A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

Pax5 loss imposes a reversible differentiation block in B-progenitor acute lymphoblastic leukemia.

Loss-of-function mutations in hematopoietic transcription factors including PAX5 occur in most cases of B-progenitor acute lymphoblastic leukemia (B-ALL), a disease characterized by the accumulation of undifferentiated lymphoblasts. Although PAX5 mutation is a critical driver of B-ALL development in mice and humans, it remains unclear how its loss contributes to leukemogenesis and whether ongoing PAX5 deficiency is required for B-ALL maintenance. Here we used transgenic RNAi to reversibly suppress endogenous Pax5 expression in the hematopoietic compartment of mice, which cooperates with activated signal transducer and activator of transcription 5 (STAT5) to induce B-ALL. In this model, restoring endogenous Pax5 expression in established B-ALL triggers immunophenotypic maturation and durable disease remission by engaging a transcriptional program reminiscent of normal B-cell differentiation. Notably, even brief Pax5 restoration in B-ALL cells causes rapid cell cycle exit and disables their leukemia-initiating capacity. These and similar findings in human B-ALL cell lines establish that Pax5 hypomorphism promotes B-ALL self-renewal by impairing a differentiation program that can be re-engaged despite the presence of additional oncogenic lesions. Our results establish a causal relationship between the hallmark genetic and phenotypic features of B-ALL and suggest that engaging the latent differentiation potential of B-ALL cells may provide new therapeutic entry points.

Lung morphogenesis is orchestrated through Grainyhead-like 2 (Grhl2) transcriptional programs.

The highly conserved transcription factor Grainyhead-like 2 (Grhl2) exhibits a dynamic expression pattern in lung epithelium throughout embryonic development. Using a conditional gene targeting approach to delete Grhl2 in the developing lung epithelium, our results demonstrate that Grhl2 plays multiple roles in lung morphogenesis that are essential for respiratory function. Loss of Grhl2 leads to impaired ciliated cell differentiation and perturbed formation of terminal saccules. Critically, a substantial increase in Sox9-positive distal tip progenitor cells was observed following loss of Grhl2, suggesting that Grhl2 plays an important role in branching morphogenesis. Gene transcription profiling of Grhl2-deficient lung epithelial cells revealed a significant down regulation of Elf5, a member of the Ets family of transcription factors. Furthermore, ChIP and comparative genomic analyzes confirmed that Elf5 is a direct transcriptional target of Grhl2. Taken together, these results support the hypothesis that Grhl2 controls normal lung morphogenesis by tightly regulating the activity of distal tip progenitor cells.

Dual roles for Id4 in the regulation of estrogen signaling in the mammary gland and ovary.

The HLH transcriptional regulator Id4 exerts important roles in different organs, including the neural compartment, where Id4 loss usually results in early lethality. To explore the role of this basally restricted transcription factor in the mammary gland, we generated a cre-inducible mouse model. MMTV- or K14-cre-mediated deletion of Id4 led to a delay in ductal morphogenesis, consistent with previous findings using a germ-line knockout mouse model. A striking increase in the expression of ERα (Esr1), PR and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands. Together with chromatin immunoprecipitation of Id4 on the Esr1 and Foxa1 promoter regions, these data imply that Id4 is a negative regulator of the ERα signaling axis. Unexpectedly, examination of the ovaries of targeted mice revealed significantly increased numbers of secondary and antral follicles, and reduced Id4 expression in the granulosa cells. Moreover, expression of the cascade of enzymes that are crucial for estrogen biosynthesis in the ovary was decreased in Id4-deficient females and uterine weights were considerably lower, indicating impaired estrogen production. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice. Collectively, these data identify Id4 as a novel regulator of estrogen signaling, where Id4 restrains ERα expression in the basal and luminal cellular compartments of the mammary gland and regulates estrogen biosynthesis in the ovary.

A pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1.

BACKGROUND: The molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation. METHODS: MaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells. RESULTS: Utilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity. CONCLUSIONS: This study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.

A Multiplexed Approach to Assess Small Cell Lung Cancer Subtype Heterogeneity in Primary and Patient-Derived Tumor Samples.

Unlocking the heterogeneity of cancers is crucial for developing therapeutic approaches that effectively eradicate disease. As our understanding of markers specific to cancer subclones or subtypes expands, there is a growing demand for advanced technologies that enable the simultaneous investigation of multiple targets within an individual tumor sample. Indeed, multiplex approaches offer distinct benefits, particularly when tumor specimens are small and scarce. Here we describe the utility of two fluorescence-based multiplex approaches; fluorescent Western blots, and multiplex immunohistochemistry (Opal™) staining to interrogate heterogeneity, using small cell lung cancer as an example. Critically, the coupling of Opal™ staining with advanced image quantitation, permits the dissection of cancer cell phenotypes at a single cell level. These approaches can be applied to patient biopsies and/or patient-derived xenograft (PDX) models and serve as powerful methodologies for assessing tumor cell heterogeneity in response to therapy or between metastatic lesions across diverse tissue sites.

The Landscape of Pediatric High-Grade Gliomas: The Virtues and Pitfalls of Pre-Clinical Models.

Pediatric high-grade gliomas (pHGG) are malignant and usually fatal central nervous system (CNS) WHO Grade 4 tumors. The majority of pHGG consist of diffuse midline gliomas (DMG), H3.3 or H3.1 K27 altered, or diffuse hemispheric gliomas (DHG) (H3.3 G34-mutant). Due to diffuse tumor infiltration of eloquent brain areas, especially for DMG, surgery has often been limited and chemotherapy has not been effective, leaving fractionated radiation to the involved field as the current standard of care. pHGG has only been classified as molecularly distinct from adult HGG since 2012 through Next-Generation sequencing approaches, which have shown pHGG to be epigenetically regulated and specific tumor sub-types to be representative of dysregulated differentiating cells. To translate discovery research into novel therapies, improved pre-clinical models that more adequately represent the tumor biology of pHGG are required. This review will summarize the molecular characteristics of different pHGG sub-types, with a specific focus on histone K27M mutations and the dysregulated gene expression profiles arising from these mutations. Current and emerging pre-clinical models for pHGG will be discussed, including commonly used patient-derived cell lines and in vivo modeling techniques, encompassing patient-derived xenograft murine models and genetically engineered mouse models (GEMMs). Lastly, emerging techniques to model CNS tumors within a human brain environment using brain organoids through co-culture will be explored. As models that more reliably represent pHGG continue to be developed, targetable biological and genetic vulnerabilities in the disease will be more rapidly identified, leading to better treatments and improved clinical outcomes.

Flow Cytometry Identification of Cell Compartments in the Murine Brain.

Glioma can be modelled in the murine brain through the induction of genetically engineered mouse models or intracranial transplantation. Gliomas (oligodendroglioma and astrocytoma) are thought to arise from neuronal and glial progenitor populations in the brain and are poorly infiltrated by immune cells. An improved understanding of oligodendrocytes, astrocytes, and the immune environment throughout tumor development will enhance the analysis and development of brain cancer models. Here, we describe the isolation and analysis of murine brain cell types using a combination of flow cytometry and quantitative RT-PCR strategies to analyze these individual cell populations in vivo.

From signalling pathways to targeted therapies: unravelling glioblastoma's secrets and harnessing two decades of progress.

Glioblastoma, a rare, and highly lethal form of brain cancer, poses significant challenges in terms of therapeutic resistance, and poor survival rates for both adult and paediatric patients alike. Despite advancements in brain cancer research driven by a technological revolution, translating our understanding of glioblastoma pathogenesis into improved clinical outcomes remains a critical unmet need. This review emphasises the intricate role of receptor tyrosine kinase signalling pathways, epigenetic mechanisms, and metabolic functions in glioblastoma tumourigenesis and therapeutic resistance. We also discuss the extensive efforts over the past two decades that have explored targeted therapies against these pathways. Emerging therapeutic approaches, such as antibody-toxin conjugates or CAR T cell therapies, offer potential by specifically targeting proteins on the glioblastoma cell surface. Combination strategies incorporating protein-targeted therapy and immune-based therapies demonstrate great promise for future clinical research. Moreover, gaining insights into the role of cell-of-origin in glioblastoma treatment response holds the potential to advance precision medicine approaches. Addressing these challenges is crucial to improving outcomes for glioblastoma patients and moving towards more effective precision therapies.

PRMT1 Inhibition Selectively Targets BNC1-Dependent Proliferation, but not Migration in Squamous Cell Carcinoma.

Squamous Cell Carcinoma (SCC) develops in stratified epithelial tissues and demonstrates frequent alterations in transcriptional regulators. We sought to discover SCC-specific transcriptional programs and identified the transcription factor Basonuclin 1 (BNC1) as highly expressed in SCC compared to other tumor types. RNA-seq and ChIP-seq analysis identified pro-proliferative genes activated by BNC1 in SCC cells and keratinocytes. Inhibition of BNC1 in SCC cells suppressed proliferation and increased migration via FRA1. In contrast, BNC1 reduction in keratinocytes caused differentiation, which was abrogated by IRF6 knockdown, leading to increased migration. Protein interactome analysis identified PRMT1 as a co-activator of BNC1-dependent proliferative genes. Inhibition of PRMT1 resulted in a dose-dependent reduction in SCC cell proliferation without increasing migration. Importantly, therapeutic inhibition of PRMT1 in SCC xenografts significantly reduced tumor size, resembling functional effects of BNC1 knockdown. Together, we identify BNC1-PRMT1 as an SCC-lineage specific transcriptional axis that promotes cancer growth, which can be therapeutically targeted to inhibit SCC tumorigenesis.

Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography-mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

Spatial architecture of high-grade glioma reveals tumor heterogeneity within distinct domains.

Background: High-grade gliomas (HGGs) are aggressive primary brain cancers with poor response to standard regimens, driven by immense heterogeneity. In isocitrate dehydrogenase (IDH) wild-type HGG (glioblastoma, GBM), increased intratumoral heterogeneity is associated with more aggressive disease. Methods: Spatial technologies can dissect complex heterogeneity within the tumor ecosystem by preserving cellular organization in situ. We employed GeoMx digital spatial profiling, CosMx spatial molecular imaging, Xenium in situ mapping and Visium spatial gene expression in experimental and validation patient cohorts to interrogate the transcriptional landscape in HGG. Results: Here, we construct a high-resolution molecular map of heterogeneity in GBM and IDH-mutant patient samples to investigate the cellular communities that compose HGG. We uncovered striking diversity in the tumor landscape and degree of spatial heterogeneity within the cellular composition of the tumors. The immune distribution was diverse between samples, however, consistently correlated spatially with distinct tumor cell phenotypes, validated across tumor cohorts. Reconstructing the tumor architecture revealed two distinct niches, one composed of tumor cells that most closely resemble normal glial cells, associated with microglia, and the other niche populated by monocytes and mesenchymal tumor cells. Conclusions: This primary study reveals high levels of intratumoral heterogeneity in HGGs, associated with a diverse immune landscape within spatially localized regions.

Tumor cell-derived spermidine promotes a pro-tumorigenic immune microenvironment in glioblastoma via CD8+ T cell inhibition.

The glioblastoma microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly impact the immune system, but the mechanisms driving these interactions are not completely clear. Here we demonstrate that the polyamine metabolite spermidine is elevated in the glioblastoma tumor microenvironment. Exogenous administration of spermidine drives tumor aggressiveness in an immune-dependent manner in pre-clinical mouse models via reduction of CD8+ T cell frequency and phenotype. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in spermidine synthesis, did not impact cancer cell growth in vitro but did result in extended survival. Furthermore, glioblastoma patients with a more favorable outcome had a significant reduction in spermidine compared to patients with a poor prognosis. Our results demonstrate that spermidine functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+T cell number and function.

Absence of pro-survival A1 has no impact on inflammatory cell survival in vivo during acute lung inflammation and peritonitis.

Inflammation is a natural defence mechanism of the body to protect against pathogens. It is induced by immune cells, such as macrophages and neutrophils, which are rapidly recruited to the site of infection, mediating host defence. The processes for eliminating inflammatory cells after pathogen clearance are critical in preventing sustained inflammation, which can instigate diverse pathologies. During chronic inflammation, the excessive and uncontrollable activity of the immune system can cause extensive tissue damage. New therapies aimed at preventing this over-activity of the immune system could have major clinical benefits. Here, we investigated the role of the pro-survival Bcl-2 family member A1 in the survival of inflammatory cells under normal and inflammatory conditions using murine models of lung and peritoneal inflammation. Despite the robust upregulation of A1 protein levels in wild-type cells upon induction of inflammation, the survival of inflammatory cells was not impacted in A1-deficient mice compared to wild-type controls. These findings indicate that A1 does not play a major role in immune cell homoeostasis during inflammation and therefore does not constitute an attractive therapeutic target for such morbidities.

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

Exploring natural killer cell immunology as a therapeutic strategy in lung cancer.

Cytotoxic immune cells are key in the control of tumor development and progression. Natural killer (NK) cells are the cytotoxic arm of the innate immune system with the capability to kill tumor cells and surveil tumor cell dissemination. As such, the interest in harnessing NK cells in tumor control is increasing in many solid tumor types, including lung cancer. Here, we review the pre-clinical models used to unveil the role of NK cells in immunosurveillance of solid tumors and highlight measures to enhance NK cell activity. Importantly, the development of NK immunotherapy is rapidly evolving. Enhancing the NK cell response can be achieved using two broad modalities: enhancing endogenous NK cell activity, or performing adoptive transfer of pre-activated NK cells to patients. Numerous clinical trials are evaluating the efficacy of NK cell immunotherapy in isolation or in combination with standard treatments, with encouraging initial results. Pre-clinical studies and early phase clinical trials suggest that patients with solid tumors, including lung cancer, have the potential to benefit from recent developments in NK cell immunotherapy.

An Integrated Molecular Grafting Approach for the Design of Keap1-Targeted Peptide Inhibitors.

Inhibiting the Nrf2:Keap1 interaction to trigger cytoprotective gene expression is a promising treatment strategy for oxidative stress-related diseases. A short linear motif from Nrf2 has the potential to directly inhibit this protein-protein interaction, but poor stability and limited cellular uptake impede its therapeutic development. To address these limitations, we utilized an integrated molecular grafting strategy to re-engineer the Nrf2 motif. We combined the motif with an engineered non-native disulfide bond and a cell-penetrating peptide onto a single multifunctionalizable and ultrastable molecular scaffold, namely, the cyclotide MCoTI-II, resulting in the grafted peptide MCNr-2c. The engineered disulfide bond enhanced the conformational rigidity of the motif, resulting in a nanomolar affinity of MCNr-2c for Keap1. The cell-penetrating peptide led to an improved cellular uptake and increased ability to enhance the intracellular expression of two well-described Nrf2-target genes NQO1 and TALDO1. Furthermore, the stability of the scaffold was inherited by the grafted peptide, which became resistant to proteolysis in serum. Overall, we have provided proof-of-concept for a strategy that enables the encapsulation of multiple desired and complementary activities into a single molecular entity to design a Keap1-targeted inhibitor. We propose that this integrated approach could have broad utility for the design of peptide drug leads that require multiple functions and/or biopharmaceutical properties to elicit a therapeutic activity.

BCKDK alters the metabolism of non-small cell lung cancer.

BACKGROUND: Metabolic reprogramming is a major feature of many tumors including non-small cell lung cancer (NSCLC). Branched-chain α-keto acid dehydrogenase kinase (BCKDK) plays an important role in diabetes, obesity, and other diseases. However, the function of BCKDK in NSCLC is unclear. This study aimed to explore the function of BCKDK in NSCLC. METHODS: Metabolites in the serum of patients with NSCLC and the supernatant of NSCLC cell cultures were detected using nuclear magnetic resonance (NMR) spectroscopy. Colony formation, cell proliferation, and cell apoptosis were assessed to investigate the function of BCKDK in the progression of NSCLC. Glucose uptake, lactate production, cellular oxygen consumption rate, extracellular acidification rate, and reactive oxygen species (ROS) were measured to examine the function of BCKDK in glucose metabolism. The expression of BCKDK was measured using reverse transcriptase-polymerase chain reaction, western blot, and immunohistochemical assay. RESULTS: Compared with healthy controls and postoperative NSCLC patients, increased branched-chain amino acid (BCAA) and decreased citrate were identified in the serum of preoperative NSCLC patients. Upregulation of BCKDK affected the metabolism of BCAAs and citrate in NSCLC cells. Knockout of BCKDK decreased the proliferation and exacerbated apoptosis of NSCLC cells ex vivo, while increased oxidative phosphorylation and, ROS levels, and inhibited glycolysis. CONCLUSIONS: BCKDK may influence glycolysis and oxidative phosphorylation by regulating the degradation of BCAA and citrate, thereby affecting the progression of NSCLC.

Consequences of Zmat3 loss in c-MYC- and mutant KRAS-driven tumorigenesis.

TP53 is a critical tumor suppressor that is mutated in approximately 50% of human cancers. Unveiling the downstream target genes of TP53 that fulfill its tumor suppressor function is an area of intense investigation. Zmat3 (also known as Wig-1 or PAG608) is one such downstream target of p53, whose loss in hemopoietic stem cells lacking the apoptosis and cell cycle regulators, Puma and p21, respectively, promotes the development of leukemia. The function of Zmat3 in tumorigenesis however remains unclear. Here, to investigate which oncogenic drivers co-operate with Zmat3 loss to promote neoplastic transformation, we utilized Zmat3 knockout mice in models of c-MYC-driven lymphomagenesis and KrasG12D-driven lung adenocarcinoma development. Interestingly, unlike loss of p53, Zmat3 germline loss had little impact on the rate of tumor development or severity of malignant disease upon either the c-MYC or KrasG12D oncogenic activation. Furthermore, loss of Zmat3 failed to rescue KrasG12D primary lung tumor cells from oncogene-induced senescence. Taken together, we conclude that in the context of c-MYC-driven lymphomagenesis or mutant KrasG12D-driven lung adenocarcinoma development, additional co-occurring mutations are required to resolve Zmat3 tumor suppressive activity.

Harnessing Natural Killer Immunity in Metastatic SCLC.

INTRODUCTION: SCLC is the most aggressive subtype of lung cancer, and though most patients initially respond to platinum-based chemotherapy, resistance develops rapidly. Immunotherapy holds promise in the treatment of lung cancer; however, patients with SCLC exhibit poor overall responses highlighting the necessity for alternative approaches. Natural killer (NK) cells are an alternative to T cell-based immunotherapies that do not require sensitization to antigens presented on the surface of tumor cells. METHODS: We investigated the immunophenotype of human SCLC tumors by both flow cytometry on fresh samples and bioinformatic analysis. Cell lines generated from murine SCLC were transplanted into mice lacking key cytotoxic immune cells. Subcutaneous tumor growth, metastatic dissemination, and activation of CD8+ T and NK cells were evaluated by histology and flow cytometry. RESULTS: Transcriptomic analysis of human SCLC tumors revealed heterogeneous immune checkpoint and cytotoxic signature profiles. Using sophisticated, genetically engineered mouse models, we reported that the absence of NK cells, but not CD8+ T cells, substantially enhanced metastatic dissemination of SCLC tumor cells in vivo. Moreover, hyperactivation of NK cell activity through augmentation of interleukin-15 or transforming growth factor-ß signaling pathways ameliorated SCLC metastases, an effect that was enhanced when combined with antiprogrammed cell death-1 therapy. CONCLUSIONS: These proof-of-principle findings provide a rationale for exploiting the antitumor functions of NK cells in the treatment of patients with SCLC. Moreover, the distinct immune profiles of SCLC subtypes reveal an unappreciated level of heterogeneity that warrants further investigation in the stratification of patients for immunotherapy.