Initiate Tumor Growth Research Articles

Accelerated tumor growth under intermittent hypoxia is associated with hypoxia-inducible factor-1-dependent adaptive responses to hypoxia.

Mounting evidence has revealed a causative role of intermittent hypoxia (IH) in cancer progression in mouse models of obstructive sleep apnea (OSA), but most studies have focused on the effects of IH following tumor implantation using an exposure to single IH frequency. Thus, we aimed to investigate 1) the potential effect of IH on the initial tumor growth in patients with OSA without an interaction with other mechanisms induced by IH in mice and 2) the influence of the IH frequency on tumor growth, which were tested using pre-conditioning with IH (Pre-IH) and 2 different IH frequencies, respectively. Pre-IH was achieved by alternatively maintaining melanoma cells between normoxia (10 min, 21% O2) and hypoxia (50 min, 1% O2) for 7 days (12 cycles per day) before administering them to mice. The conditions for IH-1 and IH-2 were 90 s of 12% FiO2 followed by 270s of 21% FiO2 (10 cycles/h), and 90 s of 12% FiO2 and 90 s of 21% FiO2 (20 cycles/h), respectively, for 8 h per day. Tumor growth was significantly higher in the Pre-IH group than in the normoxia group. In addition, the IH-2 group showed more accelerated tumor growth compared to the normoxia and IH-1 groups. Immunohistochemistry and gene-expression results consistently showed the up-regulation of molecules associated with HIF-1α-dependent hypoxic adaptation in tumors of the Pre-IH and IH-2 groups. Our findings reveal that IH increased tumor progression in a frequency-dependent manner, regardless of whether it was introduced before or after in vivo tumor cell implantation.

Whole exome sequencing and copy-number variation analysis of 20 NF2-associated spinal and cranial meningiomas.

2051 Background: Neurofibromatosis type 2 is a heritable tumor predisposition syndrome characterized by the growth of multiple tumor types in the nervous system, including bilateral vestibular schwannomas, meningiomas and ependymomas. Recent genomic sequencing studies have revealed that NF2 inactivation is the most frequent genetic event in sporadic meningiomas. In line with Knudson’s hypothesis, it is accepted that somatic inactivation of the wildtype NF2 allele initiates tumor growth in NF2 patients, but little is known of what other genes or pathways influence meningioma tumorigenesis. Methods: To investigate this question, we performed whole exome sequencing (WES) (Illumina Hi-Seq 2500 platform, 96 Mb SeqCap EZ Exome + UTR Library, NimbleGen) and SNP-array analysis (HumanOmniExpressExome-8, v1.2 arrays, Illumina) of twenty spinal and cranial meningioma samples from seven NF2 patients. Mutation validation was completed via orthogonal sequencing (IonTorrent, ThermoFisher). Results: We identified NF2 germline mutations in all patients, including five nonsense, one splice site and one likely pathogenic intronic mutation. We found that the predominant mechanism of somatic NF2 inactivation in the tumors was loss of heterozygosity (LOH): we identified large chromosome 22 deletions containing NF2 in nineteen out of twenty meningiomas. The second most frequent chromosomal aberration was a deletion within chromosome 1p, followed by entire chromosome X deletion and rearrangements in chromosome 17q. The remaining samples exhibited normal diploid genomic architecture. Somatic mutations included about twenty point substitutions and small indels. Conclusions: Our study revealed that somatic inactivation of NF2 is the most frequent and only recurrent genetic event in NF2-associated meningiomas. Large LOH events are the most prevalent second hit mechanism and may also represent a common path of meningioma progression. Somatic single nucleotide substitutions and small indels are rare in these tumors. Interestingly, we did not identify mutations in TRAF7, KLF4, AKT1, or SMO, which have been found to be critical for non-NF2 meningioma growth.

DHFR and MDR1 upregulation is associated with chemoresistance in osteosarcoma stem-like cells.

Tumor-initiating cells (TICs) are defined as a specialized subset of cells with tumor-initiating capacity that can initiate tumor growth, tumor relapse and metastasis. In the present study, osteosarcoma TICs (OS-TICs) were isolated and enriched from the osteosarcoma U2OS and MG-63 cell lines using sphere formation assays and serum-depleted media. These enriched OS-TICs showed the expression of several typical cancer stemness markers, including octamer-binding transcription factor 4, Nanog homeobox, cluster of differentiation (CD)117, Nestin and CD133, and the expression of ATP binding cassette subfamily G member 2, multidrug resistance protein 1 (MDR1) and dihydrofolate reductase (DHFR). Notably, in vitro and in vivo tumorigenic properties were enhanced in these OS-TICs. Additionally, methotrexate and doxorubicin are the most widely used anticancer agents against osteosarcoma, and the observed enhanced chemoresistance of OS-TICs to these two agents could be associated with the upregulation of DHFR and MDR1. These findings suggest that the upregulation of DHFR and MDR1 is associated with the development of chemoresistance of OS-TICs.

Erratum to: Cancer stem-like cells enriched with CD29 and CD44 markers exhibit molecular characteristics with epithelial-mesenchymal transition in squamous cell carcinoma.

Increasing evidences have indicated that only a phenotypic subset of cancer cells, termed as the cancer stem cells (CSCs), is capable of initiating tumor growth and provide a reservoir of cells that cause tumor recurrence after therapy. Epithelial–mesenchymal transition (EMT), a cell type change from an epithelial cobblestone phenotype to an elongated fibroblastic phenotype, plays a critical role not only in tumor metastasis but also in tumor recurrence and contributes to drug resistance. Accumulating evidence has shown that cells with an EMT phenotype are rich sources for CSCs, suggesting a biological link between EMT and CSCs; thus study on the link will help understand the cellular and molecular mechanisms of tumor metastasis and drug resistance. CD29 is involved in EMT through cross-talk with cadherins and CD44 has been reported as a successful used marker for CSCs. Here, we try to address whether combination of CD29 and CD44 could be used to identify cancer stem-like cells undergoing EMT in squamous cell carcinoma (SCC) and compare the molecular differences between CD29high/CD44high and CD29low/CD44low cells in SCC. Expression pattern of CD29 and CD44 was analyzed in tissues of skin SCC and cultured A431 cells by immunostaining. Subtype cells of CD29high/CD44high and CD29low/CD44low A431 were sorted by fluorescence-activated cell sorting and proliferating abilities were assayed by cell counting, colony forming and tumorigenicity in NOD/SCID mice. Finally, to probe more deeply into the molecular differences between CD29high/CD44high and CD29low/CD44low A431 cells, gene microarray analysis was applied to compare gene expression profiling. Staining of CD29 and CD44 showed similar heterogeneous expression pattern with positive cells located in the invasion front of SCC tissue as well as in cultured A431 cells. Sorted CD29high/CD44high A431 cells had higher proliferating ability in vitro and in NOD/SCID mice as compared with CD29low/CD44low cells. Gene profiling identified differentiated gene expressions between CD29high/CD44high and CD29low/CD44low A431 cells. These genes are involved in cell cycle, cell malignant transformation, metastasis, drug resistance and EMT, implying that CD29high/CD44high cells have properties of CSCs and EMT. Our present results demonstrated heterogeneous gene expression patterns and different biological behavior in SCC. Combination of CD29 and CD44 can be used as markers to enrich CSCs in human SCC. Moreover, CD29high/CD44high cells exhibit molecular characteristics of EMT, suggesting that CSC-associated pathways were involved in EMT. Studies on correlation of CSCs and the cells undergoing EMT may explain some aspects of tumor progression and drug resistance.

Involvement of Polo-like kinase 1 (Plk1) in quiescence regulation of cancer stem-like cells of the gastric cancer cell lines.

Cancer stem cells (CSCs) have been hypothesized to initiate tumor growth and be resistant to chemoradiotherapy, and these processes appear to be closely related to CSC quiescence. Here, a CSC-like cell population with a high level of CD44 expression was obtained from the human gastric cancer cell lines MKN45 and MKN74. Using a PKH26-labeling retention assay, quiescent CSC-like cells with low levels of Ki67 and PCNA expression were found in spheres formed in serum-free medium, and exhibited resistance to drug and radiation treatments. Polo-like kinase 1 (Plk1) and ribosomal S6 kinase 1 (RSK1) were silenced in the quiescent CSC-like cells. The Plk1-specific inhibitors inhibited the activation of RSK1 and induced quiescence in the CSC-like cells, but increased RSK1 activity and resulted in apoptosis in non-CSCs. Furthermore, RSK1 silencing by inhibitors activated Plk1 and had no effect on the growth of spheres in the CSC-like cells, but did not affect phosphorylation of Plk1 and led to decreased proliferation in non-CSCs. Our results showed that Plk1 and RSK1 play important roles in the conversion of CSCs between active and quiescent states.

Combined toll-like receptor 3/7/9 deficiency on host cells results in T-cell-dependent control of tumour growth

Toll-like receptors (TLRs) are located either on the cell surface or intracellularly in endosomes and their activation normally contributes to the induction of protective immune responses. However, in cancer their activation by endogenous ligands can modulate tumour progression. It is currently unknown how endosomal TLRs regulate endogenous anti-tumour immunity. Here we show that TLR3, 7 and 9 deficiencies on host cells, after initial tumour growth, result in complete tumour regression and induction of anti-tumour immunity. Tumour regression requires the combined absence of all three receptors, is dependent on both CD4 and CD8 T cells and protects the mice from subsequent tumour challenge. While tumours in control mice are infiltrated by higher numbers of regulatory T cells, tumour regression in TLR-deficient mice is paralleled by altered vascular structure and strongly induced influx of cytotoxic and cytokine-producing effector T cells. Thus, endosomal TLRs may represent a molecular link between the inflamed tumour cell phenotype, anti-tumour immunity and the regulation of T-cell activation.

MiR‑206 inhibits cancer initiating cells by targeting EHF in gastric cancer.

Cancer initiating cells (CIC) are defined as the unique subpopulation in the tumors that possess the ability to initiate tumor growth and sustain self-renewal as well as metastatic potential. In this study, we found that EHF overexpression promoted formation of CIC traits and silencing it inhibited the traits in gastric cancer NCI‑N87 cells. Overexpressing EHF downregulated the antitumor effect of 5-fluorouracil (5-FU) in NCI‑N87 cells. We found that miR‑206 downregulated EHF protein expression by targeting its 3'UTR in NCI‑N87 cells and GES-1 cells. Overexpressing miR‑206 inhibited formation of CIC in NCI‑N87 cells. In gastric cancer tissues, EHF protein expression was upregulated and miR‑206 was downregulated. We identified a negative correlation between EHF protein and miR‑206 expression in gastric cancer tissues. Thus, we concluded that miR‑206 inhibits formation of CICs by targeting EHF in gastric cancer.

Time to Progression of Pancreatic Cancer: Evaluation with Multi-Detector Computed Tomography.

The aim of this study is to evaluate the natural history of untreated pancreatic cancer, with a particular emphasis on the growth rate of primary tumor and development of metastatic disease. One hundred patients with histologically proven pancreatic ductal adenocarcinoma examined with at least two CT scans with no intervening treatment were included. Tumor diameters and volumes were measured in CT scans and tumor growth rates and volume doubling times (VDTs) were calculated. The relationship between initial tumor size, growth rate, and distant metastasis development were investigated. Included tumors were 1.0-6.2cm (mean, 2.9±1.3cm) in diameter and 5.5-1225.9cm3 (mean, 120.6±158.9cm3) in volume at the initial CT. Tumor growth rates were -0.4 to 19.9cm/year (mean, 4.2±3.8cm/year) in diameter, and 11.1-13,321.5cm3/year (mean, 727.8±1609.5cm3/year) in volume corresponding to VDT of 20.0-976.8days (mean, 132.3±132.1days). The growth rate was significantly associated with the initial diameter and volume (p<0.001). The development of distant metastasis was significantly associated with initial diameter (p<0.05), volume (p=0.015), and volume growth rate (p=0.002). The growth rate and VDTs of untreated pancreatic cancers varied widely, from less than a month to more than 4years, positively associated with tumor size. The small tumors tend to grow slowly and have low risk for developing metastasis.

Effect of delayed resection after initial surveillance and tumor growth rate on final surgical pathology in patients with small renal masses (SRMs)

ObjectiveTo understand potential harms associated with delaying resection of small renal masses (SRMs) in patients ultimately treated, and whether these patients have factors associated with adverse pathology. MethodsPatients with SRMs (≤4cm) who underwent surgical resection at our institution (2009–2015) were classified as undergoing early resection or initial surveillance with delayed resection (defined by a time from presentation to intervention of at least 6mo). Demographic and clinical variables were compared among groups. Using multivariable logistic regression, we examined the association between delayed resection and adverse pathology (Fuhrman grade 3−4, papillary type 2, sarcomatoid histology, angiomyolipoma with epithelioid features, or stage≥pT3). For patients who underwent delayed intervention, we used similar methods to examine the association between SRM growth rate and adverse pathology. ResultsOverall, 401 (81%) and 94 (19%) patients underwent early and delayed resection, respectively. Median time to resection was 84 days (interquartile range: 59−121) and 386 days (interquartile range: 272−702) (P<0.001). Patients undergoing delayed resection were older (62 vs. 58y, P = 0.01) and had smaller masses (2.3 vs. 2.7cm, P<0.001) at initial presentation. Utilization of partial vs. radical nephrectomy was similar regardless of resection timing (P = 0.5). Delayed resection was not associated with adverse pathology (P = 0.8); however, male sex was independently associated with adverse pathology (odds ratio: 1.7, 95% CI: 1.1–2.4, P = 0.009). In patients on surveillance, increasing annual SRM growth rate was associated with adverse pathology (odds ratio: 1.2, 95% CI: 1.03–1.3mm/y, P = 0.02). ConclusionsDelayed resection was not associated with adverse pathology. Patients on surveillance with increased SRM growth rates had a modest but significant increase in the risk of adverse pathology.

Retracted] Production of interleukin‑4 in CD133+ cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth.

Following the publication of this article, an investigation conducted internally within our Department has revealed that the data published in Fig. 5a are not reproducible. Consequently, confidence in the conclusions drawn from the reported results in the paper has been undermined and, as a result, all authors involved have unanimously agreed to the retraction of this article. All authors recognize the seriousness of this issue and apologize unreservedly to the editors, reviewers and readers. We are also in the process of examining other studies in the paper. We sincerely regret that this study has been compromised, and are committed to rapidly correcting the public record and implementing practices to prevent any recurrence of such a situation in the future. [the original article was published in Molecular Medicine Reports 13: 5068-5076, 2016; DOI: 10.3892/mmr.2016.5195].

β-escin selectively targets the glioblastoma-initiating cell population and reduces cell viability.

Glioblastoma multiforme (GBM) is a highly aggressive tumour of the central nervous system and is associated with an extremely poor prognosis. Within GBM exists a subpopulation of cells, glioblastoma-initiating cells (GIC), which possess the characteristics of progenitor cells, have the ability to initiate tumour growth and resist to current treatment strategies. We aimed at identifying novel specific inhibitors of GIC expansion through use of a large-scale chemical screen of approved small molecules. Here, we report the identification of the natural compound β-escin as a selective inhibitor of GIC viability. Indeed, β-escin was significantly cytotoxic in nine patient-derived GIC, whilst exhibiting no substantial effect on the other human cancer or control cell lines tested. In addition, β-escin was more effective at reducing GIC growth than current clinically used cytotoxic agents. We further show that β-escin triggers caspase-dependent cell death combined with a loss of stemness properties. However, blocking apoptosis could not rescue the β-escin-induced reduction in sphere formation or stemness marker activity, indicating that β-escin directly modifies the stem identity of GIC, independent of the induction of cell death. Thus, this study has repositioned β-escin as a promising potential candidate to selectively target the aggressive population of initiating cells within GBM.

Abstract A01: A comprehensive collection of patient derived xenografts of human melanoma with clinical, genomic, and biological characterization

Abstract Advanced melanoma has seen dramatic changes in standard of care in the last years and many novel targeted small molecules and immune checkpoint inhibitors are in development. More than 200 clinical trials are currently ongoing for metastatic melanoma. Thus, accurate pre-clinical models to predict clinical responses are urgently needed. We have established a large bank of live tumor tissue (n=500) with more than 300 models expanded as PDX. Melanoma tumor tissue is uniquely suited to establish patient-derived xenograft (PDX) models, since only one tumor cell can initiate tumor growth. Thus, our success rate is at 90% using NSG mice and matrigel for s.c. implantation of minced tissue chunks. We have opted for such a large number of models due to the genomic and clinical heterogeneity of melanoma based on available TCGA data. Although almost half of all melanomas harbor BRAF V600 hotspot mutations, followed in frequency by NRAS and NF1 mutations, many driver mutations are only found in a small subset and many mutations are occurring concurrently in various confirmations. We used a custom targeted capture of 108 genes previously implicated in melanoma, and performed massively parallel sequencing on currently 200 PDX. Samples were then clustered into groups based on deleterious mutations which were detected in all but a very small subset of samples. The biological relevance of these multiple genomic subsets was then tested against latency, growth rate, and spontaneous metastasis in PDX. Further, clinical information such as age, gender, history, and response to therapies provided additional parameters to classify this collection into meaningful subgroups. In conclusion, this collection of melanoma PDX recapitulates the breadth of advanced melanoma in the clinic and therefore a comprehensive resource for precision medicine testing in an increasingly scattered therapy landscape. Citation Format: Clemens Krepler, Katrin Sproesser, Marilda Beqiri, Min Xiao, Patricia Brafford, Wei Xu, Bradley Garman, Jennifer Wargo, Michael A. Davies, Dennie T. Frederick, Keith T. Flaherty, David Hoon, Joseph J. Bennett, Michael Guarino, Nicholas J. Petrelli, David Elder, Xiaowei Xu, Giorgos Karakousis, KATHERINE L. NATHANSON, Lynn Schuchter, Meenhard Herlyn. A comprehensive collection of patient derived xenografts of human melanoma with clinical, genomic, and biological characterization. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A01.

Abstract 3317: Tumor microenvironment of metastasis (TMEM) in breast cancer patients represents a stem cell niche; likely mediated by the Wnt pathway

Abstract Cell surface biomarkers CD44, CD24, CD133 and intracellular biomarker ALDH1 have been used to identify breast cancer stem cells (BCSC). BCSC are clinically significant because they are resistant to chemotherapy and in very small numbers can initiate tumor growth and metastasis. Recent studies suggest that juxtacrine signaling from monocytes and macrophages support the BCSC niche. Macrophages represent one of the components of the micro-anatomical sites of hematogenous dissemination of breast cancer cells called Tumor MicroEnvironment of Metastasis (TMEM) which are predictors of metastatic outcome in patients. We hypothesize that TMEM rich tumor microenvironments support BCSCs which are a prerequisite for systemic metastatic outgrowths. We used flow cytometry to quantify CD44high/CD24low cells, as well as mRNA fluorescent in situ hybridization (FISH) and qRT-PCR to quantify CD133 and ALDH1 expression in breast cancer cells from 50 invasive ductal carcinomas obtained from patients’ cancer excisions by fine needle aspiration (FNA) before formalin fixation. Formalin-fixed paraffin embedded tissue from each sample was also analyzed for TMEM score using triple immunohistochemistry and the stem cell marker expression was compared to TMEM scores for each corresponding cancer excision. We observed very strong correlation between the percentage of CD44high/CD24low cells and TMEM scores (r = 0.91), as well as the percentage of CD133 and ALDH1 expressing cells with TMEM scores (r = 0.88 and 0.86 respectively). FISH results were validated using qRT-PCR and showed very strong correlation with TMEM scores (r = 0.76 and 0.73 for CD133 and ALDH1 respectively). Wnt/β-catenin signaling pathway is involved in stem cell generation and pathogenesis of various types of cancer. Aberrant activation of Wnt-signaling pathway in normal stem cells can promote their transformation into cancer stem cells. We hypothesize that interactions between cancer cells and macrophages, and/ or cancer cells and endothelial cells (i.e. both interactions among the cellular components of TMEM) induce cancer stemness via Wnt/-β catenin pathway. Indeed, co-culture of mouse macrophages (BAC) with MCF-7 cells, as well as human endothelial cells (HUVEC) with MCF-7, significantly increased the percentage of cells expressing stem cells markers, while salinomycin, selective inhibitor of breast cancer stem cell growth and progression, significantly reduced BAC or HUVEC induced increase in the percentage of cancer stem cells. Our findings indicate that TMEM-rich microenvironments support breast cancer stem cells likely via Wnt signaling involving juxtacrine cancer cell-macrophage and cancer cell-endothelial cell interactions. Citation Format: Gargi Bandyopadhyaya, Eli Grunblatt, Sweta Roy, Nathan Agi, Esther Adler, Joan Jones, John S. Condeelis, Maja H. Oktay, Sumanta Goswami. Tumor microenvironment of metastasis (TMEM) in breast cancer patients represents a stem cell niche; likely mediated by the Wnt pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3317.

Abstract 5191: An orthotopic xenograft renal cell carcinoma Sunitinib resistant murine model

Abstract Purpose of study To develop a reproducible and reliable orthopic xenograft mouse model for Sunitinib resistant renal cell carcinoma (RCC). Introduction Clear-cell renal cell carcinoma (ccRCC) is the most common malignancy of the kidneys and has been steadily increasing at a rate of 3% yearly over the last decade. Furthermore, 30% of patients with ccRCC have de novo metastatic disease. Until the last decade, immunotherapy was the only option for metastatic ccRCC treatment. Current first-line systemic therapies for metastatic ccRCC all primarily target the angiogenesis pathway via tyrosine kinase inhibition (TKI) and have increased progression free survival dramatically. However, durable responses are rare, drug resistance invariably arises after short-term disease stabilization and metastatic ccRCC remains almost uniformly lethal. There is currently no animal model for TKI resistance in RCC. We developed a reliable and reproducible orthotopic xenograft mouse model of Suntinib resistance in RCC. Methods Luciferin expressing-CAKI-1 human renal cancer cells were injected under the renal capsule of athymic nude mice using ultrasound guidance. After initial tumor growth mice were treated with Sunitinib malate at an initial dose of 40mg/kg which was increased sequentially (60 and 80 mg/kg) at each tumor passage (Fig.1). Tumors were monitored twice weekly by ultrasound and bioluminescence (IVIS system). Tumors exceeding 200mm3 by ultrasound or 10⁁10 photons/sec by IVIS were considered end points. Before passaging, tumors were harvested and immediately dissociated with a Dispase/Collagenase mix. 5×105 cells were injected into the following batch of mice, considering all these steps as one cycle or passage. Tumors were passaged with increasing doses of Sunitinib 5 times. Results Tumor take rates were above 95% in each cycle. Initial treatment of Sunitinib at 40mg/kg resulted in a decrease in tumor size via ultrasound and IVIS of about 30%. At around 14 days of treatment tumors started to regrow despite further treatment. In the second cycle tumors did not show a response to a subsequent treatment of 40mg/kg, proving resistance. Even after increasing the dose of Suntinib in the following cycles to 60 and 80 mg/kg respectively, there was no response observed. The fifth cycle (100mg/kg) had to be terminated due to severe side effects in the animals (skin coloring, weight loss), however no decrease in tumor size was observed. We therefore created a Sunitinib resistance model in the range of the maximal tolerated long-term dose in vivo. Conclusions This study describes the first orthotopic xenograft renal cell carcinoma Sunitinib resistant mouse model created in situ. The development of Sunitinib resistance in an angiogenic environment establishes a more realistic and translational model, which opens the possibility to study not only the mechanism of Sunitinib resistance but also to find alternative therapies to overcome Sunitinib resistance. Citation Format: Sebastian K. Frees, Igor Moskalev, Betty Zhou, Peter Raven, Claudia Chavez-Munoz, Peter Black, Alan I. So. An orthotopic xenograft renal cell carcinoma Sunitinib resistant murine model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5191.

The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model.

BackgroundColorectal cancer is one of the most common types of cancers worldwide. Recent studies have identified cancer-initiating cells (CICs) as a subgroup of replication-competent cells in the development of colorectal cancer. Although it is understood that an inflammation-rich tumor microenvironment presumably supports CIC functions, the contributory factors are not very well defined. The present study advances our understanding of the role of the eicosanoids leukotriene D4 (LTD4) and prostaglandin E2 (PGE2) in the tumorigenic ability of CICs and investigates the consequential changes occurring in the tumor environment that might support tumor growth.MethodsIn this study we used human HCT-116 colon cancer ALDH+ cells in a nude mouse xenograft model. Protein expression and immune cell was determined in tumor-dispersed cells by flow cytometry and in tumor sections by immunohistochemistry. mRNA expressions were quantified using RT-q-PCR and plasma cytokine levels by Multiplex ELISA.ResultsWe observed that LTD4 and PGE2 treatment augmented CIC-induced tumor growth. LTD4-and PGE2-treated xenograft tumors revealed a robust increase in ALDH and Dclk1 protein expression, coupled with activated β-catenin signaling and COX-2 up-regulation. Furthermore, LTD4 or PGE2 accentuated the accumulation of CD45 expressing cells within xenograft tumors. Further analysis revealed that these infiltrating immune cells consisted of neutrophils (LY6G) and M2 type macrophages (CD206+). In addition, LTD4 and PGE2 treatment significantly elevated the plasma levels of cysteinyl leukotrienes and PGE2, as well as levels of IL-1β, IL-2, IL-6, TNF-α and CXCL1/KC/GRO. In addition, increased mRNA expression of IL-1β, IL-6 and IL-10 were detected in tumors from mice that had been treated with LTD4 or PGE2.ConclusionOur data suggest that both LTD4 and PGE2 promote CICs in initiating tumor growth by allowing modifications in the tumor environment. Our data indicate that new therapeutic strategies targeting eicosanoids, specifically LTD4 and PGE2, could be tested for better therapeutic management of colon cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2466-z) contains supplementary material, which is available to authorized users.

WE-FG-BRA-02: Docetaxel Eluting Brachytherapy Spacers for Local Chemo-Radiation Therapy in Prostate Cancer

Purpose: We propose an innovative combinatorial treatment strategy of Local ChemoRadiation Therapy (LCRT) using a sustained drug delivery platform in the form of a spacer to locally radio-sensitize the prostate with Docetaxel (DTX) enabling a synergistic cure with the use of lower radiation doses. These biodegradable spacers are physically similar to the inert spacers routinely used in prostate brachytherapy but are now loaded with formulations of DTX. Methods: Spacers were loaded with ∼500µg Docetaxel (DTX) for prostate cancer studies. The implants were characterized in vitro using SEM and HPLC. The release kinetic studies were carried out in buffer (pH 6.0) at 37°C. Subcutaneous PC3 tumors were xenografted in nude mice. Prostate cancer studies were done with and without radiation using SARRP at 5Gy, 10Gy, and 15Gy. Drug-loaded implants were injected once intratumorally using an 18G brachytherapy needle. Results: The release study in vitro showed a highly sustained release for multiple weeks at therapeutically relevant doses. The monotherapy with local DTX spacer showed sustained tumor inhibition compared to empty implants and an equivalent DTX dose given systemically. At 40 days, 89% survival was observed for mice treated with DTX implants compared with 0% in all other treatment groups. The combined treatment with local DTX spacer and radiation (10Gy) showed the highest degree of tumor suppression (significant tumor growth inhibition by day 90). The control mice showed continuous tumor growth and were scarified by day 56. Groups of mice treated with DTX-spacer or radiation alone showed initial tumor suppression but growth continued after day 60. A larger experiment is ongoing. Conclusion: This approach provides localized delivery of the chemotherapeutic sensitizer directly to the tumor and avoids the toxicities associated with both brachytherapy and current systemic delivery of docetaxel. Sustained release of DTX is an effective chemotherapy option alone or in combination with radiation therapy.

Reciprocal activation between STAT3 and miR-181b regulates the proliferation of esophageal cancer stem-like cells via the CYLD pathway.

BackgroundRecent studies have suggested that cancer cells contain subpopulations that can initiate tumor growth, self-renew, and maintain tumor cell growth. However, for esophageal cancer cells, the relationship between STAT3, microRNAs and cancer stem cells remains unclear.MethodsSerum-free culture was used to enrich esophageal cancer stem-like cells (ECSLC). Flow cytometry determined the proportion of ECSLC. qPCR were performed to examine expression level of stemness factors, mesenchymal markers, ATP-binding cassette (ABC) transporters, STAT3, miR-181b, CYLD. Western blot were performed to analyze the expression of STAT3, p-STAT3 and CYLD (cylindromatosis). BALB/c mice xenograft studies were conducted to evaluate the tumorigenicity of enriched ECSLC. Sphere formation assay and colony formation assays were employed to analyze the relationship between STAT3 and miR-181b. Luciferase assays were used to evaluate activity which CYLD is a target of miR-181b.ResultsSphere formation cells (SFCs) with properties of ECSLC were enriched. Enriched SFCs in serum-free suspension culture exhibited cancer stem-like cell properties and increased single-positive CD44 + CD24-, stemness factor, mesenchymal marker expression ABC transporters and tumorigenicity in vivo compared with the parental cells. Additionally, we found that reciprocal activation between STAT3 and miR-181b regulated SFCs proliferation. Moreover, STAT3 directly activated miR-181b transcription in SFCs and miR-181b then potentiated p-STAT3 activity. Luciferase assays indicated that CYLD was a direct and functional target of miR-181b.ConclusionThe mutual regulation between STAT3 and miR-181b in SFCs was required for proliferation and apoptosis resistance. STAT3 and miR-181b control each other’s expression in a positive feedback loop that regulates SFCs via CYLD pathway. These findings maybe is helpful for targeting ECSLC and providing approach for esophageal cancer treatments.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0521-7) contains supplementary material, which is available to authorized users.

The RNA Binding Protein IMP2 Preserves Glioblastoma Stem Cells by Preventing let-7 Target Gene Silencing.

Cancer stem cells (CSCs) can drive tumor growth, and their maintenance may rely on post-transcriptional regulation of gene expression, including that mediated by microRNAs (miRNAs). The let-7 miRNA family has been shown to induce differentiation by silencing stem cell programs. Let-7-mediated target gene suppression is prevented by LIN28A/B, which reduce let-7 biogenesis in normal embryonic and some cancer stem cells and ensure maintenance ofstemness. Here, we find that glioblastoma stem cells (GSCs) lack LIN28 and express both let-7 and their target genes, suggesting LIN28-independent protection from let-7 silencing. Using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we show that insulin-like growth factor 2 mRNA-binding protein 2 (IMP2) binds to let-7 miRNA recognition elements (MREs) and prevents let-7 target gene silencing. Our observations define the RNA-binding repertoire of IMP2 and identify a mechanism whereby it supports GSC and neural stem cell specification.

Production of interleukin‑4 in CD133+ cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth.

The cancer stem cell (CSC) theory suggests that cancer growth and invasion is dictated by the small population of CSCs within the heterogenous tumor. The aim of the present study was to elucidate the cause for chemotherapy failure and the resistance of CSCs to apoptosis. A total of ~2.3% cluster of differentiation (CD)133+ cancer stem-like side population (SP) cells were identified in cases of uterine cervical cancer. These CD133+ SP cells were found to potently initiate tumor growth and invasion, as they exhibit transcriptional upregulation of stemness genes, including octamer-binding transcription factor-4, B-cell-specific Moloney murine leukemia virus insertion site-1, epithelial cell adhesion molecule, (sex determining region Y)-box 2, Nestin and anti-apoptotic B cell lymphoma-2. In addition, the CD133+ SP cells showed resistance to multi-drug treatment and apoptosis. The present study further showed that the secretion of interleukin-4 (IL-4) in CD133+ cervical cancer SP cells promoted cell proliferation and prevented the SP cells from apoptosis. Following the neutralization of IL-4 with anti-IL-4 antibody, the CD133+ SP cells were more sensitive to drug treatment and apoptosis. Therefore, the data obtained in the present study suggested that the autocrine secretion of IL-4 promotes increased survival and resistance to cell death in CSCs.

Gastric Lgr5(+) stem cells are the cellular origin of invasive intestinal-type gastric cancer in mice.

The cellular origin of gastric cancer remains elusive. Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is the first identified marker of gastric stem cells. However, the role of Lgr5(+) stem cells in driving malignant gastric cancer is not fully validated. Here, we deleted Smad4 and PTEN in murine gastric Lgr5(+) stem cells by the inducible Cre-LoxP system and marked mutant Lgr5(+) stem cells and their progeny with Cre-reporter Rosa26(tdTomato). Rapid onset and progression from microadenoma and macroscopic adenoma to invasive intestinal-type gastric cancer (IGC) were found in the gastric antrum with the loss of Smad4 and PTEN. In addition, invasive IGC developed at the murine gastro-forestomach junction, where a few Lgr5(+) stem cells reside. In contrast, Smad4 and PTEN deletions in differentiated cells, including antral parietal cells, pit cells and corpus Lgr5(+) chief cells, failed to initiate tumor growth. Furthermore, mutant Lgr5(+) cells were involved in IGC growth and progression. In the TCGA (The Cancer Genome Atlas) database, an increase in LGR5 expression was manifested in the human IGC that occurred at the gastric antrum and gastro-esophageal junction. In addition, the concurrent deletion of SMAD4 and PTEN, as well as their reduced expression and deregulated downstream pathways, were associated with human IGC. Thus, we demonstrated that gastric Lgr5(+) stem cells were cancer-initiating cells and might act as cancer-propagating cells to contribute to malignant progression.