RESUMO
Essentially all bacteria secrete nano-sized (~20-200 nm) bacterial extracellular vesicles (bEVs) loaded with proteins, lipids, glycans, and nucleic acids. bEVs facilitate interactions among cells of the same species, different microbial species, and even with cells of multicellular organisms in the context of colonization or infection. Their interactions with host organism immune cell receptors vary depending on the producing bacterial species and are now being harnessed for the development of bEVs as a potential immunotherapeutic platform. Both basic/mechanistic and preclinical therapeutic development studies are thus increasing in number and require implementation of methods for multiparametric analytical characterization as well as in vivo administration in preclinical animal models of disease. We summarize a variety of analytical methods that can be used to calculate bEV dose for preparations made from diverse bacterial sources (including sterility testing, total protein concentration, particle concentration, and lipopolysaccharide concentration). We also describe basic methodology for intravenous administration of bEV preparations via tail vein injection in laboratory mice. Throughout the description of methodology, we highlight potential pitfalls and alternatives to further equip the reader for troubleshooting should challenges arise. Robust and reproducible characterization is a prerequisite of bEV preparation quality control and consistent dosing during preclinical development. This will allow for more streamlined testing of candidate therapeutic bEVs within a given research laboratory, and furthermore facilitate reproducibility of findings across laboratories.
Assuntos
Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Animais , Camundongos , Bactérias/metabolismoRESUMO
Sex differences in immune responses impact cancer outcomes and treatment response, including in glioblastoma (GBM). However, host factors underlying sex specific immune-cancer interactions are poorly understood. Here, we identify the neurotransmitter γ-aminobutyric acid (GABA) as a driver of GBM-promoting immune response in females. We demonstrated that GABA receptor B (GABBR) signaling enhances L-Arginine metabolism and nitric oxide synthase 2 (NOS2) expression in female granulocytic myeloid-derived suppressor cells (gMDSCs). GABBR agonist and GABA analog promoted GBM growth in females in an immune-dependent manner, while GABBR inhibition reduces gMDSC NOS2 production and extends survival only in females. Furthermore, female GBM patients have enriched GABA transcriptional signatures compared to males, and the use of GABA analogs in GBM patients is associated with worse short-term outcomes only in females. Collectively, these results highlight that GABA modulates anti-tumor immune response in a sex-specific manner, supporting future assessment of GABA pathway inhibitors as part of immunotherapy approaches.
RESUMO
BACKGROUND: Platinum resistance is the primary cause of poor survival in ovarian cancer (OC) patients. Targeted therapies and biomarkers of chemoresistance are critical for the treatment of OC patients. Our previous studies identified cell surface CD55, a member of the complement regulatory proteins, drives chemoresistance and maintenance of cancer stem cells (CSCs). CSCs are implicated in tumor recurrence and metastasis in multiple cancers. METHODS: Protein localization assays including immunofluorescence and subcellular fractionation were used to identify CD55 at the cell surface and nucleus of cancer cells. Protein half-life determinations were used to compare cell surface and nuclear CD55 stability. CD55 deletion mutants were generated and introduced into cancer cells to identify the nuclear trafficking code, cisplatin sensitivity, and stem cell frequency that were assayed using in vitro and in vivo models. Detection of CD55 binding proteins was analyzed by immunoprecipitation followed by mass spectrometry. Target pathways activated by CD55 were identified by RNA sequencing. RESULTS: CD55 localizes to the nucleus of a subset of OC specimens, ascites from chemoresistant patients, and enriched in chemoresistant OC cells. We determined that nuclear CD55 is glycosylated and derived from the cell surface pool of CD55. Nuclear localization is driven by a trafficking code containing the serine/threonine (S/T) domain of CD55. Nuclear CD55 is necessary for cisplatin resistance, stemness, and cell proliferation in OC cells. CD55 S/T domain is necessary for nuclear entry and inducing chemoresistance to cisplatin in both in vitro and in vivo models. Deletion of the CD55 S/T domain is sufficient to sensitize chemoresistant OC cells to cisplatin. In the nucleus, CD55 binds and attenuates the epigenetic regulator and tumor suppressor ZMYND8 with a parallel increase in H3K27 trimethylation and members of the Polycomb Repressive Complex 2. CONCLUSIONS: For the first time, we show CD55 localizes to the nucleus in OC and promotes CSC and chemoresistance. Our studies identify a therapeutic mechanism for treating platinum resistant ovarian cancer by blocking CD55 nuclear entry.
Assuntos
Antígenos CD55 , Núcleo Celular , Cromatina , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Histonas , Células-Tronco Neoplásicas , Neoplasias Ovarianas , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Feminino , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Camundongos , Antígenos CD55/metabolismo , Antígenos CD55/genética , Linhagem Celular Tumoral , Histonas/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Metilação , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacologia , Transporte ProteicoRESUMO
Hematopoietic stem cells (HSCs) adapt to organismal blood production needs by balancing self-renewal and differentiation, adjusting to physiological demands and external stimuli. Although sex differences have been implicated in differential hematopoietic function in males versus females, the mediators responsible for these effects require further study. Here, we characterized hematopoiesis at a steady state and during regeneration following hematopoietic stem cell transplantation (HST). RNA sequencing of lineage(-) bone marrow cells from C57/Bl6 mice revealed a broad transcriptional similarity between the sexes. However, we identified distinct sex differences in key biological pathways, with female cells showing reduced expression of signatures involved in inflammation and enrichment of genes related to glycolysis, hypoxia, and cell cycle regulation, suggesting a more quiescent and less inflammatory profile compared with male cells. To determine the functional impacts of the observed transcriptomic differences, we performed sex-matched and mismatched transplantation studies of lineage(-) donor cells. During short-term 56-day HST recovery, we found a male donor cell proliferative advantage, coinciding with elevated serum TNF-α, and a male recipient engraftment advantage, coinciding with increased serum CXCL12. Together, we show that sex-specific cell responses, marked by differing expression of pathways regulating metabolism, hypoxia, and inflammation, shape normal and regenerative hematopoiesis, with implications for the clinical understanding of hematopoietic function.
RESUMO
Many cancers, including glioblastoma (GBM), have a male-biased sex difference in incidence and outcome. The underlying reasons for this sex bias are unclear but likely involve differences in tumor cell state and immune response. This effect is further amplified by sex hormones, including androgens, which have been shown to inhibit anti-tumor T cell immunity. Here, we show that androgens drive anti-tumor immunity in brain tumors, in contrast to its effect in other tumor types. Upon castration, tumor growth was accelerated with attenuated T cell function in GBM and brain tumor models, but the opposite was observed when tumors were located outside the brain. Activity of the hypothalamus-pituitary-adrenal gland (HPA) axis was increased in castrated mice, particularly in those with brain tumors. Blockade of glucocorticoid receptors reversed the accelerated tumor growth in castrated mice, indicating that the effect of castration was mediated by elevated glucocorticoid signaling. Furthermore, this mechanism was not GBM specific, but brain specific, as hyperactivation of the HPA axis was observed with intracranial implantation of non-GBM tumors in the brain. Together, our findings establish that brain tumors drive distinct endocrine-mediated mechanisms in the androgen-deprived setting and highlight the importance of organ-specific effects on anti-tumor immunity.
RESUMO
Background: Biological sex is an important risk factor for glioblastoma (GBM), with males having a higher incidence and poorer prognosis. The mechanisms for this sex bias are thought to be both tumor intrinsic and tumor extrinsic. MicroRNAs (miRNAs), key post-transcriptional regulators of gene expression, have been previously linked to sex differences in various cell types and diseases, but their role in the sex bias of GBM remains unknown. Methods: We leveraged previously published paired miRNA and mRNA sequencing of 39 GBM patients (22 male, 17 female) to identify sex-biased miRNAs. We further interrogated a separate single-cell RNA sequencing dataset of 110 GBM patients to examine whether differences in miRNA target gene expression were tumor cell intrinsic or tumor cell extrinsic. Results were validated in a panel of patient-derived cell models. Results: We identified 10 sex-biased miRNAs (adjusted < 0.1), of which 3 were more highly expressed in males and 7 more highly expressed in females. Of these, miR-644a was higher in females, and increased expression of miR-644a target genes was significantly associated with decreased overall survival (HR 1.3, p = 0.02). Furthermore, analysis of an independent single-cell RNA sequencing dataset confirmed sex-specific expression of miR-644a target genes in tumor cells (p < 10-15). Among patient derived models, miR-644a was expressed a median of 4.8-fold higher in females compared to males. Conclusions: Our findings implicate miR-644a as a candidate tumor cell-intrinsic regulator of sex-biased gene expression in GBM.
RESUMO
Introduction: Sex differences in glioblastoma (GBM) have been observed in incidence, genetic and epigenetic alterations, and immune response. These differences have extended to the methylation of the MGMT promoter, which critically impacts temozolomide resistance. However, the association between sex, MGMT methylation, and survival is poorly understood, which this study sought to evaluate. Methods: A retrospective cohort study was conducted and reported following STROBE guidelines, based on adults with newly diagnosed GBM who received their first surgical intervention at Cleveland Clinic (Ohio, USA) between 2012 and 2018. Kaplan-Meier and multivariable Cox proportional hazards models were used to analyze the association between sex and MGMT promoter methylation status on overall survival (OS). MGMT was defined as methylated if the mean of CpG 1-5 ≥ 12. Propensity score matching was performed on a subset of patients to evaluate the effect of individual CpG site methylation. Results: A total of 464 patients had documented MGMT methylation status with a mean age of 63.4 (range 19-93) years. A total of 170 (36.6%) were female, and 133 (28.7%) received gross total resection as a first intervention. A total of 42.5% were MGMT methylated, with females more often having MGMT methylation than males (52.1% vs. 37.4%, p = 0.004). In univariable analysis, OS was significantly longer for MGMT promoter methylated than un-methylated groups for females (2 yr: 36.8% vs. 11.1%; median: 18.7 vs. 9.5 months; p = 0.001) but not for males (2 yr: 24.3% vs. 12.2%; median: 12.4 vs. 11.3 months; p = 0.22, p for MGMT-sex interaction = 0.02). In multivariable analysis, MGMT un-methylated versus methylated promoter females (2.07; 95% CI, 1.45-2.95; p < 0.0001) and males (1.51; 95% CI, 1.14-2.00; p = 0.004) had worse OS. Within the MGMT promoter methylated group, males had significantly worse OS than females (1.42; 95% CI: 1.01-1.99; p = 0.04). Amongst patients with data on MGMT CpG promoter site methylation values (n = 304), the median (IQR) of CpG mean methylation was 3.0% (2.0, 30.5). Females had greater mean CpG methylation than males (11.0 vs. 3.0, p < 0.002) and higher per-site CpG methylation with a significant difference at CPG 1, 2, and 4 (p < 0.008). After propensity score matching, females maintained a significant survival benefit (18.7 vs. 10.0 months, p = 0.004) compared to males (13.0 vs. 13.6 months, p = 0.76), and the pattern of difference was significant (P for CpG-sex interaction = 0.03). Conclusions: In this study, females had higher mean and individual CpG site methylation and received a greater PFS and OS benefit by MGMT methylation that was not seen in males despite equal degrees of CpG methylation.
RESUMO
Sex differences are present across multiple non-reproductive organ cancers, with male individuals generally experiencing higher incidence of cancer with poorer outcomes. Although some mechanisms underlying these differences are emerging, the immunological basis is not well understood. Observations from clinical trials also suggest a sex bias in conventional immunotherapies with male individuals experiencing a more favourable response and female individuals experiencing more severe adverse events to immune checkpoint blockade. In this Perspective article, we summarize the major biological hallmarks underlying sex bias in immuno-oncology. We focus on signalling from sex hormones and chromosome-encoded gene products, along with sex hormone-independent and chromosome-independent epigenetic mechanisms in tumour and immune cells such as myeloid cells and T cells. Finally, we highlight opportunities for future studies on sex differences that integrate sex hormones and chromosomes and other emerging cancer hallmarks such as ageing and the microbiome to provide a more comprehensive view of how sex differences underlie the response in cancer that can be leveraged for more effective immuno-oncology approaches.
Assuntos
Imunoterapia , Neoplasias , Feminino , Humanos , Masculino , Epigênese Genética , Hormônios Esteroides Gonadais/metabolismo , Hormônios Esteroides Gonadais/imunologia , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Caracteres Sexuais , Fatores Sexuais , SexismoRESUMO
BACKGROUND: Glioblastoma (GBM) commonly displays epidermal growth factor receptor (EGFR) alterations (mainly amplification and EGFRvIII) and TAT-Cx43266-283 is a Src-inhibitory peptide with antitumor properties in preclinical GBM models. Given the link between EGFR and Src, the aim of this study was to explore the role of EGFR in the antitumor effects of TAT-Cx43266-283. METHODS: The effect of TAT-Cx43266-283, temozolomide (TMZ), and erlotinib (EGFR inhibitor) was studied in patient-derived GBM stem cells (GSCs) and murine neural stem cells (NSCs) with and without EGFR alterations, in vitro and in vivo. EGFR alterations were analyzed by western blot and fluorescence in situ hybridization in these cells, and compared with Src activity and survival in GBM samples from The Cancer Genome Atlas. RESULTS: The effect of TAT-Cx43266-283 correlated with EGFR alterations in a set of patient-derived GSCs and was stronger than that exerted by TMZ and erlotinib. In fact, TAT-Cx43266-283 only affected NSCs with EGFR alterations, but not healthy NSCs. EGFR alterations correlated with Src activity and poor survival in GBM patients. Finally, tumors generated from NSCs with EGFR alterations showed a decrease in growth, invasiveness, and vascularization after treatment with TAT-Cx43266-283, which enhanced the survival of immunocompetent mice. CONCLUSIONS: Clinically relevant EGFR alterations are predictors of TAT-Cx43266-283 response and part of its mechanism of action, even in TMZ- and erlotinib-resistant GSCs. TAT-Cx43266-283 targets NSCs with GBM-driver mutations, including EGFR alterations, in an immunocompetent GBM model in vivo, suggesting a promising effect on GBM recurrence. Together, this study represents an important step toward the clinical application of TAT-Cx43266-283.
Assuntos
Neoplasias Encefálicas , Receptores ErbB , Amplificação de Genes , Glioblastoma , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Animais , Humanos , Camundongos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Temozolomida/farmacologia , Cloridrato de Erlotinib/farmacologia , Células Tumorais Cultivadas , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismoRESUMO
Cancer immunity is subjected to spatiotemporal regulation by leukocyte interaction with neoplastic and stromal cells, contributing to immune evasion and immunotherapy resistance. Here, we identify a distinct mesenchymal-like population of endothelial cells (ECs) that form an immunosuppressive vascular niche in glioblastoma (GBM). We reveal a spatially restricted, Twist1/SATB1-mediated sequential transcriptional activation mechanism, through which tumor ECs produce osteopontin to promote immunosuppressive macrophage (Mφ) phenotypes. Genetic or pharmacological ablation of Twist1 reverses Mφ-mediated immunosuppression and enhances T cell infiltration and activation, leading to reduced GBM growth and extended mouse survival, and sensitizing tumor to chimeric antigen receptor T immunotherapy. Thus, these findings uncover a spatially restricted mechanism controlling tumor immunity and suggest that targeting endothelial Twist1 may offer attractive opportunities for optimizing cancer immunotherapy.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Animais , Camundongos , Glioblastoma/genética , Células Endoteliais/patologia , Linhagem Celular Tumoral , Macrófagos , Terapia de Imunossupressão , Neoplasias Encefálicas/genéticaRESUMO
Over the past 2 decades, the cancer stem cell (CSC) hypothesis has provided insight into many malignant tumors, including glioblastoma (GBM). Cancer stem cells have been identified in patient-derived tumors and in some mouse models, allowing for a deeper understanding of cellular and molecular mechanisms underlying GBM growth and therapeutic resistance. The CSC hypothesis has been the cornerstone of cellular heterogeneity, providing a conceptual and technical framework to explain this longstanding phenotype in GBM. This hypothesis has evolved to fit recent insights into how cellular plasticity drives tumor growth to suggest that CSCs do not represent a distinct population but rather a cellular state with substantial plasticity that can be achieved by non-CSCs under specific conditions. This has further been reinforced by advances in genomics, including single-cell approaches, that have used the CSC hypothesis to identify multiple putative CSC states with unique properties, including specific developmental and metabolic programs. In this review, we provide a historical perspective on the CSC hypothesis and its recent evolution, with a focus on key functional phenotypes, and provide an update on the definition for its use in future genomic studies.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Células-Tronco Neoplásicas , Glioblastoma/patologia , Glioblastoma/metabolismo , Glioblastoma/genética , Humanos , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genéticaRESUMO
A major challenge in treating patients with glioblastoma is the inability to eliminate highly invasive cells with chemotherapy, radiation, or surgical resection. As cancer cells face the issue of replicating or invading neighboring tissue, they rewire their metabolism in a concerted effort to support necessary cellular processes and account for altered nutrient abundance. In this issue of the JCI, Garcia et al. compared an innovative 3D hydrogel-based invasion device to regional patient biopsies through a comprehensive multiomics-based approach paired with a CRISPR knockout screen. Their findings elucidate a role for cystathionine γ-lyase (CTH), an enzyme in the transsulfuration pathway, as a means of regulating the cellular response to oxidative stress. CTH-mediated conversion of cystathionine to cysteine was necessary for regulating reactive oxygen species to support invasion. Meanwhile, inhibition of CTH suppressed the invasive glioblastoma phenotype. However, inhibiting CTH resulted in a larger overall tumor mass. These findings suggest that targeting the transsulfuration pathway may serve as a means of redirecting glioblastoma to proliferate or invade.
Assuntos
Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cistationina/metabolismo , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
Background: Glioblastoma (GBM) displays alterations in iron that drive proliferation and tumor growth. Iron regulation is complex and involves many regulatory mechanisms, including the homeostatic iron regulator (HFE) gene, which encodes the homeostatic iron regulatory protein. While HFE is upregulated in GBM and correlates with poor survival outcomes, the function of HFE in GBM remains unclear. Methods: We interrogated the impact of cell-intrinsic Hfe expression on proliferation and survival of intracranially implanted animals through genetic gain- and loss-of-function approaches in syngeneic mouse glioma models, along with in vivo immune assessments. We also determined the expression of iron-associated genes and their relationship to survival in GBM using public data sets and used transcriptional profiling to identify differentially expressed pathways in control compared to Hfe-knockdown cells. Results: Overexpression of Hfe accelerated GBM proliferation and reduced animal survival, whereas suppression of Hfe induced apoptotic cell death and extended survival, which was more pronounced in females and associated with attenuation of natural killer cells and CD8+ T cell activity. Analysis of iron gene signatures in Hfe-knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Further analysis of differentially expressed pathways revealed oxidative stress as the top pathway upregulated following Hfe loss. Hfe knockdown indeed resulted in enhanced 55Fe uptake and generation of reactive oxygen species. Conclusions: These findings reveal an essential function for HFE in GBM cell growth and survival, as well as a sex-specific interaction with the immune response.
RESUMO
The association between anemia and outcomes in glioblastoma patients is unclear. We analyzed data from 1346 histologically confirmed adult glioblastoma patients in the TriNetX Research Network. Median hemoglobin and hematocrit levels were quantified for 6 months following diagnosis and used to classify patients as anemic or non-anemic. Associations of anemia and iron supplementation of anemic patients with median overall survival (median-OS) were then studied. Among 1346 glioblastoma patients, 35.9% of male and 40.5% of female patients were classified as anemic using hemoglobin-based WHO guidelines. Among males, anemia was associated with reduced median-OS compared to matched non-anemic males using hemoglobin (HR 1.24; 95% CI 1.00-1.53) or hematocrit-based cutoffs (HR 1.28; 95% CI 1.03-1.59). Among females, anemia was not associated with median-OS using hemoglobin (HR 1.00; 95% CI 0.78-1.27) or hematocrit-based cutoffs (HR: 1.10; 95% CI 0.85-1.41). Iron supplementation of anemic females trended toward increased median-OS (HR 0.61; 95% CI 0.32-1.19) although failing to reach statistical significance whereas no significant association was found in anemic males (HR 0.85; 95% CI 0.41-1.75). Functional transferrin-binding assays confirmed sexually dimorphic binding in resected patient samples indicating underlying differences in iron biology. Anemia among glioblastoma patients exhibits a sex-specific association with survival.