Intersecting Canyons and Highways (MUNICH) in simulating ozone (O3) and nitrogen oxides (NOx) concentrations within the urban street canyons in the São Paulo metropolitan area (SPMA). The MUNICH simulations are performed inside the Pinheiros neighborhood (a residential area) and Paulista Avenue (an economic hub), which are representative urban canyons in the SPMA. Both zones have air quality stations maintained by the São Paulo Environmental Agency (CETESB), providing data (both pollutant concentrations and meteorological) for model evaluation. Meteorological inputs for MUNICH are produced by a simulation with the Weather Research and Forecasting model (WRF) over triple-nested domains with the innermost domain centered over the SPMA at a spatial grid resolution of 1 km. Street coordinates and emission flux rates are retrieved from the Vehicular Emission Inventory (VEIN) emission model, representing the real fleet of the region. The VEIN model has an advantage to spatially represent emissions and present compatibility with MUNICH. Building height is estimated from the World Urban Database and Access Portal Tools (WUDAPT) local climate zone map for SPMA. Background concentrations are obtained from the Ibirapuera air quality station located in an urban park. Finally, volatile organic compound (VOC) speciation is approximated using information from the São Paulo air quality forecast emission file and non-methane hydrocarbon concentration measurements. Results show an overprediction of O3 concentrations in both study cases. NOx concentrations are underpredicted in Pinheiros but are better simulated in Paulista Avenue. Compared to O3, NO2 is better simulated in both urban zones. The O3 prediction is highly dependent on the background concentration, which is the main cause for the model O3 overprediction. The MUNICH simulations satisfy the performance criteria when emissions are calibrated. The results show the great potential of MUNICH to represent the concentrations of pollutants emitted by the fleet close to the streets. The street-scale air pollutant predictions make it possible in the future to evaluate the impacts on public health due to human exposure to primary exhaust gas pollutants emitted by the vehicles.

Coupled models using radar network database to assess vehicular emissions in current and future scenarios.

Vehicles are one of the most significant sources of air pollutant emissions in urban areas, and their real contribution always needs to be updated to predict impacts on air quality. Radar databases and traffic counts using statistical modeling is an alternative and low-cost approach to produce traffic activities data in each urban street to be used as input to predict vehicular emissions. In this work, we carried out a spatial statistical analysis of local radar data and calculated traffic flow using local radar data combined with different statistical models. Future scenarios about vehicle emission inventory to define public policies were also proposed and analyzed for Belo Horizonte (BH), a Brazilian State capital, with the third-largest metropolitan region in the country. The Normal-Neighborhood Model (i.e., the mixed effect model with random effect in the neighborhood, radar type, and in the regional area) was used to calculate traffic flow in each urban street. Results showed average reductions in CO (4.5%), NMHC (3.0%), NOx (3.0%) and PM2.5 (6.2%) emissions even with an increase in fleet composition (25% in average). The decrease is a result of the implementation of emission control programs by the government, improvements vehicles technologies, and the quality of fuels. Prediction of traffic data from radar databases has proven to be useful for avoiding the high costs of performing origin-destination surveys and traffic modeling using commercial software. Radar databases can provide many potential benefits for research and analysis in environmental and transportation planning. These findings can be incorporated in future investigations to implement public policies on vehicular emission reduction in urban areas and to advance environmental health effects research and human health risk assessment.

SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation.

INTRODUCTION: The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC. METHODS: A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing. RESULTS: Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3. CONCLUSIONS: YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.

Short-term starvation reduces IGF-1 levels to sensitize lung tumors to PD-1 immune checkpoint blockade.

Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1-programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.