Single-Cell Cloning of Breast Cancer Cells Secreting Specific Subsets of Extracellular Vesicles.

Extracellular vesicles (EVs) mediate communication in health and disease. Conventional assays are limited in profiling EVs secreted from large populations of cells and cannot map EV secretion onto individual cells and their functional profiles. We developed a high-throughput single-cell technique that enabled the mapping of dynamics of EV secretion. By utilizing breast cancer cell lines, we established that EV secretion is heterogeneous at the single-cell level and that non-metastatic cancer cells can secrete specific subsets of EVs. Single-cell RNA sequencing confirmed that pathways related to EV secretion were enriched in the non-metastatic cells compared with metastatic cells. We established isogenic clonal cell lines from non-metastatic cells with differing propensities for CD81+CD63+EV secretion and showed for the first time that specificity in EV secretion is an inheritable property preserved during cell division. Combined in vitro and animal studies with these cell lines suggested that CD81+CD63+EV secretion can impede tumor formation. In human non-metastatic breast tumors, tumors enriched in signatures of CD81+CD63+EV have a better prognosis, higher immune cytolytic activity, and enrichment of pro-inflammatory macrophages compared with tumors with low CD81+CD63+EVs signatures. Our single-cell methodology enables the direct integration of EV secretion with multiple cellular functions and enables new insights into cell/disease biology.

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

Human chimeric antigen receptor macrophages for cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1-4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity.

Author Correction: FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation.

The original version of this Article contained errors in the author affiliations. Qingnan Zhao, Xueqing Xia, Longfei Huo and Shulin Li were incorrectly associated with Beijing Institute for Brain Disorders, 100069, Beijing, China.This has now been corrected in both the PDF and HTML versions of the Article.

FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation.

Few studies implicate immunoregulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout in tumor cells did not affect tumor-cell proliferation in vitro or tumor progression in immunodeficient mice but completely impaired GBM progression in immune-competent mice. This impairment was reversed in mice with a defect in dendritic cells (DCs) or CD103+ DC differentiation in the brain and in tumor-draining lymph nodes. The presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target.

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvß5 (ITGαvß5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB-OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

Cell surface vimentin-targeted monoclonal antibody 86C increases sensitivity to temozolomide in glioma stem cells.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor. The current standard therapy, which includes radiation and chemotherapy, is frequently ineffective partially because of drug resistance and poor penetration of the blood-brain barrier. Reducing resistance and increasing sensitivity to chemotherapy may improve outcomes. Glioma stem cells (GSCs) are a source of relapse and chemoresistance in GBM; sensitization of GSCs to temozoliomide (TMZ), the primary chemotherapeutic agent used to treat GBM, is therefore integral for therapeutic efficacy. We previously discovered a unique tumor-specific target, cell surface vimentin (CSV), on patient-derived GSCs. In this study, we found that the anti-CSV monoclonal antibody 86C efficiently increased GSC sensitivity to TMZ. The combination TMZ+86C induced significantly greater antitumor effects than TMZ alone in eight of 12 GSC lines. TMZ+86C-sensitive GSCs had higher CSV expression overall and faster CSV resurfacing among CSV- GSCs compared with TMZ+86C-resistant GSCs. Finally, TMZ+86C increased apoptosis of tumor cells and prolonged survival compared with either drug alone in GBM mouse models. The combination of TMZ+86C represents a promising strategy to reverse GSC chemoresistance.

Glioblastoma stem cell-derived exosomes induce M2 macrophages and PD-L1 expression on human monocytes.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Germline polymorphisms in myeloid-associated genes are not associated with survival in glioma patients.

Immune cells of myeloid origin, including microglia, macrophages, and myeloid-derived suppressor cells adopt immunosuppressive phenotypes that support gliomagenesis. Here, we tested an a priori hypothesis that single nucleotide polymorphisms (SNPs) in genes related to glioma-associated myeloid cell regulation and function are also associated with patient survival after glioma diagnosis. Subjects for this study were 992 glioma patients treated at The University of Texas MD Anderson Cancer Center in Houston, Texas between 1992 and 2008. Haplotype-tagging SNPs in 91 myeloid-associated genes were analyzed for association with survival by Cox regression. Individual SNP- and gene-based tests were performed separately in glioblastoma (WHO grade IV, n = 511) and lower-grade glioma (WHO grade II-III, n = 481) groups. After adjustment for multiple testing, no myeloid-associated gene variants were significantly associated with survival in glioblastoma. Two SNPs, rs147960238 in CD163 (p = 2.2 × 10-5) and rs17138945 in MET (p = 5.6 × 10-5) were significantly associated with survival of patients with lower-grade glioma. However, these associations were not confirmed in an independent analysis of 563 lower-grade glioma cases from the University of California at San Francisco Adult Glioma Study (p = 0.65 and p = 0.41, respectively). The results of this study do not support a role for inherited polymorphisms in myeloid-associated genes in affecting survival of patients diagnosed with glioblastoma or lower-grade glioma.

Tumor Evolution of Glioma-Intrinsic Gene Expression Subtypes Associates with Immunological Changes in the Microenvironment.

We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single-cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ T cell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

Immune modulatory nanoparticle therapeutics for intracerebral glioma.

Background: Previously we showed therapeutic efficacy of unprotected miR-124 in preclinical murine models of glioblastoma, including in heterogeneous genetically engineered murine models by exploiting the immune system and thereby negating the need for direct tumor delivery. Although these data were promising, to implement clinical trials, we required a scalable formulation that afforded protection against circulatory RNases. Methods: We devised lipid nanoparticles that encapsulate and protect the miRs from degradation and provide enhanced delivery into the immune cell compartment and tested in vivo antitumor effects. Results: Treatment with nanoparticle-encapsulated miR-124, LUNAR-301, demonstrated a median survival exceeding 70 days, with an associated reversal of tumor-mediated immunosuppression and induction of immune memory. In both canine and murine models, the safety profile of LUNAR-301 was favorable. Conclusions: For the first time, we show that nanoparticles can direct a therapeutic response by targeting intracellular immune pathways. Although shown in the context of gliomas, this therapeutic approach would be applicable to other malignancies.

TIE2-mediated tyrosine phosphorylation of H4 regulates DNA damage response by recruiting ABL1.

DNA repair pathways enable cancer cells to survive DNA damage induced after genotoxic therapies. Tyrosine kinase receptors (TKRs) have been reported as regulators of the DNA repair machinery. TIE2 is a TKR overexpressed in human gliomas at levels that correlate with the degree of increasing malignancy. Following ionizing radiation, TIE2 translocates to the nucleus, conferring cells with an enhanced nonhomologous end-joining mechanism of DNA repair that results in a radioresistant phenotype. Nuclear TIE2 binds to key components of DNA repair and phosphorylates H4 at tyrosine 51, which, in turn, is recognized by the proto-oncogene ABL1, indicating a role for nuclear TIE2 as a sensor for genotoxic stress by action as a histone modifier. H4Y51 constitutes the first tyrosine phosphorylation of core histones recognized by ABL1, defining this histone modification as a direct signal to couple genotoxic stress with the DNA repair machinery.

Discovery of cell surface vimentin targeting mAb for direct disruption of GBM tumor initiating cells.

Intracellular vimentin overexpression has been associated with epithelial-mesenchymal transition, metastasis, invasion, and proliferation, but cell surface vimentin (CSV) is less understood. Furthermore, it remains unknown whether CSV can serve as a therapeutic target in CSV-expressing tumor cells. We found that CSV was present on glioblastoma multiforme (GBM) cancer stem cells and that CSV expression was associated with spheroid formation in those cells. A newly developed monoclonal antibody against CSV, 86C, specifically and significantly induced apoptosis and inhibited spheroid formation in GBM cells in vitro. The addition of 86C to GBM cells in vitro also led to rapid internalization of vimentin and decreased GBM cell viability. These findings were associated with an increase in caspase-3 activity, indicating activation of apoptosis. Finally, treatment with 86C inhibited GBM progression in vivo. In conclusion, CSV-expressing GBM cells have properties of tumor initiating cells, and targeting CSV with the monoclonal antibody 86C is a promising approach in the treatment of GBM.

Tipping a favorable CNS intratumoral immune response using immune stimulation combined with inhibition of tumor-mediated immune suppression.

High-grade gliomas are notoriously heterogeneous regarding antigen expression, effector responses, and immunosuppressive mechanisms. Therefore, combinational immune therapeutic approaches are more likely to impact a greater number of patients and result in longer, durable responses. We have previously demonstrated the monotherapeutic effects of miR-124, which inhibits the signal transducer and activator of transcription 3 (STAT3) immune suppressive pathway, and immune stimulatory 4-1BB aptamers against a variety of malignancies, including genetically engineered immune competent high-grade gliomas. To evaluate potential synergy, we tested an immune stimulatory aptamer together with microRNA-124 (miRNA-124), which blocks tumor-mediated immune suppression, and found survival to be markedly enhanced, including beyond that produced by monotherapy. The synergistic activity appeared to be not only secondary to enhanced CD3(+) cell numbers but also to reduced macrophage immune tumor trafficking, indicating that a greater therapeutic benefit can be achieved with approaches that both induce immune activation and inhibit tumor-mediated immune suppression within the central nervous system (CNS) tumors.

Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype.

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

Soluble Tie2 overrides the heightened invasion induced by anti-angiogenesis therapies in gliomas.

Glioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) agent bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. Our group has previously reported that Tie2-expressing monocytes (TEMs) are aberrantly present at the tumor/normal brain interface after anti-VEGF therapies and their significant role in the invasive outgrowth of these tumors. Here, we aimed to further understand the mechanisms leading to this pro-invasive tumor microenvironment. Examination of a U87MG xenogeneic glioma model and a GL261 murine syngeneic model showed increased tumor expression of angiopoietin 2 (Ang2), a natural ligand of Tie2, after anti-angiogenesis therapies targeting VEGF or VEGF receptor (VEGFR), as assessed by immunohistochemical analysis, immunofluorescence analysis, and enzyme-linked immunosorbent assays of tumor lysates. Migration and gelatinolytic assays showed that Ang2 acts as both a chemoattractant of TEMs and an enhancing signal for their tumor-remodeling properties. Accordingly, in vivo transduction of Ang2 into intracranial gliomas increased recruitment of TEMs into the tumor. To reduce invasive tumor outgrowth after anti-angiogenesis therapy, we targeted the Ang-Tie2 axis using a Tie2 decoy receptor. Using syngeneic models, we observed that overexpression of soluble Tie2 within the tumor prevented the recruitment of TEMs to the tumor and the development of invasion after anti-angiogenesis treatment. Taken together, these data indicate an active role for the Ang2-Tie2 pathway in invasive glioma recurrence after anti-angiogenesis treatment and provide a rationale for testing the combined targeting of VEGF and Ang-Tie2 pathways in patients with glioblastoma.

PD-L1 expression and prognostic impact in glioblastoma.

BACKGROUND: Therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and PD-1/PD-L1 has demonstrated tumor regression in clinical trials, and phase 2 trials are ongoing in glioblastoma (GBM). Previous reports have suggested that responses are more frequent in patients with tumors that express PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact. METHODS: Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with GBM. We categorized our results according to the total number of PD-L1-expressing cells within the GBMs and then validated this finding in ex vivo GBM flow cytometry with further analysis of the T cell populations. We then evaluated the association between PD-L1 expression and median survival time using the protein expression datasets and mRNA from The Cancer Genome Atlas. RESULTS: The median percentage of PD-L1-expressing cells in GBM by cell surface staining is 2.77% (range: 0%-86.6%; n = 92), which is similar to the percentage found by ex vivo flow cytometry. The majority of GBM patients (61%) had tumors with at least 1% or more PD-L1-positive cells, and 38% had at least 5% or greater PD-L1 expression. PD-L1 is commonly expressed on the GBM-infiltrating T cells. Expression of both PD-L1 and PD-1 are negative prognosticators for GBM outcome. CONCLUSIONS: The incidence of PD-L1 expression in GBM patients is frequent but is confined to a minority subpopulation, similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome.

MiR-138 exerts anti-glioma efficacy by targeting immune checkpoints.

BACKGROUND: Antibody therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule 4 (CTLA-4) and programmed cell death 1 (PD-1) has demonstrated marked tumor regression in clinical trials. MicroRNAs (miRNAs) can modulate multiple gene transcripts including possibly more than one immune checkpoint and could be exploited as immune therapeutics. METHODS: Using online miRNA targeting prediction algorithms, we searched for miRNAs that were predicted to target both PD-1 and CTLA-4. MiR-138 emerged as a leading candidate. The effects of miR-138 on CTLA-4 and PD-1 expression and function in T cells were determined and the therapeutic effect of intravenous administration of miR-138 was investigated in both immune-competent and -incompetent murine models of GL261 glioma. RESULTS: Target binding algorithms predicted that miR-138 could bind the 3' untranslated regions of CTLA-4 and PD-1, which was confirmed with luciferase expression assays. Transfection of human CD4+ T cells with miR-138 suppressed expression of CTLA-4, PD-1, and Forkhead box protein 3 (FoxP3) in transfected human CD4+ T cells. In vivo miR-138 treatment of GL261 gliomas in immune-competent mice demonstrated marked tumor regression, a 43% increase in median survival time (P = .011), and an associated decrease in intratumoral FoxP3+ regulatory T cells, CTLA-4, and PD-1 expression. This treatment effect was lost in nude immune-incompetent mice and with depletion of CD4+ or CD8+ T cells, and miR-138 had no suppressive effect on glioma cells when treated directly at physiological in vivo doses. CONCLUSIONS: MiR-138 exerts anti-glioma efficacy by targeting immune checkpoints which may have rapid translational potential as a novel immunotherapeutic agent.

Down-regulation of IKKß expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma.

Glioblastoma (GBM) is an aggressive malignancy associated with profound host immunosuppression. Microglia and macrophages infiltrating GBM acquire the pro-tumorigenic, M2 phenotype and support tumor invasion, proliferation, survival, angiogenesis and block immune responses both locally and systematically. Mechanisms responsible for immunological deficits in GBM patients are poorly understood. We analyzed immune/inflammatory gene expression in five datasets of low and high grade gliomas, and performed Gene Ontology and signaling pathway analyses to identify defective transcriptional responses. The expression of many immune/inflammatory response and TLR signaling pathway genes was reduced in high grade gliomas compared to low grade gliomas. In particular, we found the reduced expression of the IKBKB, a gene coding for IKKß, which phosphorylates IκB proteins and represents a convergence point for most signal transduction pathways leading to NFκB activation. The reduced IKBKB expression and IKKß levels in GBM tissues were demonstrated by qPCR, Western blotting and immunohistochemistry. The IKKß expression was down-regulated in microglia/macrophages infiltrating glioblastoma. NFκB activation, prominent in microglia/macrophages infiltrating low grade gliomas, was reduced in microglia/macrophages in glioblastoma tissues. Down-regulation of IKBKB expression and NFκB signaling in microglia/macrophages infiltrating glioblastoma correlates with defective expression of immune/inflammatory genes and M2 polarization that may result in the global impairment of anti-tumor immune responses in glioblastoma.