Single-cell spatial landscapes of the lung tumour immune microenvironment.

Single-cell technologies have revealed the complexity of the tumour immune microenvironment with unparalleled resolution1-9. Most clinical strategies rely on histopathological stratification of tumour subtypes, yet the spatial context of single-cell phenotypes within these stratified subgroups is poorly understood. Here we apply imaging mass cytometry to characterize the tumour and immunological landscape of samples from 416 patients with lung adenocarcinoma across five histological patterns. We resolve more than 1.6 million cells, enabling spatial analysis of immune lineages and activation states with distinct clinical correlates, including survival. Using deep learning, we can predict with high accuracy those patients who will progress after surgery using a single 1-mm2 tumour core, which could be informative for clinical management following surgical resection. Our dataset represents a valuable resource for the non-small cell lung cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. This study also highlights how artificial intelligence can improve our understanding of microenvironmental features that underlie cancer progression and may influence future clinical practice.

Perioperative Nivolumab in Resectable Lung Cancer.

BACKGROUND: Standard treatment with neoadjuvant nivolumab plus chemotherapy significantly improves outcomes in patients with resectable non-small-cell lung cancer (NSCLC). Perioperative treatment (i.e., neoadjuvant therapy followed by surgery and adjuvant therapy) with nivolumab may further improve clinical outcomes. METHODS: In this phase 3, randomized, double-blind trial, we assigned adults with resectable stage IIA to IIIB NSCLC to receive neoadjuvant nivolumab plus chemotherapy or neoadjuvant chemotherapy plus placebo every 3 weeks for 4 cycles, followed by surgery and adjuvant nivolumab or placebo every 4 weeks for 1 year. The primary outcome was event-free survival according to blinded independent review. Secondary outcomes were pathological complete response and major pathological response according to blinded independent review, overall survival, and safety. RESULTS: At this prespecified interim analysis (median follow-up, 25.4 months), the percentage of patients with 18-month event-free survival was 70.2% in the nivolumab group and 50.0% in the chemotherapy group (hazard ratio for disease progression or recurrence, abandoned surgery, or death, 0.58; 97.36% confidence interval [CI], 0.42 to 0.81; P<0.001). A pathological complete response occurred in 25.3% of the patients in the nivolumab group and in 4.7% of those in the chemotherapy group (odds ratio, 6.64; 95% CI, 3.40 to 12.97); a major pathological response occurred in 35.4% and 12.1%, respectively (odds ratio, 4.01; 95% CI, 2.48 to 6.49). Grade 3 or 4 treatment-related adverse events occurred in 32.5% of the patients in the nivolumab group and in 25.2% of those in the chemotherapy group. CONCLUSIONS: Perioperative treatment with nivolumab resulted in significantly longer event-free survival than chemotherapy in patients with resectable NSCLC. No new safety signals were observed. (Funded by Bristol Myers Squibb; CheckMate 77T ClinicalTrials.gov number, NCT04025879.).

Perioperative Pembrolizumab for Early-Stage Non-Small-Cell Lung Cancer.

BACKGROUND: Among patients with resectable early-stage non-small-cell lung cancer (NSCLC), a perioperative approach that includes both neoadjuvant and adjuvant immune checkpoint inhibition may provide benefit beyond either approach alone. METHODS: We conducted a randomized, double-blind, phase 3 trial to evaluate perioperative pembrolizumab in patients with early-stage NSCLC. Participants with resectable stage II, IIIA, or IIIB (N2 stage) NSCLC were assigned in a 1:1 ratio to receive neoadjuvant pembrolizumab (200 mg) or placebo once every 3 weeks, each of which was given with cisplatin-based chemotherapy for 4 cycles, followed by surgery and adjuvant pembrolizumab (200 mg) or placebo once every 3 weeks for up to 13 cycles. The dual primary end points were event-free survival (the time from randomization to the first occurrence of local progression that precluded the planned surgery, unresectable tumor, progression or recurrence, or death) and overall survival. Secondary end points included major pathological response, pathological complete response, and safety. RESULTS: A total of 397 participants were assigned to the pembrolizumab group, and 400 to the placebo group. At the prespecified first interim analysis, the median follow-up was 25.2 months. Event-free survival at 24 months was 62.4% in the pembrolizumab group and 40.6% in the placebo group (hazard ratio for progression, recurrence, or death, 0.58; 95% confidence interval [CI], 0.46 to 0.72; P<0.001). The estimated 24-month overall survival was 80.9% in the pembrolizumab group and 77.6% in the placebo group (P = 0.02, which did not meet the significance criterion). A major pathological response occurred in 30.2% of the participants in the pembrolizumab group and in 11.0% of those in the placebo group (difference, 19.2 percentage points; 95% CI, 13.9 to 24.7; P<0.0001; threshold, P = 0.0001), and a pathological complete response occurred in 18.1% and 4.0%, respectively (difference, 14.2 percentage points; 95% CI, 10.1 to 18.7; P<0.0001; threshold, P = 0.0001). Across all treatment phases, 44.9% of the participants in the pembrolizumab group and 37.3% of those in the placebo group had treatment-related adverse events of grade 3 or higher, including 1.0% and 0.8%, respectively, who had grade 5 events. CONCLUSIONS: Among patients with resectable, early-stage NSCLC, neoadjuvant pembrolizumab plus chemotherapy followed by resection and adjuvant pembrolizumab significantly improved event-free survival, major pathological response, and pathological complete response as compared with neoadjuvant chemotherapy alone followed by surgery. Overall survival did not differ significantly between the groups in this analysis. (Funded by Merck Sharp and Dohme; KEYNOTE-671 ClinicalTrials.gov number, NCT03425643.).

The role of neutrophil extracellular traps in cancer progression and metastasis.

Neutrophil extracellular traps (NET) are the extracellular release of decondensed chromatin and granules known for their role in host defense against foreign pathogens. However, a novel and predominantly pro-tumorigenic role of NETs in cancer is emerging. Tumors induce NET formation through the secretion of an array of tumor and infection-derived factors. NET deposition favors tumor cell proliferation, immunosuppression and cancer-associated thrombosis. Moreover, NETs enhance metastasis by contributing to epithelial-to-mesenchymal transition. A mesenchymal transition in tumor cells potentiates their migratory and invasive abilities. Circulating NETs capture circulating tumor cells and increase vascular permeability, thereby promoting tumor cell intravasation and establishment of micrometastasis. NETs present in pre-metastatic niches serve as cytokines in the recruitment of tumor cells through CCDC25. Through NET-mediated laminin remodelling, dormant cells are reawakened favoring metastatic growth. Moreover, post-operative infection results in high NET deposition and exacerbates post-surgical cancer progression and recurrence. Anti-NET therapies are in use for autoimmune diseases and are now being investigated in the context of cancer. The hope is that anti-NET therapies may synergize with existing anti-cancer therapy to increase rate of response given their multi-faceted interplay in several domains of cancer progression.

Modern era systemic therapies: Expanding concepts of cure in early and locally advanced non-small cell lung cancer.

Cure of cancer is a sensitive and multidimensional concept that is challenging to define, difficult to assert at the individual patient level, and often surrounded by controversy. The notion of cure in non-small cell lung cancer (NSCLC) has changed and continues to evolve with improvements in diagnosis and treatment. Targeted and immune therapies have recently entered the treatment landscape of stage I-III NSCLC. While some initial pivotal trials of such agents failed to improve survival, recently approved epidermal growth factor receptor (EGFR) inhibitors (in EGFR-mutated NSCLC) and immune checkpoint inhibitors have shown delays in disease recurrence or progression and unprecedented survival gains compared to previous standards of care. Additional data is now emerging supporting the benefit of treatment strategies based on alternation-matched targeting (anaplastic lymphoma kinase [ALK] inhibition in ALK-altered disease) and immune checkpoint inhibition in stage I-III NSCLC. Similar to previous developments in the treatment of early and locally advanced NSCLC, it is expected that statistically significant and clinically meaningful trial-level benefits will translate into real-world benefits, including improvements in cure measures. Parallel advances in molecular testing (e.g., circulating tumor DNA analyses) are also allowing for a deeper and more comprehensive characterization of disease status and treatment response. Given the impact that curative-intent treatments have on survival, it is critical that various stakeholders, including clinicians and patients, are aware of new opportunities to pursue cure in stage I-III NSCLC.

Soluble factors in malignant ascites promote the metastatic adhesion of gastric adenocarcinoma cells.

BACKGROUND: Adenocarcinoma of the proximal stomach is the fastest rising malignancy in North America. It is commonly associated with peritoneal accumulation of malignant ascites (MA), a fluid containing cancer and inflammatory cells and soluble proteins. Peritoneal metastasis (PM) is the most common site of gastric cancer (GC) progression after curative-intent surgery and is the leading cause of death among GC patients. METHODS/RESULTS: Using a panel of gastric adenocarcinoma cell lines (human: MKN 45, SNU-5; murine: NCC-S1M), we demonstrate that prior incubation of GC cells with MA results in a significant (> 1.7-fold) increase in the number of cells capable of adhering to human peritoneal mesothelial cells (HPMC) (p < 0.05). We then corroborate these findings using an ex vivo PM model and show that MA also significantly enhances the ability of GC cells to adhere to strips of human peritoneum (p < 0.05). Using a multiplex ELISA, we identify MIF and VEGF as consistently elevated across MA samples from GC patients (p < 0.05). We demonstrate that agents that block the effects of MIF or VEGF abrogate the ability of MA to stimulate the adhesion of GC cells to adhere to human peritoneum and promote both ex vivo and in vivo metastases. CONCLUSION: Agents targeting MIF or VEGF may be relevant to the treatment or prevention of PM in GC patients.

Murine fecal microbiota transfer models selectively colonize human microbes and reveal transcriptional programs associated with response to neoadjuvant checkpoint inhibitors.

Human gut microbial species found to associate with clinical responses to immune checkpoint inhibitors (ICIs) are often tested in mice using fecal microbiota transfer (FMT), wherein tumor responses in recipient mice may recapitulate human responses to ICI treatment. However, many FMT studies have reported only limited methodological description, details of murine cohorts, and statistical methods. To investigate the reproducibility and robustness of gut microbial species that impact ICI responses, we performed human to germ-free mouse FMT using fecal samples from patients with non-small cell lung cancer who had a pathological response or nonresponse after neoadjuvant ICI treatment. R-FMT mice yielded greater anti-tumor responses in combination with anti-PD-L1 treatment compared to NR-FMT, although the magnitude varied depending on mouse cell line, sex, and individual experiment. Detailed investigation of post-FMT mouse microbiota using 16S rRNA amplicon sequencing, with models to classify and correct for biological variables, revealed a shared presence of the most highly abundant taxa between the human inocula and mice, though low abundance human taxa colonized mice more variably after FMT. Multiple Clostridium species also correlated with tumor outcome in individual anti-PD-L1-treated R-FMT mice. RNAseq analysis revealed differential expression of T and NK cell-related pathways in responding tumors, irrespective of FMT source, with enrichment of these cell types confirmed by immunohistochemistry. This study identifies several human gut microbial species that may play a role in clinical responses to ICIs and suggests attention to biological variables is needed to improve reproducibility and limit variability across experimental murine cohorts.

Neutrophil Extracellular Trap-Associated CEACAM1 as a Putative Therapeutic Target to Prevent Metastatic Progression of Colon Carcinoma.

Neutrophils promote tumor growth and metastasis at multiple stages of cancer progression. One mechanism through which this occurs is via release of neutrophil extracellular traps (NETs). We have previously shown that NETs trap tumor cells in both the liver and the lung, increasing their adhesion and metastasis following postoperative complications. Multiple studies have since shown that NETs play a role in tumor progression and metastasis. NETs are composed of nuclear DNA-derived web-like structures decorated with neutrophil-derived proteins. However, it is unknown which, if any, of these NET-affiliated proteins is responsible for inducing the metastatic phenotype. In this study, we identify the NET-associated carcinoembryonic Ag cell adhesion molecule 1 (CEACAM1) as an essential element for this interaction. Indeed, blocking CEACAM1 on NETs, or knocking it out in a murine model, leads to a significant decrease in colon carcinoma cell adhesion, migration and metastasis. Thus, this work identifies NET-associated CEACAM1 as a putative therapeutic target to prevent the metastatic progression of colon carcinoma.

Casting A Wide Net On Surgery: The Central Role of Neutrophil Extracellular Traps.

: Since their discovery, neutrophil extracellular traps (NETs) have been implicated in a broad array of functions, both beneficial and detrimental to the host. Indeed, NETs have roles in infection, sepsis, wound healing, thrombotic disease, and cancer propagation, all of which are directly implicated in the care of surgical patients. Here we provide an updated review on the role of NETs in the perioperative period with specific emphasis on perioperative infections, wound healing, vascular complications, cancer propagation, as well as discussing ongoing, and future therapeutic targets. Surgeons will benefit from understanding the latest discoveries in neutrophil biology and how these novel functions affect the care of surgical patients. Furthermore, novel anti-NET therapies are being developed which may have profound effects on the care of surgical patients.

The innate immune architecture of lung tumors and its implication in disease progression.

Lung malignancies are the leading cause of cancer-related mortality. By virtue of its unique physiological function, the lung microenvironment is highly dynamic and constantly subjected to mechanical, chemical and pathogenic stimuli. Thus, the airways rely on highly organized innate defense mechanisms to rapidly protect against pathogens and maintain pulmonary homeostasis. However, in the context of lung malignancy, these defenses often provide collateral inflammatory insults that can foster tumor progression. This review summarizes the interactions between cancer cells, recruited immune cells and tissue-resident cell subpopulations, such as airway epithelial cells and alveolar macrophages, during homeostasis and disease. Furthermore, we examine the role of the lung immune landscape in response to current therapeutic interventions for cancer. Given the prevalence of lung malignancies, we propose that consideration of lung physiology as a whole is necessary to understand and treat these lethal diseases. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neutrophil extracellular traps sequester circulating tumor cells via ß1-integrin mediated interactions.

Despite advances in cancer treatment, metastasis remains today the main cause of cancer death. Local control through complete surgical resection of the primary tumor continues to be a key principle in cancer treatment. However, surgical interventions themselves lead to adverse oncologic outcomes and are associated with significantly increased rates of metastasis. Neutrophils through release of neutrophil extracellular traps (NETs) in response to infections were shown to be able to capture circulating cancer cells, and in doing so, support the development of metastatic disease. To be able to intervene on this process, understanding the exact molecular nature of these mechanisms is crucial. We therefore hypothesize and demonstrate that ß1-integrin is an important factor mediating the interactions between circulating tumor cells and NETs. We show that ß1-integrin expression on both cancer cells and NETs is important for the adhesion of circulating tumor cells to NETs both in vitro and in vivo. Using a murine model of intra-abdominal sepsis to mimic the postoperative inflammatory environment, we show that ß1-integrin expression is upregulated in the context of inflammation in vivo. Ultimately, we show that this increased early cancer cell adhesion to NETs in vivo and this effect is abrogated when mice are administered DNAse 1. Our data therefore sheds light on the first molecular mechanism by which NETs can trap circulating tumor cells (CTCs), broadening our understanding of this process.

Gram-positive pneumonia augments non-small cell lung cancer metastasis via host toll-like receptor 2 activation.

Surgical resection of early stage nonsmall cell lung cancer (NSCLC) is necessary for cure. However, rates of postoperative bacterial pneumonias remain high and may confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory cascade by recognizing microbial products at the surface of numerous cell types in the lung; however, little is known about how host TLRs influence NSCLC metastasis. TLR2 recognizes gram-positive bacterial cell wall components activating innate immunity. We demonstrate that lower respiratory tract infection with Streptococcus pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through host TLR2 activation. Infected mice demonstrate increased H59 and human A549 NSCLC adhesion to hepatic sinusoids in vivo compared with noninfected controls, a response that is significantly diminished in TLR2 knock-out mice. Intra-tracheal injection of purified TLR2 ligand lipoteichoic acid into mice similarly augments in vivo adhesion of H59 cells to hepatic sinusoids. Additionally, H59 and A549 NSCLC cells incubated with bronchoepithelial conditioned media show increased cell adhesion to extracellular matrix components in vitro and hepatic sinusoids in vivo in a manner that is dependent on bronchoepithelial TLR2 activation and interleukin-6 secretion. TLR2 is therefore a potential therapeutic target for gram-positive pneumonia-driven NSCLC metastasis.

Perioperative Immunotherapy for Node-Negative Non-Small Cell Lung Cancer-Current Evidence and Future Directions.

Neoadjuvant immunotherapy has gone from an idea to an indication in locally advanced lung cancer. Several phase III trials have demonstrated the superiority of neoadjuvant chemoimmunotherapy compared with chemotherapy in this setting. Although such progress has revolutionized the treatment of locally advanced disease, the unmet needs of stage I and stage II patients without lymph node disease have largely been underrepresented in existing trials. Up-front resection with few patients going on to complete adjuvant therapy remains the norm for most stage I and II patients. Emerging evidence now supports the exploration of supplemental checkpoint blockade in well-selected early-stage, node-negative patients with large tumors and no actionable driver mutations. Although concerns surrounding safety and risk exist, patient selection could be substantially improved using novel biomarker approaches that leverage our understanding of the tumor immune microenvironment of lung cancer. This review provides a comprehensive overview of the opportunities and controversies of perioperative immunotherapy in node-negative lung cancer.

Neoadjuvant Chemoimmunotherapy for NSCLC: A Systematic Review and Meta-Analysis.

Importance: To date, no meta-analyses have comprehensively assessed the association of neoadjuvant chemoimmunotherapy with clinical outcomes in non-small cell lung cancer (NSCLC) in randomized and nonrandomized settings. In addition, there exists controversy concerning the efficacy of neoadjuvant chemoimmunotherapy for patients with NSCLC with programmed cell death 1 ligand 1 (PD-L1) levels less than 1%. Objective: To compare neoadjuvant chemoimmunotherapy with chemotherapy by adverse events and surgical, pathological, and efficacy outcomes using recently published randomized clinical trials and nonrandomized trials. Data Sources: MEDLINE and Embase were systematically searched from January 1, 2013, to October 25, 2023, for all clinical trials of neoadjuvant chemoimmunotherapy and chemotherapy that included at least 10 patients. Study Selection: Observational studies and trials reporting the use of neoadjuvant radiotherapy, including chemoradiotherapy, molecular targeted therapy, or immunotherapy monotherapy, were excluded. Main Outcomes and Measures: Surgical, pathological, and efficacy end points and adverse events were pooled using a random-effects meta-analysis. Results: Among 43 eligible trials comprising 5431 patients (4020 males [74.0%]; median age range, 55-70 years), there were 8 randomized clinical trials with 3387 patients. For randomized clinical trials, pooled overall survival (hazard ratio, 0.65; 95% CI, 0.54-0.79; I2 = 0%), event-free survival (hazard ratio, 0.59; 95% CI, 0.52-0.67; I2 = 14.9%), major pathological response (risk ratio, 3.42; 95% CI, 2.83-4.15; I2 = 31.2%), and complete pathological response (risk ratio, 5.52; 95% CI, 4.25-7.15; I2 = 27.4%) favored neoadjuvant chemoimmunotherapy over neoadjuvant chemotherapy. For patients with baseline tumor PD-L1 levels less than 1%, there was a significant benefit in event-free survival for neoadjuvant chemoimmunotherapy compared with chemotherapy (hazard ratio, 0.74; 95% CI, 0.62-0.89; I2 = 0%). Conclusion and Relevance: This study found that neoadjuvant chemoimmunotherapy was superior to neoadjuvant chemotherapy across surgical, pathological, and efficacy outcomes. These findings suggest that patients with resectable NSCLC with tumor PD-L1 levels less than 1% may have an event-free survival benefit with neoadjuvant chemoimmunotherapy.

Neoadjuvant and Adjuvant Treatment for Early-Stage Resectable Non-small Cell Lung Cancer (NSCLC): Consensus Recommendations from the International Association for the Study of Lung Cancer (IASLC).

Advances in the multidisciplinary care of early-stage resectable non-small cell lung cancer (rNSCLC) are emerging at an unprecedented pace. Numerous phase 3 trials produced results that have transformed patient outcomes for the better, yet these findings also require important modifications to the patient treatment journey trajectory and re-organization of care pathways. Perhaps most notably, the need for multispecialty collaboration for this patient population has never been greater. These rapid advances have inevitably left us with important gaps in knowledge for which definitive answers will only become available in several years. To this end, the IASLC commissioned a diverse multidisciplinary international expert panel to evaluate the current landscape and provide diagnostic, staging, and therapeutic recommendations for patients with rNSCLC, with particular emphasis on patients with AJCC/UICC TNM 8th edition stage II and III disease. Using a team-based approach, we generated 19 recommendations, of which all but one achieved greater than 85% consensus amongst panel members. A public voting process was initiated, which successfully validated and provided qualitative nuance to our recommendations. Highlights include: 1) the critical importance of a multidisciplinary approach to the evaluation of patients with rNSCLC driven by shared clinical decision making of a multispecialty team of expert providers; 2) biomarker testing for rNSCLC; 3) a preference for neoadjuvant chemoimmunotherapy for stage III rNSCLC; 4) equipoise regarding the optimal management of patients with stage II between up-front surgery followed by adjuvant therapy and neoadjuvant/perioperative strategies; and 5) the robust preference for adjuvant targeted therapy for patients with rNSCLC and sensitizing EGFR and ALK tumor alterations. Our primary goals were to provide practical recommendations sensitive to the global differences in biology and resources for patients with rNSCLC, and to provide expert consensus guidance tailored to the individualized patient needs, goals, and preferences in their cancer care journey as these are areas where physicians must make daily clinical decisions in the absence of definitive data. These recommendations will continue to evolve as the treatment landscape for rNSCLC expands and more knowledge is acquired on the best therapeutic approach in specific patient and disease subgroups.

Transitioning to Neoadjuvant Therapy for Resectable Non-Small Cell Lung Cancer: Trends and Surgical Outcomes in a Regionalized Pulmonary Oncology Network.

BACKGROUND: Several regulatory agencies have approved the use of the neoadjuvant chemo-immunotherapy for resectable stage II and III of non-small cell lung cancer (NSCLC) and numerous trials investigating novel agents are underway. However, significant concerns exist around the feasibility and safety of offering curative surgery to patients treated within such pathways. The goal in this study was to evaluate the impact of a transition towards a large-scale neoadjuvant therapy program for NSCLC. METHODS: Medical charts of patients with clinical stage II and III NSCLC who underwent resection from January 2015 to December 2020 were reviewed. The primary outcome was perioperative complication rate between neoadjuvant-treated versus upfront surgery patients. Multivariable logistic regression estimated occurrence of postoperative complications and overall survival was assessed as an exploratory secondary outcome by Kaplan-Meier and Cox-regression analyses. RESULTS: Of the 428 patients included, 106 (24.8%) received neoadjuvant therapy and 322 (75.2%) upfront surgery. Frequency of minor and major postoperative complications was similar between groups (P = .22). Occurrence in postoperative complication was similar in both cohort (aOR = 1.31, 95% CI 0.73-2.34). Neoadjuvant therapy administration increased from 10% to 45% with a rise in targeted and immuno-therapies over time, accompanied by a reduced rate of preoperative radiation therapy use. 1-, 2-, and 5-year overall survival was higher in neoadjuvant therapy compared to upfront surgery patients (Log-Rank P = .017). CONCLUSIONS: No significant differences in perioperative outcomes and survival were observed in resectable NSCLC patients treated by neoadjuvant therapy versus upfront surgery. Transition to neoadjuvant therapy among resectable NSCLC patients is safe and feasible from a surgical perspective.

Cotargeting CDK4/6 and BRD4 Promotes Senescence and Ferroptosis Sensitivity in Cancer.

Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are approved for breast cancer treatment and show activity against other malignancies, including KRAS-mutant non-small cell lung cancer (NSCLC). However, the clinical efficacy of CDK4/6 inhibitors is limited due to frequent drug resistance and their largely cytostatic effects. Through a genome-wide cDNA screen, we identified that bromodomain-containing protein 4 (BRD4) overexpression conferred resistance to the CDK4/6 inhibitor palbociclib in KRAS-mutant NSCLC cells. Inhibition of BRD4, either by RNA interference or small-molecule inhibitors, synergized with palbociclib to induce senescence in NSCLC cells and tumors, and the combination prolonged survival in a KRAS-mutant NSCLC mouse model. Mechanistically, BRD4-inhibition enhanced cell-cycle arrest and reactive oxygen species (ROS) accumulation, both of which are necessary for senescence induction; this in turn elevated GPX4, a peroxidase that suppresses ROS-triggered ferroptosis. Consequently, GPX4 inhibitor treatment selectively induced ferroptotic cell death in the senescent cancer cells, resulting in tumor regression. Cotargeting CDK4/6 and BRD4 also promoted senescence and ferroptosis vulnerability in pancreatic and breast cancer cells. Together, these findings reveal therapeutic vulnerabilities and effective combinations to enhance the clinical utility of CDK4/6 inhibitors. SIGNIFICANCE: The combination of cytostatic CDK4/6 and BRD4 inhibitors induces senescent cancer cells that are primed for activation of ferroptotic cell death by targeting GPX4, providing an effective strategy for treating cancer.

Visualizing NETosis Using a Novel Neutrophil Extracellular Trap-Specific Marker.

As the most abundant leukocyte in circulation, the neutrophil plays a far-reaching role in maintaining homeostasis. Within the context of disease, however, neutrophils can potentiate various pathophysiological mechanisms with disastrous consequences for patients. The role of the neutrophil in disease is complex with mechanisms like NETosis driving the progression of several pathologies. NETosis involves neutrophils extruding protein-decorated DNA webs called neutrophil extracellular traps (NETs), which facilitate the progression of inflammatory, non-infectious, and neoplastic pathologies. The need to visualize NETs has thus never been greater. Current approaches for visualizing NETs are limited in specificity and sensitivity, involving non-specific fluorescent DNA dyes or co-stains of neutrophil and DNA markers. Improved methodologies are needed to robustly distinguish NETs from other cell-free DNA. Excitingly, a novel NET-specific posttranslational modification involving cleavage on the N-terminus of histone H3 has recently been identified. Here, we demonstrate that this single marker is superior to the conventional use of the co-stain of the neutrophil marker, myeloperoxidase, and, the DNA marker, histone H3 citrullination in visualizing neutrophil NETosis. This is due to this single marker's unparalleled ability to identify, not only more NETs but also their formation at earlier stages of NETosis. Moreover, we additionally propose a stepwise mechanism of neutrophil NETosis in which a histone H3 cleavage event precedes histone H3 citrullination. Taken together, these results demonstrate a novel method for visualizing NETs, allowing for continued exploration of their multifaceted roles in immunity and disease.

Single-cell spatial landscape of immunotherapy response reveals mechanisms of CXCL13 enhanced antitumor immunity.

BACKGROUND: Immunotherapy has revolutionized clinical outcomes for patients suffering from lung cancer, yet relatively few patients sustain long-term durable responses. Recent studies have demonstrated that the tumor immune microenvironment fosters tumorous heterogeneity and mediates both disease progression and response to immune checkpoint inhibitors (ICI). As such, there is an unmet need to elucidate the spatially defined single-cell landscape of the lung cancer microenvironment to understand the mechanisms of disease progression and identify biomarkers of response to ICI. METHODS: Here, in this study, we applied imaging mass cytometry to characterize the tumor and immunological landscape of immunotherapy response in non-small cell lung cancer by describing activated cell states, cellular interactions and neighborhoods associated with improved efficacy. We functionally validated our findings using preclinical mouse models of cancer treated with anti-programmed cell death protein-1 (PD-1) immune checkpoint blockade. RESULTS: We resolved 114,524 single cells in 27 patients treated with ICI, enabling spatial resolution of immune lineages and activation states with distinct clinical outcomes. We demonstrated that CXCL13 expression is associated with ICI efficacy in patients, and that recombinant CXCL13 potentiates anti-PD-1 response in vivo in association with increased antigen experienced T cell subsets and reduced CCR2+ monocytes. DISCUSSION: Our results provide a high-resolution molecular resource and illustrate the importance of major immune lineages as well as their functional substates in understanding the role of the tumor immune microenvironment in response to ICIs.