bims-malgli 2021-01-17 papers

bims-malgli

Biomed News

on Biology of malignant gliomas

Issue of 2021–01–17
fiveteen papers selected by
Oltea Sampetrean, Keio University

A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors.
ANO1 regulates the maintenance of stemness in glioblastoma stem cells by stabilizing EGFRvIII.
Pten Deficiency Leads To Proteasome Addiction, A Novel Vulnerability In Glioblastoma.
Investigation of Inter- and Intratumoral Heterogeneity of Glioblastoma Using TOF-SIMS.
PARP mediated PARylation of MGMT is critical to promote repair of temozolomide-induced O6-methylguanine DNA damage in glioblastoma.
BATF2 prevents glioblastoma multiforme progression by inhibiting recruitment of myeloid-derived suppressor cells.
Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma.
Adenosinergic Pathway: A Hope in the Immunotherapy of Glioblastoma.
An Arf/Rab cascade controls the growth and invasiveness of glioblastoma.
Nanotechnology and Nanocarrier-Based Drug Delivery as the Potential Therapeutic Strategy for Glioblastoma Multiforme: An Update.
Single-cell chromatin accessibility profiling of glioblastoma identifies an Invasive cancer stem cell population associated with lower survival.
Updated Insights on EGFR Signaling Pathways in Glioma.
Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes.
Mapping the Intratumoral Heterogeneity in Glioblastomas with Hyperspectral Stimulated Raman Scattering Microscopy.
Very low mutation burden is a feature of inflamed recurrent glioblastomas responsive to cancer immunotherapy.

Cancers (Basel). 2021 Jan 10. pii: E230. [Epub ahead of print]13(2):

A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors.

Barbara Costa, Michael N C Fletcher, Pavle Boskovic, Ekaterina L Ivanova, Tanja Eisemann, Sabrina Lohr, Lukas Bunse, Martin Löwer, Stefanie Burchard, Andrey Korshunov, Nadia Coltella, Melania Cusimano, Luigi Naldini, Hai-Kun Liu, Michael Platten, Bernhard Radlwimmer, Peter Angel, Heike Peterziel.

  Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific Pten/p53 double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.

Keywords:  glioblastoma; mouse model; syngeneic cell line

DOI:  https://doi.org/10.3390/cancers13020230
Oncogene. 2021 Jan 15.

ANO1 regulates the maintenance of stemness in glioblastoma stem cells by stabilizing EGFRvIII.

Hee-Jin Kim, Jeong-Yub Kim, Chan-Woong Jung, Young-Sun Lee, Joon-Yong An, Eun Ho Kim, Ki-Hong Kim, Sang Pyung Lee, Jae-Yong Park, Myung-Jin Park.

Glioblastoma multiforme (GBM) or glioblastoma is the most deadly malignant brain tumor in adults. GBM is difficult to treat mainly due to the presence of glioblastoma stem cells (GSCs). Epidermal growth factor receptor variant III (EGFRvIII) has been linked to stemness and malignancy of GSCs; however, the regulatory mechanism of EGFRvIII is largely unknown. Here, we demonstrated that Anoctamin-1 (ANO1), a Ca2+-activated Cl- channel, interacts with EGFRvIII, increases its protein stability, and supports the maintenance of stemness and tumor progression in GSCs. Specifically, shRNA-mediated knockdown and pharmacological inhibition of ANO1 suppressed the self-renewal, invasion activities, and expression of EGFRvIII and related stem cell factors, including NOTCH1, nestin, and SOX2 in GSCs. Conversely, ANO1 overexpression enhanced the above phenomena. Mechanistically, ANO1 protected EGFRvIII from proteasomal degradation by directly binding to it. ANO1 knockdown significantly increased survival in mice and strongly suppressed local invasion of GSCs in an in vivo intracranial mouse model. Collectively, these results suggest that ANO1 plays a crucial role in the maintenance of stemness and invasiveness of GSCs by regulating the expression of EGFRvIII and related signaling molecules, and can be considered a promising therapeutic target for GBM treatment.

DOI: https://doi.org/10.1038/s41388-020-01612-5
Neuro Oncol. 2021 Jan 11. pii: noab001. [Epub ahead of print]

Pten Deficiency Leads To Proteasome Addiction, A Novel Vulnerability In Glioblastoma.

Jorge A Benitez, Darren Finlay, Anthony Castanza, Alison D Parisian, Jianhui Ma, Ciro Longobardi, Alex Campos, Raghavendra Vadla, Alejandro Izurieta, Gianluca Scerra, Tomoyuki Koga, Tao Long, Lukas Chavez, Jill P Mesirov, Kristiina Vuori, Frank Furnari.

   BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults with a median survival of approximately 15 months, therefore, more effective treatment options for GBM are required. To identify new drugs targeting glioblastomas, we performed a high throughput drug screen using patient-derived neurospheres cultured to preferentially retain their glioblastoma stem cell (GSC) phenotype.
METHODS: High throughput drug screening was performed on GSCs followed by a dose response assay of the 5 identified original "hits". A PI3K/mTOR dependency to a proteasome inhibitor (carfilzomib), was confirmed by genetic and pharmacologic experiments. Proteasome inhibition response signatures were derived from proteomic and bioinformatic analysis. Molecular mechanism of action was determined using 3-dimensional (3D) GBM organoid and preclinical orthotopic models.
RESULTS: We found that GSCs were highly sensitive to proteasome inhibition due to an underlying dependency on an increased protein synthesis rate, and loss of autophagy, associated with PTEN loss and activation of the PI3K/mTOR pathway. In contrast, combinatory inhibition of autophagy and the proteasome, resulted in enhanced cytotoxicity specifically in GSCs that did express PTEN. Finally, proteasome inhibition specifically increased cell death markers in 3D glioblastoma organoids, suppressed tumor growth, and increased survival of mice orthotopically engrafted with GSCs. As perturbations of the PI3K/mTOR pathway occur in nearly 50% of GBMs, these findings suggest that a significant fraction of these tumors could be vulnerable to proteasome inhibition.
CONCLUSIONS: Proteasome inhibition is a potential synthetic lethal therapeutic strategy for GBM with proteasome addiction due to a high protein synthesis rate and autophagy deficiency.

Keywords:  Glioblastoma; PTEN; neurospheres; organoids; proteasome

DOI:  https://doi.org/10.1093/neuonc/noab001
Mol Cell Proteomics. 2020 Jun;pii: S1535-9476(20)34991-4. [Epub ahead of print]19(6): 960-970

Investigation of Inter- and Intratumoral Heterogeneity of Glioblastoma Using TOF-SIMS.

Samvel K Gularyan, Alexander A Gulin, Ksenia S Anufrieva, Victoria O Shender, Michail I Shakhparonov, Soniya Bastola, Nadezhda V Antipova, Tatiana F Kovalenko, Yury P Rubtsov, Yaroslav A Latyshev, Alexander A Potapov, Marat S Pavlyukov.

  Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity because of the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

Keywords:  Glioblastoma; TOF-SIMS; cancer biomarker(s); caveolin-1; glioma; imaging visualization tools; mass spectrometry; stem cells

DOI:  https://doi.org/10.1074/mcp.RA120.001986
Neuro Oncol. 2021 Jan 12. pii: noab003. [Epub ahead of print]

PARP mediated PARylation of MGMT is critical to promote repair of temozolomide-induced O6-methylguanine DNA damage in glioblastoma.

Shaofang Wu, Xiaolong Li, Feng Gao, John F de Groot, Dimpy Koul, W K Alfred Yung.

   BACKGROUND: Temozolomide (TMZ) resistance in Glioblastoma (GBM) is mediated by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). MGMT promoter methylation (occurs in about 40% patients) is associated with loss of MGMT expression (MGMT-) that compromises DNA repair, leading to a favorable response to TMZ therapy. The 60% of patients with unmethylated MGMT (MGMT+) GBM experience resistance to TMZ; in these patients, understanding the mechanism of MGMT mediated repair and modulating MGMT activity may lead to enhanced TMZ activity. Here, we report a novel mode of regulation of MGMT protein activity by poly-ADP-ribose polymerase (PARP).
METHODS: MGMT-PARP interaction was detected by co-immunoprecipitation (IP). PARylation of MGMT and PARP was detected by co-immunoprecipitation with anti-PAR antibody. O 6-methylguanine (O 6-MetG) adducts were quantified by immunofluorescence assay. In vivo studies were conducted in mice to determine the effectiveness of PARP inhibition in sensitizing GBM to TMZ.
RESULTS: We demonstrated that PARP physically binds with MGMT and PARylates MGMT in response to TMZ treatment. In addition, PARylation of MGMT by PARP is required for MGMT binding to chromatin to enhance the removal of O 6-MetG adducts from DNA after TMZ treatment. PARP inhibitors reduced PARP-MGMT binding and MGMT PARylation, silencing MGMT activity to repair O 6-MetG. PARP inhibition restored TMZ sensitivity in vivo in MGMT expressing GBM.
CONCLUSION: This study demonstrated that PARylation of MGMT by PARP is critical for repairing TMZ-induced O6-MetG, and inhibition of PARylation by PARP inhibitor reduces MGMT function rendering sensitization to TMZ, providing a rationale for combining PARP inhibitors to sensitize TMZ in MGMT unmethylated GBM.

Keywords:  DNA damage repair; MGMT PARylation; PARP; TMZ resistance

DOI:  https://doi.org/10.1093/neuonc/noab003
Oncogene. 2021 Jan 15.

BATF2 prevents glioblastoma multiforme progression by inhibiting recruitment of myeloid-derived suppressor cells.

Xin Zhang, Yi Liu, Lei Dai, Gang Shi, Jie Deng, Qiang Luo, Qian Xie, Lin Cheng, Chunlei Li, Yi Lin, Qingnan Wang, Ping Fan, Hantao Zhang, Xiaolan Su, Shuang Zhang, Yang Yang, Xun Hu, Qiyong Gong, Dechao Yu, Lei Zheng, Hongxin Deng.

The basic leucine zipper ATF-like transcription factor 2 (BATF2) has been implicated in inflammatory responses and anti-tumour effects. Little, however, is known regarding its extracellular role in maintaining a non-supportive cancer microenvironment. Here, we show that BATF2 inhibits glioma growth and myeloid-derived suppressor cells (MDSCs) recruitment. Interestingly, extracellular vesicles (EVs) from BATF2-overexpressing glioma cell lines (BATF2-EVs) inhibited MDSCs chemotaxis in vitro. Moreover, BATF2 inhibited intracellular SDF-1α and contributes to decreased SDF-1α in EVs. In addition, BATF2 downregulation-induced MDSCs recruitment were reversed by blocking SDF-1α/CXCR4 signalling upon AMD3100 treatment. Specifically, detection of EVs in 24 pairs of gliomas and healthy donors at different stages revealed that the abundance of BATF2-positive EVs in plasma (BATF2+ plEVs) can distinguish stage III-IV glioma from stage I-II glioma and healthy donors. Taken together, our study identified novel regulatory functions of BATF2 in regulating MDSCs recruitment, providing a prognostic value in terms of the number of BATF2+ plEVs in glioma stage.

DOI: https://doi.org/10.1038/s41388-020-01627-y
Oncotarget. 2020 Dec 29. 11(52): 4822-4835

Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma.

Hector A Cuello, Gretel M Ferreira, Cynthia A Gulino, Alejandro Gomez Toledo, Valeria I Segatori, Mariano R Gabri.

  Gliomas are the most common intracranial primary tumors, for which very few therapeutic options are available. The most malignant subtype is the glioblastoma, a disease associated with a 5-year survival rate lower than 5%. Given that research in glycobiology continues highlighting the role of glycans in tumor cell biology, it offers an interesting niche for the search of new therapeutic targets. In this study, we characterized aberrant glycosylation and its impact on cell biology over a broad panel of high- and low-grade glioma cell lines. Results show high expression of terminal Lewis glycans, mainly SLex, and overexpression of sialyl- and fucosyltransferases involved in their biosynthesis in high-grade glioma cell lines. Moreover, we report an association of complex multi-antennary N-glycans presenting β1,6-GlcNAc branches with the high-grade glioma cells, which also overexpressed the gene responsible for these assemblies, MGAT5. In addition, downmodulation of N-glycosylation by treatment with the inhibitors Tunicamycin/Swainsonine or MGAT5 silencing decreased SLex expression, adhesion and migration in high-grade glioma cells. In contrast, no significant changes in these cell capacities were observed in low-grade glioma after treatment with the N-glycosylation inhibitors. Furthermore, inhibition of histone deacetylases by Trichostatin A provoked an increase in the expression of SLex and its biosynthetic related glycosyltransferases in low-grade glioma cells. Our results describe that aggressive glioma cells show high expression of Lewis glycans anchored to complex multi-antennary N-glycans. This glycophenotype plays a key role in malignant cell behavior and is regulated by histone acetylation dependent mechanisms.

Keywords:  Lewis glycans; N-glycans; glioblastoma; glioma; histone acetylation

DOI:  https://doi.org/10.18632/oncotarget.27850
Cancers (Basel). 2021 Jan 10. pii: E229. [Epub ahead of print]13(2):

Adenosinergic Pathway: A Hope in the Immunotherapy of Glioblastoma.

Ketao Jin, Chunsen Mao, Lin Chen, Lude Wang, Yuyao Liu, Jianlie Yuan.

  Brain tumors comprise different types of malignancies, most of which are originated from glial cells. Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor with a poor response to conventional therapies and dismal survival rates (15 months) despite multimodal therapies. The development of immunotherapeutic strategies seems to be necessary to enhance the overall survival of GBM patients. So far, the immunotherapies applied in GBM had promising results in the primary phases of clinical trials but failed to continue their beneficial effects in later phases. GBM-microenvironment (GME) is a heterogenic and rigorously immunosuppressive milieu wrapping by an impenetrable blood-brain barrier. Hence, in-depth knowledge about the dominant immunosuppressive mechanisms in the GME could foster GBM immunotherapy. Recently, the adenosinergic pathway (AP) is found to be a major player in the suppression of antitumor immune responses in the GME. Tumor cells evolve to metabolize pro-inflammatory ATP to anti-inflammatory adenosine. Adenosine can suppress immune responses through the signaling of adenosine receptors on immune cells. The preclinical results targeting AP in GBM showed promising results in reinvigorating antitumor responses, overriding chemoresistance, and increasing survival. We reviewed the current GBM immunotherapies and elaborated on the role of AP in the immunopathogenesis, treatment, and even prognosis of GBM. We suggest that future clinical studies should consider this pathway in their combination therapies along with other immunotherapeutic approaches.

Keywords:  CD39; CD73; adenosine; brain tumor; glioblastoma; immunotherapy

DOI:  https://doi.org/10.3390/cancers13020229
J Cell Biol. 2021 Feb 01. pii: e202004229. [Epub ahead of print]220(2):

An Arf/Rab cascade controls the growth and invasiveness of glioblastoma.

Gopinath Kulasekaran, Mathilde Chaineau, Valerio Emilio Crescenzo Piscopo, Federica Verginelli, Maryam Fotouhi, Martine Girard, Yeman Tang, Rola Dali, Rita Lo, Stefano Stifani, Peter S McPherson.

Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

DOI: https://doi.org/10.1083/jcb.202004229
Cancers (Basel). 2021 Jan 07. pii: E195. [Epub ahead of print]13(2):

Nanotechnology and Nanocarrier-Based Drug Delivery as the Potential Therapeutic Strategy for Glioblastoma Multiforme: An Update.

Jen-Fu Hsu, Shih-Ming Chu, Chen-Chu Liao, Chao-Jan Wang, Yi-Shan Wang, Mei-Yin Lai, Hsiao-Chin Wang, Hsuan-Rong Huang, Ming-Horng Tsai.

  Glioblastoma multiforme (GBM) is the most common and malignant brain tumor with poor prognosis. The heterogeneous and aggressive nature of GBMs increases the difficulty of current standard treatment. The presence of GBM stem cells and the blood brain barrier (BBB) further contribute to the most important compromise of chemotherapy and radiation therapy. Current suggestions to optimize GBM patients' outcomes favor controlled targeted delivery of chemotherapeutic agents to GBM cells through the BBB using nanoparticles and monoclonal antibodies. Nanotechnology and nanocarrier-based drug delivery have recently gained attention due to the characteristics of biosafety, sustained drug release, increased solubility, and enhanced drug bioactivity and BBB penetrability. In this review, we focused on recently developed nanoparticles and emerging strategies using nanocarriers for the treatment of GBMs. Current studies using nanoparticles or nanocarrier-based drug delivery system for treatment of GBMs in clinical trials, as well as the advantages and limitations, were also reviewed.

Keywords:  glioblastoma multiforme; immunotherapy; liposomal doxorubicin; nanoparticle; target therapy

DOI:  https://doi.org/10.3390/cancers13020195
Elife. 2021 Jan 11. pii: e64090. [Epub ahead of print]10

Single-cell chromatin accessibility profiling of glioblastoma identifies an Invasive cancer stem cell population associated with lower survival.

Paul Guilhamon, Charles Chesnelong, Michelle M Kushida, Ana Nikolic, Divya Singhal, Graham MacLeod, Seyed Ali Madani Tonekaboni, Florence Mg Cavalli, Christopher Arlidge, Nishani Rajakulendran, Naghmeh Rastegar, Xiaoguang Hao, Rozina Hassam, Laura J Smith, Heather Whetstone, Fiona J Coutinho, Bettina Nadorp, Katrina I Ellestad, Artee H Luchman, Jennifer Ai-Wen Chan, Molly S Shoichet, Michael D Taylor, Benjamin Haibe-Kains, Sam Weiss, Stephane Angers, Marco Gallo, Peter B Dirks, Mathieu Lupien.

  Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as Glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.

Keywords:  cancer biology; human

DOI:  https://doi.org/10.7554/eLife.64090
Int J Mol Sci. 2021 Jan 08. pii: E587. [Epub ahead of print]22(2):

Updated Insights on EGFR Signaling Pathways in Glioma.

Alexandru Oprita, Stefania-Carina Baloi, Georgiana-Adeline Staicu, Oana Alexandru, Daniela Elise Tache, Suzana Danoiu, Elena Simona Micu, Ani-Simona Sevastre.

  Nowadays, due to recent advances in molecular biology, the pathogenesis of glioblastoma is better understood. For the newly diagnosed, the current standard of care is represented by resection followed by radiotherapy and temozolomide administration, but because median overall survival remains poor, new diagnosis and treatment strategies are needed. Due to the quick progression, even with aggressive multimodal treatment, glioblastoma remains almost incurable. It is known that epidermal growth factor receptor (EGFR) amplification is a characteristic of the classical subtype of glioma. However, targeted therapies against this type of receptor have not yet shown a clear clinical benefit. Many factors contribute to resistance, such as ineffective blood-brain barrier penetration, heterogeneity, mutations, as well as compensatory signaling pathways. A better understanding of the EGFR signaling network, and its interrelations with other pathways, are essential to clarify the mechanisms of resistance and create better therapeutic agents.

Keywords:  EGFR; clinical trials; glioma; pathways

DOI:  https://doi.org/10.3390/ijms22020587
Sci Rep. 2021 Jan 14. 11(1): 1333

Late p65 nuclear translocation in glioblastoma cells indicates non-canonical TLR4 signaling and activation of DNA repair genes.

Isabele F Moretti, Antonio M Lerario, Marina Trombetta-Lima, Paula R Sola, Roseli da Silva Soares, Sueli M Oba-Shinjo, Suely K N Marie.

Glioblastoma (GBM) is the most aggressive brain primary malignancy. Toll-like receptor 4 (TLR4) has a dual role in cell fate, promoting cell survival or death depending on the context. Here, we analyzed TLR4 expression in different grades of astrocytoma, and observed increased expression in tumors, mainly in GBM, compared to non-neoplastic brain tissue. TLR4 role was investigated in U87MG, a GBM mesenchymal subtype cell line, upon LPS stimulation. p65 nuclear translocation was observed in late phase, suggesting TLR4-non-canonical pathway activation. In fact, components of ripoptosome and inflammasome cascades were upregulated and they were significantly correlated in GBMs of the TCGA-RNASeq dataset. Moreover, an increased apoptotic rate was observed when the GBM-derived U87MG cells were co-treated with LPS and Temozolomide (TMZ) in comparison to TMZ alone. Increased TLR4 immunostaining was detected in nuclei of U87MG cells 12 h after LPS treatment, concomitant to activation of DNA repair genes. Time-dependent increased RAD51, FEN1 and UNG expression levels were confirmed after LPS stimulation, which may contribute to tumor cell fitness. Moreover, the combined treatment with the RAD51 inhibitor, Amuvatinib in combination with, TMZ after LPS stimulation reduced tumor cell viability more than with each treatment alone. In conclusion, our results suggest that stimulation of TLR4 combined with pharmacological inhibition of the DNA repair pathway may be an alternative treatment for GBM patients.

DOI: https://doi.org/10.1038/s41598-020-79356-1
Anal Chem. 2021 Jan 14.

Mapping the Intratumoral Heterogeneity in Glioblastomas with Hyperspectral Stimulated Raman Scattering Microscopy.

Kideog Bae, Le Xin, Wei Zheng, Carol Tang, Beng-Ti Ang, Zhiwei Huang.

Recent genomic studies on the glioblastoma (GBM) subtypes (e.g., mesenchymal, proneural, and classical) pave a way for effective clinical treatments of the recurrent brain tumor. However, identification of the GBM subtype is complicated by the intratumoral heterogeneity that results in coexistence of multiple subtypes within the tissue specimen. Here, we present the use of hyperspectral stimulated Raman scattering (SRS) microscopy for rapid, label-free molecular assessment of GBM intratumoral heterogeneity with submicron resolution. We develop a unique label-free Raman imaging diagnostic platform consisting of the spectral focusing hyperspectral SRS imaging of the large-area GBM tissue specimens, SRS images, and spectrum retrieval using the multivariate curve resolution algorithm and subtype classification based on the quadratic support vector machine model for rapid molecular subtyping of GBMs. Both the stain-free SRS histological images and 2D subtype maps can be obtained within 20-30 min which is superior to the days of the conventional single-cell RNA sequencing. While the SRS histology assesses the demyelination status as a new diagnostic feature, the SRS mapping provides a new insight into intratumoral heterogeneity across GBM tissue specimens. We find that the major proportions of the GBM tissues agree with the diagnostic results of the genomic analysis, but nontrivial portions of the remaining SRS image tiles in the specimens are found to belong to other molecular subtypes, implying the substantial degree of GBM heterogeneity. The rapid SRS imaging diagnostic platform developed has shown the ability of unveiling tumor heterogeneity in GBM tissues accurately, which would promote the improvement of the GBM-targeted therapy in near future.

DOI: https://doi.org/10.1021/acs.analchem.0c04262
Nat Commun. 2021 01 13. 12(1): 352

Very low mutation burden is a feature of inflamed recurrent glioblastomas responsive to cancer immunotherapy.

Matthias Gromeier, Michael C Brown, Gao Zhang, Xiang Lin, Yeqing Chen, Zhi Wei, Nike Beaubier, Hai Yan, Yiping He, Annick Desjardins, James E Herndon, Frederick S Varn, Roel G Verhaak, Junfei Zhao, Dani P Bolognesi, Allan H Friedman, Henry S Friedman, Frances McSherry, Andrea M Muscat, Eric S Lipp, Smita K Nair, Mustafa Khasraw, Katherine B Peters, Dina Randazzo, John H Sampson, Roger E McLendon, Darell D Bigner, David M Ashley.

Several immunotherapy clinical trials in recurrent glioblastoma have reported long-term survival benefits in 10-20% of patients. Here we perform genomic analysis of tumor tissue from recurrent WHO grade IV glioblastoma patients acquired prior to immunotherapy intervention. We report that very low tumor mutation burden is associated with longer survival after recombinant polio virotherapy or after immune checkpoint blockade in recurrent glioblastoma patients. A relationship between tumor mutation burden and survival is not observed in cohorts of immunotherapy naïve newly diagnosed or recurrent glioblastoma patients. Transcriptomic analyses reveal an inverse relationship between tumor mutation burden and enrichment of inflammatory gene signatures in cohorts of recurrent, but not newly diagnosed glioblastoma tumors, implying that a relationship between tumor mutation burden and tumor-intrinsic inflammation evolves upon recurrence.

DOI: https://doi.org/10.1038/s41467-020-20469-6