Abstract
Inducing an immunosuppressive environment has been one of the most notable subversive tactics employed by aggressive primary brain tumors like Glioblastoma (GBM). It is vital that we garner ...a better understanding of the cell-cell interactions occurring within the tumor microenvironment (TME) in situ. Current in vitro and in vivo models sorely limit our ability to study the cell-cell interactions within the TME while accounting for its complexity and the spatial orientation of its components. Unfilled, this gap in physiologically relevant models greatly hinders the translation of impactful research findings to successful treatment options for patients diagnosed with GBM and other primary CNS tumors. We have devised a novel organotypic brain slice culture (BSC) model study using tumor-bearing C57BL/6J mice which brings the potential to not only determine the cell-cell interactions within the TME but also investigate the underlined mechanisms which govern them. Obtaining BSCs from tumor-bearing mice at various time points post-implantation also allows for analysis of the trafficking of various immune cells across the blood-brain barrier (BBB). We demonstrated outcomes similar to those seen in vivo following the introduction of parallel conditions, further supporting the physiological relevancy of this model. Thus, organotypic brain slice cultures from tumor-bearing mouse models may serve as a credible and incredibly versatile model for the study of primary CNS tumors which may bridge the gap between in vitro and in vivo studies.
We identified 2 cases of Salmonella enterica serovar Vitkin infection linked by whole-genome sequencing in infants in Ontario, Canada, during 2022. Both households of the infants reported having ...bearded dragons as pets. The outbreak strain was also isolated from an environmental sample collected from a patient’s bearded dragon enclosure. Twelve cases were detected in the United States, and onset dates occurred during March 2021–September 2022 (isolates related to isolates from Canada within 0–9 allele differences by core-genome multilocus sequence typing). Most US patients (66.7%) were <1 year of age, and most (72.7%) had reported bearded dragon exposure. Hospitalization was reported for 5 (38.5%) of 13 patients. Traceback of bearded dragons identified at least 1 potential common supplier in Southeast Asia. Sharing rare serovar information and whole-genome sequencing data between Canada and the United States can assist in timely identification of outbreaks, including those that might not be detected through routine surveillance.
BackgroundDendritic cell (DC) plays crucial role in eliciting anti-tumor immune response through both innate and adaptive immune system. Our group has developed an adoptive cellular therapy (ACT) ...against high grade gliomas, which significantly improves survival in murine models of CNS malignancies. Our studies have shown that the efficacy is associated with the observed increase in intratumoral dendritic cells (DC) which arise from transferred hematopoietic stem cells (HSC).1 However, tumors develop diverse mechanisms to restrict DC function to evade immune surveillance. In this study, we will address the various DC dysfunctional mechanism in both primary and ACT escaped tumors.MethodsACT treatment was administered to mice bearing the KR158B glioblastoma cell line to get ACT escaped tumor. The mice received 9 Gy of irradiation prior to the transfer of hematopoietic stem cells and tumor-reactive T cells, followed by three doses of BMDC vaccine. Tumor associated DCs sorted from primary and ACT escaped tumors were subjected to assess DC function or gene expression profiling. T cell proliferation, T cell activation markers, and IFNγ secretion were measured by flow cytometry and ELISA as a readout for DC-T cell co-culture functional assays. Brain tumor slices were prepared using a vibratome, and the conditioned medium from the slice culture was used to pre-treat DCs to determine the impact of brain tumor secretion on DCs.ResultsFunctional evidence demonstrated that DCs from ACT-escaped tumors is impaired in activating tumor-reactive T cells. By comparing the gene expressing profiles between tumor associated DCs in primary and ACT escaped tumors,we found a significant decrease in cDC marker genes, antigen assembly genes, and MHC molecules in ACT-escaped tumor DCs, compared to primary tumor DCs, suggesting a decreased cDC population and impaired antigen presentation in ACT-escaped tumor DCs. To determine whether secreted factors from tumor microenvironment that drives DC dysfunction, conditioned medium(CM) from tumor brain slices were tested in inducing DC dysfunction in vitro. CM from primary tumor brain slices suppressed DC function compared to healthy brain slice CM. Further results showed that non-tumor cell-derived secretion from the slices drives DC dysfunction in primary tumors. Strikingly, unlike primary tumor brain CM, ACT-escaped tumor brain slice CM did not decrease DC function capacity, suggesting different tumor-driven DC dysfunctional mechanisms in primary and ACT-escaped tumors.ConclusionsThe primary glioma tumor drives DC dysfunction through non-tumor cell-derived secretion, whereas the ACT-escaped tumor employs a different mechanism involving cDC exclusion and impaired MHC expression and antigen loading.ReferencesWildes TJ, et al. Cross-talk between T Cells and Hematopoietic Stem Cells during Adoptive Cellular Therapy for Malignant Glioma. Clin Cancer Res, 2018; 24(16): 3955–3966.Ethics ApprovalThe study is approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) and are covered under protocol number IACUC202100000053. All participants were given informed consent before taking part.
BackgroundGlioma-induced immune dysregulation of the hematopoietic system has been described in a limited number of studies. In this study, our group further demonstrates that gliomas interrupt the ...cellular differentiation programming and outcomes of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. HSPCs from glioma-bearing mice are reprogrammed and driven towards expansion of myeloid lineage precursors and myeloid-derived suppressor cells (MDSCs) in secondary lymphoid organs. However, we found this expansion is reversed by immunotherapy. Adoptive cellular therapy (ACT) has been demonstrably efficacious in multiple preclinical models of central nervous system (CNS) malignancies, and here we describe how glioma-induced dysfunction is reversed by this immunotherapeutic platform.MethodsThe impact of orthotopic KR158B-luc glioma on HSPCs was evaluated in an unbiased fashion using single cell RNAseq (scRNAseq) of lineage− cells and phenotypically using flow cytometry. Mature myeloid cell frequencies and function were also evaluated using flow cytometry. Finally, ACT containing total body irradiation, tumor RNA-pulsed dendritic cells, tumor-reactive T cells and HSPCs isolated from glioma-bearing or non-tumor-bearing mice were used to evaluate cell fate differentiation and survival.ResultsUsing scRNAseq, we observed an altered HSPC landscape in glioma-bearing versus non-tumor-bearing mice . In addition, an expansion of myeloid lineage subsets, including granulocyte macrophage precursors (GMPs) and MDSCs, were observed in glioma-bearing mice relative to non-tumor-bearing controls. Furthermore, MDSCs from glioma-bearing mice demonstrated increased suppressive capacity toward tumor-specific T cells as compared with MDSCs from non-tumor-bearing hosts. Interestingly, treatment with ACT overcame these suppressive properties. When HSPCs from glioma-bearing mice were transferred in the context of ACT, we observed significant survival benefit and long-term cures in orthotopic glioma models compared with mice treated with ACT using non-glioma-bearing HSPCs.
Food-producing animals are recognized to play a role in the epidemiology of antimicrobial resistance in Canada. However, the presence of resistant organisms in particular groups of animals, such as ...chickens raised in small-holder flocks, has not been studied. The purpose of this study was, therefore, to identify and characterize
possessing broad-spectrum β-lactamase genes among a collection of third-generation cephalosporin-resistant isolates recovered from 205 small flocks in southern Ontario. Extended-spectrum β-lactamase (ESBL; CTX-M-1) positive strains were isolated from 26 out of 205 flocks (12.7%), whereas 39 strains possessing AmpC (CMY-2) were grown from 31 out of 205 flocks (15.1%). Pulsed-field gel electrophoresis (PFGE) revealed that the isolates were genetically heterogeneous. Further testing by multi-locus sequence typing confirmed that none of the PFGE-defined clusters belonged to ST131. Our results suggest that the dissemination of this resistance in bacteria isolated from chickens in small-holder flocks may be associated with the spread of plasmids rather than particular
clones and that these isolates do not possess the ESBL types most commonly associated with human infections (CTX-M-15).
Abstract
Glioblastoma (GBM) is an incredibly aggressive and prevalent primary CNS tumor with dismal survival outcomes. GBM’s intra-tumor heterogeneity and the powerful regulatory mechanisms within ...the tumor microenvironment (TME) that hamper immune activation have proved to be formidable challenges to the development of effective therapies. Fortunately, immunotherapy has emerged as a potentially powerful approach to achieve of immune activation within the TME through adoptive immunotherapy and checkpoint inhibition Our previous work has demonstrated that the survival benefit of adoptive cellular therapy (ACT) is significantly enhanced by concomitant transfer of bone marrow derived hematopoietic stem cells (HSC) with tumor-reactive T cells in preclinical models. Using a novel organotypic brain slice culture (BSC) model study using glioma-bearing C57BL/6J mice, we interrogate tumor microenvironment and directly demonstrate that the presence of HSC-derived cells in the microenvironment increases T cell migration to tumor, and increased tumor cell death. In addition, we can investigate reprogramming of the TME as a result of treatment with this ACT+HSC platform. These studies are unique in that through this model system, we are capable of studying the cell-cell interactions occurring within the tumor microenvironment TME longitudinally while accounting for the complexity and the spatial orientation of the components within the TME that promote immunosuppression and tumor escape.
Abstract
INTRODUCTION
A multitude of recently published papers have found immunosuppression caused by glioblastoma (GBM) is not limited to the tumor microenvironment and impacts the peripheral immune ...system. Our group has recently reported an expansion of granulocyte macrophage precursors, a subset of hematopoietic stem and progenitor populations (HSPCs), in glioma-bearing mice relative to healthy, age-matched controls. Thus we sought to determine if GBM causes cell fate dysfunction in CD34+ HSPCs in patients.
METHODS
CD34+ cells were magnetically isolated from peripheral blood of newly-diagnosed GBM patients and healthy age-matched donors and processed for single-cell RNA sequencing. CD34+ cells were magnetically isolated from peripheral blood of newly-diagnosed GBM patients and healthy age-matched donors and processed for single-cell RNA sequencing using the 10X Genomics platform. CellRanger was used for primary analysis with default parameters. After quality control removal of doublets, dead, and low quality cells automated cell type annotation was performed using the Azimuth Hubmap Consortium human bone marrow reference. The annotated single cell object was then read into R and Seurat was used for differential expression analysis and visualization.
RESULTS
AND
CONCLUSIONS
There we no significant expansions of stem and progenitor populations. GBM patient CD34+ samples had upregulation of VWF, a marker of myeloid-bias cell subset. Within the mature immune cell compartments, we observed a significant expansion of NK cells in GBM patients including 1,997 genes differentially expressed in the NK cell cluster between cohorts. This includes downregulation of SLAMF6, which triggers cytolytic activity in NK cells, in GBM patients. Upregulation of early NK cell activation markers CD69 and JUN were seen in GBM patient samples. These results strongly suggest NK are likely activated in the context of GBM, but may not be functional.
Abstract
Among pediatric cancers, malignancies of the brain carry significant morbidity and mortality, harboring dismal prognosis and poor outcomes. Our group has previously shown that adoptive ...cellular therapy (ACT) provides therapeutic benefit against central nervous system (CNS) malignancies, including medulloblastoma and high-grade glioma. In this study, we characterized cell cycling differences of immune sub-populations in peripheral lymphoid and tumor tissue compartments in vivo. ACT was conducted in orthotopic KR158 luciferase tumor-bearing mice, a syngeneic murine glioma model. Five days following intracranial implantation of tumor cells, mice were treated with ACT which included the following elements: total body irradiation, hematopoietic stem cell transplantation, one dose of tumor-activated splenocytes and three weekly doses of dendritic cells that had been electroporated with tumor-derived ribonucleic acid. One week after the completion of ACT, bromodeoxyuridine (BRDU) was administered to mice 12-hours prior to tissue collection and flow cytometry was utilized to measure the level of BRDU incorporation. 7-aminoactinomycin-D was used in conjunction with BRDU to provide insight into all phases of the cell cycle. We observed significant differences in cycling populations of myeloid derived suppressor cells (MDSC) and T-cells. In ACT-treated-mice, MDSCs found in secondary lymphoid tissues were significantly more proliferative. In the tumor microenvironment (TME), proliferating MDSCs were significantly reduced compared to mice that did not receive treatment. Frequencies of proliferating CD4+ and CD8+ T-cells were significantly increased in cervical lymph node and TME but reduced in spleen. These findings suggest that ACT alters immune cycling in peripheral lymphoid tissues and TME to potentially promote antitumor T-cell responses and minimize MDSC-mediated immunosuppression. While ACT-mediated alterations to immune cell cycling are likely a major contributor to the therapy’s benefit, it is unlikely to be the sole mechanism. Further exploration into other functional pathways, namely migration, is needed to explain changes to immune populations following ACT.
Abstract
Despite advances in solid tumor biology and oncology, pediatric and adult primary brain cancer and metastasis to the brain harbor dismal prognosis and poor outcomes. One avenue to overcome ...these clinical challenges is through developing robust immunotherapy to better target the tumor. Our group has previously shown that adoptive cellular therapy (ACT) provides therapeutic benefit against central nervous system (CNS) malignancies, namely medulloblastoma, brain stem glioma and glioblastoma. In this study, we characterized cell cycling differences of immune cellular sub-populations in peripheral lymphoid and tumor tissue compartments in vivo. ACT was conducted in orthotopic KR158 luciferase tumor-bearing mice, a syngeneic murine glioma model. Following completion of ACT, bromodeoxyuridine (BRDU) was administered to mice 12-hours prior to tissue collection and flow cytometry was utilized to measure the level of BRDU incorporation. 7-aminoactinomycin D was used in conjunction with BRDU to provide insight into all phases of the cell cycle. We observed significant differences in cycling populations of myeloid derived suppressor cells (MDSC) and T cells. In mice receiving ACT, MDSCs found in secondary lymphoid tissues were significantly more proliferative. In the tumor microenvironment (TME), proliferating MDSCs were significantly reduced compared to mice that did not receive treatment. Frequencies of proliferating CD4+ and CD8+ T-cells were significantly increased in cervical lymph node and TME, but reduced in spleen. These findings suggest that ACT changes immune cycling and migration dynamics in lymphoid tissue and TME to potentially promote antitumor T-cell responses and minimize tumor-mediated immunosuppression.
5R01NS112315-04
Abstract
BACKGROUND
Mutated neoantigens have shown promise as targets for cancer immunotherapy but embryonal pediatric brain tumors with notoriously low mutational burden have more limited ...opportunities for neoantigen directed therapies. Evidence suggests that embryonal brain tumors such as medulloblastoma (MB) and brain stem gliomas (BSG) arise from the aberrant reactivation of fetal developmental programming. We explored the hypothesis that neonatal mouse cerebellum and brainstem express developmentally regulated proteins that could serve as potent tumor rejection antigens for a preclinical model of Group 3 MB (NSC) and H3.3K27M positive BSG (K2), respectively. We thus generated tumor-reactive T cells by using P5 cerebellum and P4 brain stem RNA as sources of antigens. P5-cerebellum-specific T cells (cDevAg-T cells) and P4-brain stem-specific T cells (bsDevAg-T cells) demonstrated effector function against respective MB and BSG tumor cells with exquisite specificity.
METHODS
RNA was isolated from P5 cerebellum and P4 brain stem and pulsed into bone marrow-derived dendritic cells. These were then used to in vitro activate splenocytes from previously immunized mice, generating either cDevAg-T cells or bsDevAg-T cells. In vitro functionality assays against tumor targets were conducted to determine reactivity and specificity. DevAg-T cells were used in adoptive cellular therapy in orthotopic models to determine therapeutic efficacy of DevAg-specific T cells in vivo. Results and
CONCLUSIONS
DevAg-T cells produce high levels of Th1-type cytokines that recognize distinct subtypes of MB and BSG, show no cross-reactivity with normal brain. Adoptive cellular therapy employing DevAg-specific T cells demonstrate a significant survival benefit in orthotopic models of established Group 3 NSC MB and H3.3K27M mutation positive BSG. Our studies demonstrate that RNA encoding non-mutated and organ-specific developmental antigens can serve as novel tumor rejection antigens for pediatric brain cancers.
