Role of gut microbiota in regulating immune checkpoint inhibitor therapy for glioblastoma.

Glioblastoma (GBM) is a highly malignant, invasive, and poorly prognosed brain tumor. Unfortunately, active comprehensive treatment does not significantly prolong patient survival. With the deepening of research, it has been found that gut microbiota plays a certain role in GBM, and can directly or indirectly affect the efficacy of immune checkpoint inhibitors (ICIs) in various ways. (1) The metabolites produced by gut microbiota directly affect the host's immune homeostasis, and these metabolites can affect the function and distribution of immune cells, promote or inhibit inflammatory responses, affect the phenotype, angiogenesis, inflammatory response, and immune cell infiltration of GBM cells, thereby affecting the effectiveness of ICIs. (2) Some members of the gut microbiota may reverse T cell function inhibition, increase T cell anti-tumor activity, and ultimately improve the efficacy of ICIs by targeting specific immunosuppressive metabolites and cytokines. (3) Some members of the gut microbiota directly participate in the metabolic process of drugs, which can degrade, transform, or produce metabolites, affecting the effective concentration and bioavailability of drugs. Optimizing the structure of the gut microbiota may help improve the efficacy of ICIs. (4) The gut microbiota can also regulate immune cell function and inflammatory status in the brain through gut brain axis communication, indirectly affecting the progression of GBM and the therapeutic response to ICIs. (5) Given the importance of gut microbiota for ICI therapy, researchers have begun exploring the use of fecal microbiota transplantation (FMT) to transplant healthy or optimized gut microbiota to GBM patients, in order to improve their immune status and enhance their response to ICI therapy. Preliminary studies suggest that FMT may enhance the efficacy of ICI therapy in some patients. In summary, gut microbiota plays a crucial role in regulating ICIs in GBM, and with a deeper understanding of the relationship between gut microbiota and tumor immunity, it is expected to develop more precise and effective personalized ICI therapy strategies for GBM, in order to improve patient prognosis.

Inflammatory activation of microglia by Staphylococcus aureus caused phenotypic alterations and affected glioblastoma growth.

As the most common type of tumour in brain, glioma has a high rate of morbidity and mortality and easily penetrates the surrounding normal brain parenchyma. The immunosuppressive microenvironment, which is similar to that in other neoplasms, is believed to participate in the tumorigenesis of glioma. Thus, many experts are seeking to exploit microenvironment as a therapeutic target. In the present study, we focused on microglia polarization to investigate the anti-glioma response of microglia inflammatory activation by Staphylococcus aureus in vitro and in vivo. First, we found that intratumor injection of S. aureus delayed glioma growth in C57/BL6 mice. Then, we showed that inflammatory microglia activated by S. aureus inhibited glioma cell proliferation, migration, and invasion. This inhibition was likely related to the phenotypic switch observed in microglia. In intracranial tumour models, the microglia activated by S. aureus exerted antitumour efficacy and prolonged animal survival. Taken together, our results suggest that microglia activated by S. aureus have antitumour efficacy, which may be a potential therapeutic agent for glioma. SIGNIFICANCE OF THE STUDY: In this study, we mainly demonstrated the antitumour efficacy of microglia after activated by S. aureus. Firstly, we found that intratumor injection of S. aureus inhibited the tumour growth in intracranial orthotopic glioma model. In addition, we found that the microglia around the glioma may exert antitumour efficacy, and its phenotype may be altered by stimulation of S. aureus. Our data manifested that S. aureus did not directly suppress cell proliferative, migration, and invasion capacity, but by activating microglia. And in mice GL261 GBMs, injection of microglia after cocultured with S. aureus inhibited tumour growth and prolonged animal survival.

Distinguishing potential bacteria-tumor associations from contamination in a secondary data analysis of public cancer genome sequence data.

BACKGROUND: A variety of bacteria are known to influence carcinogenesis. Therefore, we sought to investigate if publicly available whole genome and whole transcriptome sequencing data generated by large public cancer genome efforts, like The Cancer Genome Atlas (TCGA), could be used to identify bacteria associated with cancer. The Burrows-Wheeler aligner (BWA) was used to align a subset of Illumina paired-end sequencing data from TCGA to the human reference genome and all complete bacterial genomes in the RefSeq database in an effort to identify bacterial read pairs from the microbiome. RESULTS: Through careful consideration of all of the bacterial taxa present in the cancer types investigated, their relative abundance, and batch effects, we were able to identify some read pairs from certain taxa as likely resulting from contamination. In particular, the presence of Mycobacterium tuberculosis complex in the ovarian serous cystadenocarcinoma (OV) and glioblastoma multiforme (GBM) samples was correlated with the sequencing center of the samples. Additionally, there was a correlation between the presence of Ralstonia spp. and two specific plates of acute myeloid leukemia (AML) samples. At the end, associations remained between Pseudomonas-like and Acinetobacter-like read pairs in AML, and Pseudomonas-like read pairs in stomach adenocarcinoma (STAD) that could not be explained through batch effects or systematic contamination as seen in other samples. CONCLUSIONS: This approach suggests that it is possible to identify bacteria that may be present in human tumor samples from public genome sequencing data that can be examined further experimentally. More weight should be given to this approach in the future when bacterial associations with diseases are suspected.

Clostridium novyi-NT can cause regression of orthotopically implanted glioblastomas in rats.

Glioblastoma (GBM) is a highly aggressive primary brain tumor that is especially difficult to treat. The tumor's ability to withstand hypoxia leads to enhanced cancer cell survival and therapy resistance, but also yields a microenvironment that is in many aspects unique within the human body, thus offering potential therapeutic opportunities. The spore-forming anaerobic bacterium Clostridium novyi-NT(C. novyi-NT) has the ability to propagate in tumor-generated hypoxia, leading to oncolysis. Here, we show that intravenously injected spores of C. novyi-NT led to dramatic tumor destructions and significant survival increases in implanted, intracranial syngeneic F98 and human xenograft 060919 rat GBM models. C. novyi-NT germination was specific and confined to the neoplasm, with sparing of the normal brain parenchyma. All animals tolerated the bacteriolytic treatment, but edema and increased intracranial pressure could quickly be lethal if not monitored and medically managed with hydration and antibiotics. These results provide pre-clinical data supporting the development of this therapeutic approach for the treatment of patients with GBM.

Expression of insulin-like growth factor binding protein-2 (IGFBP-2) gene in negative and positive human cytomegalovirus glioblastoma multiforme tissues.

Glioblastoma multiforme (GBM), a WHO grade IV malignant glioma, is the most common and lethal primary brain tumor in adults and has but few treatments. The median survival of glioblastoma patients is 12 months. The (possible) relationship between human cytomegalovirus (HCMV) infection and cancer has been investigated for decades. Detection of viral DNA, mRNA and/or antigens in tumor tissues suggests that HCMV infection has a role to play in the etiology of several human malignancies. HCMV gene products can promote the various signaling pathways critical to tumor growth, including platelet derived growth factor receptor, phosphatidyl inositol 3-kinases (PI3K/AKT), signal transducer and activator of transcription 3 and glycogen synthase kinase 3 beta that are involved in apoptosis, angiogenesis, invasion and immune evasion. Insulin-like growth factor binding protein 2 (IGFBP2) is a biomarker of the PI3K/AKT pathway so we decided to evaluate the expression of this gene in 3 groups: HCMV-negative GBM tissues, HCMV-positive GBM tissues and non-tumor tissues. The presence of HCMV was assessed according to our previous article. HCMV was present in %75 of glioblastoma tissues. Then RNA was extracted, cDNA was synthesized, and real-time PCR was performed. Then, the rate of increased expression was calculated using the Livac or 2(-ΔΔCt). ΔCt of samples in the three groups were compared using analysis of variance (ANOVA). The expression of IGFBP2 gene relative to GAPDH gene in HCMV-negative glioblastoma tissues and HCMV-positive glioblastoma tissues, respectively, was increased 5.486 and 15.032 times compared to non-neoplastic brain tissues. ANOVA tests showed that the difference of mean ΔCt for IGFBP2 gene between healthy subjects and patients with HCMV-positive and HCMV-negative glioblastoma tumors statistically significant.

High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (µCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and µCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.

Concomitant presentation of a glioblastoma multiforme with superimposed abscess.

BACKGROUND: The coexistence of a primary brain tumor such as high-grade glioma and superimposed abscess is a rare entity and can present a diagnostic and therapeutic challenge. The concomitant abscess may not be recognized until surgery, and the overall course of treatment may require adjustment in the presence of a coinciding infection. In the present report we evaluate the diagnosis and treatment of a glioblastoma multiforme with an intratumoral abscess. METHODS: A patient was diagnosed with a glioblastoma multiforme with a concomitantly superimposed multimicrobial abscess containing coagulase-negative Staphylococcus, Acinetobacter iwofii, and Propionibacterium species. The suspected infectious source was a dental abscess with presumed secondary seeding. The patient underwent a left anterior temporal lobectomy with debulking of the lesion. Although the adjuvant therapy schedule was adjusted to accommodate the course of antibiotics, the existence of a concurrent abscess did not preclude adjuvant radiation and chemotherapy. RESULTS: The patient responded well to antibiotic treatment with no evidence of recurrent infection. He underwent a second operation for additional debulking of the lesion approximately half a year after his initial surgery. The patient died 2 years after the initial diagnosis. CONCLUSIONS: There are insufficient guidelines on the treatment of a primary brain tumor with intratumoral abscess. In this report we present our therapeutic decisions in this rare case.

Expression of the reporter genes lacZ and EGFP in human glioblastoma cells using Listeria monocytogenes as vector for gene transfer.

OBJECTIVE: Glioblastoma has become a model target for viral gene therapy approaches. To circumvent some of the inherent problems of viral vectors, we examined the feasibility of Listeria monocytogenes for foreign gene expression in glioblastoma cells. METHODS: The internalin-dependent uptake of L. monocytogenes into human glioma cell lines and into 22 cell cultures obtained during neurosurgery was studied by in vitro infection assays. Internalization rates of wild-type L. monocytogenes were compared with a deletion mutant and to four strains with plasmid-based recomplementation of the internalin A/B operon. For gene transfer experiments, plasmids encoding lacZ or EGFP under control of the cytomegalovirus promotor were transfected into Listeriae. Incubation times in cell culture were studied regarding infection of cells, intracellular listerial replication and reporter gene translation. Gene transfer efficiency was determined by beta-galactosidase activity and by fluorescence microscopy. RESULTS: The internalin-defective strain (DeltainlAB2) showed less uptake compared with the wild type. An increased invasion rate was observed in strain DeltainlAB2 + inlB(PinlA) overexpressing internalin B; complementation of the entire operon (DeltainlAB2 + inlAB(PinlA)) revealed a synergistic effect. For gene transfer experiments into glioblastoma cells, the pathogenic strain 4bL312 was employed, and efficiencies up to 5% were achieved. DISCUSSION: Internalins A and B are major determinants for listerial uptake into neuroepithelial tumors. The L. monocytogenes vector system enables foreign gene expression in glioblastoma cells. Ongoing research deals with optimization of transfection efficiencies, with the use of less pathogenic substrains and the cloning of suicide gene vectors.

Identification of Staphylococcus aureus brain abscesses: rat and human studies with 1H MR spectroscopy.

PURPOSE: To determine the feasibility of a statistical classification strategy (SCS) and the identity of metabolites of bacterial and host origins that potentially contributed to the most discriminatory regions of magnetic resonance (MR) spectra from Staphylococcus aureus abscesses of biopsy material from controls, gliomas, and staphylococcal abscesses. MATERIALS AND METHODS: Human and animal study received ethics committee approval, and informed patient consent was obtained. A rat model of S aureus brain abscess was developed. Histologic and microbiologic examination was performed to assess abscess development 3-4, 6-8, and 10-15 days after initiation. Metabolite profiles in pus (n = 62) and controls (n = 37) were characterized with ex vivo MR spectroscopy and compared with data from rat gliomas (n = 27). SCS, optimal region selection, and development of pairwise classifiers allowed MR spectra of abscesses (n = 42, day 6-8) to be distinguished from those of glioblastoma multiforme and controls. MR spectroscopy profiles of pus from animal abscesses were compared with in vivo MR spectra from patients with staphylococcal brain abscesses (n = 7, aged 6-67 years) and ex vivo pus MR spectra from patients with S aureus abscesses. RESULTS: Histologically confirmed abscesses were present 6-8 days after stereotactic injection of S aureus in 42 of 47 rats (89%). MR spectra of abscesses and glioblastoma multiforme in the animal model were similar. Typical metabolites of abscesses due to anaerobe bacteria (acetate, succinate, amino acids) were not detectable in S aureus abscesses in rats or humans. MR spectroscopic findings from controls, abscesses, and gliomas were distinguished by means of SCS with an accuracy of 99%. Analysis of the most discriminatory regions with two-dimensional correlation spectra indicated that glutamine and/or glutamate and aspartate potentially contributed to successful classification. CONCLUSION: S aureus is detectable in abscesses with a non-culture-based method in an animal model.

Reduced levels of IE2 gene expression and shutdown of early and late viral genes during latent infection of the glioblastoma cell line U138-MG with selectable recombinants of human cytomegalovirus.

To establish stable culture conditions which support persistence of the human cytomegalovirus (HCMV) genome in a latent state, the expression of the bacterial neomycin phosphotransferase (neo) from HCMV recombinants was used for selection. Different cell lines were infected with HCMV recombinants. The human glioblastoma line U138-MG was rendered resistant to G418 and retained the viral genome. More than 90% of the cells expressed the viral IE1 protein of 72 kDa for a culture period of 18 months. Many fewer cells expressed IE2-encoded proteins. No late gene expression or infectious virus was detectable. IE2 gene expression in latently infected cells appeared to be restricted at the level of RNA accumulation. Treatment with TPA or retinoic acid led to enhanced expression of the IE2 gene and the early genes encoding pp65 (UL83) and p52 (UL44). Superinfection with wild-type HCMV led to replication of neo-recombinant virus, indicating that replication-competent virus had been retained in latently infected U138-MG and that the cells had kept their permissive phenotype. Latent HCMV infection in U138-MG cells provides a useful model system for studying the role of particular viral and cellular genes in latent and permissive infections.

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.

Studies on the SV40-like papovavirus SV40-GBM. II. Molecular cloning in Escherichia coli of variant DNA molecules from glioblastoma-derived virus.

The complete DNA genomes of the SV40-like GBM virus (GBM1), isolated from a human glioblastoma multiforme, and of two discrete classes of GBM DNA molecules that appear following three passages in CV-1 monkey cells at low multiplicities (GBM3-H and GBM3-L), were cloned in Escherichia coli using plasmid vector pBR322. The cloned viral DNAs were characterized (i) by digestion of the chimeric plasmid DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose with that of similarly digested uncloned DNA, (ii) by hybridization of digested chimeric plasmid DNAs to 32P-labeled uncloned GBM DNA, and (iii) by electron microscopy. In restriction enzyme analysis the cloned GBM DNAs showed the same cleavage pattern as the uncloned DNAs, indicating that no major insertions or deletions were present. The electrophoretic data were confirmed by electron microscopic heteroduplex analysis.

Glial and divergent cells in primate central nervous system tumors induced by JC virus isolated from human progressive multifocal leukoencephalopathy (PML).

Immunofluorescent stains for fibronectin (FN) and glial fibrillary acidic protein (GFAP) were used in conjunction with routine histologic stains to study tumors induced in squirrel and owl monkeys by JC virus from progressive multifocal leukoencephalopathy (PML). Three varieties of glioma were identified. The first and most common variety was a neoplasm similar to grade 4 astrocytoma in humans. The second had thin, normal-appearing FN-positive vessel walls and a vastly expanded neuroectodermal parenchyma which could not be characterized by routine histologic stains. Anti-GFAP revealed the glial nature of the parenchyma. Isolating glial parenchymal cells from divergent FN-positive cells has become important to neurooncology. This type of tumor may be of particular interest for such isolations due to its high ratio of glial cells to divergent cells. The third variety was not a homogeneous neoplasm. It occurred as focal regions within tumors of the first type, and consisted of giant cells with huge nuclei. These cells resemble the cells of a human giant cell glioblastoma and bear a slight similarity to the bizarre glial cells seen in PML. The rare human giant cell glioblastoma might have an association with JC virus or with PML.

Diverse familial malignant tumors and Epstein-Barr virus.

Continued monitoring of a family for new malignant tumors has revealed diverse immunological and neoplastic disorders during a 15-year period. In 1966, the proband developed lymphoma. In 1975, his antibody titers to Epstein-Barr virus (EBV) became elevated, and again, he developed a malignant lymphoma. He also had borderline hypo-immunoglobulin A, died of glioblastoma multiforme in 1977, and at autopsy, had adenomatous colonic polyps. His eldest brother has normal immunoglobulin levels, but developed immune thrombocytopenia in 1973 and had elevated EBV antibody titers in 1980. Another brother had hypo-immunoglobulin A, thymoma in 1965, and adenomas and adenocarcinoma of the colon. Two other brothers succumbed to glioblastoma in 1968 and 1969. The father of the proband had bronchiectasis in 1952, hypo-immunoglobulin M documented in 1972, and elevated EBV antibody titers 5 years preceding development of a malignant lymphoma. The latter contained 10 EBV genome equivalents/cell by EBV viral DNA/DNA reassociation kinetics analysis. The proband's grandmother had died of an immunoglobulin G-secreting myeloma in 1977, and his grandfather had borderline low immunoglobulin M, elevated EBV antibody titers, and hypopharyngeal carcinoma in 1980. Predisposition to oncogenesis in this family was probably inherited.

Isolation of a SV40-like Papovavirus from a human glioblastoma.

A human glioblastoma multiforme (M27) tested in early cell cultures by indirect immunofluorescence staining showed SV40-related tumor (T)-antigen, 95% of the cells being positive. SV40-related viral capsid (V)-antigen was absent in all cells tested. Experiments to rescue this virus were performed by fusing M27 cells with CV-I monkey cells, which were permissive for SV40, using polyethylene glycol (PEG) as fusion factor. We succeeded in isolating virus particles SV40-GBM which electron microscopy showed to correspond in size and morphology to papovaviruses. Serological tests (hemagglutination, neutralization, fluorescent antibody) revealed that the virus is indistinguishable from SV40. Despite this apparent antigenic identity SV40-GBM differs slightly from SV40 wild type. This virus can propagate and produce CPE in both CV-I cells and primary fetal human kidney cells. Furthermore digestion of SV40-GBM DNA with the HindII/III restriction endonucleases revealed minor differences compared with the SV40 DNA. Therefore the virus SV40-GBM obtained from glioblastoma cells seems to be closely related to the SV40-PML viruses described earlier.

Particles with RNA of high molecular weight and RNA-directed DNA polymerase in human brain tumors.

We have previously shown that neoplastic cells of human breast cancers, leukemias, lymphomas, and sarcomas contain particles similar to the viruses that have been established as etiologic agents of these diseases in mice. The present paper concerns tumors of the central nervous system for which no suitable animal model or corresponding virus exists. Nevertheless, using the simultaneous detection test, we showed that human brain tumors contain 70S RNA and RNA-directed DNA polymerase encapsulated in a particulate component possessing a density of 1.17 g/ml. These particles satisfy the three diagnostic criteria that characterize RNA tumor viruses of animals. 24 Out of 26 (92%) of the most malignant (glioblastoma and medulloblastoma) brain tumors examined contained these virus-like entities.