Spatiotemporal modeling reveals high-resolution invasion states in glioblastoma.

BACKGROUND: Diffuse invasion of glioblastoma cells through normal brain tissue is a key contributor to tumor aggressiveness, resistance to conventional therapies, and dismal prognosis in patients. A deeper understanding of how components of the tumor microenvironment (TME) contribute to overall tumor organization and to programs of invasion may reveal opportunities for improved therapeutic strategies. RESULTS: Towards this goal, we apply a novel computational workflow to a spatiotemporally profiled GBM xenograft cohort, leveraging the ability to distinguish human tumor from mouse TME to overcome previous limitations in the analysis of diffuse invasion. Our analytic approach, based on unsupervised deconvolution, performs reference-free discovery of cell types and cell activities within the complete GBM ecosystem. We present a comprehensive catalogue of 15 tumor cell programs set within the spatiotemporal context of 90 mouse brain and TME cell types, cell activities, and anatomic structures. Distinct tumor programs related to invasion align with routes of perivascular, white matter, and parenchymal invasion. Furthermore, sub-modules of genes serving as program network hubs are highly prognostic in GBM patients. CONCLUSION: The compendium of programs presented here provides a basis for rational targeting of tumor and/or TME components. We anticipate that our approach will facilitate an ecosystem-level understanding of the immediate and long-term consequences of such perturbations, including the identification of compensatory programs that will inform improved combinatorial therapies.

Single-cell landscapes of primary glioblastomas and matched explants and cell lines show variable retention of inter- and intratumor heterogeneity.

Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive inter- and intratumor heterogeneity. Patient-derived models, such as organoids and explants, have recently emerged as useful models to study such heterogeneity, although the extent to which they can recapitulate GBM genomic features remains unclear. Here, we analyze bulk exome and single-cell genome and transcriptome profiles of 12 IDH wild-type GBMs, including two recurrent tumors, and of patient-derived explants (PDEs) and gliomasphere (GS) lines derived from these tumors. We find that PDEs are genetically similar to, and variably retain gene expression characteristics of, their parent tumors. Notably, PDEs appear to exhibit similar levels of transcriptional heterogeneity compared with their parent tumors, whereas GS lines tend to be enriched for cells in a more uniform transcriptional state. The approaches and datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived models, especially in the context of studying cellular heterogeneity.

Glioma-derived IL-33 orchestrates an inflammatory brain tumor microenvironment that accelerates glioma progression.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.

Development of a peptide-based delivery platform for targeting malignant brain tumors.

Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.

Eukaryotic initiation factor 5B (eIF5B) regulates temozolomide-mediated apoptosis in brain tumour stem cells (BTSCs).

Glioblastoma multiforme (GBM) is among the deadliest cancers, owing in part to complex inter- and intra-tumor heterogeneity and the presence of a population of stem-like cells called brain tumour stem cells (BTSCs/BTICs). These cancer stem cells survive treatment and confer resistance to the current therapies - namely, radiation and the chemotherapeutic, temozolomide (TMZ). TMZ induces cell death by alkylating DNA, and BTSCs resist this mechanism via a robust DNA damage response. Hence, recent studies aimed to sensitize BTSCs to TMZ using combination therapy, such as inhibition of DNA repair machinery. We have previously demonstrated in established GBM cell lines that eukaryotic initiation factor 5B (eIF5B) promotes the translation of pro-survival and anti-apoptotic proteins. Consequently, silencing eIF5B sensitizes these cells to TRAIL-induced apoptosis. However, established cell lines do not always recapitulate the features of human glioma. Therefore, we investigated this mechanism in patient-derived BTSCs. We show that silencing eIF5B leads to increased TMZ sensitivity in two BTSC lines: BT25 and BT48. Depletion of eIF5B decreases the levels of anti-apoptotic proteins in BT48 and sensitizes these cells to TMZ-induced activation of caspase-3, cleavage of PARP, and apoptosis. We suggest that eIF5B represents a rational target to sensitize GBM tumors to the current standard-of-care.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

A Small Molecule Targeting the Transmembrane Domain of Death Receptor p75NTR Induces Melanoma Cell Death and Reduces Tumor Growth.

Small molecules offer powerful ways to alter protein function. However, most proteins in the human proteome lack small-molecule probes, including the large class of non-catalytic transmembrane receptors, such as death receptors. We hypothesized that small molecules targeting the interfaces between transmembrane domains (TMDs) in receptor complexes may induce conformational changes that alter receptor function. Applying this concept in a screening assay, we identified a compound targeting the TMD of death receptor p75NTR that induced profound conformational changes and receptor activity. The compound triggered apoptotic cell death dependent on p75NTR and JNK activity in neurons and melanoma cells, and inhibited tumor growth in a melanoma mouse model. Due to their small size and crucial role in receptor activation, TMDs represent attractive targets for small-molecule manipulation of receptor function.

Inclusion body myopathy with Paget disease of bone and frontotemporal dementia linked to VCP p.Arg155Cys in a Korean family.

BACKGROUND: Missense mutations in the valosin-containing protein (VCP) gene on chromosome 9p13.3-p12 cause inclusion body myopathy with Paget disease of bone and frontotemporal dementia (hereafter referred to as IBMPFD; OMIM 167320). OBJECTIVE: To describe detailed clinical, electrophysiological, biochemical, and neuroimaging findings in IBMPFD linked to VCP p.Arg155Cys in a Korean family. DESIGN: Case series. Clinical, electrophysiological, biochemical, and neuroimaging findings were obtained by direct evaluation and from previous medical records. SETTING: Tertiary referral hospital. PARTICIPANTS: Three affected family members in a Korean family. RESULTS: The clinical features of myopathy, Paget disease of bone, and semantic dementia (a clinical subtype of frontotemporal dementia) in our patients were similar to those of previously reported cases. However, the brain magnetic resonance imaging features in our patients, including asymmetric anterior and lateral temporal and inferior parietal atrophy with ventricular dilatation on the affected side, differed from those of previously published features in patients with IBMPFD and in patients with typical semantic dementia who show anterior temporal and frontal atrophy. CONCLUSION: To our knowledge, this report provides the first documented IBMPFD family in Asia and broadens the phenotypic spectrum of VCP mutation-associated frontotemporal dementia.

Genetic screen identifies insulin-like growth factor binding protein 5 as a modulator of tamoxifen resistance in breast cancer.

Tamoxifen resistance is one of the overarching challenges in the treatment of patients with estrogen receptor (ER)-positive breast cancer. Through a genome-wide RNA interference screen to discover genes responsible for tamoxifen resistance in vitro, we identified insulin-like growth factor binding protein 5 (IGFBP5) as a determinant of drug sensitivity. Specific knockdown of IGFBP5 by retroviral infection with short hairpin RNA-expressing cassette in MCF7 human breast cancer cells (pRS-shIGFBP5) conferred tamoxifen resistance in vitro due to concomitant loss of ERalpha expression and signaling. IGFBP5 expression was also reduced in MCF7 cells selected for tamoxifen resistance in culture (TAMR). Both tamoxifen-resistant MCF7-TAMR and MCF7-pRS-shIGFBP5 cells could be resensitized to drug by treatment with exogenous recombinant IGFBP5 (rIGFBP5) protein. Treatment with rIGFBP5 protein in mouse tumor xenografts reversed the in vivo tamoxifen resistance of MCF7-pRS-shIGFBP5 cell-derived tumors by reducing tumor cell proliferation. IGFBP5 immunohistochemical staining in a cohort of 153 breast cancer patients showed that low IGFBP5 expression was associated with shorter overall survival after tamoxifen therapy. Thus, IGFBP5 warrants investigation as an agent to reverse tamoxifen resistance.

Adjuvant effect of CIA07, a combination of Escherichia coli DNA fragments and modified lipopolysaccharides, on the immune response to hepatitis B virus surface antigen.

CIA07 is an immunostimulatory agent composed of bacterial DNA fragments and modified lipopolysaccharide, which has antitumor activity against bladder cancer in mice. In this study, the adjuvant activity of CIA07 was evaluated using hepatitis B virus surface antigen (HBsAg) as the immunogen. Mice were immunized intramuscularly three times at 1-week intervals with HBsAg alone or in combination with alum, bacterial DNA fragments, modified lipopolysaccharide, CIA07 or CpG1826, and immune responses were assessed. At 1 week after the final injection, the HBsAg-specific total serum IgG antibody titer in CIA07-treated mice was 14 times higher than that in animals administered antigen alone, six times higher than in mice given alum or bacterial DNA fragments and twice as high as those treated with modified lipopolysaccharide or CpG1826, and remained maximal until 8 weeks postimmunization. Animals receiving antigen alone or plus alum displayed barely detectable HBsAg-specific serum IgG2a antibody responses. However, coadministration of CIA07 with antigen led to markedly enhanced serum IgG2a antibody titer and IFN-gamma(+) production in splenocytes, indicating that CIA07 effectively induces Th1-type immune responses. In addition, the number of HBsAg-specific CD8(+) T cells in peripheral blood mononuclear cells was elevated in CIA07-treated mice. These data clearly demonstrate that CIA07 is able to induce both cellular and humoral immune responses to HBsAg, and confirm its potential as an adjuvant in therapeutic vaccines for hepatitis B virus infections.

Isolated monocular visual loss as an initial manifestation of polycythemia vera.

A 25-year-old man developed prolonged loss of vision in the left eye. Examination revealed that visual acuity was 20/20 in the right eye and 10/20 in the left eye, with a left relative afferent pupillary defect. Fundoscopy showed multiple cotton wool spots in the left whole retina with normal optic disc. Fluorescein angiography (FA) revealed markedly delayed arterial, venous and recirculation time in the left eye without retinal arterial or venous occlusion. Bone marrow aspirate confirmed polycythemia vera. After the patient underwent phlebotomy, his visual acuity markedly improved and cotton wool spots in the retina disappeared. On follow-up FA, delayed arterial and venous filling, and recirculation time also became normalized. This case suggests that ischemic damage of the retina due to the great viscosity of blood may be a possible mechanism of monocular visual loss in polycythemia vera. Clinicians should be aware that isolated monocular visual loss may be an initial manifestation of polycythemia vera, since if untreated, polycythemia vera carries a high risk of permanent complications due to intravascular thrombosis.

Bacterial DNA containing methylated CpG motifs retains immunostimulatory activity in synergy with modified lipopolysaccharides.

We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF-kappaB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs.

A combination of E. coli DNA fragments and modified lipopolysaccharides as a cancer immunotherapy.

The use of Escherichia coli DNA or lipopolysaccharide (LPS) as an immunotherapy is often associated with unacceptable toxicity and insufficient therapeutic effects. In this study, we investigated the efficacy of using a combination of bacterial DNA fragments and LPS as an anticancer agent. LPS was isolated from an E. coli strain expressing short-carbohydrate-chain-containing LPS and subjected to alkaline hydrolysis to remove lipid A. The ability to induce tumor necrosis factor-alpha (TNF-alpha) release in human whole blood cells was significantly lower for the LPS devoid of lipid A than for its parent form. The immunostimulating activity of E. coli DNA fragments of various sizes were tested. Those of 0.2-0.5 kb in size exhibited the highest activity in whole blood assays, whereas those of size 0.5-2.0 kb exhibited the highest adjuvant activity in mice. A combination of 0.5-2.0-kb DNA fragments and modified LPS at a ratio of 100:1, designated CIA07, exhibited higher immunostimulating activity than each substance alone, and its antitumor activity was significantly higher than that of Bacillus Calmette-Guerin in a mouse bladder cancer model. An intraperitoneal injection of CIA07 at a dose of 25mg/kg body weight caused no apparent adverse effects in mice and guinea pigs. Taken together, these data demonstrate that CIA07 exhibits potent immunostimulating activity with no apparent toxicity, and therefore warrant the further development of CIA07 as an immunotherapy for cancer treatment.

Comparative proteomic analysis of Helicobacter pylori strains associated with iron deficiency anemia.

Helicobacter pylori is known to cause chronic gastritis, peptic ulcer, and gastric cancer, and has also been linked to iron deficiency anemia (IDA). To determine whether H. pylori clinical isolates correlate with the prevalence of H. pylori-associated IDA, we compared the proteomic profiles of H. pylori strains isolated from antral biopsy specimens of H. pylori-positive patients with or without IDA. Fifteen strains, including eight non-IDA and seven IDA strains, were cultured under iron-rich and iron-depleted conditions and then analyzed for protein expression profiles by 2-DE. The distances between two H. pylori strains were determined on the basis of similarities between their expression patterns of 189 protein spots, and a phylogenetic tree was constructed. The results revealed that the IDA strains formed a cluster separate from that of six non-IDA strains, with two non-IDA strains between the clusters. H. pylori strain 26695 was located in the non-IDA cluster. Protein spots displaying similar expression patterns were clustered, and 18 spots predominantly expressed in IDA strains were identified by MALDI-TOF analysis. These data indicate that the non-IDA and IDA strains can be distinguished by their protein expression profiles, suggesting that the polymorphism of H. pylori strains may be one of the factors determining the occurrence of H. pylori-associated IDA.

Pregnancy- and lactation-associated breast cancer: mammographic and sonographic findings.

OBJECTIVE: To evaluate the mammographic and sonographic findings of pregnancy-associated breast cancer. METHODS: A total of 22 consecutive patients with breast cancer pathologically diagnosed during pregnancy (n = 10) or lactation (n = 12) were included in this study. The ages of the patients ranged from 26 to 49 years. Both mammography and sonography were performed on 12 patients; sonography only was performed on 7 patients; and mammography only was performed on 3 patients. Mammographic and sonographic findings were evaluated retrospectively. RESULTS: Mammography revealed positive findings in 13 (86.7%) of 15 patients, even though all 15 patients had dense breasts. Mammographic findings included masses (n = 5), masses with calcifications (n = 2), calcifications with axillary lymphadenopathy (n = 2), a mass with axillary lymphadenopathy (n = 1), calcifications alone (n = 1), asymmetric density alone (n = 1), and diffuse skin and trabecular thickening alone (n = 1). Sonographic findings were positive and showed masses for all 19 patients (100%). The common sonographic findings of masses were irregular shapes (n = 15), irregular margins (n = 16), parallel orientation (n = 11), complex echo patterns (n = 14, including marked cystic [anechoic] components [n = 4]), and posterior acoustic enhancement (n = 12). Surrounding tissue effects could be seen in 5 patients, including ductal changes (n = 2), Cooper ligament thickening (n = 1), edema (n = 3), and skin thickening (n = 3). Calcifications within or outside a mass (n = 7) and axillary lymphadenopathy (n = 8) were also detected. CONCLUSIONS: Although a mass could not be discernible by mammography because of increased radiodensity during pregnancy or lactation, calcification, asymmetric density, axillary lymphadenopathy, and skin and trabecular thickening were helpful for diagnosis of pregnancy-associated breast cancer. Sonographic findings of a solid mass with posterior acoustic enhancement and a marked cystic component were somewhat different from the appearance of breast cancer in nonpregnant women, possibly because of the physiologic changes of pregnancy and lactation.