Patient-derived Glioblastoma Cell Lines Research Articles

Targeting intracranial patient-derived glioblastoma (GBM) with a NIR-I fluorescent immunoconjugate for facilitating its image-guided resection.

Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor type and is associated with a high mortality rate borne out of such affording a survival rate of only 15 months. GBM aggressiveness is associated with the overexpression of epidermal growth factor receptor (EGFR) and its mutants. Targeting GBM with therapeutics is challenging because the blood-brain barrier (BBB) permits primarily select small-molecule entities across its semipermeable blockade. However, recent preclinical data suggest that large biomolecules, such as the anti-EGFR antibody therapeutic, cetuximab, could be capable of bypassing the BBB despite the relative enormity of its size. As such, we set forth to establish the feasibility of utilizing an EGFR-targeting near-infrared-I (NIR-I) fluorescent construct in the form of an immunoconjugate (cetuxmimab-IRDye800) to achieve visual differentiation between diseased brain tissue arising from a low-passage patient-derived GBM cell line (GBM39) and healthy brain tissue via utilizing orthotopic intracranial murine GBM39 tumor models for in vivo and ex vivo evaluation such that by doing so would establish proof of concept for ultimately facilitating its in vivo fluorescence-guided resection and ex vivo surgical back-table pathological confirmation in the clinic. As anticipated, we were not capable of distinguishing between malignant tumor tissue and healthy tissue in resected intact and slices of whole brain ex vivo under white-light illumination (WLI) due to both the diseased tissue and healthy tissue appearing virtually identical to the unaided eye. However, we readily observed over an average 6-fold enhancement in the fluorescence emission in the resected intact whole brain ex vivo when performing NIR-I fluorescence imaging (FLI) on the cohort of GBM39 tumor models that were administered the immunoconjugate compared to controls. In all, we laid the initial groundwork for establishing that NIR-I fluorescent immunoconjugates (theranostics) such as cetuximab–IRDye800 can bypass the BBB to visually afford GBM39 tumor tissue differentiation for its image-guided surgical removal.

BET and Aurora Kinase A inhibitors synergize against MYCN-positive human glioblastoma cells

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. Patients usually undergo surgery followed by aggressive radio- and chemotherapy with the alkylating agent temozolomide (TMZ). Still, median survival is only 12–15 months after diagnosis. Many human cancers including GBMs demonstrate addiction to MYC transcription factor signaling and can become susceptible to inhibition of MYC downstream genes. JQ1 is an effective inhibitor of BET Bromodomains, a class of epigenetic readers regulating expression of downstream MYC targets. Here, we show that BET inhibition decreases viability of patient-derived GBM cell lines. We propose a distinct expression signature of MYCN-elevated GBM cells that correlates with significant sensitivity to BET inhibition. In tumors showing JQ1 sensitivity, we found enrichment of pathways regulating cell cycle, DNA damage response and repair. As DNA repair leads to acquired chemoresistance to TMZ, JQ1 treatment in combination with TMZ synergistically inhibited proliferation of MYCN-elevated cells. Bioinformatic analyses further showed that the expression of MYCN correlates with Aurora Kinase A levels and Aurora Kinase inhibitors indeed showed synergistic efficacy in combination with BET inhibition. Collectively, our data suggest that BET inhibitors could potentiate the efficacy of either TMZ or Aurora Kinase inhibitors in GBM treatment.

TMIC-47. INHIBITION OF GLIOBLASTOMA GROWTH THROUGH TUMOR-MICROENVIRONMENT CROSSTALK USING CLINICALLY SUITABLE NANOBIOCONJUGATE

Abstract Tumor environment in glioblastoma (GBM) is a dynamic interactive complex between tumor, immune and stem cells and extracellular matrix (ECM). In 226 patient glioma samples, we found a clinical correlation between expression of tumor vascular laminin-411 (α4β1γ1) and cancer stem cell (CSC) markers: Notch pathway members, CD133, Nestin, and c-Myc, with faster tumor recurrence and shorter survival of patients (Tao S, et al, Cancer Res., 2019). Novel nanotechnology approach to block trimeric ECM laminin-411 was developed for GBM treatment in experimental and preclinical models on human U87MG and LN229, and patient-derived TS543 and TS576 GBM cell lines. Nanobioconjugates (NBC) based on natural polymer, poly(β-L-malic acid), with antisense against laminin-411 α4 and β-chains, endosome escape unit (Ding H, et al, PNAS, USA, 2010), antibodies for blood-brain barrier (BBB) crossing and tumor targeting was characterized for toxicity and biodistribution according to FDA guidelines. Ex vivo depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of intracranial tumors in mice, and significantly increased survival in hosts compared to mice with untreated cells. A nanobioconjugate potentially suitable for clinical use and capable of crossing BBB was designed to block laminin-411 expression. In biodistribution studies the NBC labeled with 125I on tyrosines of attached antibodies was accumulated in GBM but not in healthy brain tissue. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, Notch signaling system through the b1 integrin-Dll4 (Notch ligand) pathway. No toxicity revealed in 4 naïve Cynomolgus macaques after administration of three therapeutic 1X and acute 10X dosages of NBC. Conclusion: An efficient strategy of GBM treatment was developed via targeting a critical component of the tumor microenvironment, laminin-411, which is independent of heterogeneous genetic mutations in glioblastomas. Support: NIH grants U01CA151815, R01CA188743, R01CA206220, R01CA209921.

EXTH-34. IN VITRO TUMOR TREATING FIELDS (TTFIELDS) APPLIED PRIOR TO RADIATION ENHANCES THE RESPONSE TO RADIATION IN PATIENT-DERIVED GLIOBLASTOMA CELL LINES

Abstract Tumor treating fields are FDA-approved for treatment of newly-diagnosed and recurrent glioblastoma. Adding TTFields therapy to standard-of-care extended progression-free survival and overall survival in newly-diagnosed glioblastoma patients. In this study, cell lines generated from newly-diagnosed glioblastoma patients were treated in vitro with TTFields prior to irradiation to determine if the response to radiation would be altered. This study was approved by the WSU Institutional Review Board and written consent obtained from the patients. Single-cell suspensions generated from tumor tissues obtained from newly-diagnosed patients were cultured in DMEM/F12 media with 10% fetal bovine serum and gentamicin. Prior to TTFields initiation, cells were plated on plastic coverslips (5×104cells/coverslip) and incubated overnight. Then, TTFields were applied at 200 kHz with a field intensity of ~1.6 V/cm for 3 days. After TTFields application, cells were irradiated with 2, 4, or 6 Gy, or were untreated. After radiation delivery, cells were harvested and replated in a clonogenic assay. From each group, 3 coverslips were each replated in triplicate. After 3 days (3 doubling times), cells were stained with crystal violet and plates were scanned to determine cell counts. Treatment groups were compared to their control group with t-test. For both patient-derived GBM cell lines tested, TTFields prior to radiation decreased the number of crystal violet-stained cells in the clonogenic assay plates. In cell line 15–037, pretreatment with TTFields decreased cell counts by 16, 29, and 56% after 2, 4, or 6 Gy radiation, respectively, compared to the control cells with no TTFields pretreatment (p< 0.05). The response in 14-015S cells was less radiation dose-dependent, with the decrease in cell counts ranging from 33–47% control across the radiation doses (p< 0.05). These data suggest that the use of TTFields therapy prior to radiotherapy may enhance the response to radiotherapy in GBM patients.

Cholesterol protects glioblastoma cells against premature mitosis

Abstract Glioblastoma is the most common malignant primary tumour with a dismal prognosis. So far, no inhibitors targeting frequently altered pathways in GBM have improved patient survival. Premature entry to mitosis by small molecules that promote cancer cells to bypass the cell-cycle checkpoints have shown potent anti-tumour activity in vitro in a variety of cancers including GBM. We have reproduced these cytotoxic effects in our patient-derived GBM cell lines with the small molecule ME-344 and the Wee1-specific inhibitor AZ1775 both by by clonogenic survival and cell viability assays (EC50 values ranging from 0.003–0.02 μM and 0.2–0.4 μM, respectively). ME-344 and AZ1775 triggered profound morphological and cell cycle effects including mitotic induction, arrest and mitotic catastrophe. Bioinformatic analysis of global mRNA expression of our GBM cell lines stratified by ME-344 sensitivity showed a correlation between high ABCA1 and low cholesterol pathway gene expression with high sensitivity, and vice-versa. Cholesterol is a main component of membranes and is critical for cell growth and mitosis progression. GBM cells rely on cholesterol for survival. Due to the unique metabolic environment of the brain where a nearly unlimited supply of cholesterol is provided by astrocytes, targeting cellular activities regulated by cholesterol might lose their anti-tumour activity. Here we report that cholesterol confers cytoprotection to AZ1775 and ME344 in all GBM cell lines tested. These results suggest that cholesterol can override premature mitotic anti-tumour activity, indicating that mitotic induction and cholesterol inhibition might be a better therapeutic strategy for GBM than either treatment alone.

Identifying the Zika Virus Target Cell in Malignant Glioma

Abstract The Zika Virus epidemic of 2015–2016 was associated with striking failure of forebrain development in infants born to infected mothers, resulting in microcephaly. Studies from numerous labs subsequently confirmed a selective effect of the virus on neural stem cell survival, self-renewal and differentiation. The glioma stem cells which drive glioblastoma and other malignant gliomas depend on neural stem cell transcription programs, so that understanding Zika infection in these cells promises valuable insights into future therapy. We describe here: 1) Study of low passage patient-derived glioblastoma cell lines and normal neural stem cells (CRUK Glioma Cellular Genetics Resource) in adherent culture to address the influence of glioma subtype on infectability 2) Application of genetically modified mCherry reporter Zika Virus strains to address the Zika target cell in glioblastoma. 3) Development of a cerebral organoid model to interrogate the differential effects of the virus on tumour cells and surrounding normal brain. 4) Demonstration of Zika infection on primary patient derived tissue in slice culture format Using these tools we find that Zika targets a common stem cell population across tumour subtypes and neural stem cell controls, and in embryonic and primary tumour explants, and that infection is influenced by activity of cholesterol biosynthesis pathways.

Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion

Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.

P11.33 Characterization of a new patient-derived metastatic glioblastoma cell line and response to sodium selenite anticancer agent: in vitro results and preclinical data in mice

Abstract BACKGROUND Glioblastoma (GBM) are very heterogeneous, organized in a hierarchical pattern, including cancer stem cells (CSC), and are responsible for development, maintenance, and cancer relapse. Therefore, it is relevant to establish new GBM cell lines with CSC characteristics to develop new treatments. MATERIAL AND METHODS A new human GBM cell line, named R2J, was established from the cerebro-spinal fluid of a patient affected by GBM with leptomeningeal metastasis. Cells were cultured both in monolayer and in spheres in a medium without serum and characterized before being implanted into the striatum of nude mice. The tumor progression was followed by MRI and brains were collected to perform IHC analyses. Sodium selenite (SS), previously described for its anticancer potential in GBM cell lines, was tested in the R2J cells prior to delimitate its toxicity and absorption in Balb/c mice. To be achieved, we tested different doses of SS (2.25, 4.5, 6.75 and 10.125 mg/kg-dissolve in water-) given orally for 5 days followed by a wash-out of 2 days followed by 5 supplementary days. At the end of this protocol, mice were sacrificed to collect blood, brain, kidney, liver and lungs. We evaluated the behavior and the weight of the mice along the study and we performed biochemical measurements including the dosage of selenium (Se) in blood and organs by ICP-MS. RESULTS R2J cells displays an abnormal karyotype and about 2% of the population is able to form self-renewable spheres in a define medium. Original tumor, R2J cells cultured in monolayer (2D) and in spheres showed a persistence expression of CD44, CD56 (except in monolayer), EGFR, Ki67, Nestin, and vimentin. The R2J cell line is tumorigenic and possesses CSC properties. We tested in vitro the anticancer effects of sodium selenite (SS) compared to temozolomide (TMZ). SS was absorbed by R2J cells, was cytotoxic, induced an oxidative stress, and arrested cell growth in G2M before inducing both necrosis and apoptosis via caspase-3. SS also changed dimethyl-histone-3-lysine-9 (H3K9m2) levels and reduced histone deacetylase (HDAC) activity, suggesting anti-invasiveness potential. Mouse orthotopic xenografts of R2J cells were able to form tumor within 14 days, even when implanted at 1000 cells as spheres. IHC revealed a persistent CD56 and nestin expression as in the parental tumor. After the period of treatment with SS, mice lost about 5% of their weight at 6.75 mg/kg. Se concentration significantly increased in plasma in a dose dependent manner, in kidney (at 2.25 mg/kg), in liver (at 4.5mg/kg), in brain and lung (at 6.75 mg/kg). CONCLUSION This study highlights the value of this new GBM cell line for preclinical modeling and allowed us to test SS as a potential anticancer agent. All these data plus the fact that Se crosses the blood brain barrier are the first step to further investigations in orthotopic patient-derived glioblastoma xenografts in mice.

The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived glioblastoma cell lines.

We describe the synthesis of drug-dye conjugate 1 between anaplastic lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed potent cytotoxic activity with nanomolar potency (EC50: 50.9 nM). We also demonstrate evidence for antiproliferative activity of 1 with single digit nanomolar potency (IC50: 4.7 nM). Furthermore, the cytotoxic effects conveyed a dramatic, 110-fold improvement over Crizotinib. This improvement was even more pronounced (492-fold) when 1 was combined with Temozolomide, the standard drug for treatment for glioblastoma. This work lays the foundation for future exploration of similar tyrosine kinase inhibitor drug-dye conjugates for the treatment of glioblastoma.

Abstract 1153: Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells

Abstract Glioblastoma (GBM) is a highly malignant primary brain tumor and has a poor prognosis and is resistant to the most advanced therapies. The development of highly hypoxic centrally located necrotic regions is strongly associated with accelerated GBM progression, yet mechanistic explanations are not complete. Hypoxic regions within GBM are highly enriched for glioma stem cells (GSC), which are critical for tumor initiation, recurrence and therapeutic resistance due to their self-renewing capabilites. While most GBM cells migrate outward, away from hypoxia, GSCs accumulate there, forming a hypercellular border called pseudopalisades that promote their survival. Mechanisms that mediate GSC accumulation within pseudopalisades are unknown. We hypothesize that GSCs are enriched in the pseudopalisades due to the homing of a subpopulation towards the supportive hypoxic microenvironment. This in turn activates critical hypoxia-induced drivers to promote stemness, survival and migratory behavior encouraging the recruitment of more GSCs. Using two genetically distinct patient-derived GBM cell lines, we found that exposure to hypoxia in vitro led to enrichment of GSCs among a heterogeneous population of GSCs and non-GSCs. We found that hypoxia enhanced the expression of stem cell, pro-survival and pro-migratory factors in GSCs, such as the pro-migratory chemokine SDF-1α, and its cognate receptor CXCR4. Importantly, we found hypoxia also induced upregulation of OLIG2, one of four core transcription factors necessary and sufficient to induce stemness, in GSCs and non-GSCs exposed to hypoxia. We determined that OLIG2 was necessary for the expression of stem cell markers, and that its knockdown reduced cellular migration in hypoxia vs normoxia in GSCs. This suggests a critical role for OLIG2 in both hypoxia-induced stemness and homing behavior. We also generated a unique model of localized hypoxia in one brain hemisphere through vaso-cclusion and observed the homing of xenografted GBM cells towards hypoxia, regulated by OLIG2 in vivo. These data shed light on how hypoxic tumor microenvironments facilitate mechanisms vital to GBM progression and therapeutic resistance. Citation Format: Nitya V. Sharma, Monica Chau, Subhas Mukherjee, James Ross, Brandon Miller, Changming Zhang, Jun Kong, Carol Tucker-Burden, Emily Kaissi, John W. Reitnauer, Bilge Dundar, Cheryl L. Olson, Gregory Bix, Daniel J. Brat. Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1153.

Temporary blood-brain barrier disruption by low intensity pulsed ultrasound increases carboplatin delivery and efficacy in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary brain cancer in adults. Few cytotoxic chemotherapies have been shown to be effective against GBM, due in part to the presence of the blood-brain barrier (BBB), which reduces the penetration of chemotherapies from the blood to the brain. Ultrasound-induced BBB opening (US-BBB) has been shown to increase the penetration of multiple chemotherapeutic agents in the brain in animal models. In the current study, the anti-tumor activity of carboplatin chemotherapy with and without US-BBB was investigated in several GBM mouse models. First, the IC50 of two commercial (U87 and U251) and six patient-derived GBM cell lines (PDCL) to carboplatin was measured. Next, U87 was subcutaneously grafted to a nude mouse model to test the in vivo response of the tumor to carboplatin in the absence of the BBB. Lastly, nude mice bearing orthotopically xenografted GBM cell lines (U87 or a PDCL) were randomized to four experimental groups: (i) untreated, (ii) US-BBB alone, (iii) carboplatin alone and, (iv) carboplatin + US-BBB. Mice were treated once weekly for 4 weeks and monitored for toxicity, tumor growth, and survival. Carboplatin plus US-BBB enhanced survival (p = 0.03) and delayed tumor growth (p < 0.05) of GBM-bearing mice compared to carboplatin alone, with a 4.2-fold increase of carboplatin penetration in the brain, without evidence of significant neurological or systemic toxicity. Carboplatin efficacy was enhanced in GBM mouse models with US-BBB and appears to be a promising chemotherapy for this approach.

PELP1 promotes glioblastoma progression by enhancing Wnt/β-catenin signaling.

BackgroundGlioblastoma (GBM) is a deadly neoplasm of the central nervous system. The molecular mechanisms and players that contribute to GBM development is incompletely understood.MethodsThe expression of PELP1 in different grades of glioma and normal brain tissues was analyzed using immunohistochemistry on a tumor tissue array. PELP1 expression in established and primary GBM cell lines was analyzed by Western blotting. The effect of PELP1 knockdown was studied using cell proliferation, colony formation, migration, and invasion assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, immunoprecipitation, reporter gene assays, and signaling analysis. Mouse orthotopic models were used for preclinical evaluation of PELP1 knock down.ResultsNuclear receptor coregulator PELP1 is highly expressed in gliomas compared to normal brain tissues, with the highest expression in GBM. PELP1 expression was elevated in established and patient-derived GBM cell lines compared to normal astrocytes. Knockdown of PELP1 resulted in a significant decrease in cell viability, survival, migration, and invasion. Global RNA-sequencing studies demonstrated that PELP1 knockdown significantly reduced the expression of genes involved in the Wnt/β-catenin pathway. Mechanistic studies demonstrated that PELP1 interacts with and functions as a coactivator of β-catenin. Knockdown of PELP1 resulted in a significant increase in survival of mice implanted with U87 and GBM PDX models.ConclusionsPELP1 expression is upregulated in GBM and PELP1 signaling via β-catenin axis contributes to GBM progression. Thus, PELP1 could be a potential target for the development of therapeutic intervention in GBM.

A New Patient-Derived Metastatic Glioblastoma Cell Line: Characterisation and Response to Sodium Selenite Anticancer Agent.

Glioblastoma multiform (GBM) tumors are very heterogeneous, organized in a hierarchical pattern, including cancer stem cells (CSC), and are responsible for development, maintenance, and cancer relapse. Therefore, it is relevant to establish new GBM cell lines with CSC characteristics to develop new treatments. A new human GBM cell line, named R2J, was established from the cerebro-spinal fluid (CSF) of a patient affected by GBM with leptomeningeal metastasis. R2J cells exhibits an abnormal karyotype and form self-renewable spheres in a serum-free medium. Original tumor, R2J, cultured in monolayer (2D) and in spheres showed a persistence expression of CD44, CD56 (except in monolayer), EGFR, Ki67, Nestin, and vimentin. The R2J cell line is tumorigenic and possesses CSC properties. We tested in vitro the anticancer effects of sodium selenite (SS) compared to temozolomide TMZ. SS was absorbed by R2J cells, was cytotoxic, induced an oxidative stress, and arrested cell growth in G2M before inducing both necrosis and apoptosis via caspase-3. SS also modified dimethyl-histone-3-lysine-9 (H3K9m2) levels and decreased histone deacetylase (HDAC) activity, suggesting anti-invasiveness potential. This study highlights the value of this new GBM cell line for preclinical modeling of clinically relevant, patient specific GBM and opens a therapeutic window to test SS to target resistant and recurrent GBM.

Quantitative histopathologic assessment of perfusion MRI as a marker of glioblastoma cell infiltration in and beyond the peritumoral edema region.

Conventional MRI fails to detect regions of glioblastoma cell infiltration beyond the contrast-enhanced T1 solid tumor region, with infiltrating tumor cells often migrating along host blood vessels. To quantitatively and qualitatively analyze the correlation between perfusion MRI signal and tumor cell density in order to assess whether local perfusion perturbation could provide a useful biomarker of glioblastoma cell infiltration. Animal model. Mice bearing orthotopic glioblastoma xenografts generated from a patient-derived glioblastoma cell line. 7T perfusion images acquired using a high signal-to-noise ratio (SNR) multiple boli arterial spin labeling sequence were compared with conventional MRI (T1 /T2 weighted, contrast-enhanced T1 , diffusion-weighted, and apparent diffusion coefficient). Immunohistochemistry sections were stained for human leukocyte antigen (probing human-derived tumor cells). To achieve quantitative MRI-tissue comparison, multiple histological slices cut in the MRI plane were stacked to produce tumor cell density maps acting as a "ground truth." Sensitivity, specificity, accuracy, and Dice similarity indices were calculated and a two-tailed, paired t-test used for statistical analysis. High comparison test results (Dice 0.62-0.72, Accuracy 0.86-0.88, Sensitivity 0.51-0.7, and Specificity 0.92-0.97) indicate a good segmentation for all imaging modalities and highlight the quality of the MRI tissue assessment protocol. Perfusion imaging exhibits higher sensitivity (0.7) than conventional MRI (0.51-0.61). MRI/histology voxel-to-voxel comparison revealed a negative correlation between tumor cell infiltration and perfusion at the tumor margins (P = 0.0004). These results demonstrate the ability of perfusion imaging to probe regions of low tumor cell infiltration while confirming the sensitivity limitations of conventional imaging modalities. The quantitative relationship between tumor cell density and perfusion identified in and beyond the edematous T2 hyperintensity region surrounding macroscopic tumor could be used to detect marginal tumor cell infiltration with greater accuracy. 1 Technical stage: 2 J. Magn. Reson. Imaging 2019;50:529-540.

Linking single-cell measurements of mass, growth rate, and gene expression

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.

Glioma cell proliferation is enhanced in the presence of tumor-derived cilia vesicles

BackgroundThe mechanisms by which primary cilia affect glioma pathogenesis are unclear. Depending on the glioma cell line, primary cilia can promote or inhibit tumor development. Here, we used piggyBac-mediated transgenesis to generate patient-derived glioblastoma (GBM) cell lines that stably express Arl13b:GFP in their cilia. This allowed us to visualize and analyze the behavior of cilia and ciliated cells during live GBM cell proliferation.ResultsTime-lapse imaging of Arl13b:GFP+ cilia revealed their dynamic behaviors, including distal tip excision into the extracellular milieu. Recent studies of non-cancerous cells indicate that this process occurs during the G0 phase, prior to cilia resorption and cell cycle re-entry, and requires ciliary recruitment of F-actin and actin regulators. Similarly, we observed ciliary buds associated with Ki67− cells as well as scattered F-actin+ cilia, suggesting that quiescent GBM cells may also utilize an actin network-based mechanism for ciliary tip excision. Notably, we found that the proliferation of ciliated GBM cells was promoted by exposing them to conditioned media obtained from ciliated cell cultures when compared to conditioned media collected from cilia-defective cell cultures (depleted in either KIF3A or IFT88 using CRISPR/Cas9). These results suggest that GBM cilia may release mitogenic vesicles carrying factors that promote tumor cell proliferation. Although Arl13b is implicated in tumor growth, our data suggest that Arl13b released from GBM cilia does not mediate tumor cell proliferation.ConclusionCollectively, our results indicate that ciliary vesicles may represent a novel mode of intercellular communication within tumors that contributes to GBM pathogenesis. The mitogenic capacity of GBM ciliary vesicles and the molecular mediators of this phenomenon requires further investigation.

Abstract 1399: Passenger deletion vulnerability in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common, aggressive and lethal primary brain tumor in humans. Despite aggressive therapy the median survival of GBM patients is approximately 15 months. Monoallelic loss of PTEN occurs in ~70% of GBM. Through experimental validation studies in patient-derived primary GBM cell lines, we discovered that the lipogenic gene SCD undergoes unintended co-deletion as a passenger to PTEN, resulting in hemizygous loss of SCD in a subset of GBM. Additional in silico, genetic and biochemical analysis lead to the identification of two distinct PTEN-deleted subgroups: one with hemizygous co-deletion of PTEN and SCD (hereafter SCD-expressing), and another with very little or no PTEN/SCD expression due to PTEN mutation/homozygous loss and epigenetic suppression of the remaining SCD allele (hereafter SCD-non-expressing). SCD is an integral membrane protein of the endoplasmic reticulum (ER), involved in mediating the rate-limiting step of unsaturated fatty acid biosynthesis. SCD catalyzes the desaturation of the saturated fatty acids stearic (C18:0) and palmitic acids (C16:0) to the monounsaturated fatty acids oleic (C18:1) and palmitoleic acids (C16:1), respectively. We showed that the SCD-expressing lines are highly sensitive to SCD inhibition in the absence of dietary oleate and that the SCD-non-expressing lines are resistant to SCD inhibition. In vivo, we showed that mice treated with the SCD inhibitor survive longer than mice treated with vehicle. We also showed that SCD-expressing GBM lines acquire inhibitor resistance upon long-term exposure to the inhibitor in vitro. To understand the mechanism of resistance, we performed Reverse Phase Protein Array (RRPA) and RNA-Seq. Results illustrated the up-regulation of SCD among other genes. To identify the gene/s that provide SCD inhibitor resistance, we are currently performing a targeted shRNA screen of the highly expressed genes that we determined through RPPA and RNA-Seq. Citation Format: Nicole Oatman, Kakajan Komurov, Priyanka Arora, Pankaj Desai, Nupur Dasgupta, Kwangmin Choi, Biplab Dasgupta. Passenger deletion vulnerability in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1399.

61 Functional characterization of ribosomal RNA methyltransferase NSUN5 in glioblastoma

Glioblastoma is the most common and malignant brain tumor with a median overall survival of 20.5 months. There is an urgent need to develop novel therapeutic strategies. Using a glioblastoma TCGA dataset, we have determined that high NSUN5 mRNA expression is strongly associated with poor survival in glioblastoma patients. NSUN5 is a ribosomal RNA (rRNA) cytosine methyltransferase. Human NSUN5 is located in chromosome 7 and is completely deleted in the Williams-Beurren syndrome, a complex neurodevelopmental disorder. However, RNA targets of NSUN5 in mammals and its role in cancer are unknown. The objective of this project is to determine whether elevated NSUN5 changes rRNA methylation pattern and thereby leads to pro-tumorigenic translational reprogramming and pro-tumorigenic phenotypes in glioblastoma. Western blotting showed that NSUN5 is expressed in 7 out of 9 established glioblastoma cell lines and in 8 out of 12 primary patient-derived glioblastoma cell lines. Bisulfite sequencing confirmed that NSUN5 methylates C3782 of human 28S rRNA in glioblastoma cells. Functionally, overexpression of NSUN5 increases, whereas NSUN5 knockout decreases global protein synthesis and sphere formation in glioblastoma cells. More importantly, mice bearing intracranial NSUN5-expressing U87 tumors survived for a shorter time than mice bearing tumors derived from U87 control cells. Our results suggest that NSUN5 methylates 28S rRNA and may enhance cancer stem cell phenotypes and tumor formation and/or progression in glioblastoma. Experiments are ongoing to determine whether NSUN5 promotes tumor formation and/or progression through translational reprogramming in glioblastoma. This study may help identify novel therapeutic targets for glioblastoma.

Presence of stromal cells in a bioengineered tumor microenvironment alters glioblastoma migration and response to STAT3 inhibition.

Despite the increasingly recognized importance of the tumor microenvironment (TME) as a regulator of tumor progression, only few in vitro models have been developed to systematically study the effects of TME on tumor behavior in a controlled manner. Here we developed a three-dimensional (3D) in vitro model that recapitulates the physical and compositional characteristics of Glioblastoma (GBM) extracellular matrix (ECM) and incorporates brain stromal cells such as astrocytes and endothelial cell precursors. The model was used to evaluate the effect of TME components on migration and survival of various patient-derived GBM cell lines (GBM10, GBM43 and GBAM1) in the context of STAT3 inhibition. Migration analysis of GBM within the 3D in vitro model demonstrated that the presence of astrocytes significantly increases the migration of GBM, while presence of endothelial precursors has varied effects on the migration of different GBM cell lines. Given the role of the tumor microenvironment as a regulator of STAT3 activity, we tested the effect of the STAT3 inhibitor SH-4-54 on GBM migration and survival. SH-4-54 inhibited STAT3 activity and reduced 3D migration and survival of GBM43 but had no effect on GBM10. SH-4-54 treatment drastically reduced the viability of the stem-like line GBAM1 in liquid culture, but its effect lessened in presence of a 3D ECM and stromal cells. Our results highlight the interplay between the ECM and stromal cells in the microenvironment with the cancer cells and indicate that the impact of these relationships may differ for GBM cells of varying genetic and clinical histories.

ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma.

ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p < 0.01) and multivariate analyses including MGMT promoter methylation (p = 0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM-PDCL. ABCA13 expression is an independent prognostic factor in newly diagnosed GBM patients. Further prospective studies are warranted to investigate whether ABCA13 expression can be used to further personalize treatments for GBM.