Tumor recurrence following treatment is the major cause of mortality for glioblastoma multiforme (GBM) patients. Thus, insights on the evolutionary process at recurrence are critical for improved ...patient care. Here, we describe our genomic analyses of the initial and recurrent tumor specimens from each of 38 GBM patients. A substantial divergence in the landscape of driver alterations was associated with distant appearance of a recurrent tumor from the initial tumor, suggesting that the genomic profile of the initial tumor can mislead targeted therapies for the distally recurred tumor. In addition, in contrast to IDH1-mutated gliomas, IDH1-wild-type primary GBMs rarely developed hypermutation following temozolomide (TMZ) treatment, indicating low risk for TMZ-induced hypermutation for these tumors under the standard regimen.
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•Distant recurrence predicts branched evolution of the paired tumors•Distant recurrence frequently involves divergence in key GBM driver alterations•Recurrent GBMs have more aberrations in core GBM driver pathways than initial GBMs•TMZ-induced hypermutation is rare in IDH1-wild-type primary GBMs
Kim et al. find that glioblastomas recurring at distant sites have driver genetic alterations very different from those of matched initial tumors. They also show that, in contrast to IDH1-mutated tumors, IDH1-wild-type primary glioblastomas rarely develop hypermutation following temozolomide treatment.
Metabolic associated fatty liver disease (MAFLD) is a new concept where the presence of both fatty liver and metabolic abnormality are necessary for diagnosis. Several studies have reported that ...altered gut microbiome is closely associated with metabolic diseases and non-alcoholic fatty liver disease. However, the studies on MAFLD population are scarce. This prospective study aimed to identify differences in gut microbiome between patients with MAFLD and healthy controls in Korean population. In this study, patients with MAFLD and age, sex-matched healthy controls were included, and their stool samples were collected. Taxonomic composition of gut microbiota was analyzed using 16S ribosomal ribonucleic acid pyrosequencing. Twenty-two MAFLD patients and 44 healthy controls were included. Taxonomic diversity was lower in patients with MAFLD in the aspect of alpha and beta diversity. The differences were also found at phylum, class, family, and genus levels between the two groups. Phylum
, family
, genus
abundance was significantly increased and genus
was significantly decreased in patients with MAFLD. In addition, butyrate-producing bacteria were decreased and ethanol-producing bacteria were increased in patients with MAFLD. The composition of gut microbiome was different between MAFLD and healthy controls in Korean population. This could offer potential targets for therapeutic intervention in MAFLD.
Transfusion-associated graft-versus-host disease (TA-GvHD) is caused by leukocytes, specifically T cells within a transfused blood product. Currently, the prevention of transfusion-associated ...graft-versus-host disease is performed by irradiation of blood products. With a sufficient reduction of leukocytes, the risk for TA-GvHD can be decreased. With consistent advances in current state-of-the-art blood filters, we herein propose that double filtration can sufficiently reduce leukocytes to reduce the risk for TA-GvHD.
Thirty RBC concentrates were filtered with leukocyte filters, followed by storage at 1-6 oC for 72 hours, and then a second filtration was performed. Residual leukocytes in the double-filtered RBC units (n = 30) were assessed with flow cytometric methods, and an additional assay with isolated peripheral blood mononuclear cells (PBMCs) (n = 6) was done by both flow cytometric methods and an automated hematology analyzer. Quality of the RBCs after filtration was evaluated by hematological and biochemical tests. In vitro T cell expansion was performed using anti-CD3/CD28-coated Dynabeads or anti-CD3 (OKT3). In vivo experiment for GvHD was performed by using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice.
Double-filtered blood products showed residual leukocyte levels below detection limits, which calculated to be below 1200-2500 cells per blood unit. In vitro expansion rate of T cells showed that 6x103 and 1x103 cell-seeded specimens showed 60.8±10.6 fold and 10.2±9.7-fold expansion, respectively. Cell expansion was not sufficiently observed in wells planted with 1x102 or 10 cells. In vivo experiments showed that mice injected with 1x105 or more cells cause fatal GvHD. GvHD induced inflammation was observed in mice injected with 1x104 or more cells. No evidence of GvHD was found in mice injected with 103 cells.
Our study suggests that additional removal of contaminating lymphocytes by a second leukodepletion step may further reduce the risk for TA-GvHD.
Recent studies indicate that signaling molecules traditionally associated with central nervous system function play critical roles in cancer. Dopamine receptor signaling is implicated in various ...cancers including glioblastoma (GBM) and it is a recognized therapeutic target, as evidenced by recent clinical trials with a selective dopamine receptor D2 (DRD2) inhibitor ONC201. Understanding the molecular mechanism(s) of the dopamine receptor signaling will be critical for development of potent therapeutic options. Using the human GBM patient-derived tumors treated with dopamine receptor agonists and antagonists, we identified the proteins that interact with DRD2. DRD2 signaling promotes glioblastoma (GBM) stem-like cells and GBM growth by activating MET. In contrast, pharmacological inhibition of DRD2 induces DRD2-TRAIL receptor interaction and subsequent cell death. Thus, our findings demonstrate a molecular circuitry of oncogenic DRD2 signaling in which MET and TRAIL receptors, critical factors for tumor cell survival and cell death, respectively, govern GBM survival and death. Finally, tumor-derived dopamine and expression of dopamine biosynthesis enzymes in a subset of GBM may guide patient stratification for DRD2 targeting therapy.
Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural ...conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.
Although temozolomide (TMZ) has been used as a standard adjuvant chemotherapeutic agent for primary glioblastoma (GBM), treating isocitrate dehydrogenase wild-type (IDH-wt) cases remains challenging ...due to intrinsic and acquired drug resistance. Therefore, elucidation of the molecular mechanisms of TMZ resistance is critical for its precision application.
We stratified 69 primary IDH-wt GBM patients into TMZ-resistant (n = 29) and sensitive (n = 40) groups, using TMZ screening of the corresponding patient-derived glioma stem-like cells (GSCs). Genomic and transcriptomic features were then examined to identify TMZ-associated molecular alterations. Subsequently, we developed a machine learning (ML) model to predict TMZ response from combined signatures. Moreover, TMZ response in multisector samples (52 tumor sectors from 18 cases) was evaluated to validate findings and investigate the impact of intra-tumoral heterogeneity on TMZ efficacy.
In vitro TMZ sensitivity of patient-derived GSCs classified patients into groups with different survival outcomes (P = 1.12e-4 for progression-free survival (PFS) and 3.63e-4 for overall survival (OS)). Moreover, we found that elevated gene expression of EGR4, PAPPA, LRRC3, and ANXA3 was associated to intrinsic TMZ resistance. In addition, other features such as 5-aminolevulinic acid negative, mesenchymal/proneural expression subtypes, and hypermutation phenomena were prone to promote TMZ resistance. In contrast, concurrent copy-number-alteration in PTEN, EGFR, and CDKN2A/B was more frequent in TMZ-sensitive samples (Fisher's exact P = 0.0102), subsequently consolidated by multi-sector sequencing analyses. Integrating all features, we trained a ML tool to segregate TMZ-resistant and sensitive groups. Notably, our method segregated IDH-wt GBM patients from The Cancer Genome Atlas (TCGA) into two groups with divergent survival outcomes (P = 4.58e-4 for PFS and 3.66e-4 for OS). Furthermore, we showed a highly heterogeneous TMZ-response pattern within each GBM patient using in vitro TMZ screening and genomic characterization of multisector GSCs. Lastly, the prediction model that evaluates the TMZ efficacy for primary IDH-wt GBMs was developed into a webserver for public usage ( http://www.wang-lab-hkust.com:3838/TMZEP ).
We identified molecular characteristics associated to TMZ sensitivity, and illustrate the potential clinical value of a ML model trained from pharmacogenomic profiling of patient-derived GSC against IDH-wt GBMs.
Abstract The influenza virus, a mucosal pathogen that infects the respiratory tract, is a major global health issue. There have been attempts to mucosally administer inactivated influenza vaccines to ...induce both mucosal and systemic immune responses. However, mucosally administered inactivated influenza vaccine has low immunogenicity, which is partially due to the lack of an effective mucosal adjuvant. The development of a safe and effective mucosal adjuvant is a prerequisite to the practical use of a mucosal inactivated influenza vaccine. We have previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, when mixed with antigen and administered intranasally, exerts a strong mucosal adjuvant activity by stimulating the Toll-like receptor 5 (TLR5). In this study, we tested whether the FlaB protein could serve as an effective mucosal adjuvant for an inactivated trivalent influenza vaccine (TIV) manufactured for humans; in a murine vaccination model, this vaccine consists of A/Brisbane/59/07 (H1N1 subtype), A/Uruguay/716/07 (H3N2 subtype), and B/Florida/4/06 (B type). Intranasal co-administration of the TIV with FlaB induced prominent humoral responses as demonstrated by high influenza-specific IgA levels in both the mucosal secretions and serum and significant specific IgG induction in the systemic compartment. The FlaB protein significantly potentiated influenza-specific cytokine production by draining lymph node cells and splenocytes. The FlaB mucosal adjuvant conferred excellent protection against a lethal challenge with a live virulent virus with high hemagglutination inhibition (HAI) antibody (Ab) titers. The FlaB did not accumulate in the olfactory nerve and epithelium, guaranteeing against a retrograde uptake into the central nervous system. These results suggest that FlaB can be used as a promising mucosal adjuvant for nasal inactivated influenza vaccine development.
The physiological conditions of small animals are an essential component to be considered when acquiring images for pre-clinical studies, and they play a vital role in the overall results of a study. ...However, several previous studies did not consider these conditions. In this study, a new animal cradle that can be modified and adjusted to suit multiple imaging modalities such as positron emission tomography (PET)/computed tomography (CT) and magnetic resonance imaging (MRI) was developed. Unlike previous cradles where only one mouse can be imaged at a time, a total of four mice can be imaged simultaneously using this new cradle. Additionally, fusion images with high-throughput multiple-mouse imaging (MMI) of PET/MRI and PET/CT images can be acquired using this newly developed cradle. The dynamic brain images were also acquired simultaneously by applying PET dynamic imaging technology to high-throughput MMI methods. The results of this study suggest that the newly developed small animal cradle can be widely used in pre-clinical studies.
Hepatocyte growth factor receptor (HGFR, c-Met) is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic ...state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth.
Oncolytic viruses are of significant clinical interest due to their ability to directly infect and kill tumors and enhance the anti-tumor immune response. Previously, we developed KLS-3010, a novel ...oncolytic virus derived from the International Health Department-White (IHD-W) strain vaccinia virus, which has robust tumoricidal effects. In the present study, we generated a recombinant oncolytic virus, KLS-3020, by inserting three transgenes (hyaluronidase PH-20, interleukin-12 IL-12, and soluble programmed cell death 1 fused to the Fc domain sPD1-Fc) into KLS-3010 and investigated its anti-tumor efficacy and ability to induce anti-tumor immune responses in CT26.WT and B16F10 mouse tumor models. A single injection of KLS-3020 significantly decreased tumor growth. The roles of the transgenes were investigated using viruses expressing each single transgene alone and KLS-3020. PH-20 promoted virus spread and tumor immune cell infiltration, IL-12 activated and reprogrammed T cells to inflammatory phenotypes, and sPD1-Fc increased intra-tumoral populations of activated T cells. The tumor-specific systemic immune response and the abscopal tumor control elicited by KLS-3020 were demonstrated in the CT26.WT tumor model. The insertion of transgenes into KLS-3020 increased its anti-tumor efficacy, supporting further clinical investigation of KLS-3020 as a novel oncolytic immunotherapy.
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Kim and colleagues improved their previous recombinant oncolytic vaccinia virus by arming it with PH-20, IL-12, and soluble PD1-Fc, leading to the significantly further increase of the number of cytotoxic T cells activated in tumor and marked suppression of tumor growth against solid tumors such as colon cancer and melanoma.
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