Aptamer-based strategies for glioblastoma: From SELEX to preclinical success.

P-glycoprotein (Pgp) is highly expressed on blood-brain barrier (BBB) and glioblastoma (GB) cells, particularly on cancer stem cells (SC). Pgp recognizes a broad spectrum of substrates, limiting the therapeutic efficacy of several chemotherapeutic drugs in eradicating GB SC. Finding effective and safe inhibitors of Pgp that improve drug delivery across the BBB and target GB SC is open to investigation. We previously identified a series of thiosemicarbazone compounds that inhibit Pgp with an EC50 in the nanomolar range, and herein, we investigate the efficacy of three of them in bypassing Pgp-mediated drug efflux in primary human BBB and GB cells. At 10 nM, the compounds were not cytotoxic for the brain microvascular endothelial hCMEC/D3 cell line, but they markedly enhanced the permeability of the Pgp-substrate doxorubicin through the BBB. Thiosemicarbazone derivatives increased doxorubicin uptake in GB, with greater effects in the Pgp-rich SC clones than in the differentiated clones derived from the same tumor. All compounds increased intratumor doxorubicin accumulation and consequent toxicity in GB growing under competent BBB, producing significant killing of GB SC. The compounds crossed the BBB monolayer. The most stable derivative, 10a, had a half-life in serum of 4.2 h. The coadministration of doxorubicin plus 10a significantly reduced the growth of orthotopic GB-SC xenografts, without eliciting toxic side effects. Our work suggests that the thiosemicarbazone compounds are able to transform doxorubicin, a prototype BBB-impermeable drug, into a BBB-permeable drug. Bypassing Pgp-mediated drug efflux in both BBB and GB SC, thiosemicarbazones might increase the success of chemotherapy in targeting GB SC, which represent the most aggressive and difficult components to eradicate.

GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. Despite advances in immunotherapy, current treatment modalities have only provided a modest benefit to GBM patients. We propose that enhancing the efficacy of immunotherapy for GBM requires approaches that increase T cell infiltration and reprogram the myeloid cells to a more pro-inflammatory state. We previously reported that S100A4 is a critical regulator of immune suppressive myeloid and T cell phenotypes and also a major regulator of glioma stem cells in GBM. Our preliminary data show that targeting S100A4 in either glioma cells or stromal cells is sufficient to reprogram the tumor immune landscape and extend survival of glioma bearing mice, indicating that S100A4 is a promising therapeutic target. We recently developed a S100A4 blocking monoclonal antibody; however, this antibody does not cross the blood brain barrier (BBB) efficiently to confer significant therapeutic benefit in glioma bearing mice. To overcome this challenge, we generated a bispecific S100A4-TfR antibody (BsA) that allows robust BBB penetration and accumulation in the brain. We assessed efficacy of BsA treatment in a highly aggressive and therapy-resistant, syngeneic mouse model of GBM (SVP). Using ELISA, confocal immunofluorescent microscopy, flow cytometry, and bulk RNA sequencing, we report that BsA treatment accumulates in the gliomas and reprograms the tumor immune microenvironment to increase T cell infiltration and pro-inflammatory activation. Furthermore, BsA treatment in combination with αPD1 checkpoint blockade significantly extends survival in the SVP mouse glioma model. Combination treated tumors exhibit extensive CD8 T cell infiltration and increased tumor reactive CD4 and CD8 T cells compared to controls. In addition, the BsA is taken up intracellularly within GBM cells and depletes S100A4 and nuclear SOX2 protein. In vitro treatment of GBM stem cell cultures with the BsA reduced self-renewal, as measured by the secondary sphere formation assay. These results indicate that the BsA is a promising agent to reprogram the GBM immune landscape and target GBM cell stemness through inhibition of both extracellular and intracellular S100A4 protein. We are currently performing in vivo efficacy studies in additional murine glioma models, and deeper mechanistic studies using single cell RNA sequencing and functional assays. Citation Format: Thomas Wong, Jia-Shiun Leu, Xuejun Fan, Fransisca Leonard, Maryam Faisal, Reece Kang, Yaqoob Ali, Nourhan Abdelfattah, Ningyan Zhang, Kyuson Yun. A bispecific S100A4/TFR antibody to reprogram the GBM tumor microenvironment and target GBM stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6069.

Glioblastoma (GBM) is an aggressive Grade IV brain tumor with a poor prognosis. It results from genetic mutations, epigenetic changes, and factors within the tumor microenvironment (TME). Traditional treatments like surgery, radiotherapy, and chemotherapy provide limited survival benefits due to the tumor's heterogeneity and resistance mechanisms. This review examines novel approaches for treating GBM, focusing on repurposing existing medications such as antipsychotics, antidepressants, and statins for their potential anti-GBM effects. Advances in molecular profiling, including next-generation sequencing, artificial intelligence (AI), and nanotechnology-based drug delivery, are transforming GBM diagnosis and treatment. The TME, particularly GBM stem cells and immune evasion, plays a key role in therapeutic resistance. Integrating multi-omics data and applying precision medicine show promise, especially in combination therapies and immunotherapies, to enhance clinical outcomes. Addressing challenges such as drug resistance, targeting GBM stem cells, and crossing the blood–brain barrier is essential for improving treatment efficacy. While current treatments offer limited benefits, emerging strategies such as immunotherapies, precision medicine, and drug repurposing show significant potential. Technologies like liquid biopsies, AI-powered diagnostics, and nanotechnology could help overcome obstacles like the blood–brain barrier and GBM stem cells. Ongoing research into combination therapies, targeted drug delivery, and personalized treatments is crucial. Collaborative efforts and robust clinical trials are necessary to translate these innovations into effective therapies, offering hope for improved survival and quality of life for GBM patients.

Background: Glioblastoma multiforme (GBM) is the most frequent malignant central nervous system tumor in adults, with an average survival rate of approximately 15 months. In children, high grade gliomas, such as diffuse intrinsic pontine glioma (DIPG), are equally aggressive and present unique challenges due to developmental differences in the brain and the limited treatment options available. One model for studying high grade gliomas lies within a population of stem-like cells, known as glioblastoma stem cells (GSCs). GSCs exhibit self-renewal capabilities, resistance to conventional therapies, and the ability to drive tumor recurrence and progression. Understanding and targeting GSCs is necessary for developing therapies that can improve treatment outcomes in both adult and pediatric patients suffering from high grade gliomas. A family of neurotrophin receptors known as the tropomyosin receptor kinases (TRKs), including TrkA, TrkB, and TrkC, encoded by the NTRK1, NTRK2, and NTRK3 genes respectively, are critical for normal neuronal development and survival. In brain tumors, the canonical signaling of these receptors is often corrupted through gene fusions or overexpression, leading to abnormal activation of signaling pathways that promote tumor cell survival, self-renewal, and resistance to treatment. Here, we investigate the use of novel agonists 231 and 237, developed by our team, to specifically target TrkA, in effort to better understand its role in GSC biology, with the goal of finding ways to inhibit tumor growth and improve therapeutic outcomes in high grade glioma models. GSC lines 448T and 559T were cultured under standard conditions to investigate the effects of novel TrkA agonists (231 and 237) on proliferation, differentiation, and TrkA/TrkB expression. Synergy with targeted therapies, including Abemaciclib (a CDK4/6 inhibitor) and Altiratinib (a multi-kinase inhibitor targeting MET, VEGFR2, and Trk pathways), was also evaluated. For proliferation and Western blot analysis, cells were treated with PBS (control), 231, or 237 for 72 hours, followed by cell counting and lysate preparation. Differentiation was assessed using neurite outgrowth assays with Incucyte Live-Cell Imaging and Neurotrack software, measuring neurite length, branch points, and neurite-bearing cells. Western blot analysis demonstrated a reduction in TrkA expression in 559T glioblastoma stem cell lines treated with novel TrkA-specific agonists 231 and 237 compared to the PBS control. This decrease suggests potential downregulation of TrkA or feedback inhibition in response to agonist treatment. Ongoing studies aim to further elucidate the mechanisms underlying TrkA modulation and its role in maintaining GSCs in vitro. Additionally, investigations are exploring the potential synergistic effects of TrkA-specific agonists with targeted therapies to enhance GSC susceptibility to treatment. Citation Format: Taylor Simone Jackson, David E. Johnson, Hawa L. Jagana, Leyre Merino-Galan, Siobhan S. Pattwell, Thomas Schlichthaerle, Natasha Edman, David Baker. Evaluating the therapeutic potential of novel TrkA agonists in targeting glioblastoma stem cells (GSCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB267.

Glioblastoma multiforme (GBM) is the most common primary brain tumor, claiming the lives of roughly 10,000 Americans each year. Despite being the first cancer analyzed through the Cancer Genome Atlas, treatment success remains minimal, resulting average survival time of patients is 15 months following diagnosis and a five-year survival rate is 6.8%. One of the major challenges in treating GBM is the presence of GBM stem cells (GSCs) that are resistant to temozolomide (TMZ) chemotherapy and radiation, which is part of the current standard of care for GBM following maximal surgical resection. We found, however, that GSCs have a unique dependence on core circadian clock proteins, Brain and Muscle ARNTL-Like 1 (BMAL1) and Circadian Locomoter Output Cycles Protein Kaput (CLOCK), which is not observed in differentiated GBM cells or normal neuronal stem cells. Here we explore the use of novel small molecule circadian clock compounds that either lower BMAL1 transcription (REV-ERB agonists) or inhibit BMAL1::CLOCK heterodimer transcriptional activity (Cryptochrome (CRY) stabilizers or Casein Kinase (CK) 1/2 inhibitors) in targeting GSCs in in vitro patient derived cell lines and in in vivo GBM patient-derived xenograft (PDX) models. GSCs display increased sensitivity to clock compounds at single agent and combinations of clock compounds compared to non-cancerous cells, U2OS human osteosarcoma cells, and differentiated GSCs. Additionally, clock compounds are significantly more effective in targeting GSCs than TMZ. The clock compound SHP1705 increased over survival and delayed tumor growth in GBM PDX models. These results highlight the therapeutic potential small molecule circadian clock compounds have against GBM as both a single agent and adjuvant to existing therapies by specifically targeting the GSC population. Citation Format: Priscilla Chan, Lian Wu, Anahit Hovsepyan, Seda Mkhitaryan, Gevorg Karapetyan, Khalid Shah, Hiroaki Wakimoto, Theodore Kamenecka, Laura A. Solt, Jamie Cope, Rex A. Moats, Tsuyoshi Hirota, Jeremy N. Rich, Steve A. Kay. Small molecule circadian clock compounds display therapeutic potential in targeting glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 505.

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults, characterized by complex genetic changes and a poor prognosis. Current standard therapies, including surgery, chemotherapy, and radiotherapy, have limited effectiveness. Emerging therapeutic strategies aim to address the high recurrence rate and improve outcomes by targeting glioblastoma stem cells (GSCs), the blood-brain barrier, and utilizing advanced drug delivery systems. This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. An electronic search was conducted across several databases, including PubMed, Embase, Scopus, Web of Science, and Cochrane, covering studies published from January 2019 to May 2024. The inclusion criteria encompassed primary research studies in English focusing on emerging therapies for treating GB in adults. Eligible studies included experimental and observational studies. Only peer-reviewed journal articles were considered. Exclusion criteria included non-human studies, pediatric studies, non-peer-reviewed articles, systematic reviews, case reports, conference abstracts, and editorials. The search identified 755 articles and, finally, 24 of them met the inclusion criteria. The key findings highlight various promising therapies. Despite advances in treatment approaches, the complexity and heterogeneity of GB necessitate ongoing research to optimize these innovative strategies. The study has limitations that should be considered. The inclusion of only English-language articles may introduce language bias, and the focus on peer-reviewed articles could exclude valuable data from non-peer-reviewed sources. Heterogeneity among studies, particularly in sample sizes and designs, complicates comparison and synthesis, while the reliance on preclinical models limits generalizability to clinical practice. Nonetheless, this review provides a comprehensive overview of the emerging therapies that hold promise for improving patient outcomes in GB treatment.

Lipid-based nanoparticles to address the limitations of GBM therapy by overcoming the blood-brain barrier, targeting glioblastoma stem cells, and counteracting the immunosuppressive tumor microenvironment

Background: Glioblastoma (GBM) is the most common and aggressive human brain cancer with high relapse rate, lacking any targeted therapy. The current therapy consisting of surgery in combination with radiotherapy and the DNA-alkylating agent temozolomide has led to median survival increase. Nevertheless, many patients, after early treatment response, experience drug resistance and tumor progression. Recent advances identified cancer stem cell subpopulation as the cause of treatment failure, suggesting that the direct targeting of this cell subset may represent an innovative approach to improve the therapy efficacy. Based on studies indicating the role of the tyrosine kinase receptor MET in GBM stem cells, we examined the effects of the rational combination of radiotherapy with a MET inhibitor against stem-like cells from a human GBM cell line. Methods: Stem-like cells from U87MG cell line were sorted using PKH-67-labeling method for isolating slow-dividing cells (PKH67+), and analyzed for cell proliferation index by WST assay, clonogenic potential, and expression of stemness-related transcription factors by RT-PCR. Effects by the MET inhibitor alone and in combination with radiotherapy (IR) were evaluated on cell proliferation by Trypan Blue exclusion test, on apoptosis, cell cycle progression, GFAP protein expression by flow cytometry and on neurosphere-forming efficiency. The mechanisms underlying the effects induced by the combined treatment were dissected by biochemical and RT-PCR assays. Results: The PKH-67+ cells recapitulated the functional properties of stem-like cells, as slower cell proliferation index, higher clonogenic capacity, and expression of stemness-transcription factors than the negative counterpart. They also displayed a marked radioresistance dependent on MET signalling activation and characterized by the enrichment in stem-like cells as indicated by the upregulation of stemness-related transcription factor expression Nanog and Sox2, and enhanced susceptibility to MET inhibitor. The single-agent therapy with the inhibitor induced cell proliferation inhibition with an early cell cycle arrest in G2/M phase and a late cytotoxic effect. The drug alone negatively affected the neurosphere-forming efficiency and the clonogenic capacity. Following IR, the daily treatment with the inhibitor of MET significantly caused time-dependent cell proliferation inhibition, associated with an increased percentage of apoptotic cells and cells positive for GFAP, a glial differentiation marker, as compared to single-agent treatments. The biochemical analysis confirmed that MET inhibition remarkably impaired the radioresistance, and parallelly reduced the content of stem-like cells, as indicated either by the negative regulation of Nanog and Sox2 genes or the positive regulation of GFAP. Conclusions: Our results provide evidence that targeting MET with a specific inhibitor might interfere with MET-mediated radiotherapy resistance, thus sensitizing the GBM-stem-like cells to radiation, depleting the pool, and parallelly inducing their reprogramming towards more differentiated subtype. Citation Format: Anna Rossini, Evelyn Oliva Savoia, Eleonora Cicoria, Lorenzo Castagnoli, Serenella Pupa, Filippo de Braud, Massimo Di Nicola. Targeting glioblastoma stem cells through a MET inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A068.

Glioblastoma stem cells (GSC) have been implicated in tumor resistance to radio- and chemotherapy. T type calcium channels (Cav3.2) regulate cell cycle progression by mediating the necessary influx of calcium for transit past the G1/S cell cycle checkpoint. We hypothesized that treating GSCs and GSC-derived xenografts with the FDA-approved Cav3.2 channel blocker mibefradil would synchronize GSCs to enter the S phase, and consequently sensitize them to cytotoxic therapies. We demonstrated that Cav3.2 is highly expressed in the majority of human GBM specimens and all GCSs, compared to normal brain tissue or glioma cell lines, respectively. Mibefradil treatment inhibited GSC proliferation and induced cell death. Furthermore, mibefradil sensitized GSCs to temozolomide treatment (TMZ) and increased TMZ-induced cell death by 25-63% (p < 0.05) in vitro. To determine the effect of mibefradil on glioblastoma xenograft growth, we implanted GSCs in the brains of immunodeficient mice and treated the mice with mibefradil and/or TMZ and monitored tumor growth by MRI. We found that mibefradil increased TMZ-induced tumor growth inhibition by 60% (p < 0.05). Mibefradil also significantly improved the survival of TMZ-treated mice bearing GSC-derived xenografts. To further investigate the mechanism of action of mibefradil, we performed reverse phase protein arrays on GSCs treated with mibefradil. We found that mibefradil strongly regulated GSC apoptosis by regulating BCL2, PUMA and BAX expressions/activations as well as caspase cleavage. Mibefradil also altered proteins involved in autophagy and invasion including LC3, FAK and other. We are currently performing RNA-seq to determine the transcriptome wide changes that are induced by Cav3.2 inhibition. Altogether, the data provide mechanistic and functional rationales for the use of Cav3.2 inhibitors such as mibefradil as a new adjuvant therapy that enhances the efficacy of cytotoxic therapies in glioblastoma by targeting glioblastoma stem cells.

Glioblastoma (GBM) is the most devastating and least treatable brain tumor with median survival <15 months and extremely high recurrence rates. Promising results of immune checkpoint blockade obtained from pre-clinical studies in mice did not translate to clinic, and new strategies are urgently needed, particularly those targeting GBM stem cells (GSCs) that are held responsible for drug resistance and tumor recurrence. Patient-derived GSC cultures are critical for finding effective brain tumor therapies. Here, we investigated the ability of the recently described monoclonal antibody Nilo1 to specifically recognize GSCs isolated from GBM surgical samples. We employed five patient-derived GSC cultures with different stemness marker expression and differentiation potential, able to recapitulate original tumors when xenotransplanted in vivo. To answer whether Nilo1 has any functional effects in patient-derived GSCs lines, we treated the cells with Nilo1 in vitro and analyzed cell proliferation, cell cycle, apoptosis, sphere formation, as well as the expression of stem vs. differentiation markers. All tested GSCs stained positively for Nilo1, and the ability of Nilo1 to recognize GSCs strongly relied on their stem-like phenotype. Our results showed that a subset of patient-derived GSCs were sensitive to Nilo1 treatment. In three GSC lines Nilo1 triggered differentiation accompanied by the induction of p21. Most strikingly, in one GSC line Nilo1 completely abrogated self-renewal and led to Bax-associated apoptosis. Our data suggest that Nilo1 targets a molecule functionally relevant for stemness maintenance and pinpoint Nilo1 as a novel antibody-based therapeutical strategy to be used either alone or in combination with cytotoxic drugs for GSC targeting. Further pre-clinical studies are needed to validate the effectiveness of GSC-specific Nilo1 targeting in vivo.

&lt;div&gt;Abstract&lt;p&gt;Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of &lt;i&gt;BMAL1&lt;/i&gt; or &lt;i&gt;CLOCK&lt;/i&gt; in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting &lt;i&gt;BMAL1&lt;/i&gt; or &lt;i&gt;CLOCK&lt;/i&gt; attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1–CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor efficacy. Collectively, these findings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms.&lt;/p&gt;Significance:&lt;p&gt;Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal.&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1469&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of &lt;i&gt;BMAL1&lt;/i&gt; or &lt;i&gt;CLOCK&lt;/i&gt; in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting &lt;i&gt;BMAL1&lt;/i&gt; or &lt;i&gt;CLOCK&lt;/i&gt; attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1–CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor efficacy. Collectively, these findings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms.&lt;/p&gt;Significance:&lt;p&gt;Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal.&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 1469&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of BMAL1 or CLOCK in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting BMAL1 or CLOCK attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1-CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor efficacy. Collectively, these findings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms. SIGNIFICANCE: Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal.This article is highlighted in the In This Issue feature, p. 1469.

Background Glioblastoma (GBM) represents the most common primary malignant brain tumor in adults, with a median survival rate of less than two years. GBM presents with therapeutic resistance and high rates of recurrence, largely attributed to the presence of glioblastoma stem cells (GSCs). GSCs are frequently located in specific niches, including a hypoxic niche and a normoxic, perivascular niche. Targeting GSCs is a potential therapeutic avenue for treating GBM, but the details of GSC function in hypoxic conditions remains poorly understood. Further, most gene expression findings are limited to epigenetic or transcriptional regulation assays, overlooking the importance of the translatome. This study expands on conclusions made using steady-state mRNA levels by including ribosome profiling to evaluate translational regulation mechanisms. Methods We selected two patient-derived GSC lines (GSC 215, GSC 316) and cultured them in both hypoxic (5% O2) and normoxic (21% O2) conditions. RNA-seq libraries were prepared in parallel with RIBO-seq libraries for the lines in both conditions, with biological replicates for each treatment group, after which Illumina sequencing was performed. Raw sequencing reads were assessed for quality. Single-end reads were aligned and counted using command line versions of STAR, BEDTools, and SAMTools. Differential changes in ribosome-protected fragments (∆RPF) and changes in transcript levels (∆RNA) were calculated using DESeq2. GO term analyses for transcripts was performed with Reactome using a hypergeometric statistical test and BH FDR correction. Results Comparing ∆RNA and ∆RPF levels between hypoxic and normoxic GSCs reveals 9 functional clusters of genes. These categories indicate genes which are post-transcriptionally controlled in 3 manners: translationally suppressed (28.19%), translationally upregulated (32.87%), or concurrently translated (38.93%). Altogether, more than 60% of mRNA show marked regulation on the translational level. Significant enrichment of genes in the NOTCH signaling pathway (p = 1.09e-02), WNT signaling pathway (p = 1.37e-04), and RHO GTPase cycle (p = 2.37e-07) are present among the transcripts that experience translational upregulation in hypoxic conditions. Conclusion This study may lead to the identification of potential therapeutic targets as evidenced by their function as regulatory hubs in cellular networks responsible for hypoxic adaptation. These observations are largely undetected by RNA-seq measurements.

Background Glioblastoma is the most common and lethal brain tumor in the adult population and immunotherapy is playing an increasingly central role in the treatment of many cancers. Nevertheless, the search for effective immunotherapeutic approaches for glioblastoma patients continues. In this study, we aimed to explore the therapeutic potential of allogeneic highly activated super-charged natural killer (NK) cells in glioblastoma. Material and Methods Chromium release- and calcein release-based cytotoxicity assays, ELISA, ELISPOT, and multiplex cytokine assays were used to determine NK cell cytotoxicity against glioblastoma stem cells (GSCs) and secretion of cytokines. Cell surface marker expression using flow cytometry and cell growth in vitro and in vivo were measured to determine GSC phenotype. NK cell killing and penetration in 3D were measured using confocal microscopy of GSC tumorospheres. Results Super-charged NK cells efficiently lysed patient-derived GSCs in 2D and 3D models potentially reversing the immunosuppression observed in patients. NK-cells secreted IFN-γ, upregulated GSC surface expression of CD54 and MHC class I and increased sensitivity of GSCs to chemotherapeutic drugs. Co-localization of NK cells with GBM cells in perivascular niches in glioblastoma tissues and their direct contact with GSCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSCs. Conclusion Allogeneic super-charged NK cells appear to be a potential therapeutic approach for glioblastoma by selectively killing therapy-resistant cancer stem cell population, increasing their immune-related surface markers and enhancing their sensitivity to chemotherapy. Due to GSC heterogeneity and plasticity personalized immunotherapeutic strategies should be developed to effectively target glioblastomas.