Organoid cultures derived from patients with advanced prostate cancer.

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, and CHD1 loss. Whole-exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.

Diagnosing destabilization risk in global land carbon sinks.

Global net land carbon uptake or net biome production (NBP) has increased during recent decades1. Whether its temporal variability and autocorrelation have changed during this period, however, remains elusive, even though an increase in both could indicate an increased potential for a destabilized carbon sink2,3. Here, we investigate the trends and controls of net terrestrial carbon uptake and its temporal variability and autocorrelation from 1981 to 2018 using two atmospheric-inversion models, the amplitude of the seasonal cycle of atmospheric CO2 concentration derived from nine monitoring stations distributed across the Pacific Ocean and dynamic global vegetation models. We find that annual NBP and its interdecadal variability increased globally whereas temporal autocorrelation decreased. We observe a separation of regions characterized by increasingly variable NBP, associated with warm regions and increasingly variable temperatures, lower and weaker positive trends in NBP and regions where NBP became stronger and less variable. Plant species richness presented a concave-down parabolic spatial relationship with NBP and its variability at the global scale whereas nitrogen deposition generally increased NBP. Increasing temperature and its increasing variability appear as the most important drivers of declining and increasingly variable NBP. Our results show increasing variability of NBP regionally that can be mostly attributed to climate change and that may point to destabilization of the coupled carbon-climate system.

Glucocorticoid receptor confers resistance to antiandrogens by bypassing androgen receptor blockade.

The treatment of advanced prostate cancer has been transformed by novel antiandrogen therapies such as enzalutamide. Here, we identify induction of glucocorticoid receptor (GR) expression as a common feature of drug-resistant tumors in a credentialed preclinical model, a finding also confirmed in patient samples. GR substituted for the androgen receptor (AR) to activate a similar but distinguishable set of target genes and was necessary for maintenance of the resistant phenotype. The GR agonist dexamethasone was sufficient to confer enzalutamide resistance, whereas a GR antagonist restored sensitivity. Acute AR inhibition resulted in GR upregulation in a subset of prostate cancer cells due to relief of AR-mediated feedback repression of GR expression. These findings establish a mechanism of escape from AR blockade through expansion of cells primed to drive AR target genes via an alternative nuclear receptor upon drug exposure.

Postanesthesia Recovery Unit Optimization for Patients With Postictal Agitation Secondary to Electroconvulsive Therapy.

OBJECTIVES: The occurrence of postictal agitation (PIA) can rapidly alter and intensify the level of care that electroconvulsive therapy (ECT) patients require during their recovery in the postanesthesia care unit (PACU). This operational analysis was undertaken to determine the impact PIA has on phase 1 PACU resources. METHODS: This operational analysis was undertaken at the Seattle Division of the US Department of Veterans Affairs Puget Sound Health Care System. From August 2019 to April 2020, we prospectively collected data on the recovery from ECT of 61 unique patients who underwent a total of 334 ECT sessions. Utilization of PACU resources was assessed by determining the PACU length of stay (LOS), onset of PIA, severity of PIA, and duration of agitation in encounters complicated by PIA. RESULTS: Seventy-nine occurrences of PIA occurred during the 334 ECT encounters. The mean ± SD PACU LOS was longer in encounters complicated by the occurrence of PIA compared with those not complicated by PIA (72 ± 32 and 59 ± 18 minutes respectively; P -value <0.05). Postanesthesia care unit LOS and mean duration of agitation increased as severity of PIA increased. CONCLUSIONS: The occurrence of PIA can rapidly alter and intensify the level of care that ECT patients may require. Postictal agitation has a significant impact on the phase 1 PACU LOS of patients undergoing ECT. Phase 1 PACU staffing models should factor in the acute and prolonged care needs of patients who develop PIA during the recovery from ECT.

Quality of life from cytoreductive surgery in advanced ovarian cancer: Investigating the association between disease burden and surgical complexity in the international, prospective, SOCQER-2 cohort study.

OBJECTIVE: To investigate quality of life (QoL) and association with surgical complexity and disease burden after surgical resection for advanced ovarian cancer in centres with variation in surgical approach. DESIGN: Prospective multicentre observational study. SETTING: Gynaecological cancer surgery centres in the UK, Kolkata, India, and Melbourne, Australia. SAMPLE: Patients undergoing surgical resection (with low, intermediate or high surgical complexity score, SCS) for late-stage ovarian cancer. MAIN OUTCOME MEASURES: Primary: change in global score on the European Organisation for Research and Treatment of Cancer (EORTC) core quality-of-life questionnaire (QLQ-C30). Secondary: EORTC ovarian cancer module (OV28), progression-free survival. RESULTS: Patients' preoperative disease burden and SCS varied between centres, confirming differences in surgical ethos. QoL response rates were 90% up to 18 months. Mean change from the pre-surgical baseline in the EORTC QLQ-C30 was 3.4 (SD 1.8, n = 88) in the low, 4.0 (SD 2.1, n = 55) in the intermediate and 4.3 (SD 2.1, n = 52) in the high-SCS group after 6 weeks (p = 0.048), and 4.3 (SD 2.1, n = 51), 5.1 (SD 2.2, n = 41) and 5.1 (SD 2.2, n = 35), respectively, after 12 months (p = 0.133). In a repeated-measures model, there were no clinically or statistically meaningful differences in EORTC QLQ-C30 global scores between the three SCS groups (p = 0.840), but there was a small statistically significant improvement in all groups over time (p < 0.001). The high-SCS group experienced small to moderate decreases in physical (p = 0.004), role (p = 0.016) and emotional (p = 0.001) function at 6 weeks post-surgery, which resolved by 6-12 months. CONCLUSIONS: The global QoL of patients undergoing low-, intermediate- and high-SCS surgery improved at 12 months after surgery and was no worse in patients undergoing extensive surgery. TWEETABLE ABSTRACT: Compared with surgery of lower complexity, extensive surgery does not result in poorer quality of life in patients with advanced ovarian cancer.

Correction: Urine tumor DNA detection of minimal residual disease in muscle-invasive bladder cancer treated with curative-intent radical cystectomy: A cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1003732.].

Urine tumor DNA detection of minimal residual disease in muscle-invasive bladder cancer treated with curative-intent radical cystectomy: A cohort study.

BACKGROUND: The standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. METHODS AND FINDINGS: We applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD-positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4-38.9; p = 0.02) compared to utDNA MRD-negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD-positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point. CONCLUSIONS: utDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future.

Noncoding RNA Ginir functions as an oncogene by associating with centrosomal proteins.

Long noncoding RNAs constitute a major fraction of the eukaryotic transcriptome, and together with proteins, they intricately fine-tune various growth regulatory signals to control cellular homeostasis. Here, we describe the functional characterisation of a novel pair of long intergenic noncoding RNAs (lincRNAs) comprised of complementary, fully overlapping sense and antisense transcripts Genomic Instability Inducing RNA (Ginir) and antisense RNA of Ginir (Giniras), respectively, from mouse cells. This transcript pair is expressed in a spatiotemporal manner during embryonic development. The individual levels of the sense and antisense transcripts are finely balanced during embryonic growth and in adult tissues. Functional studies of the individual transcripts performed using overexpression and knock-down strategies in mouse cells has led to the discovery that Ginir RNA is a regulator of cellular proliferation and can act as an oncogene having a preeminent role in malignant transformation. Mechanistically, we demonstrate that the oncogenic function of Ginir is mediated by its interaction with centrosomal protein 112 (Cep112). Additionally, we establish here a specific interaction between Cep112 with breast cancer type 1 susceptibility protein (Brca1), another centrosome-associated protein. Next, we prove that the mutual interaction between Cep112 with Brca1 is significant for mitotic regulation and maintenance of genomic stability. Furthermore, we demonstrate that the Cep112 protein interaction with Brca1 protein is impaired when an elevated level of Ginir RNA is present in the cells, resulting in severe deregulation and abnormality in mitosis, leading to malignant transformation. Inhibiting the Ginir RNA function in transformed cells attenuates transformation and restores genomic stability. Together, these findings unravel, to our knowledge, a hitherto-unknown mechanism of oncogenesis mediated by a long noncoding RNA and establishes a unique role of Cep112-Brca1 interaction being modulated by Ginir RNA in maintaining mitotic fidelity.

Cell-free DNA is abundant in ascites and represents a liquid biopsy of ovarian cancer.

OBJECTIVE: Malignant ascites is a common clinical feature of ovarian cancer and represents a readily accessible sample of tumour cells and tumour DNA. This study aimed to characterise the cell-free DNA (cfDNA) in ascites in terms of its size profile, stability and cell-free tumour DNA (cftDNA) content. METHODS: Cell spheroids, loose cells and cell-free fluid was collected from ascites from 18 patients with ovarian cancer. cfDNA was isolated and assessed for size by electrophoresis, concentration by fluorometry,cftDNA content by methylation specific qPCR of HOXA9 and IFFO1 promoter regions and by targeted sequencing. Stability was assessed after ascites fluid was stored at 4 °C for 24 and 72 h before fractionating. RESULTS: The concentration of cfDNA in ascites ranged from 6.6 to 300 ng/mL. cfDNA size distribution resembled blood plasma-derived cfDNA, with major peaks corresponding to mono- and di-nucleosome DNA fragments. High molecular weight cfDNA was observed in 7 of 18 patients and appeared to be associated with extracellular vesicles. IFFO1 and HOXA9 methylation was proportionately higher in cfDNA than spheroid- and loose-cell fractions and was not observed in healthy primary cells. Variant allele frequency was highest in cfDNA compared to single cells and spheroids from ascites. Though cancer cell numbers in ascites declined to near zero in recurrent ascites from one patient undertaking chemotherapy, cftDNA could still be sampled. cfDNA size, concentration and tumour content was stable over 72 h. CONCLUSION: cfDNA in ovarian cancer ascites demonstrates inter-patient variability, yet is consistently a rich source of cftDNA, which is a stable substrate. This supports the wider clinical use of ascites in the molecular analysis of ovarian cancer.

Comparison of forest above-ground biomass from dynamic global vegetation models with spatially explicit remotely sensed observation-based estimates.

Gaps in our current understanding and quantification of biomass carbon stocks, particularly in tropics, lead to large uncertainty in future projections of the terrestrial carbon balance. We use the recently published GlobBiomass data set of forest above-ground biomass (AGB) density for the year 2010, obtained from multiple remote sensing and in situ observations at 100 m spatial resolution to evaluate AGB estimated by nine dynamic global vegetation models (DGVMs). The global total forest AGB of the nine DGVMs is 365 ± 66 Pg C, the spread corresponding to the standard deviation between models, compared to 275 Pg C with an uncertainty of ~13.5% from GlobBiomass. Model-data discrepancy in total forest AGB can be attributed to their discrepancies in the AGB density and/or forest area. While DGVMs represent the global spatial gradients of AGB density reasonably well, they only have modest ability to reproduce the regional spatial gradients of AGB density at scales below 1000 km. The 95th percentile of AGB density (AGB95 ) in tropics can be considered as the potential maximum of AGB density which can be reached for a given annual precipitation. GlobBiomass data show local deficits of AGB density compared to the AGB95 , particularly in transitional and/or wet regions in tropics. We hypothesize that local human disturbances cause more AGB density deficits from GlobBiomass than from DGVMs, which rarely represent human disturbances. We then analyse empirical relationships between AGB density deficits and forest cover changes, population density, burned areas and livestock density. Regression analysis indicated that more than 40% of the spatial variance of AGB density deficits in South America and Africa can be explained; in Southeast Asia, these factors explain only ~25%. This result suggests TRENDY v6 DGVMs tend to underestimate biomass loss from diverse and widespread anthropogenic disturbances, and as a result overestimate turnover time in AGB.

Causes of slowing-down seasonal CO2 amplitude at Mauna Loa.

Changing amplitude of the seasonal cycle of atmospheric CO2 (SCA) in the northern hemisphere is an emerging carbon cycle property. Mauna Loa (MLO) station (20°N, 156°W), which has the longest continuous northern hemisphere CO2 record, shows an increasing SCA before the 1980s (p < .01), followed by no significant change thereafter. We analyzed the potential driving factors of SCA slowing-down, with an ensemble of dynamic global vegetation models (DGVMs) coupled with an atmospheric transport model. We found that slowing-down of SCA at MLO is primarily explained by response of net biome productivity (NBP) to climate change, and by changes in atmospheric circulations. Through NBP, climate change increases SCA at MLO before the 1980s and decreases it afterwards. The effect of climate change on the slowing-down of SCA at MLO is mainly exerted by intensified drought stress acting to offset the acceleration driven by CO2 fertilization. This challenges the view that CO2 fertilization is the dominant cause of emergent SCA trends at northern sites south of 40°N. The contribution of agricultural intensification on the deceleration of SCA at MLO was elusive according to land-atmosphere CO2 flux estimated by DGVMs and atmospheric inversions. Our results also show the necessity to adequately account for changing circulation patterns in understanding carbon cycle dynamics observed from atmospheric observations and in using these observations to benchmark DGVMs.

Climate-Driven Variability and Trends in Plant Productivity Over Recent Decades Based on Three Global Products.

Variability in climate exerts a strong influence on vegetation productivity (gross primary productivity; GPP), and therefore has a large impact on the land carbon sink. However, no direct observations of global GPP exist, and estimates rely on models that are constrained by observations at various spatial and temporal scales. Here, we assess the consistency in GPP from global products which extend for more than three decades; two observation-based approaches, the upscaling of FLUXNET site observations (FLUXCOM) and a remote sensing derived light use efficiency model (RS-LUE), and from a suite of terrestrial biosphere models (TRENDYv6). At local scales, we find high correlations in annual GPP among the products, with exceptions in tropical and high northern latitudes. On longer time scales, the products agree on the direction of trends over 58% of the land, with large increases across northern latitudes driven by warming trends. Further, tropical regions exhibit the largest interannual variability in GPP, with both rainforests and savannas contributing substantially. Variability in savanna GPP is likely predominantly driven by water availability, although temperature could play a role via soil moisture-atmosphere feedbacks. There is, however, no consensus on the magnitude and driver of variability of tropical forests, which suggest uncertainties in process representations and underlying observations remain. These results emphasize the need for more direct long-term observations of GPP along with an extension of in situ networks in underrepresented regions (e.g., tropical forests). Such capabilities would support efforts to better validate relevant processes in models, to more accurately estimate GPP.

Current evidence on the use of sugammadex for neuromuscular blockade antagonism during electroconvulsive therapy - a narrative review.

Depression is a common mental health problem that is associated with significant disability and mortality. Electroconvulsive therapy (ECT) has been demonstrated to be effective at resolving expression of suicidal intent in patients with depression. In less acute situations, patients are usually referred for ECT after several medication trials. Neuromuscular blocking agents (NMBAs) are used to block tonic-clonic motor activity and associated physical harm during the delivery of ECT. Succinylcholine (Sch), with its rapid onset of muscle relaxation, short self-terminating duration of action, and rapid subsequent return of spontaneous ventilation, is the NMBA of choice for ECT. However, the use of Sch is problematic or contraindicated is some situations. Although non-depolarizing NMBAs can be used, the variable time to onset of adequate muscle relaxation and prolonged duration of action have limited their widespread acceptance as alternatives to Sch. Recently, however, with the widespread availability of sugammadex, a chemically modified γ- cyclodextrin that rapidly and predictably reverses the effect of non-depolarizing NMBAs, the muscle relaxation achieved by rocuronium can predictably and effectively be reversed. In situations where Sch is contraindicated or otherwise problematic, rocuronium, followed by pharmacological antagonism with sugammadex, can provide a safe and effective muscle relaxation approach comparable to that of Sch in terms of duration of action. This review provides a summary of the current state of evidence for the use of sugammadex during ECT, which should lend support to further acceptance and future studies of sugammadex in the context of ECT.

Global climate change below 2 °C avoids large end century increases in burned area in Canada.

Wildfire impacts the global carbon cycle, property, harvestable timber, and public health. Canada saw a record fire season in 2023 with 14.9 Mha burned-over seven times the 1986-2022 average of 2.1 Mha. Here we utilize a new process-based wildfire module that explicitly represents fire weather, fuel type and availability, ignition sources, fire suppression, and vegetation's climate response to project the future of wildfire in Canada. Under rapid climate change (shared socioeconomic pathway [SSP] 370 & 585) simulated annual burned area in the 2090 s reaches 10.2 ± 2.1 to 11.7 ± 2.4 Mha, approaching the 2023 fire season total. However, climate change below a 2 °C global target (SSP126), keeps the 2090 s area burned near modern (2004-2014) norms. The simulated area burned and carbon emissions are most sensitive to climate drivers and lightning but future lightning activity is a key uncertainty.

Cell-free DNA from ascites identifies clinically relevant variants and tumour evolution in patients with advanced ovarian cancer.

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour-enriched tissue samples. The use of cell-free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next-generation sequencing was performed on ascites-derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites-derived tumour cells (n = 5) and archived formalin-fixed paraffin-embedded (FFPE) tissue (n = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large-scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy.

Antimicrobial resistance in bacterial isolates from a delhi hospital: one year outcomes.

Antimicrobial resistance and hospital acquired infections have become an important public health issue. Data on pathogen and antibiotic resistance is important for physicians, microbiologists and infection control officials but limited information on antibiotic resistance prevents pathogen specific therapy and propels antibiotic misuse. A retrospective review of bacterial isolation and antimicrobial susceptibility profiles in the in- and outpatients of a Delhi hospital between January 2009-December 2009 was performed. A total of 1772 pathogens causing bacterial infections were recorded during the study period January 2009-December 2009. The most frequently encountered bacterial pathogens were Escherichia coli (40.51%), Klebsiella spp. (14.84%) and Staphylococcus aureus (13.99%). We encountered high resistance to ciprofloxacin in Enterobactereaceae family, i.e., 32.5%. Aminoglycosides, once considered optimum for broad spectrum coverage of pathogens for almost all systemic infections, are now showing high rate of resistance as was noted in Acinetobacter sp. (57.14%) and Pseudomonas aeruginosa (69.2%). Antibiotic susceptibility results show a higher level of resistance to cotrimoxazole, cephalosporins and ciprofloxacin which are easily available, orally administered and cheaper and thus are considered a better option for the patients. This study provides insight into the problem of resistance in bacterial pathogens in Delhi. Our results demonstrated that, in general, isolates have high rates of resistance to antibiotics commonly used in developing countries. Guidelines for surveillance and prevention of nosocomial infections must be implemented in order to reduce the rate of hospital acquired infections.

RBM47 is a Critical Regulator of Mouse Embryonic Stem Cell Differentiation.

RNA-binding proteins (RBPs) are pivotal for regulating gene expression as they are involved in each step of RNA metabolism. Several RBPs are essential for viable growth and development in mammals. RNA-binding motif 47 (RBM47) is an RRM-containing RBP whose role in mammalian embryonic development is poorly understood yet deemed to be essential since its loss in mouse embryos leads to perinatal lethality. In this study, we attempted to elucidate the significance of RBM47 in cell-fate decisions of mouse embryonic stem cells (mESCs). Downregulation of Rbm47 did not affect mESC maintenance and the cell cycle but perturbed the expression of primitive endoderm (PrE) markers and increased GATA4 + PrE-like cells. However, the PrE misregulation could be reversed by either overexpressing Rbm47 or treating the knockdown mESCs with the inhibitors of FGFR or MEK, suggesting an implication of RBM47 in regulating FGF-ERK signaling. Rbm47 knockdown affected the multi-lineage differentiation potential of mESCs as it regressed teratoma in NSG mice and led to a skewed expression of differentiation markers in serum-induced monolayer differentiation. Further, lineage-specific differentiation revealed that Rbm47 is essential for proper differentiation of mESCs towards neuroectodermal and endodermal fate. Taken together, we assign a hitherto unknown role(s) to RBM47 in a subtle regulation of mESC differentiation.

Endothelial cells are a key target of IFN-g during response to combined PD-1/CTLA-4 ICB treatment in a mouse model of bladder cancer.

To explore mechanisms of response to combined PD-1/CTLA-4 immune checkpoint blockade (ICB) treatment in individual cell types, we generated scRNA-seq using a mouse model of invasive urothelial carcinoma with three conditions: untreated tumor, treated tumor, and tumor treated after CD4+ T cell depletion. After classifying tumor cells based on detection of somatic variants and assigning non-tumor cell types using SingleR, we performed differential expression analysis, overrepresentation analysis, and gene set enrichment analysis (GSEA) within each cell type. GSEA revealed that endothelial cells were enriched for upregulated IFN-g response genes when comparing treated cells to both untreated cells and cells treated after CD4+ T cell depletion. Functional analysis showed that knocking out IFNgR1 in endothelial cells inhibited treatment response. Together, these results indicated that IFN-g signaling in endothelial cells is a key mediator of ICB induced anti-tumor activity.