Presence Of Cancer Stem-like Cells Research Articles

Evaluation of the theranostic potential of [64Cu]CuCl2 in glioblastoma spheroids

BackgroundGlioblastoma is an extremely aggressive malignant tumor with a very poor prognosis. Due to the increased proliferation rate of glioblastoma, there is the development of hypoxic regions, characterized by an increased concentration of copper (Cu). Considering this, 64Cu has attracted attention as a possible theranostic radionuclide for glioblastoma. In particular, [64Cu]CuCl2 accumulates in glioblastoma, being considered a suitable agent for positron emission tomography. Here, we explore further the theranostic potential of [64Cu]CuCl2, by studying its therapeutic effects in advanced three-dimensional glioblastoma cellular models. First, we established spheroids from three glioblastoma (T98G, U373, and U87) and a non-tumoral astrocytic cell line. Then, we evaluated the therapeutic responses of spheroids to [64Cu]CuCl2 exposure by analyzing spheroids' growth, viability, and cells' proliferative capacity. Afterward, we studied possible mechanisms responsible for the therapeutic outcomes, including the uptake of 64Cu, the expression levels of a copper transporter (CTR1), the presence of a cancer stem cell population, and the production of reactive oxygen species (ROS).ResultsResults revealed that [64Cu]CuCl2 is able to significantly reduce spheroids' growth and viability, while also affecting cells' proliferation capacity. The uptake of 64Cu, the presence of cancer stem-like cells and the production of ROS were in accordance with the therapeutic response. However, expression levels of CTR1 were not in agreement with uptake levels, revealing that other mechanisms could be involved in the uptake of 64Cu.ConclusionsOverall, our results further support [64Cu]CuCl2 potential as a theranostic agent for glioblastoma, unveiling potential mechanisms that could be involved in the therapeutic response.

Fumagillin regulates stemness and malignancies in cancer stem-like cells derived from liver cancer via targeting to MetAP-2.

Cancer relapse is associated with the presence of cancer stem-like cells (CSCs), which lead to multidirectional differentiation and unrestricted proliferative replication. Fumagillin, a myocotoxin produced by the saprophytic filamentous fungus Aspergillus fumigatus, has been reported to affect malignant characteristics in hepatocellular cancer cells. However, its exact role in CSCs is still unknown. CSCs were enriched by culturing cancer cells in serum-free medium. The effects of fumagillin on malignant cell characteristics and mitochondrial function were measured. The regulatory role of fumagillin on methionine aminopeptidase-2 (MetAP-2) was assessed. When it was supplemented in medium, fumagillin treatment inhibited sphere formation and the maintenance of stemness of CSCs without disturbing cell growth. Fumagillin also decreased stemness-related markers and the aldehyde dehydrogenase 1 (ALDH1)-positive proportion, which demonstrated that fumagillin decreases stemness in CSCs. It was also found to inhibit malignant traits in CSCs, including cell proliferation, invasion, and tumor formation, and sensitize CSCs to chemoagents, including sorafenib and doxorubicin, by promoting chemoagent-induced apoptosis. Moreover, fumagillin treatment was found to disturb mitochondrial membrane homeostasis, ATP synthesis and mitochondrial transcriptional activity. In addition, we found that fumagillin decreased MetAP-2 protein levels and exerted anti-CSC effects potentially by regulating MetAP-2. We also found that fumagillin treatment activated p53 and its transcriptional activity and thus caused cell cycle blockade. Moreover, fumagillin treatment significantly decreased tumor formation in nude mice. This work offers evidence for fumagillin as a specific inhibitor of liver cancer CSCs and proposes a novel strategy for cancer therapy.

Abstract 1638: Validation of a novel pan-Ras inhibitor, ADT-007, against lethal variants of prostate cancer

Abstract Metastatic prostate cancer is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and are therefore sensitive to androgen deprivation therapies. However, eventual resistance to standard medical castration and secondary chemotherapies including next-generation AR-targeting agents like abiraterone or enzalutamide and taxanes (Docetaxel or cabazitaxel) is nearly universal. Further, neuroendocrine PCa or NEPC, a poorly differentiated aggressive variant of PCa that lacks AR expression, and the presence of cancer stem-like cells with self-renewal and differentiation (epithelial to mesenchymal trans-differentiation or EMT) capacities significantly contribute to the development of clinically most aggressive and lethal variants of PCa. Endogenous Ras/ERK/MAPK signaling is known to drive prostate tumor formation, cancer progression, cell migration, and the acquisition of mesenchymal phenotype (EMT) in metastatic prostate cancers. Ras family members are responsible for modulating the function of most of the receptors upregulated in advanced PCa, including AR, so that AR can be functional even in the absence of physiologic levels of androgen. Previous studies have reported that the NSAID, sulindac, inhibits Ras-induced transformation and directly binds Ras. In this study, we show that a non-COX inhibitory sulindac derivative, ADT-007, is a potent drug candidate against lethal PCa. First, we performed single-cell RNA sequencing as a biomarker-based drug screen and showed that chemo-resistant, drug-tolerant, stem-cell-like PCa cell clusters had high expression of Ras genes, indicating that the Ras inhibitor, ADT-007, may be effective in targeting these aggressive subclones. Next, using several cell-based assays on a large panel of lethal PCa cell lines representing metastatic castration-resistant PCa (mCRPC), NEPC as well as African American ancestry, we showed that ADT-007 is highly effective as a single-agent and in combination with taxanes. Combination index values were <0.7, and BLISS scores were >10, indicating very high synergy. Finally, using pre-vs-post-treatment bulk RNAseq, we demonstrated that ADT-007 is indeed effective in downregulating KRas and NRas in a dose-dependent manner. In addition, ingenuity pathway analysis (IPA) predicted significant downregulation of cell cycle, DNA replication, CCND1, AR, and the lncRNA ELDR, and upregulation of autophagy and interferon pathway genes, and the microRNA miR-96. Finally, using PCa patient datasets, we showed that ADT-007 is potentially capable of reversing the expression of several genes associated with biochemical recurrence. Thus, our results show that ADT-007 is a novel drug candidate that circumvents subclonal aggressiveness, drug resistance, and stemness in lethal PCa through simultaneous inhibition of multiple oncogenic factors/pathways. Citation Format: Taraswi Mitra Ghosh, Adam B. Keeton, Xi Chen, Salsabil Ahmed, Razan S. Waliagha, Gary A. Piazza, Amit K. Mitra. Validation of a novel pan-Ras inhibitor, ADT-007, against lethal variants of prostate cancer [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 1638.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

Evaluation of anticancer effects of frankincense on breast cancer stem-like cells.

Relapse and metastasis in breast cancer are linked to cancer stem cells (CSCs) resistant to anticancer therapies. The presence of cancer stem-like cells (CSLCs) and their ability to self-renew is determined by in vitro spheroid formation. Many studies have found that frankincense has anticancer impacts, although these effects on breast CSLCs have never been evaluated. A population of heterogeneous breast tumor cells was extracted from the tumor mass after generating an animal model of triple-negative breast cancer (TNBC). Spheroid formation was used as an in vitro assay to determine the existence of CSLCs in these cells. MTT assay was used to determine frankincense's cytotoxic activity. An annexin V- propidium iodide (PI) staining and scratch test were used to assess the induction of apoptosis and antimetastatic effects of frankincense. The frankincense extract has significant cytotoxic and apoptotic effects on breast CSLCs. Although, the breast CSLCs are more resistant to these impacts than other breast cancer cells. Our study is the first report that indicates that frankincense extract has anticancer properties in breast CSLCs. Compared to many anticancer chemicals, which have limited potential to battle cancer stem cells, frankincense is an appropriate option to combat breast CSCs.

Chelerythrine Chloride Inhibits Stemness of Melanoma Cancer Stem-Like Cells (CSCs) Potentially via Inducing Reactive Oxygen Species and Causing Mitochondria Dysfunction.

Growing evidence has demonstrated that high heterogeneity contributes to poor prognosis and malignancies. The existence of melanoma cancer stem-like cells (CSCs), which are a small subpopulation of melanoma cells, is responsible for tumour resistance to therapies. Recently, plant secondary metabolites have attracted attention because they are considered promising compounds that are isolated from herbs that could help to target different subpopulations of tumours. In the present study, we aimed to identify the antitumourigenic activities of the medicinal compound chelerythrine chloride (CHE) on melanoma CSCs. CHE (30-40 μmol/L) induced apoptosis in A375 and A2058 CSCs. A relatively low dose of CHE (1-5 μmol/L) inhibited the stemness of melanoma CSCs without inducing apoptosis. Coculture of CHE with A375 and A2058 cells also inhibited sphere formation and decreased stemness factors, including Nanog, Oct4, and Sox2. In functional characterizations, we observed that CHE treatment increased both cellular reactive oxygen species (ROS) and mitochondrial ROS, which resulted in a decrease in mitochondrial energy production and sphere formation. Abolishing CHE-induced ROS by N-acetyl-L-cysteine (NAC), a ROS scavenger, reversed the inhibitory effects of CHE on sphere formation, suggesting that CHE-induced ROS are the potential cause of the inhibition of sphere formation. In conclusion, CHE may exert its antitumour effect as an antistem cell natural compound, suggesting that selection of the antistem cell effects of natural compounds might be a promising strategy to overcome the poor prognosis of melanoma due to the presence of CSCs.

Association of ALDH1 with Response to Radiotherapy and Its Impact on Survival in Patients with Advanced Stage of Head and Neck Squamous Cell Carcinoma (HNSCC).

Background:The presence of cancer stem-like cells within tumor microenvironment distinctly governs response to chemo-radiotherapy. The ALDH1 (Aldehyde dehydrogenase 1) has emerged as a cancer stem cell (CSC) marker in various tumors. The aim of the study was to examine the expression of ALDH1 in HNSCC patients undergoing radiotherapy to evaluate its correlation with clinicopathological parameter, treatment response and survival. Methods:Expression of ALDH1 was evaluated by immunohistochemistry in 90 histopathologically confirmed HNSCC patients and 90 matched controls. The association between ALDH1 expression, clinicopathological parameters and treatment response was determined. Results:The immunohistochemistry results showed that ALDH1 was consistently expressed in all the HNSCC specimens although at different intensities. On the other hand, control specimens did not show similar expression of ALDH1. ALDH1 expression demonstrated statistically significant association with tumor size (p<0.001), lymph node status (p<0.001), stage (p<0.001), grade (p<0.001) and treatment response (p<0.001). Multivariate ordinal logistic regression analysis indicated alcohol and ALDH1 as an independent predictor of responsiveness to radiotherapy in HNSCC patients. Multivariate Cox regression analysis indicated that lymph node status (p=0.020), grade (p=0.006) and recurrence (p=0.002) were potential independent predictors of overall survival. Conclusion:From previous studies, ALDH1 has been contemplated not only as a promising prognostic and diagnostic marker but also as a likely drug target. Our study gives new understanding regarding the association between ALDH1, cancer prognosis and radioresistance. Our findings suggest that ALDH1, lymph node status, grade and alcohol could be the viable targets for HNSCC and it also provides new prospects for radiotherapy sensitivity in HNSCC.

Temozolomide: An Updated Overview of Resistance Mechanisms, Nanotechnology Advances and Clinical Applications.

Temozolomide (TMZ), an oral alkylating prodrug which delivers a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine), is frequently used together with radiotherapy as part of the first-line treatment of high-grade gliomas. The main advantages are its high oral bioavailability (almost 100% although the concentration found in the cerebrospinal fluid was approximately 20% of the plasma concentration of TMZ), its lipophilic properties, and small size that confer the ability to cross the blood-brain barrier. Furthermore, this agent has demonstrated activity not only in brain tumors but also in a variety of solid tumors. However, conventional therapy using surgery, radiation, and TMZ in glioblastoma results in a median patient survival of 14.6 months. Treatment failure has been associated with tumor drug resistance. This phenomenon has been linked to the expression of O6-methylguanine-DNA methyltransferase, but the mismatch repair system and the presence of cancer stem-like cells in tumors have also been related to TMZ resistance. The understanding of these mechanisms is essential for the development of new therapeutic strategies in the clinical use of TMZ, including the use of nanomaterial delivery systems and the association with other chemotherapy agents. The aim of this review is to summarize the resistance mechanisms of TMZ and the current advances to improve its clinical use.

Cyclopamine sensitizes glioblastoma cells to temozolomide treatment through Sonic hedgehog pathway

AimGlioblastoma is an extremely aggressive glioma, resistant to radio and chemotherapy usually performed with temozolomide. One of the main reasons for glioblastoma resistance to conventional therapies is due to the presence of cancer stem-like cells. These cells could recapitulate some signaling pathways important for embryonic development, such as Sonic hedgehog. Here, we investigated if the inhibitor of the Sonic hedgehog pathway, cyclopamine, could potentiate the temozolomide effect in cancer stem-like cells and glioblastoma cell lines in vitro. Main methodsThe viability of glioblastoma cells exposed to cyclopamine and temozolomide treatment was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while the induction of apoptosis was assessed by western blot. The stemness properties of glioma cells were verified by clonogenic and differentiation assay and the expression of stem cell markers were measured by fluorescence microscopy and western blot. Key findingsThe glioblastoma viability was reduced by cyclopamine treatment. Cyclopamine potentiated temozolomide treatment in glioblastoma cell lines by inducing apoptosis through activation of caspase-3 cleaved. Conversely, the combined treatment of cyclopamine and temozolomide potentiated the stemness properties of glioblastoma cells by inducing the expression of SOX-2 and OCT-4. SignificanceCyclopamine plays an effect on glioblastoma cell lines but also sensibilize them to temozolomide treatment. Thus, first-line treatment with Sonic hedgehog inhibitor followed by temozolomide could be used as a new therapeutic strategy for glioblastoma patients.

Abstract P6-03-22: MUC1 oncoprotein confers trastuzumab resistance in HER2-positive breast cancer cells by maintaining cancer stem cell phenotype

Abstract Background: Although trastuzumab has greatly improved the outcome of HER2-positive breast cancer, the emergence of resistance hampers its clinical benefits. Trastuzumab resistance is a multi-factorial consequence predominantly due to the presence of cancer stem-like cells (CSCs). Interestingly, upregulation of the MUC1 gene has been reported to be associated with CSC growth, but its function in regulating CSC proliferation has not been studied in the trastuzumab-resistant (TrR) breast cancer cohort. Purpose: To evaluate the function of MUC1 in regulating cancer stem cell phenotype in TrR HER2-positive breast cancer cohort Methods 20,000 cells of each HER2-positive cell line (BT474R, T47D, HCC1954) were plated in ultralow attachment dishes and cultured in complete Mammocult Media for two weeks. Bright-field images were acquired. For CD44/CD24 staining, cells were incubated with Cy7-conjugated anti-CD44 and CF594-conujugated anti-CD24 per the previously described protocol. Cy7- and CF594-conjugated anti-mouse IgG were used as negative controls. For cell cycle analysis, cells were harvested and fixed with 70% cold ethanol for 24 H, followed by incubation with propidium iodide (PI, 50 μg/ml) and RNase (50 μg/ml) for 30 min, and analyzed by flow cytometry using Flowjo software. To silence MUC1 expression, 1 × 106 cells were transfected using the Neon electroporation system with 5 ng siMUC1 or 5 ng control siRNA vectors. The cells were electroporated for 30 ms at 1100 volts with two pulses. The cells were plated in 6-well plates with the appropriate media, incubated at 37°C for 48 hours and processed for further experiments. Results: Our results show that BT474R, the TrR cell line, forms more mammospheres and has a greater inherent CD44high/CD24low CSC population than BT474, the trastuzumab-sensitive (TrS) cell line, and that MUC1 is significantly upregulated in TrR compare to TrS HER2-positive breast cancer cells. We further demonstrate the significant inhibition of the CSC population upon silencing MUC1 expression by performing both mammosphere formation assay and CD44high/CD24low phenotype characterization and that this regulation is independent of cell cycle arrest or HER2 expression levels. Conclusion: MUC1 oncoprotein confers trastuzumab resistance in HER2-Positive breast cancer cells by maintaining cancer stem cell phenotype, indicating that it could be a potential predictive and targetable biomarker to overcome trastuzumab resistance in HER2-Positive breast cancer patients. Citation Format: Jun Yin, Andrea Sand, Mitchell Piacsek, Chaoyang Sun, Logan S. Friedrich, Richard A. Rovin, Judy A. Tjoe. MUC1 oncoprotein confers trastuzumab resistance in HER2-positive breast cancer cells by maintaining cancer stem cell phenotype [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-22.

WEE1 inhibitor, AZD1775, overcomes trastuzumab resistance by targeting cancer stem-like properties in HER2-positive breast cancer.

Although trastuzumab has greatly improved the outcome of HER2-positive breast cancer, the emergence of resistance hampers its clinical benefits. Trastuzumab resistance is a multi-factorial consequence predominantly due to presence of cancer stem-like cells (CSCs). AZD1775, a potent anti-cancer agent targeting WEE1 kinase to drive tumor cells with DNA damage to premature mitosis, has previously shown high efficacies when targeting different cancers with a well-tolerated cytotoxic profile, but has not been evaluated in trastuzumab-resistant (TrR) breast cancer. We sought to investigate the effect of AZD1775 on cancer stem-like cell (CSC) properties, apoptosis, cell cycle regulation in TrR breast cancer. Our study for the first time demonstrated that AZD1775 induces apoptosis and arrests TrR cells at G2/M phase. More importantly, AZD1775 effectively targeted CSC properties by suppressing MUC1 expression levels. AZD1775 administration also induced apoptosis in our in-house patient-derived tumor cell line at passage 0, implying its significant clinical relevance. These findings highlight the potential clinical application of AZD1775 in overcoming trastuzumab resistance in breast cancer.

Abstract GMM-019: MEK INHIBITOR TRAMETINIB IN HIGH GRADE OVARIAN CANCER PROLIFERATION

Abstract A major challenge in ovarian cancer therapy is very high recurrence that is currently attributed to the presence of cancer stem-like cells (CSCs). CSCs are characterized by pronounced chemoresistance and high clonogenic ability that allow them to survive treatment and trigger relapse. The search for new treatment approaches capable of eradicating CSCs is crucial to prevent recurrent disease. Despite lack of BRAF or KRAS mutations in high grade serous ovarian cancer, MEK1/2-ERK1/2 pathway is activated in ovarian cancer cell lines and primary tumors. The aim of present work was to evaluate MEK inhibitor trametinib on cell proliferation and tumorigenesis. Trametinib is approved by FDA for melanoma and other solid tumors with specific BRAF mutations, thus can be used in a timely manner in clinic if found effective. We used cisplatin-resistant OVCAR8 and PEO4 cell lines displaying very high fraction of CD133-positive cells exhibiting CSC-like properties as main experimental model. Treatment of cells with trametinib caused very efficient inhibition of MEK-ERK pathway activity (up to 100% inhibition of ERK1/2 phosphorylation). Inhibition of pathway activity was confirmed by MEK-ERK-associated genes downregulation as assessed by RT-qPCR. PHLDA1, SPRY2 and DUSP4 genes were identified as early MEK responders whereas SPRY4, ETV4 and ETV5 were responsive to prolonged MEK inhibition. Trametinib treatment resulted in significant reduction of cell growth rate and number of viable cells (5-fold reduction with 100nM trametinib, p&amp;lt;0.05) in both cell lines. Cell cycle assay demonstrated cell cycle arrest in G1/0 -phase indicating that MEK-ERK activation is required for transition from G1 to S-phase. To further elaborate associations between MEK pathway activity and cell proliferation, we studied cells enriched in different stages of S- and G2-phase. MEK pathway activity was subject to prominent activation during S-phase reflected by significant increase in phosphorylation levels of ERK1/2, its direct targets FRA1 and p90RSK and subsequent upregulation of reporter genes PHLDA1, SPRY2 and DUSP4. Our data indicate that trametinib treatment may induce efficient inhibition of ovarian cancer cell proliferation and therefore may have positive impact upon duration of remission for ovarian cancer patients. While trametinib alone is not capable of eradicating ovarian CSCs, its combinations with other drugs may provide a promising tool for ovarian cancer treatment, especially for tumors with high level of MEK-ERK pathway activity. One of such drugs to use in conjunction with trametinib can be Aldehyde Dehydrogenase (ALDH) inhibitor 673A that was previously characterized in our research as a compound capable of inducing cell programed necroptosis in ovarian CSCs. Citation Format: Mikhail Chesnokov, Anmbreen Jamroze, Wootae Ha, Ilana Chefetz. MEK INHIBITOR TRAMETINIB IN HIGH GRADE OVARIAN CANCER PROLIFERATION [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-019.

Insights in the immunobiology of glioblastoma.

Glioblastoma, a grade IV astrocytoma, is considered as the most malignant intracranial tumor, characterized by poor prognosis and therapy resistance. Tumor heterogeneity that often leads to distinct functional phenotypes contributes to glioblastoma (GB) indispensable growth and aggressiveness. The complex interaction of neoplastic cells with tumor microenvironment (TME) along with the presence of cancer stem-like cells (CSCs) largely confers to extrinsic and intrinsic GB heterogeneity. Recent data indicate that glioma cells secrete a variety of soluble immunoregulatory factors to attract different cell types to TME including astrocytes, endothelial cells, circulating stem cells, and a range of immune cells. These further induce a local production of cytokines, chemokines, and growth factors which upon crosstalk with extracellular matrix (ECM) components reprogram immune cells to inflammatory or anti-inflammatory phenotypes and manipulate host's immune response in favor of cancer growth and metastasis. Herein, we provide an overview of the immunobiologic factors that orchestrate the complex network of glioma cells and TME interactions in an effort to identify potential therapeutic targets for GB malignancy. Current therapeutic schemes and advances in targeting GB-TME crosstalk are further discussed. KEY MESSAGES: • Intrinsic and extrinsic tumor heterogeneity affects GB growth and aggressiveness. • GB cells secrete growth factors and chemoattractants to recruit immune cells to TME. • GAMs are a critical cell type in promoting GB growth. • GAMs change from pro-inflammatory, anti-tumor M1 phenotype to pro-tumorigenic M2. • Novel therapeutic agents target the crosstalk of neoplastic cells with TME.

Endothelial cells promote 3D invasion of GBM by IL-8-dependent induction of cancer stem cell properties

Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids were co-cultured with brain endothelial cells (ECs) in microfabricated collagen gels. Integrating these systems with 3D imaging and biochemical assays revealed that ECs increase GBM invasiveness and growth through interleukin-8 (IL-8)-mediated enrichment of CSCs. Blockade of IL-8 inhibited these effects in GBM-EC co-cultures, while IL-8 supplementation increased CSC-mediated growth and invasion in GBM-monocultures. Experiments in mice confirmed that ECs and IL-8 stimulate intracranial tumor growth and invasion in vivo. Collectively, perivascular niche conditions promote GBM growth and invasion by increasing CSC frequency, and IL-8 may be explored clinically to inhibit these interactions.

SOX9 Stem-Cell Factor: Clinical and Functional Relevance in Cancer.

Transcriptional and epigenetic embryonic programs can be reactivated in cancer cells. As result, a specific subset of undifferentiated cells with stem-cells properties emerges and drives tumorigenesis. Recent findings have shown that ectoderm- and endoderm-derived tissues continue expressing stem-cells related transcription factors of the SOX-family of proteins such as SOX2 and SOX9 which have been implicated in the presence of cancer stem-like cells (CSCs) in tumors. Currently, there is enough evidence suggesting an oncogenic role for SOX9 in different types of human cancers. This review provides a summary of the current knowledge about the involvement of SOX9 in development and progression of cancer. Understanding the functional roles of SOX9 and clinical relevance is crucial for developing novel treatments targeting CSCs in cancer.

Radiation-induced DNA damage response and resistance in colorectal cancer stem-like cells

Purpose: Radiation therapy is an integral part of current treatment modality for colorectal cancer. Recent studies have revealed the presence of cancer stem-like cells (CSCs) population, in different tumors are responsible for therapeutic resistance and disease relapse, including colorectal cancer with poorer survival rate. Hence, characterization of the effect of Ionizing Radiation (IR) in colorectal cancer may serve to explain possible mechanisms.Material and methods: Parental HCT116 and HCT-15 cells and derived colonospheres were irradiated and dose was optimized based on cell survival assay and cell cycle analysis. DNA damage response (DDR) was elucidated by γH2AX foci formation, COMET assay, and ATM, p-ATM, ERCC1 expression post-treatment. The expression level of developmental marker (β-catenin), CSC markers (CD44, KLF4) and telomeric components (TRF2, RAP1, hTERT) were evaluated.Results: We observed cell survival was more in colonospheres post-irradiation and also exhibited decreased γH2AX foci, olive tail moment, increased ERCC1, and p-ATM expression than its parental counterpart which corresponds to efficient DDR. Differential expression of developmental marker, CSC markers, and telomeric components were observed after irradiation.Conclusion: This study highlighted the presence of CSC phenotype in colonospheres having increased DNA repair capacity. Differential expression of developmental marker, CSC markers and telomeric components between parental and colonospheres may contribute in radio-resistance property of CSCs.

KLF4 expression in the surgical cut margin is associated with disease relapse of oral squamous cell carcinoma

The presence of cancer stem-like cells (CSCs) in the majority of tumors is one of the factors responsible for disease relapse in oral squamous cell carcinoma (OSCC). In this study, we investigated the role of octamer-binding transcription factor 4 (OCT4) and Kruppel-like factor 4 (KLF4) in OSCC progression and disease relapse. In this study, 102 patients with OSCC were included. The expression of β-catenin and CSC markers (KLF4 and OCT4) in surgical cut margin and tumor were investigated through Western blot analysis, immunohistochemistry, and quantitative polymerase chain reaction analysis. The χ2 test was used to evaluate the association of β-catenin, OCT4, and KLF4 expression with clinicopathologic characteristics. Kaplan-Meier and Cox regression analyses were performed to correlate different clinical factors with the prognoses of patients with OSCC. We observed increased expression of OCT4, KLF4, and β-catenin in the cut margins (CMs) in recurrent OSCC. The χ2 test exhibited recurrence as one of the key factors associated with high expression of these markers. Kaplan-Meier and COX regression analyses demonstrated that increased expression of KLF4 in the CM region of recurrent patients was independently associated with a poor prognosis. Our findings indicated that expression of KLF4 can be used for monitoring disease progression and may serve as prognostic marker to predict recurrence.

Simvastatin modulates β-catenin/MDR1 expression on spheres derived from CF41.Mg canine mammary carcinoma cells.

The presence of cancer stem-like cells (CSC) within canine mammary tumors, may explain partly local recurrence and spreading, since their ability to resist conventional antitumor treatments as chemo and radiotherapy. It has been recently described that simvastatin - a drug that inhibits synthesis of cholesterol - attenuates the proliferation of canine mammary CSC derived from CF41.Mg canine mammary carcinoma cells, promoting their chemosensitizing and apoptosis. The canonical Wnt/β-catenin pathway is usually activated at CSC and up-regulates multidrug resistance protein 1 (MDR1), triggering chemoresistance. In the present study, we analyze the effect of simvastatin on β-catenin/MDR1 expression in spheres obtained from the CF41.Mg cell line as a model of CSC. Simvastatin increased phosphorylation of β-catenin without affecting its total expression. Moreover, MDR1 expression was decreased by simvastatin. These results suggest that simvastatin would facilitate the degradation of β-catenin, decreasing MDR1 expression and contributing to the chemosensitizing effects of the statin on canine mammary CSC.

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs).

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The presence of cancer stem-like cells (CSCs) is believed to be among the underlying reasons for the aggressiveness of PDAC, which contributes to chemoresistance and recurrence. However, the mechanisms that induce chemoresistance and inhibit apoptosis remain largely unknown.MethodsWe used serum-free medium to enrich CSCs from panc-1 human pancreatic cancer cells and performed sphere formation testing, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and semi-quantitative western blotting to confirm the stemness of panc-1 CSCs. Hallmarks of endoplasmic reticulum (ER) stress, including IRE1, PERK, ATF4, ATF6α, GRP78 and uPA expression, were detected after gemcitabine treatment. Effects of gemcitabine-induced uPA expression on cell invasion, sphere formation, colony formation and gemcitabine sensitivity were detected. Electrophoretic mobility shift assays (EMSAs) and RNA-immunoprecipitation (RIP) were performed to detect interaction between the uPA mRNA 3’-UTR and mutant p53-R273H expressed by panc-1 CSCs. The effects of upregulated uPA by gemcitabine on apoptosis were detected by Annexin V-FITC/PI staining, and the impact of uPA on small molecule CP-31398-restored mutant p53 transcriptional activity was measured by a luciferase reporter assay.ResultsEnriched panc-1 CSCs expressing high levels of CD44 and CD133 also produced significantly higher amounts of Oct4 and Nanog. Compared with panc-1 cells, panc-1 CSCs presented chemoresistance to gemcitabine. ER stress gene detections demonstrated effects of gemcitabine-induced ER stress on both the pro-apoptotic and pro-survival branches. ER stress-induced ATF6α upregulated level of uPA by transcriptionally activating GRP78. Gemcitabine-induced uPA promoted invasion, sphere formation and colony formation and attenuated apoptosis induced by gemcitabine in panc-1 CSCs, depending on interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs.ConclusionGemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed.

A phase II clinical trial of metformin as a cancer stem cell targeting agent in stage IIc/III/IV ovarian, fallopian tube, and primary peritoneal cancer.

5556 Background: Epidemiologic and preclinical studies suggest that Metformin has antitumor effects which may be due to an impact on cancer stem-like cells (CSC). We present a phase II trial of metformin administered in combination with chemotherapy for patients with advanced stage epithelial ovarian cancer (EOC). Primary endpoints were 18 month progression free survival (PFS) and CSC number in Metformin treated tumors. Methods: Thirty-eight patients with confirmed stage IIC(n=1)/III(n=25)/IV(n=12) EOC were treated with either neoadjuvant metformin followed primary debulking surgery and adjuvant Metformin+chemotherapy, or neo-adjuvant metformin+chemotherapy, followed by interval debulking and adjuvant chemotherapy+Metformin. Patients were evaluated for side effects, PFS and overall survival (OS). Metformin treated tumors were evaluated for the presence of CSC via FACS and sphere assays. Results: Thirty-two patients (84%) completed at least six cycles of metformin+chemotherapy. Metformin was well tolerated with only one grade III/IV treatment-related adverse event (3%) noted. Common adverse effects were diarrhea (18%) and nausea (16%). Eighteen month PFS was 65.4% (95% confidence interval 47.9-78.3), Median PFS was 21.7 months (CI-17-26.7). Estimated three year OS was 73.5% (CI-54.7-84.3) with median OS not reached after a media follow-up of 33 months. Finally, tumors treated with metformin were noted to have a 3-fold decrease in ALDH+ CSC at baseline, increased sensitivity to Cisplatin in vitro, and a reduced ability to amplify ALDH+ CSC with passage in vitro. Conclusions: This is the first prospective study of Metformin in EOC patients. Translational studies confirm an impact of metformin on CSC. Metformin was well tolerated and outcome results were favorable, supporting the use of Metformin in phase-III studies. Clinical trial information: NCT01579812.