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Hepatocellular carcinoma presents strong sexual dimorphism, being 2-3 times more frequent in males than in females; however, the role of sex in response to immunotherapies in HCC remains unknown. We demonstrate that NOTCH1, an understudied oncogene in HCC, elicits sexually dimorphic anti-tumor immunity and response to FDA-approved immunotherapies. Surprisingly, males harboring NOTCH1-driven tumors displayed enhanced anti-tumor immune responses, which, in mice, were mediated by dendritic and T cells. Conversely, females harboring NOTCH1-driven tumors presented immune evasion and resistance to immunotherapies through a defect in DC-mediated priming and activation of CD8+ T cells in mice, which was restored therapeutically with CD40 agonism. Mechanistically, the sexually dimorphic immunity was mediated by genes in the sex chromosomes but not by sex hormones. Together, our study unravels an unexpected association between NOTCH1 and sex in cancer immunity and highlights the potential of restoring the DC-CD8+ T cell axis with CD40 agonism to improve outcomes.

Immunotherapies have shown great promise in pleural mesothelioma (PM), yet most patients still do not achieve significant clinical response, highlighting the importance of improving the understanding of the tumor microenvironment (TME). Here, we utilized high-throughput, single-cell RNA sequencing (scRNA-seq) to de novo identify 54 expression programs and construct a comprehensive cellular catalog of the PM TME. We found four cancer-intrinsic programs associated with poor disease outcome and a novel fetal-like, endothelial cell population that likely responds to VEGF signaling and promotes angiogenesis. Across cellular compartments, we observe substantial difference in the TME associated with a cancer-intrinsic sarcomatoid signature, including enrichment in fetal-like endothelial cells, CXCL9+ macrophages, and cytotoxic, exhausted, and regulatory T cells, which we validated using imaging and bulk deconvolution analyses on independent cohorts. Finally, we show, both computationally and experimentally, that NKG2A:HLA-E interaction between NK and tumor cells represents an important new therapeutic axis in PM, especially for epithelioid cases. Significance: This manuscript presents the first single-cell RNA sequencing atlas of PM tumor microenvironment. Findings of translational relevance, validated experimentally and using independent bulk cohorts, include identification of gene programs predictive of survival, a fetal-like endothelial cell population, and NKG2A blockade as a promising new immunotherapeutic intervention in PM., (©2024 American Association for Cancer Research.)

Liver cancer, the third leading cause of cancer-related mortality worldwide, has two main subtypes: hepatocellular carcinoma (HCC), accounting most of the cases, and cholangiocarcinoma (CAA). NOTCH pathway regulates the intrahepatic development of bile ducts, which are lined with cholangiocytes, but it can also be upregulated in 1/3 of HCCs. To better understand the role of NOTCH in HCC, we developed a novel mouse model driven by activated NOTCH1 intracellular domain (NICD1) and MYC overexpression in hepatocytes. Using the hydrodynamic tail vein injection method for establishing primary liver tumors, we generated a novel murine model of liver cancer harboring MYC overexpression and NOTCH1 activation. We characterized this model histopathologically as well as transcriptomically, utilizing both bulk and single cell RNA-sequencing. MYC;NICD1 tumors displayed a combined HCC-CCA phenotype with temporal plasticity. At early time-points, histology was predominantly cholangiocellular, which then progressed to mainly hepatocellular. The hepatocellular component was enriched in mesenchymal genes and gave rise to lung metastasis. Metastatic cells were enriched in the TGFB and VEGF pathways and their inhibition significantly reduced the metastatic burden. Our novel mouse model uncovered NOTCH1 as a driver of temporal plasticity and metastasis in HCC, the latter of which is, in part, mediated by angiogenesis and TGFß pathways.

CMTR2 is an mRNA cap methyltransferase with poorly understood physiological functions. It catalyzes 2'-O-ribose methylation of the second transcribed nucleotide of mRNAs, potentially serving to mark RNAs as "self" to evade the cellular innate immune response. Here we analyze the consequences of Cmtr2 deficiency in mice. We discover that constitutive deletion of Cmtr2 results in mouse embryos that die during mid-gestation, exhibiting defects in embryo size, placental malformation and yolk sac vascularization. Endothelial cell deletion of Cmtr2 in mice results in vascular and hematopoietic defects, and perinatal lethality. Detailed characterization of the constitutive Cmtr2 KO phenotype shows an activation of the p53 pathway and decreased proliferation, but no evidence of interferon pathway activation. In summary, our study reveals the essential roles of Cmtr2 in mammalian cells beyond its immunoregulatory function., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Monocyte-derived macrophages (mo-macs) drive immunosuppression in the tumor microenvironment (TME) and tumor-enhanced myelopoiesis in the bone marrow (BM) fuels these populations. Here, we performed paired transcriptome and chromatin analysis over the continuum of BM myeloid progenitors, circulating monocytes, and tumor-infiltrating mo-macs in mice and in patients with lung cancer to identify myeloid progenitor programs that fuel pro-tumorigenic mo-macs. Analyzing chromatin accessibility and histone mark changes, we show that lung tumors prime accessibility for Nfe2l2 (NRF2) in BM myeloid progenitors as a cytoprotective response to oxidative stress. NRF2 activity is sustained and increased during monocyte differentiation into mo-macs in the lung TME to regulate oxidative stress, in turn promoting metabolic adaptation, resistance to cell death, and contributing to immunosuppressive phenotype. NRF2 genetic deletion and pharmacological inhibition significantly reduced mo-macs' survival and immunosuppression in the TME, enabling NK and T cell therapeutic antitumor immunity and synergizing with checkpoint blockade strategies. Altogether, our study identifies a targetable epigenetic node of myeloid progenitor dysregulation that sustains immunoregulatory mo-macs in the TME.

Monocytes and monocyte-derived macrophages (MDMs) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here, we show that PDGFB-driven GBM cells induce the expression of the potent proinflammatory cytokine IL-1β in MDM, which engages IL-1R1 in tumor cells, activates the NF-κB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1β/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1β/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, and reduced exhausted CD8+ T cells and thereby extends the survival of tumor-bearing mice. In contrast to IL-1β, IL-1α exhibits antitumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss-of-interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively active NF-κB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1β could be considered as an effective therapy specifically for proneural GBM.

Glioblastoma (GBM), a highly lethal brain cancer, is notorious for immunosuppression, but the mechanisms remain unclear. Here, we documented a temporospatial patterning of tumor-associated myeloid cells (TAMs) corresponding to vascular changes during GBM progression. As tumor vessels transitioned from the initial dense regular network to later scant and engorged vasculature, TAMs shifted away from perivascular regions and trafficked to vascular-poor areas. This process was heavily influenced by the immunocompetence state of the host. Utilizing a sensitive fluorescent UnaG reporter to track tumor hypoxia, coupled with single-cell transcriptomics, we revealed that hypoxic niches attracted and sequestered TAMs and cytotoxic T lymphocytes (CTLs), where they were reprogrammed toward an immunosuppressive state. Mechanistically, we identified chemokine CCL8 and cytokine IL-1β as two hypoxic-niche factors critical for TAM trafficking and co-evolution of hypoxic zones into pseudopalisading patterns. Therefore, perturbation of TAM patterning in hypoxic zones may improve tumor control., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13 + CH25H + IL-21 + PD-1 + CD4 + T helper cells ("CXCL13 + T H ") and Granzyme K + PD-1 + effector-like CD8 + T cells, whereas terminally exhausted CD39 hi TOX hi PD-1 hi CD8 + T cells dominated in nonresponders. CD4 + and CD8 + T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1 + TCF-1 + (Progenitor-exhausted) CD8 + T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8 + T cell differentiation occurs upon ICB. We found that these Progenitor CD8 + T cells interact with CXCL13 + T H within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13 + T H control the differentiation of tumor-specific Progenitor exhasuted CD8 + T cells following ICB., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Myeloid cells comprise the majority of immune cells in tumors, contributing to tumor growth and therapeutic resistance. Incomplete understanding of myeloid cells response to tumor driver mutation and therapeutic intervention impedes effective therapeutic design. Here, by leveraging CRISPR/Cas9-based genome editing, we generate a mouse model that is deficient of all monocyte chemoattractant proteins. Using this strain, we effectively abolish monocyte infiltration in genetically engineered murine models of de novo glioblastoma (GBM) and hepatocellular carcinoma (HCC), which show differential enrichment patterns for monocytes and neutrophils. Eliminating monocyte chemoattraction in monocyte enriched PDGFB-driven GBM invokes a compensatory neutrophil influx, while having no effect on Nf1-silenced GBM model. Single-cell RNA sequencing reveals that intratumoral neutrophils promote proneural-to-mesenchymal transition and increase hypoxia in PDGFB-driven GBM. We further demonstrate neutrophil-derived TNF-a directly drives mesenchymal transition in PDGFB-driven primary GBM cells. Genetic or pharmacological inhibiting neutrophils in HCC or monocyte-deficient PDGFB-driven and Nf1-silenced GBM models extend the survival of tumor-bearing mice. Our findings demonstrate tumor-type and genotype dependent infiltration and function of monocytes and neutrophils and highlight the importance of targeting them simultaneously for cancer treatments., (© 2023. The Author(s).)

Late prenatal development of the human neocortex encompasses a critical period of gliogenesis and cortical expansion. However, systematic single-cell analyses to resolve cellular diversity and gliogenic lineages of the third trimester are lacking. Here, we present a comprehensive single-nucleus RNA sequencing atlas of over 200,000 nuclei derived from the proliferative germinal matrix and laminating cortical plate of 15 prenatal, non-pathological postmortem samples from 17 to 41 gestational weeks, and 3 adult controls. This dataset captures prenatal gliogenesis with high temporal resolution and is provided as a resource for further interrogation. Our computational analysis resolves greater complexity of glial progenitors, including transient glial intermediate progenitor cell (gIPC) and nascent astrocyte populations in the third trimester of human gestation. We use lineage trajectory and RNA velocity inference to further characterize specific gIPC subpopulations preceding both oligodendrocyte (gIPC-O) and astrocyte (gIPC-A) lineage differentiation. We infer unique transcriptional drivers and biological pathways associated with each developmental state, validate gIPC-A and gIPC-O presence within the human germinal matrix and cortical plate in situ, and demonstrate gIPC states being recapitulated across adult and pediatric glioblastoma tumors., (© 2022. The Author(s).)

Tumors exhibit enhancer reprogramming compared to normal tissue. The etiology is largely attributed to cell-intrinsic genomic alterations. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon, we find divergent epigenetic landscapes between CRC tumors and cell lines. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over normal. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating cell-extrinsic etiology. We demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway. We show this activation enables efficient growth under oxidative conditions, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the significance of epigenomic profiling on primary specimens., (© 2022. The Author(s).)

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2 + TMPRSS2 + cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.

The set of proteins required for mitotic division remains poorly characterized. Here, an extensive series of correlation analyses of human and mouse transcriptomics data were performed to identify genes strongly and reproducibly associated with cells undergoing S/G2-M phases of the cell cycle. In so doing, 701 cell cycle-associated genes were defined and while it was shown that many are only expressed during these phases, the expression of others is also driven by alternative promoters. Of this list, 496 genes have known cell cycle functions, whereas 205 were assigned as putative cell cycle genes, 53 of which are functionally uncharacterized. Among these, 27 were screened for subcellular localization revealing many to be nuclear localized and at least three to be novel centrosomal proteins. Furthermore, 10 others inhibited cell proliferation upon siRNA knockdown. This study presents the first comprehensive list of human cell cycle proteins, identifying many new candidate proteins., (© The Author(s) 2018. Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

In aggressive non-Hodgkin's lymphoma (NHL), constitutive apoptosis of a proportion of the tumor cell population can promote net tumor growth. This is associated with the accumulation of tumor-associated macrophages (TAMs) that clear apoptotic cells and exhibit pro-oncogenic transcriptional activation profiles characteristic of reparatory, anti-inflammatory and angiogenic programs. Here we consider further the activation status of these TAMs. We compare their transcriptomic profile with that of a range of other macrophage types from various tissues noting especially their expression of classically activated (IFN-γ and LPS) gene clusters - typically antitumor - in addition to their previously described protumor phenotype. To understand the impact of apoptotic cells on the macrophage activation state, we cocultured apoptotic lymphoma cells with classically activated macrophages (M (IFN-γ/LPS) , also known as M1, macrophages). Although untreated and M (IFN-γ/LPS) macrophages were able to bind apoptotic lymphoma cells equally well, M (IFN-γ/LPS) macrophages displayed enhanced ability to phagocytose them. We found that direct exposure of M (IFN-γ/LPS) macrophages to apoptotic lymphoma cells caused switching towards a protumor activation state (often referred to as M2-like) with concomitant inhibition of antitumor activity that was a characteristic feature of M (IFN-γ/LPS) macrophages. Indeed, M (IFN-γ/LPS) macrophages exposed to apoptotic lymphoma cells displayed increased lymphoma growth-promoting activities. Antilymphoma activity by M (IFN-γ/LPS) macrophages was mediated, in part, by galectin-3, a pleiotropic glycoprotein involved in apoptotic cell clearance that is strongly expressed by lymphoma TAMs but not lymphoma cells. Intriguingly, aggressive lymphoma growth was markedly impaired in mice deficient in galectin-3, suggesting either that host galectin-3-mediated antilymphoma activity is required to sustain net tumor growth or that additional functions of galectin-3 drive key oncogenic mechanisms in NHL. These findings have important implications for anticancer therapeutic approaches aimed at polarizing macrophages towards an antitumor state and identify galectin-3 as a potentially important novel target in aggressive NHL.

Background: Cell division is central to the physiology and pathology of all eukaryotic organisms. The molecular machinery underpinning the cell cycle has been studied extensively in a number of species and core aspects of it have been found to be highly conserved. Similarly, the transcriptional changes associated with this pathway have been studied in different organisms and different cell types. In each case hundreds of genes have been reported to be regulated, however there seems to be little consensus in the genes identified across different studies. In a recent comparison of transcriptomic studies of the cell cycle in different human cell types, only 96 cell cycle genes were reported to be the same across all studies examined., Results: Here we perform a systematic re-examination of published human cell cycle expression data by using a network-based approach to identify groups of genes with a similar expression profile and therefore function. Two clusters in particular, containing 298 transcripts, showed patterns of expression consistent with cell cycle occurrence across the four human cell types assessed., Conclusions: Our analysis shows that there is a far greater conservation of cell cycle-associated gene expression across human cell types than reported previously, which can be separated into two distinct transcriptional networks associated with the G 1 /S-S and G 2 -M phases of the cell cycle. This work also highlights the benefits of performing a re-analysis on combined datasets.