Tumor Stem-like Cells Research Articles

EPCO-55. SPATIAL HETEROGENEITY IN THE TUMOR CORE AND INFILTRATING MARGIN OFIDHWT GLIOBLASTOMA

Abstract We integrated single-cell RNA-sequencing, Visium spatial transcriptomic, and Xenium in situ transcriptomic data to identify differences in cell composition between the tumor core and infiltrating edge in IDHWT glioblastoma (GBM). Using CellTrek, an algorithm that maps single cells to their most-likely locations in spatial transcriptomic data, we identified extensive spatial organization in GBM. Cell mapping revealed distinct regions enriched for tumor, immune, endothelial, neuronal, and glial cells. These were associated with the location of the tissue sample – edge regions were enriched for neurons and glia (as expected), while core regions were enriched for tumor cells and infiltrating immune cells, particularly macrophages. Within the core samples, the tumor and immune cells tended to occupy distinct spatial niches. Spatial clustering of mapped tumor cells across all samples revealed five spatially segregated tumor cell states, each associated with specific biological processes, including (1) histone methylation, (2) proliferation, (3) wound-healing, EMT, and antigen presentation, (4) hypoxia response, and (5) stem cell differentiation. These tumor cell states exhibited preferential colocalization with specific non-malignant cell types. For instance, antigen-presenting/wound-healing tumor cells tended to colocalize with diverse immune cells, while stem-like tumor cells tended to reside in neuron-rich regions at the tumor edge. Next, to identify ligand-receptor interactions governing this spatial organization, we analyzed known ligand-receptor pairs within all three data types. This revealed known and novel mediators of tumor cell interactions with vasculature, lymphoid cells, myeloid cells, neurons, and glia. While some interactions were shared across all tumor cell states (e.g. all tumor cell states interacted with myeloid cells via APP/CD74 interactions), others were specific to particular tumor cell states (e.g. wound-healing/antigen-presenting tumor cells interacted with macrophages via CSF1/CSF1R, while proliferating and stem-like tumor cells interacted with neurons through multiple known NRXN/NLGN interactions). These interactions suggest potential targets for clinical disruption of the tumor ecosystem.

DNAR-04. TUMOUR TREATING FIELDS (TTFIELDS) AND DNA DAMAGE RESPONSE (DDR) INHIBITORS ENHANCE GLIOMA CELL DEATH THROUGH CHEMO-/RADIOSENSITISATION

Abstract High grade gliomas are the most common primary brain cancer malignancies with ~300,000 global diagnoses each year with glioblastomas (WHO grade IV) conferring the worst prognoses. This arises from their treatment resistance, rationalised by the presence of glioma stem cell-like (GSC) tumour sub-regions and high intratumoural spatio-heterogeneity. TTFields are clinically approved alternating electric fields of intermediate frequency that exert anti-mitotic effects and invoke downregulation of the DDR. Therefore, TTFields alongside clinically developed, and blood brain barrier (BBB) penetrating DDR inhibitors (DDRi) could boost cell death and overcome treatment resistance through chemo-/radiosensitisation. Primary GSC cell lines were derived from surgically resected and clinically defined tumour core and invasive edge regions and grown in more biologically relevant 3D-printed AlvetexTM scaffolds, offering highly clinically relevant models. Cells were then pre-treated with DDRi, subjected to chemo-/radiotherapy and then incubated under TTFields (200 kHz, 72 hrs) with their survival measured by clonogenic assays after 3 weeks. Immunofluorescence and western blot analysis were used to analyse the combination treatment effects on the DDR. In multiple 3D-grown core/edge GSC models, western blot analyses revealed that the DDR was altered by TTFields and clonogenic survival assays revealed that TTFields alongside DDRi led to significant chemo-/radiosensitisation. TTFields alongside DDRi effectively enhances cell death by significant treatment sensitisation effects in clinically relevant 3D GSCs which exhibit extensive intra and intertumoral spatio-heterogeneity. Given that all the DDRi used are BBB penetrable and are either clinically approved or in clinical trials, TTFields could additionally compliment these treatment regimens in gliomas to boost their efficacy.

Metabolic regulation of the glioblastoma stem cell epitranscriptome by malate dehydrogenase 2

Tumors reprogram their metabolism to generate complex neoplastic ecosystems. Here, we demonstrate that glioblastoma (GBM) stem cells (GSCs) display elevated activity of the malate-aspartate shuttle (MAS) and expression of malate dehydrogenase 2 (MDH2). Genetic and pharmacologic targeting of MDH2 attenuated GSC proliferation, self-renewal, and invivo tumor growth, partially rescued by aspartate. Targeting MDH2 induced accumulation of alpha-ketoglutarate (αKG), a critical co-factor for dioxygenases, including the N6-methyladenosine (m6A) RNA demethylase AlkB homolog 5, RNA demethylase (ALKBH5). Forced expression of MDH2 increased m6A levels and inhibited ALKBH5 activity, both rescued by αKG supplementation. Reciprocally, targeting MDH2 reduced global m6A levels with platelet-derived growth factor receptor-β (PDGFRβ) as a regulated transcript. Pharmacological inhibition of MDH2 in GSCs augmented efficacy of dasatinib, an orally bioavailable multi-kinase inhibitor, including PDGFRβ. Collectively, stem-like tumor cells reprogram their metabolism to induce changes in their epitranscriptomes and reveal possible therapeutic paradigms.

Integration of single-cell sequencing and bulk transcriptome data develops prognostic markers based on PCLAF+ stem-like tumor cells using artificial neural network in gastric cancer

BackgroundGastric cancer stem cells (GCSCs) are important tumour cells involved in tumourigenesis and gastric cancer development. However, their clinical value remains unclear due to the limitations of the available technologies. This study aims to explore the clinical significance of GCSCs, their connection to the tumour microenvironment, and their underlying molecular mechanisms. MethodsStem-like tumour cells were identified by mining single-cell transcriptomic data from multiple samples. Integrated analysis of single-cell and bulk transcriptome data was performed to analyse the role of stem-like tumour cells in predicting clinical outcomes by introducing the intermediate variable mRNA stemness degree (SD). Consensus clustering analysis was performed to develop an SD-related molecular classification strategy to assess the clinical characteristics in gastric cancer. A prognostic model was constructed using a customized approach that comprehensively considered SD-related gene signatures based on an artificial neural network. ResultsBy analysing single-cell data and validating immunofluorescence results, we identified a PCLAF+ stem-like tumour cell population in GC. By calculating SD, we observed that PCLAF+ stem-like tumour cells were associated with poor prognosis and certain clinical features. The SD was negatively correlated with the abundance of most immune cell types. Furthermore, we proposed an SD-related classification method and prognostic model. In addition, the customised prognostic model can be used to predict whether a patient respond to PD-1/PD-L1 immunotherapy. ConclusionWe identified a cluster of stem-like cells and elucidated their clinical significance, highlighting the possibility of their use as immunotherapeutic targets.

Spatial analysis reveals targetable macrophage-mediated mechanisms of immune evasion in hepatocellular carcinoma minimal residual disease.

Hepatocellular carcinoma (HCC) frequently recurs from minimal residual disease (MRD), which persists after therapy. Here, we identified mechanisms of persistence of residual tumor cells using post-chemoembolization human HCC (n = 108 patients, 1.07 million cells) and a transgenic mouse model of MRD. Through single-cell high-plex cytometric imaging, we identified a spatial neighborhood within which PD-L1 + M2-like macrophages interact with stem-like tumor cells, correlating with CD8+ T cell exhaustion and poor survival. Further, through spatial transcriptomics of residual HCC, we showed that macrophage-derived TGFβ1 mediates the persistence of stem-like tumor cells. Last, we demonstrate that combined blockade of Pdl1 and Tgfβ excluded immunosuppressive macrophages, recruited activated CD8+ T cells and eliminated residual stem-like tumor cells in two mouse models: a transgenic model of MRD and a syngeneic orthotopic model of doxorubicin-resistant HCC. Thus, our spatial analyses reveal that PD-L1+ macrophages sustain MRD by activating the TGFβ pathway in stem-like cancer cells and targeting this interaction may prevent HCC recurrence from MRD.

Immunotherapy drives mesenchymal tumor cell state shift and TME immune response in glioblastoma patients.

Glioblastoma is a highly aggressive type of brain tumor for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected patients, and there is an urgent need to identify mechanisms of treatment resistance to design novel therapy strategies. Here we investigated the phenotypic and transcriptional dynamics at single-cell resolution during nivolumab immune checkpoint treatment of glioblastoma patients. We present the integrative paired single-cell RNA-seq analysis of 76 tumor samples from patients in a clinical trial of the PD-1 inhibitor nivolumab and untreated patients. We identify a distinct aggressive phenotypic signature in both tumor cells and the tumor microenvironment in response to nivolumab. Moreover, nivolumab-treatment was associated with an increased transition to mesenchymal stem-like tumor cells, and an increase in TAMs and exhausted and proliferative T cells. We verify and extend our findings in large external glioblastoma dataset (n = 298), develop a latent immune signature and find 18% of primary glioblastoma samples to be latent immune, associated with mesenchymal tumor cell state and TME immune response. Finally, we show that latent immune glioblastoma patients are associated with shorter overall survival following immune checkpoint treatment (P = .0041). We find a resistance mechanism signature in one fifth of glioblastoma patients associated with a tumor-cell transition to a more aggressive mesenchymal-like state, increase in TAMs and proliferative and exhausted T cells in response to immunotherapy. These patients may instead benefit from neuro-oncology therapies targeting mesenchymal tumor cells.

Engineering Nanomedicine for Non-Viral RNA-Based Gene Therapy of Glioblastoma.

Glioblastoma multiforme (GBM) is the most common type of malignant tumor of the central nervous system, characterized by aggressiveness, genetic instability, heterogenesis, and unpredictable clinical behavior. Disappointing results from the current clinical therapeutic methods have fueled a search for new therapeutic targets and treatment modalities. GBM is characterized by various genetic alterations, and RNA-based gene therapy has raised particular attention in GBM therapy. Here, we review the recent advances in engineered non-viral nanocarriers for RNA drug delivery to treat GBM. Therapeutic strategies concerning the brain-targeted delivery of various RNA drugs involving siRNA, microRNA, mRNA, ASO, and short-length RNA and the therapeutical mechanisms of these drugs to tackle the challenges of chemo-/radiotherapy resistance, recurrence, and incurable stem cell-like tumor cells of GBM are herein outlined. We also highlight the progress, prospects, and remaining challenges of non-viral nanocarriers-mediated RNA-based gene therapy.

Abstract 5444: EGFR missense mutation(s) induce OLIG2 expression to regulate glioma stem cells maintenance and therapeutic resistance

Abstract Glioblastoma (GBM) tumors contain a subpopulation of glioma stem cells (GSC) which can escape therapeutic effects through self-renewal and invasion. Epidermal growth factor receptor (EGFR) mutations are the most common alteration in GBM (50%). One example includes extracellular domain missense mutations like G598V, A289V, and R108K (10-15%). These extracellular domain missense mutations increase ligand binding affinity, inducing a highly active receptor. Earlier reports have shown these EGFR missense mutations exhibit therapeutic resistance; however, the molecular mechanism of these missense mutations in GBM remains unknown. In this study, we show high oligodendrocyte transcription factor 2 (OLIG2) expression in GSCs with EGFR-G598V and EGFR-A289V missense mutations. OLIG2 is universally expressed in glioma and essential for reprogramming GBM cells into stem-like tumor cells. Here, we report GSCs with these point mutations display increased self-renewal, growth, invasion, and therapeutic resistance. In addition, EGFR-G598V and EGFR-A289V trigger distinct downstream signaling pathways in GSCs to regulate OLIG2 function. These missense mutation(s) also represent different pathway-based GBM subclassifications, where A289V induces a Neuronal subtype and G598V induces a Glycolytic/Plurimetabolic subtype. We hypothesize that GBM tumors harboring EGFR missense mutations (G598V, A289V) influence stem cell properties via multiple pathways to evade therapeutic effects and inhibiting these different signaling pathways will decrease therapeutic resistance in GBM. Understanding the mechanism of these EGFR mutations may lead to improved therapeutic strategies for GBM patients. Citation Format: Sidney Weaver, Aunay Miller, Mylan Blomquist, James McNamara, Keely Orndorff, Chris Sereduk, Jean Kloss, Joseph Loftus, Shwetal Mehta, Nhan L. Tran. EGFR missense mutation(s) induce OLIG2 expression to regulate glioma stem cells maintenance and therapeutic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5444.

Abstract 4264: The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment

Abstract High grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy but a majority relapse with chemoresistant disease. Research supports the notion that tumor-initiating cells (TICs), a subpopulation of drug resistant stem-like tumor cells, are responsible for facilitating relapse and therapies targeting these fecund cells may prolong remission. The tumor microenvironment (TME) plays a crucial role in therapy resistance, and tumor relapse. Cytotoxic chemotherapies have been shown to modify the stroma of the TME which may favor the survival of TICs displaying specific adhesion proteins. We and others have shown that NF-kB transcription factors RelA and RelB respond to signals from the ovarian TME to promote proliferation, chemoresistance, and survival. Our results further show that RelA and RelB regulate expression of specific integrins. Recently we found that TNF-like inducer of apoptosis (TWEAK), a cytokine involved in tissue repair, and its only known receptor Fn14, are enriched following chemotherapy, leading to enhanced chemoresistance and survival of TICs. TWEAK also induces expression of specific integrins in ovarian TICs. Therefore, we hypothesize that NF-kB mediated integrin expression enables adhesion and survival of TICs to a chemotherapy modified extracellular matrix (ECM) to facilitate tumor repopulation and relapse. Using magnetic activated cell sorting (MACS) for CD117+ TICs, we found that a significant majority are enriched for integrin AVB3. Supporting a role for TWEAK-Fn14-NF-kB in mediating integrin expression we further show that integrin AVB3, but not AVB5 or A5B1, was significantly increased in CD117+ TICs in the presence of TWEAK and was dependent on Fn14 as demonstrated through knockdown experiments. RNA-sequencing analysis of tumor cells grown in spheroid conditions with RelA or RelB shRNA knockdown relative to control, show ITGAV is dependent on RelA while ITGB3 expression is dependent on RelB. Taken together, our findings suggest CD117+ TICs have enhanced expression of AVB3 and is further induced by TWEAK mediated NF-kB activity for adaptation to a stressful post-chemotherapy environment. Ongoing in vitro and in vivo studies are investigating the therapeutic benefit of targeting AVB3 or TWEAK in CD117+ TICs for prevention of adhesion and tumor repopulation following chemotherapy. These studies will uncover interactions between TICs and ECM through their unique expression of integrins and clarifies the role of NF-kB transcription factors RelA and RelB in regulating TIC survival in the post-chemotherapy TME. Citation Format: Omar Lujano Olazaba, Mikella Robinson, Cecilia Gallo, Mena Shammas, Katelyn Shelby, Luisjesus Cruz, Samuel F. Gilbert, Carrie D. House. The TWEAK-Fn14-NF-kB signaling axis enhances expression of integrin AVB3 in a post-chemotherapy tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4264.

Exosomal circZNF800 Derived from Glioma Stem-like Cells Regulates Glioblastoma Tumorigenicity via the PIEZO1/Akt Axis

Exosomes play a crucial role in regulating crosstalk between tumor and tumor stem-like cells through their cargo molecules. Circular RNAs (circRNAs) have recently been demonstrated to be critical factors in tumorigenesis. This study focuses on the molecular mechanism by which circRNAs from glioma stem-like cell (GSLC) exosomes regulate glioblastoma (GBM) tumorigenicity. In this study, we validated that GSLC exosomes accelerated the malignant phenotype of GBM. Subsequently, we found that circZNF800 was highly expressed in GSLC exosomes and was negatively associated with GBM patients. CircZNF800 promoted GBM cell proliferation and migration and inhibited GBM cell apoptosis in vitro. Silencing circZNF800 could improve the GBM xenograft model survival rate. Mechanistic studies revealed that circZNF800 activated the PIEZO1/Akt signaling pathway by sponging miR-139-5p. CircZNF800 derived from GSLC exosomes promoted GBM cell tumorigenicity and predicted poor prognosis in GBM patients. CircZNF800 has the potential to serve as a promising target for further therapeutic exploration.

Role of catecholamine synthases in the maintenance of cancer stem-like cells in malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are malignant tumors that are derived from Schwann cell lineage around peripheral nerves. As in many other cancer types, cancer stem cells (CSCs) have been identified in MPNSTs, and they are considered the cause of treatment resistance, recurrence, and metastasis. As an element defining the cancer stemness of MPNSTs, we previously reported a molecular mechanism by which exogenous adrenaline activates a core cancer stemness factor, YAP/TAZ, through β2 adrenoceptor (ADRB2). In this study, we found that MPNST cells express catecholamine synthases and that these enzymes are essential for maintaining cancer stemness, such as the ability to self-renew and maintain an undifferentiated state. Through gene knockdown and inhibition of these enzymes, we confirmed that catecholamines are indeed synthesized in MPNST cells. The results confirmed that catecholamine synthase knockdown in MPNST cells reduces the activity of YAP/TAZ. These data suggest that a mechanism of YAP/TAZ activation by de novo synthesized adrenaline, as well as exogenous adrenaline, may exist in the maintenance of cancer stemness of MPNST cells. This mechanism not only helps to understand the pathology of MPNST, but could also contribute to the development of therapeutic strategies for MPNST.

A SOX9-B7x axis safeguards dedifferentiated tumor cells from immune surveillance to drive breast cancer progression

How dedifferentiated stem-like tumor cells evade immunosurveillance remains poorly understood. We show that the lineage-plasticity regulator SOX9, which is upregulated in dedifferentiated tumor cells, limits the number of infiltrating T lymphocytes in premalignant lesions of mouse basal-like breast cancer. SOX9-mediated immunosuppression is required for the progression of in situ tumors to invasive carcinoma. SOX9 induces the expression of immune checkpoint B7x/B7-H4 through STAT3 activation and direct transcriptional regulation. B7x is upregulated in dedifferentiated tumor cells and protects them from immunosurveillance. B7x also protects mammary gland regeneration in immunocompetent mice. In advanced tumors, B7x targeting inhibits tumor growth and overcomes resistance to anti-PD-L1 immunotherapy. In human breast cancer, SOX9 and B7x expression are correlated and associated with reduced CD8+ Tcell infiltration. This study, using mouse models, cell lines, and patient samples, identifies a dedifferentiation-associated immunosuppression mechanism and demonstrates the therapeutic potential of targeting the SOX9-B7x pathway in basal-like breast cancer.

STEM-20. REGULATION OF GLIOMA STEM CELL MAINTENANCE AND THERAPEUTIC RESISTANCE BY EGFR MISSENSE MUTATIONS

Abstract Glioblastoma (GBM) tumors contain a subpopulation of glioma stem cells (GSC) which can escape therapeutic effects through self-renewal and invasion. Epidermal growth factor receptor (EGFR) mutations are the most common alteration in GBM. EGFRvIII (deletion of exons 2-7) is observed in GBM (20-25%) resulting in a constitutively activated EGFR receptor. In addition, extracellular domain missense point mutations, (e.g. G598V, A289V, R108K) are observed in GBM (14.4%). These missense mutations in the EGFR extracellular domain also induces a highly active receptor by enhancing ligand binding. Earlier reports have shown that these EGFR missense point mutations have exhibited therapeutic resistance; however, the molecular mechanism of these missense point mutations in GBM remains unknown. Previous studies in our lab showed that EGFRvIII promotes GSC maintenance and therapeutic resistance via oligodendrocyte transcription factor 2 (OLIG2) signaling. OLIG2 is universally expressed in glioma and essential for reprogramming GBM cells into stem-like tumor cells. Increased OLIG2 expression promotes invasion, growth, proliferation, and survival. Here we report an increase in OLIG2 protein expression in GSCs harboring EGFR-G598V or EGFR-A289V mutations, suggesting these mutations may support stemness properties in GBM tumors. GSC expressing these point mutations display increased self-renewal, growth, and survival. In addition, EGFR-G598V and EGFR-A289V trigger distinct downstream signaling pathways in GSCs to regulate OLIG2 function. Hence, we hypothesize that GBM tumors harboring EGFR missense mutations (G598V, A289V) influence stem cell properties via multiple pathways to evade therapeutic effects and inhibiting these different signaling pathways will decrease therapeutic resistance in GBM. Understanding the mechanism of these EGFR mutations may lead to improved therapeutic strategies for GBM patients.

STEM-18. HYPOXIA-REGULATED EXPRESSION OF CD44 AND OSTEOPONTIN CAN CHANGE THE PHENOTYPE OF GLIOMA STEM-LIKE CELLS FROM HIGHLY INVASIVE TO LESS INVASIVE/PROLIFERATIVE TUMORS IN GLIOBLASTOMA

Abstract BACKGROUND The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive and highly migratory glioma stem-like cells(GSCs) in tumors. Upon GBM recurrence or progression, the highly invasive phenotype of GSCs changes to a less-motile, proliferative phenotype, thus generating tumor mass. Elucidating the molecular mechanism underlying this phenotypic transition could lead to the identification of effective molecular targets for treating GBM. METHODS We examined the effects of hypoxia (1% O2: severe or 5% O2: moderate) on the expression and functions of hypoxia-inducible factor(HIF), CD44 and osteopontin (OPN) to clarify the role of these molecules in GBM tumor invasion and proliferation. We also investigated the anti-tumor effects of CD44 knockdown in vitro and in vivo using a GSC-transplantation mouse model. RESULTS Severe Hypoxia upregulates CD44 expression via activation of HIF-1α, inducing GSCs to assume a highly invasive phenotype. In contrast, moderate hypoxia upregulates OPN expression via activation of HIF-2α. OPN inhibits CD44-promoted GSC migration and invasion and stimulates GSC proliferation, resulting in GSCs assuming a less-invasive, highly proliferative phenotype. CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo, and the mice survived significantly longer than control mice. CONCLUSION The highly invasive phenotype of GSCs can be reversed by switching from severe to moderate hypoxia, leading to less-invasive and proliferative tumors. CD44 and OPN play a central role in regulating GSC invasion and proliferation by inducing a phenotypic transition, suggesting that these molecules could be effective targets for treating GBM.

Tumor microenvironment-derived exosomes-assisted single-cell-derived tumor-sphere formation and drug-resistance by a microfluidic system

Without changing the cell biological characteristics, single-cell-derived tumor-sphere (SCDTS) is encouraged to be applied in exploring tumor stem-like cells (TSCs). Whereas, tumor microenvironment simulation is required for promoting SCDTS formation as that TSCs are under rigid regulation from their microenvironment. Tumor microenvironment-derived exosomes (TMDEs) have been employed as a potential approach for simulating tumor microenvironment. Herein, we developed a microfluidic system for TMDEs isolation, the analysis of TMDEs effect on SCDTS formation and the SCDTS drug-resistance. As a proof of concept, the exosomes of primary breast cancer associated fibroblasts (CAFs) were isolated, and the effects of CAF exosomes on SCDTS formation of single breast cells (MCF-7 and MDA-MB-231) with different sizes and deformations were perceived. Then, the SCDTS co-cultured with paclitaxel were adopted for anticancer drug assessment. Results showed that CAF exosomes could be isolated with a simple method, and promote the formation rate and size of SCDTS. Moreover, the smaller and/or more deformable TSCs could be more sensitive to CAF exosomes and more resistant to chemotherapeutics than the larger and/or lesser deformable TSC. Our device supplies a novel modality for promoting SCDTS formation and TSC heterogeneity study.

CELF2 Sustains a Proliferating/OLIG2+ Glioblastoma Cell Phenotype via the Epigenetic Repression of SOX3.

Glioblastomas (GBs) are incurable brain tumors. The persistence of aggressive stem-like tumor cells after cytotoxic treatments compromises therapeutic efficacy, leading to GBM recurrence. Forcing the GBM cells to irreversibly abandon their aggressive stem-like phenotype may offer an alternative to conventional cytotoxic treatments. Here, we show that the RNA binding protein CELF2 is strongly expressed in mitotic and OLIG2-positive GBM cells, while it is downregulated in differentiated and non-mitotic cells by miR-199a-3p, exemplifying GBM intra-tumor heterogeneity. Using patient-derived cells and human GBM samples, we demonstrate that CELF2 plays a key role in maintaining the proliferative/OLIG2 cell phenotype with clonal and tumorigenic properties. Indeed, we show that CELF2 deficiency in patient-derived GSCs drastically reduced tumor growth in the brains of nude mice. We further show that CELF2 promotes TRIM28 and G9a expression, which drive a H3K9me3 epigenetic profile responsible for the silencing of the SOX3 gene. Thus, CELF2, which is positively correlated with OLIG2 and Ki67 expression in human GBM samples, is inversely correlated with SOX3 and miR-199a-3p. Accordingly, the invalidation of SOX3 in CELF2-deficient patient-derived cells rescued proliferation and OLIG2 expression. Finally, patients expressing SOX3 above the median level of expression tend to have a longer life expectancy. CELF2 is therefore a crucial target for the malignant potential of GBM and warrants attention when developing novel anticancer strategies.

Isolation of tumor stem-like cells from primary laryngeal squamous cell carcinoma cells (FD-LS-6).

The development of efficient treatments for laryngeal squamous cell carcinoma (LSCC) is hindered by the lack of applicable tumor cell lines and animal models of the disease, especially those related to cancer stem-like cells (CSCs). CSCs play critical roles in tumor propagation and pathogenesis whereas no CSCs lines have been developed to date. In this study, we establish an LSCC cell line (FD-LS-6) from primary LSCC tumor tissue (not experienced single-cell cloning) and adapted a culturing condition for the expansion of potential stem cells (EPSCs) to isolate CSCs from FD-LS-6. We successfully derived novel CSCs and named them as LSCC sphere-forming cells (LSCSCs) which were subsequently characterized for their CSC properties. We showed that LSCSCs shared many properties of CSCs, including CSC marker, robust self-renewal capacity, tumorigenesis ability, potential to generate other cell types such as adipocytes and osteoblasts, and resistance to chemotherapy. Compared to parental cells, LSCSCs were significantly more potent in forming tumors in vivo in mice and more resistant to chemotherapy. LSCSCs have higher expressions of epithelial-mesenchymal transition proteins and chemotherapy resistance factors, and exhibit an activated COX2/PEG2 signaling pathway. Altogether, our work establishes the first CSCs of LSCC (FD-LS-6) and provides a tool to study tumorigenesis and metastasis of LSCC and help the development of anticancer therapies.

TUMOUR TREATING FIELDS WITH DDR INHIBITORS EFFECTIVELY LEADS TO CHEMO-/RADIO-SENSITISATION OF GLIOMAS

Abstract AIMS High grade gliomas are the most common CNS malignancies and cause ~200,000 deaths/year globally. Glioblastoma (WHO grade IV) confer the worst prognoses with their treatment being hindered by glioma stem cell-like (GSC) tumour sub-regions and high intratumoural spatio-heterogeneity. Tumour Treating Fields (TTFields) exert clinically approved alternating electric fields of intermediate frequency which induces anti-mitotic effects within concomitant deleterious effects on cellular responses to DNA damage (DDR). Therefore, treatment resistance could be overcome with TTFields with clinically developed DDR inhibitors (DDRi) to cause chemo-/radio- sensitisation. METHOD Surgically resected samples derived from the tumour core and invasive edge were processed to create GSC cell lines, offering highly clinically relevant models. These were grown in either 2D or in 3D-printed AlvetexTM scaffolds, treated with DDRi, subjected to chemo-/radio-therapy and then placed under TTFields (200 kHz, 48- 72 hrs). Survival was measured by clonogenic assays after 3 weeks. Immunofluorescence and western blot analysis were used to analyse the combination treatment effects on the DDR. RESULTS TTFields with DDRi led to significant chemo/radio-sensitisation in both 2D and 3D intratumoral core/edge GSC models. Western blot and immunofluorescence analyses revealed that DNA damage was heightened by the combination treatments with lesion resolution being impaired. CONCLUSIONS DDRi with concurrent TTFields effectively leads to significant chemo-/radio-sensitisation in clinically relevant 3D GSCs which model spatio-heterogeneity which is thought to impede treatment effcacy. Given that all the DDRi used are BBB penetrable and are either clinically approved or in clinical trials, TTFields could additionally compliment these treatment regimens in gliomas to boost their effcacy.

P02.19.A ASSESSMENT OF HEAT SHOCK PROTEIN HSP70 EXPRESSION IN THE GLIOBLASTOMA STEM-LIKE CELLS

Abstract BACKGROUND Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by poor prognosis. The search of biomarkers for early diagnosis and as targets and for tumor theranostics due to the aggressiveness of the disease is urgently needed. One of the promising targets for therapy of brain tumors is 70 kDa heat shock protein (Hsp70), which has been found on the plasma membrane of various tumor cells (including GBM) but not on normal cells. GBM, like other types of tumors, has cellular heterogeneity. In particular, it contains tumor stem-like cells (TSLCs), which are considered analogues of healthy stem cells and are largely responsible for disease progression, recurrence and mediate resistance to ongoing therapy. In the present work, the histological sections of human GBM were analyzed to identify and quantify the colocalization of the heat shock protein Hsp70 with TSLC markers using the Opal Multiplex analysis system. MATERIAL AND METHODS Histological sections were prepared in accordance with the standard protocol using postoperative material from patients with newly diagnosed GBM (n=15). Zones of necrosis and living tissue were identified on tumor sections. Based on a phenotyping map using the Pathology Views™ fluorescence image analysis software, Hsp70 colocalization was quantified with Nestin and Sox2 in the areas of necrosis and viable tissue employing primary antibodies (anti-Hsp70, anti-Nestin and anti-Sox2). The preparations were stained using an Opal 3-Plex Manual Detection Kit according to the manufacturer's protocol. RESULTS The expression of Hsp70 and TSLC markers (Nestin and Sox2) was detected. The obtained quantitative data did not reveal significant differences in the distribution of cells expressing Hsp70 in the areas of necrosis and viable tissue (36 and 41%, respectively), which may indicate their uniform distribution in the tumor. Sufficiently high colocalization of Hsp70 with Nestin and Sox2 factors was detected mainly in the zone of viable tissue (57 and 30%, respectively). CONCLUSION Colocalization of Hsp70 with TSLC markers indicates the prospects for further studies of Hsp70, as well as its use for targeted therapy of malignant brain tumors.

Radiated tumor cell-derived microparticles effectively kill stem-like tumor cells by increasing reactive oxygen species.

Stem-like tumor cells (SLTCs) are thought to be the cellular entity responsible for clinical recurrence and subsequent metastasis. Inhibiting or killing SLTCs can effectively reduce recurrence and metastasis, yet little has been done to clear SLTCs because they are usually resistant to chemotherapy, radiotherapy, and even immunotherapy. In this study, we established SLTCs by low-serum culture and confirmed that the low-serum-cultured tumor cells were in a quiescent state and resistant to chemotherapy, showing features of SLTCs, consistent with the reported data. We demonstrated that SLTCs had high levels of reactive oxygen species (ROS). Based on the finding that radiated tumor cell-derived microparticles (RT-MPs) contained ROS, we used RT-MPs to kill SLTCs. We found that RT-MPs could further increase ROS levels and kill SLTCs in vivo and in vitro partially by ROS carried by the RT-MPs themselves, providing a new method for eliminating SLTCs.