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SUMMARYMyeloid 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 genomic editing, we generated a mouse model that is deficient of all monocyte chemoattractant proteins (MCP). Using this strain, we effectively abolished monocyte infiltration in glioblastoma (GBM) and hepatocellular carcinoma (HCC) murine models, which were enriched for monocytes or neutrophils, respectively. Remarkably, eliminating monocyte chemoattraction invokes a significant compensatory neutrophil influx in GBM, but not in HCC. Single-cell RNA sequencing revealed that intratumoral neutrophils promoted proneural-to-mesenchymal transition in GBM, and supported tumor aggression by facilitating hypoxia response via TNF production. Importantly, genetic or pharmacological inhibiting neutrophil in HCC or qMCP-KO GBM extended the survival of tumor-bearing mice. Our findings emphasize the importance of targeting both monocytes and neutrophils simultaneously for cancer immunotherapy.In BriefEliminating monocyte chemoattraction invokes compensatory neutrophil influx in tumor, and vice versa, rendering current myeloid-targeted therapies ineffective. Using genetic and pharmacological approaches combined with novel mouse models of GBM and HCC, we provide credence advocating for combinational therapies aiming at inhibiting both monocytes and neutrophils simultaneously.Highlights•Blocking monocyte chemoattraction results in increased neutrophil infiltration.•Increased neutrophil recruitment induces GBM PN to MES transition.•Inhibiting neutrophil infiltration in monocyte-deficient tumors improves mouse GBM survival.•Blocking neutrophil, but not monocyte, infiltration in HCC prolongs mouse survival.

SummaryMonocytes and monocyte-derived macrophages (MDM) from blood circulation infiltrate and promote glioblastoma growth. Here we discover that glioma cells induce the expression of potent pro-inflammatory cytokine IL-1β in MDM, which engages IL-1R1 in glioma cells, activates NF-κB pathway, and subsequently leads to the induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1β/IL-1R1 between the tumors and MDM creates an interdependence driving glioblastoma progression. Locally antagonizing IL-1β/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, reduced exhausted CD8+T cells, and thereby extends the survival of tumor-bearing mice. In contrast to IL-1β, IL-1a exhibits anti-tumor effects. Genetic deletion ofIl1ais associated with decreased recruitment of lymphoid cells and loss of interferon (IFN) signaling in various immune populations and subsets of malignant cells. IL-1β antagonism of IL-1β should be considered as an effective anti-glioblastoma therapy.Graphical Abstract

Glioblastoma (GBM), a highly lethal brain cancer, is notorious for its immunosuppressive microenvironment, yet current immunotherapies are ineffective. Thus, understanding the immune contexture and governing factors of immunosuppression is crucial. Here, we identified a highly dynamic temporospatial patterning of tumor-associated macrophages (TAMs) corresponding to vascular changes in GBM: as tumor vessels transition from an initial dense regular network to later scant engorged vasculature, CD68+ TAMs shift away from perivascular regions to poorly vascularized areas. Remarkably, this process is heavily influenced by the immunocompetency of host animal, as tumor vessels in immunodeficient hosts remained dense and regular while TAMs evenly distributed. Utilizing a sensitive fluorescent reporter to track tumor hypoxia, we revealed that hypoxic niche controls immunosuppression by at least two mechanisms: first, attracting and sequestering activated TAMs in hypoxic zones, and second, reprograming entrapped TAMs towards an immunotolerant state. Indeed, entrapped TAMs also experience hypoxia and upregulate phagocytic marker Cd68 and immunotolerant genes Mrc1 and Arg1, thereby facilitating debris clearing, inflammatory containment, and immunosuppression in hypoxic zones. Mechanistically, we identified Ccl8 and IL-1β as two hypoxic niche factors released by TAMs in response to cues from hypoxic GBM cells, functioning to reinforce TAM retainment. Reciprocally, niche factors also shape the transcriptional responses of hypoxic tumor cells that exhibit quiescence and mesenchymal shift. Moreover, hypoxic niche factors are highly enriched in human GBMs, particularly mesenchymal subtype, and predict poor survival. Importantly, perturbing hypoxic niches resulted in reduced TAM sequestration and better tumor control. Together, understanding the mutual influence of immune contexture and metabolic landscape has important ramifications for improving efficacy of immunotherapies against GBM.

SummaryThe rise in aging population worldwide is increasing death from cancer, including glioblastoma. Here, we explore the impact of brain aging on glioma tumorigenesis. We find that glioblastoma in older patients and older mice displayed reduced neuronal signaling, including a decline of NTRK-like family member 6 (SLITRK6), a receptor for neurotrophic factor BDNF. This reduction was linked to the systemic decline of nicotinamide adenine dinucleotide (NAD+) with aging, as old mice exposed to young blood via parabiosis or supplemented with the NAD+ precursor NMN (nicotinamide mononucleotide) reverted phenotypically to young-brain responses to glioma, with reactivated neuronal signaling and reduced death from tumor burden. Interestingly, the phenotypic reversal by NMN was largely absent in old mice undergoing parabiosis with BDNF+/- young mice and in BDNF+/- mice undergoing tumor challenge, supporting the notion that the lower NAD+-BDNF signaling in the aging brain aggravated glioma tumorigenesis. We propose that the aging-associated decline in brain NAD+ worsens glioma outcomes at least in part by decreasing neuronal/synaptic activity and increasing neuroinflammation.

Mutation in isocitrate dehydrogenase (mIDH) is a gain of function mutation resulting in the production of the oncometabolite, R-2-hydroxyglutarate, that inhibits DNA and histone demethylases. The resultant hypermethylation phenotype reprograms the glioma cells’ transcriptome and elicits profound effects on glioma immunity. We report that in mouse models and human gliomas, mIDH1 in the context of ATRX and TP53 inactivation results in global expansion of the granulocytic myeloid cells’ compartment. Single-cell RNA-sequencing coupled with mass cytometry analysis revealed that these granulocytes are mainly non-immunosuppressive neutrophils and pre-neutrophils; with a small fraction of polymorphonuclear myeloid-derived suppressor cells. The mechanism of mIDH1 mediated pre-neutrophils expansion involves epigenetic reprogramming which leads to enhanced expression of the granulocyte colony-stimulating factor (G-CSF). Blocking G-CSF restored the inhibitory potential of PMN-MDSCs and enhanced tumor progression. Thus, G-CSF induces remodeling of the inhibitory PMN-MDSCs in mIDH1 glioma rendering them non-immunosuppressive; and having significant therapeutic implications.SIGNIFICANCEmIDH1 is the most common mutation in gliomas associated with improved prognosis. Gliomas harboring mIDH1, together with ATRX and TP53 inactivation, exhibit higher circulating levels of G-CSF, ensuing the recruitment and expansion of non-suppressive neutrophils, pre-neutrophils and small fraction of PMN-MDSCs to the TME leading to an immune permissive phenotype.

Based on the clonal evolution theory of cancer formation, a single cell within a tissue gains a cancer-driving mutation and thus a growth advantage. From this expanded cellular mass, another cell gains a new mutation allowing this newly mutated cell to gain new competitive advantage and to expand in number (thus clonal expansion). Another clone then emerges. Eventually all required mutations are gained, and a cancer forms. Consequently, while a primary lesion may harbor divergent subclones, all the subclones within the primary cancer as well as all metastatic growths in secondary organs share at least the very first oncogenic mutation that initiates the primary cancer. However, by tracking genetically marked mammary epithelial cells that suffered the initiating oncogenic mutation—and their neighboring mammary cells that did not-in several mouse models of human breast cancer, we found that genetically unrelated mammary epithelial cells can be colluded by neighboring mutated cells to disseminate, and that they can even undergo de novo tumorigenic transformation and form distant metastases. Therefore, clonally unrelated epithelial cells may contribute to cancer progression and to the heterogeneity of the systemic disease. The non-linear cancer spread has important implications in cancer prevention, treatment, and therapeutic resistance.

Medulloblastomas are brain tumors that arise in the cerebellum of children and contain stem cells in a perivascular niche thought to give rise to recurrence following radiation. We used several mouse models of medulloblastomas in parallel to better understand how the critical cell types in these tumors respond to therapy. In our models, the proliferating cells in the tumor bulk undergo radiation-induced, p53-dependent apoptotic cell death. Activation of Akt signaling via PTEN loss transforms these cells to a nonproliferating extensive nodularity morphology. By contrast, the nestin-expressing perivascular stem cells survive radiation, activate PI3K/Akt pathway, undergo p53-dependent cell cycle arrest, and re-enter the cell cycle at 72 h. Furthermore, the ability of these cells to induce p53 is dependent on the presence of PTEN. These cellular characteristics are similar to human medulloblastomas. Finally, inhibition of Akt signaling sensitizes cells in the perivascular region to radiation-induced apoptosis.