Tissues Of Tumor-bearing Mice Research Articles

Tumor-Derived Oxidative Stress Triggers Ovarian Follicle Loss in Breast Cancer

Breast cancer is a common indication for ovarian cryopreservation. However, whether the grafting ovarian tissue meets functional requirements, as well as the need for additional interventions, remains unclear. The current study demonstrates abnormal serum hormones in breast cancer in humans and breast cancer cell line-derived tumor-bearing mice, and for the first time shows tumor-induced loss of primordial and growing follicles, and the number of follicles being lost to either growth or atresia. A gene signature of tumor-bearing mice demonstrates the disturbed regulatory network of steroidogenesis, which links to mitochondria dysfunction in oocytes and granulosa cells via the phosphatidylinositol 3-kinase signaling pathway. Notably, increased reactive oxygen species were identified in serum and ovarian tissues in tumor-bearing mice. Furthermore, supplementation withvitamin C promoted follicular quiescence, repairing tumor-induced follicle loss via inactivation of the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway, indicating the potential ofantioxidants as a fertility therapy to achieve higher numbers of healthy follicles ready for ovarian cryopreservation.

Delicaflavone Represses Lung Cancer Growth by Activating Antitumor Immune Response through N6-Methyladenosine Transferases and Oxidative Stress.

Our previous studies have shown that delicaflavone (DLL), a biocomponent extracted from Selaginella doederleinii Hieron, has antitumor activity. However, the role of DLL in the antitumor immune response is unknown. In this study, we tested the potential roles of DLL in antitumor immune response. An animal tumor model with Lewis lung cancer cell line (3LL) in C57BL/6 mice was established to determine whether DLL induced the tumor-bearing host's antitumor immune response. m6A-MeRIP-qPCR, western blot, and flow cytometry were performed to explore the underlying mechanisms. DLL inhibited the proliferation of 3LL lung cancer cells in vitro and in vivo and induced tumor cell oxidative stress. DLL significantly inhibited tumor growth in immunocompetent mice compared with nude mice. DLL treatment significantly increased Th1 cytokine production and CD8+ T cell infiltration into tumor tissues in tumor-bearing mice. DLL-mediated antitumor immune effects were reversed by overexpression of the N6-methyladenosine (m6A) transferase Mettl3/Mettl14. Mechanistically, DLL upregulated the expression of Stat1 and Irf1 and the secretion of cytokines by inhibiting Mettl3 and Mettl14 in lung cancer cells. In conclusion, DLL inhibited lung cancer cell growth by suppressing Mettl3/Mettl14 to activate antitumor immunity. These findings provided an opportunity to enhance lung cancer immunotherapy.

Carbon spheres with high photothermal conversion efficiency for photothermal therapy of tumor

Development of high-performance photothermal agent plays a decisive role in photothermal therapy of tumor. Herein, carbon spheres are rapidly synthesized by pyrolysis of pyrogallol-formaldehyde spheres at 400–1000 °C under nitrogen atmosphere. As-prepared carbon spheres exhibit strong absorption in near-infrared region and good photothermal effect. The results reveal that the photothermal conversion efficiency of carbon spheres has a tight association with carbon defects caused by pyrolysis. The carbon defects serve as the nonradiative recombination centers of the excited electrons to promote photothermal conversion efficiency. Strikingly, carbon spheres after calcination at 700 °C exhibit the photothermal conversion efficiency of 54.2% under 808 nm laser irradiation, which is superior to the reported carbon-based photothermal agents. The prepared carbon spheres have good cell biocompatibility and can ablate the tumor tissues in tumor-bearing mice through the conversion of near-infrared laser energy into thermal energy.

Effect of fruquintinib on programmed death receptor-1 blockade antitumor immune responses in colorectal cancer.

150 Background: Programmed death receptor-1 (PD-1) blockade shows little benefit in the patients with microsatellite-stable colorectal cancer (MSS-CRC).Fruquintinib, a China-made anti-angiogenic drug, applied for third line therapy in mCRC. However, the effects of combination of fruquintinib and PD-1 blockade on MSS-CRC and its relative mechanisms are not well determined. Methods: Syngeneic xenograft mouse models were establish using murine MC38 and CT26 colon cancer cells to assess treatment efficacy. The percentages of immune cells were detected in peripheral blood, spleen and tumor tissues in tumor-bearing mice by flow cytometry analysis. Angiogenesis in tumor tissues was detected by immunofluorescence. The safety of drug treatment was evaluated by histopathology analysis in murine main organs. The efficacy of combination of fruquintinib and sintilimab were verified in the treatment of MSS-CRC patients. Results: Our results showed the combination of fruquintinib and sintilimab exhibited strongest inhibition of tumor growth and achieved longest survival time in mice bearing MC38 or CT26 xenograft tumors, compared to fruquintinib and sintilimab alone. Mechanistically, the combination of fruquintinib and sintilimab reduced angiogenesis, reprogramed the vascular structure, enhanced the infiltration of CD8+T cells, CD8+TNFα+T cells and CD8+IFNγ+T cells and reduced the ratios of MDSCs and macrophages in mice. No obvious damage was observed in main organs in tumor-bearing mice with the combined treatment. Moreover, the treatment of combination of fruquintinib and sintilimab anti-PD-1 antibodies achieved effective response in five refractory advanced MSS CRC patients. Conclusions: Our results show that combination of fruquintinib and sintilimab synergistically inhibits CRC growth by alterating antitumor immune microenvironment.

The Effect of Sirtuin 2 (Sirt2) Overexpressing Bone Marrow Mesenchymal Stem Cells on the Growth of Human Epidermal Growth Factor Receptor 2 (Her-2) Breast Cancer Cells and Its Mechanism

Our study investigates the effect of high expression of Sirt2 in MSCs (MSCs-Sirt2) on Her-2 breast cancer cell proliferation. A mouse subcutaneous xenograft tumor model was established and MSCssirt2 analysis was performed on nude mice. TUNEL staining, flow cytometry, western-blot, real-time PCR and immunohistochemistry were used to detect cancer cell apoptosis. The number of NK cells infiltrated by flow cytometry detected the tumor tissue of tumor-bearing mice, and its killing activity on tumor-bearing mice was detected by isotope labeling and release method. The levels of TNF-α, IFN-γ, IL-8, IL-6 and IL-10 were detected by ELISA. Caspase-3 level was decreased in the MSCs group (P <0.01) while increased in the MSCs-sirt2 group (P <0.001). However, PCNA expression showed an opposite profile in the Her-2 group and MSCs-sirt2 group compared to Caspase-3 level (P <0.01). The tumor volume and weight in the MSCs-sirt2 group was significantly reduced (P < 0.01), while increased in the MSCs group significantly (P < 0.05). The number of Ki-67-positive tumor cells in MSCs-sirt2 group was significantly reduced (P <0.01) and increased in MSCs group (P < 0.001) with oppositive number of TUNEL-positive tumor cells in the MSCs-sirt2 group and MSCs group (P <0.01). IFN-γ level showed an upward trend (P <0.001). The NK cell toxicity of MSCs-Sirt2 group was significantly higher (P <0.001). MSCs-Sirt2 has an inhibitory effect on Her-2 breast cancer cell growth by enhancing the local inflammatory response of NK cells.

Role of Sulforaphane on Histone Deacetylase Activity in Solid Ehrlich Carcinoma

Histone acylation is one in every of the posttranslational modification that incorporates a role within the regulation of sequence expression. This study was aimed to ameliorate neoplasm cell model Solid Ehrlich malignant neoplastic disease with sulforaphane extracted from cabbage alone and together with immune suppressant drug (MTX) by study the activity of simple protein deacetylase accelerator (HDAC). In this study, sulforaphane was extracted from cabbage leaves and evaluated victimization GC-MS and ultraviolet illumination spectrophotometry. 60 white male rats were divided into six equal groups. Group I: management ordinarily. The remaining mice were subjected to Ehrlich neoplasm cells. Group II: Tumor-bearing group. Group III: immune suppressant drug “MTX” treated group. Group IV, V treated with SFN before and when Paul Ehrlich cells implantation group VI (Methotrexate and sulforaphane-treated group). This result showed that sulforaphane was extracted from cabbage (Brassica oleracea) in concentration of 833 μg/g leave. HDAC was attenuated when treatment with sulforaphane after treatment with methotrexate or sulforaphane alone. The desoxyribonucleic acid injury was attenuated in neoplasm tissue of tumor-bearing mice when treatment with immune suppressant drug and hyperbolic considerably in tumor tissue of tumor-bearing mice treated with sulforaphane before and after carcinogenesis and together with methotrexate treatment after carcinogenesis.

Uptake and release profiles of PEGylated liposomal doxorubicin nanoparticles: A comprehensive picture based on separate determination of encapsulated and total drug concentrations in tissues of tumor-bearing mice

The PEGylated liposomal nanoparticle has been widely used as a carrier in drug delivery system. To become biologically active, the encapsulated drug must be released from the nanoparticle vehicle. However, due to limitations of current bioanalytical methods, the characterization of this release process has been restricted to determination of total drug in tissues and tumor. As a result, the fate of liposomal nanoparticles including their uptake into target tissue has not been fully characterized. In this study, we developed a novel two-step solid phase extraction on two separated columns procedure to separate liposomes from tissues and tumors without liposomal leakage. This allowed us to determine encapsulated drug, total drug and, by difference, released drug and compare the release and uptake profiles of PEGylated liposomal doxorubicin in tissues and tumor of tumor-bearing mice with corresponding profiles for free doxorubicin. The liposomal nanoparticles released doxorubicin into tumor efficiently and, compared with administration of free drug, increased doxorubicin uptake into tumor by 1.8-fold. It also decreased doxorubicin uptake into heart (0.78-fold lower) with the potential to reduce doxorubicin cardiotoxicity. Drug release reached constant levels in tissues and tumor after 12 h with released doxorubicin concentration remaining at 70–80% of total doxorubicin concentration and in tumor at 86% of total drug concentration. The assay also included determination of the main doxorubicin metabolites. Determination of the metabolites showed that liposomal entrapment delays and decreases the metabolism of doxorubicin but does not alter the metabolic pathway. These results provide a clear and comprehensive picture of the biodistribution of doxorubicin administered in liposomal nanoparticles which may assist in the rational design of other liposomal nanoparticles.

Magnetic And pH Dual-Responsive Nanoparticles For Synergistic Drug-Resistant Breast Cancer Chemo/Photodynamic Therapy.

BackgroundDrug resistance is one of the prime reasons of chemotherapy failure in breast cancer and is also an important factor affecting prognosis.PurposeIn this study, we constructed a functional magnetic mesoporous silica-based nanocomposite (MMSN) for breast cancer chemotherapy/photodynamic therapy.MethodsMMSN was characterized by scanning electron microscopy and transmission electron microscopy to observe the morphology. The size distribution and zeta potential of the MSNs were determined using Malvern Particle Size Analyzer. Anti-tumor activity in vitro was investigated by CCK-8 assay, flow cytometry and transwell experiment, and the anti-tumor activity in vivo was probed into by magnetic targeting, toxicity, and antitumor effects in breast cancer-bearing BABL/c nude mice.ResultsThe results showed that the release of doxorubicin in the nanocomposites was pH sensitive, and the cumulative release rate reached 80.53% at 60 h under acidic conditions. The nanocomposites had a high cellular uptake ability in MCF-7/ADR cells, and the IC50 value of the nanocomposites on MCF-7/ADR cells was 4.23 μg/mL, much smaller than that of free DOX (363.2 μg/mL). The nanocomposites could effectively reverse resistance and induce apoptosis of MCF-7/ADR cells. The blood biochemistry parameters and H&E staining results showed no serious adverse effects after treatment with the nanocomposites. Prussian blue staining showed that the nanocomposites were able to target tumor tissues in tumor-bearing mice under a magnetic field. The combined chemical/photodynamic therapy significantly inhibited tumor growth in vivo.ConclusionNanocomposites with magnetic and pH dual-responsive performance has shown a promising platform for enhanced drug-resistant breast cancer treatment.

Mathematical simulation of temperature distribution in tumor tissue and surrounding healthy tissue treated by laser combined with indocyanine green

BackgroundPhotothermal therapy is a local treatment method for cancer and the heat energy generated from it could destroy the tumor cells. This study is aimed to investigate the temperature distribution in tumor tissue and surrounding health tissue of tumor bearing mice applying mathematical simulation model. Tumor bearing mice treated by laser combined with or without indocyanine green. Monte Carlo method and the Pennes bio-heat equation were used to calculate the light distribution and heat energy. COMSOL Multiphysic was adopted to construct three dimensional temperature distribution model.ResultsThis study revealed that the data calculated by simulation model is in good agreement with the surface temperature monitored by infrared thermometer. Effected by the optical parameters and boundary conditions of tissue, the highest temperature of tissue treated by laser combined with indocyanine green was about 65 °C which located in tumor tissue and the highest temperature of tissue treated by laser was about 43 °C which located under the tumor tissue. The temperature difference was about 20 °C. Temperature distribution in tissue was not uniform. The temperature difference in different parts of tumor tissue raised up to 15 °C. The temperature of tumor tissue treated by laser combined with indocyanine green was about 20 °C higher than that of the surrounding healthy tissue.ConclusionsReasonably good matching between the calculated temperature and the measured temperature was achieved, thus demonstrated great utility of our modeling method and approaches for deepening understand in the temperature distribution in tumor tissue and surrounding healthy tissue during the laser combined with photosensitizer. The simulation model could provide guidance and reference function for the effect of photothermal therapy.

Lycorine inhibits melanoma cell migration and metastasis mainly through reducing intracellular levels of β-catenin and matrix metallopeptidase 9.

Metastatic melanoma accounts for 60% of death for skin cancer. Although great efforts have been made to treat the disease, effective drugs against metastatic melanoma still lack at the clinical setting. In the current study, we found that lycorine, a small molecule of isoquinoline alkaloid, significantly suppressed melanoma cell migration and invasion in vitro, and decreased the metastasis of melanoma cells to lung tissues in tumor-bearing mice, resulting in significant prolongation of the survival of the mice without obvious toxicity. Molecular mechanistic studies revealed that lycorine significantly reduced intracellular levels of β-catenin protein through degradation of the protein via the ubiquitin-proteasome pathway, and decreased the expression of β-catenin downstream prometastatic matrix metallopeptidase 9 and Axin2 genes. Collectively, our findings support the notion that targeting the oncogenic β-catenin by lycorine is a new option to inhibit melanoma cell metastasis, providing a good drug candidate potential for development novel therapeutics against metastatic melanoma.

IL-33 Promotes the Development of Colorectal Cancer Through Inducing Tumor-Infiltrating ST2L+ Regulatory T Cells in Mice.

Colorectal cancer, one of the most commonly diagnosed and lethal cancers worldwide, is accompanied by the disorders of immune system. However, the underlying mechanism is still not fully understood. In this study, our goal was to determine whether interleukin 33 promotes tumorigenesis and progression of colorectal cancer through increased recruitment of tumor-infiltrating ST2+ regulatory T cells in CT26 tumor-bearing mice. We found that the mRNA or protein levels of interleukin 33, soluble ST2, and membrane ST2 were elevated in the serum of tumor-bearing mice when compared to WT mice. The mRNA levels of interleukin 33, soluble ST2, and membrane ST2 were also elevated in the tissue of tumor-bearing mice when compared to surrounding nontumor muscular tissues. In addition, the frequency of ST2L+ regulatory T cells was significantly increased in both tumor tissue and spleen of tumor-bearing mice. Higher protein levels of interleukin-4, -10, and -13 were also observed in the serum or the tumor homogenates of tumor-bearing mice. We found exogenously administered recombinant mouse interleukin 33 promoted tumor size and induced tumor-infiltrating ST2L+ regulatory T cells in tumor-bearing mice while neutralizing interleukin-33 or ST2L inhibited tumor size and decreased ST2L+ regulatory T cells. Furthermore, ST2L+ regulatory T cells from tumor tissue were also able to suppress CD4+CD25−T cell proliferation and interferon γ production. Altogether, our findings demonstrate the critical roles of interleukin 33 in promoting colorectal cancer development through inducing tumor-infiltrating ST2L+ regulatory T cells, and inhibition of interleukin-33/ST2L signaling maybe a potential target for the prevention of colorectal cancer.

Abstract 1678: A flow-based immunoprofiling strategy for interrogating system-wide leukocyte composition in response to glioblastoma

Abstract Glioblastoma (GBM) is a deadly brain cancer that suppresses cellular immunity through the production of anti-inflammatory cytokines/catabolites, expression of immune checkpoint molecules, and induction of tolerogenic immune cell subsets. While preclinical models have shown each of these mechanisms to negatively impact survival outcome, it remains unclear how these distinct processes manifest themselves within the context of a coordinated immune system and whether additional immunosuppressive mechanisms exist. To address these questions, we have developed an extensible in vivo screening platform to holistically determine how GBM affects cellular immune organization. The method allows for temporal assessment of leukocyte population dynamics in response to tumor progression. Leukocytes derived from lymphoid tissues of tumor-bearing mice are isolated and immunolabeled with a 12-color optimized immunofluorescence panel (OMIP) targeting several major leukocyte lineages. Single-cell data is acquired by flow cytometry and is clustered in an unsupervised manner based on cell-surface protein expression. A vector-based classification system is then used to map phenotypically distinct clusters onto known immune cell lineages. The platform allows for the extraction of immune signatures whose divergence from tumor-naïve standards may be quantified and statistically analyzed; such signatures may also be correlated with response/resistance to therapy. The approach has been validated on orthotopic GL261 glioma in syngeneic C57BL6/J mice, yet it is readily amenable to the study of diverse model systems, including de novo tumors driven by clinically-relevant oncogenes. Alternative flow cytometry antibody panels may easily be substituted to probe more specific immune cell subsets of interest. Systems biology approaches applied to the field of tumor-induced immunosuppression hold promise in identifying novel network-level immune signatures, facilitating a better understanding of immunotherapeutic drug action, and driving next generation high-dimensional biomarker discovery. Citation Format: Gregory J. Baker, P.S. Thiagarajan, Sucheendra K. Palaniappan, Stephanie H. Davis, Jodene K. Moore, Peter K. Sorger. A flow-based immunoprofiling strategy for interrogating system-wide leukocyte composition in response to glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1678. doi:10.1158/1538-7445.AM2017-1678

Non-oncogenic Acute Viral Infection Reduces Tumor Growth in Hosts with Established Cancer

Abstract The ability of the immune system to recognize cancer cells as abnormal and to enact effector activity to destroy such cells is a delicate maneuver dependent upon multiple factors. Cancer patients exhibit higher rates of infection than the general population, prompting the question of whether non-oncogenic acute infections impact the ability of patients to mount an anti-tumor immune response, potentially affecting tumor growth and survival outcomes. To assess this, we challenged C57BL/6 mice with B16-F10 melanoma and A/H1N1/PR8 influenza. Influenza was administered upon development of a palpable tumor. We observed that mice bearing an established melanoma that were infected with influenza exhibited reduced tumor growth as compared to mice bearing melanoma alone. Tissues in tumor-bearing mice were interrogated via flow cytometry and a 9-fold reduction in myeloid-derived suppressor cells (MDSCs) was observed in mice concomitantly bearing melanoma and infected with influenza, relative to that observed in the mice bearing melanoma alone. These findings are in sharp contrast to our previous report of increased cancer incidence in cancer-prone mice infected with influenza prior to tumor emergence. These findings suggest that non-oncogenic acute viral infection may play a dual role (increasing the emergence of immune-controlled cancer but decreasing the progression of established cancer). Further, our findings suggest that viral infection in cancer-bearing hosts can be utilized as an immunotherapy for cancer. Future studies will focus on elucidating both the immune-related and cancer cell-related mechanisms that lead to altered tumor growth and survival outcomes in cancer-bearing hosts in the context of viral infection.

Nucleolin-targeted Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer

Small interfering RNAs (siRNA)/microRNAs (miRNA) have promising therapeutic potential, yet their clinical application has been hampered by the lack of appropriate delivery systems. Herein, we employed extracellular vesicles (EVs) as a targeted delivery system for small RNAs. EVs are cell-derived small vesicles that participate in cell-to-cell communication for protein and RNA delivery. We used the aptamer AS1411-modified EVs for targeted delivery of siRNA/microRNA to breast cancer tissues. Tumor targeting was facilitated via AS1411 binding to nucleolin, which is highly expressed on the surface membrane of breast cancer cells. This delivery vesicle targeted let-7 miRNA delivery to MDA-MB-231 cells in vitro as confirmed with fluorescent microscopic imaging and flow cytometry. Also, intravenously delivered AS1411-EVs loaded with miRNA let-7 labeled with the fluorescent marker, Cy5, selectively targeted tumor tissues in tumor-bearing mice and inhibited tumor growth. Importantly, the modified EVs were well tolerated and showed no evidence of nonspecific side effects or immune response. Thus, the RNAi nanoplatform is versatile and can deliver siRNA or miRNA to breast cancer cells both in vitro and in vivo. Our results suggest that the AS1411-EVs have a great potential as drug delivery vehicles to treat cancers.

Abstract 2058: Identification of a specific RNA aptamer targeting non-small cell lung cancer by in vivo SELEX

Abstract In vivo selection of the RNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX) has demonstrated a great targeting tool for diagnostics and treatments in the clinic. In this study, in vivo selection of specific RNA aptamer for cells was established through the use of tumor-bearing xenograft mice injected with human non-small cell lung cancer NCI-H460 cells. Briefly, a synthetic DNA pool was first transcribed in vitro using mutant T7 RNA polymerase (Y639F), which is capable of recognizing 2’-fluoropyrimidines to produce nuclease-resistant RNA. PEGylated RNA library was then constructed to produce stable PEG-RNA molecules. A cDNA library was produced and was further transcribed for an RNA pool. The enriched nuclease-resistant PEG-RNA pool was re-injected into NCI-H460 tumor-bearing mice again. After multiple cycle selections in vivo, the cDNA pool was cloned into T-vector for sequencing analysis for enriched RNA motifs. On round 8 of SELEX selection, an enriched RNA sequence (RA16) was identified at a frequency of 21.2%. The RA16 preparation was further enriched up to 94.7% on round 11. To characterize the specificity in vitro, NCI-H460 cells, along with additional tumor cells or normal control cells, were incubated with Cy3-labeled RA16 or the initial RNA library. The specific fluorescent binding was observed for HCI-H460 and H1299 cells, but not for HEK293T, Hela, and H226 cells. Binding affinity (Kd 9±2 nM) for RA16 was determined by flow cytometry using FITC-labeled RA16 to HCI-H460 cells. Meanwhile, cryosections from various organs of tumor-bearing mice also revealed strong binding of RA16 with HCI-H460 tumors but only slightly with lung tissue. No any significant binding was detected in other tissues of tumor-bearing mice or in any normal tissues of control mice. Furthermore, a quantitative-RT-PCR was performed to trap specific RA16 sequence from various tissues of control or tumor-bearing mice. The selected RNA aptamer RA16 or the initial RNA library was injected into tumor-bearing(n = 4) and normal control mice (n = 4). Total RNAs were extracted from the mouse tumor or from heart, liver, spleen, lung and kidney 3.5 hrs post injection. There was an enrichment of 44.3±3.6-folds for the injected RA16 in mouse tumors treated by RA16 preparation comparing with mouse tumors treated by the initial RNA library. No significant enrichment of RA16 was detected in any other tissues of tumor-bearing mice or tissues from normal control mice. Significantly, in vivo tumor imaging study with Cy5.5-labeled RA16 showed that aptamer RA16 was gradually migrated toward tumor site and enriched with time at tumor area in mice. Our studies thus strongly suggests that RNA aptamer RA16 was specifically selected for targeting human NCI-H460 cells, as well as xenograft tumor tissues with high affinity both in vitro and in vivo. Citation Format: Hanlu Wang, Yibang Zhang, Chang Chu, Wei Dai, Rihe Liu, Yongping Jiang. Identification of a specific RNA aptamer targeting non-small cell lung cancer by in vivo SELEX. [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 2058.

Transcriptional Activation of Human CDCA8 Gene Regulated by Transcription Factor NF-Y in Embryonic Stem Cells and Cancer Cells

The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

Echogenic Glycol Chitosan Nanoparticles for Ultrasound-Triggered Cancer Theranostics.

Theranostic nanoparticles hold great promise for simultaneous diagnosis of diseases, targeted drug delivery with minimal toxicity, and monitoring of therapeutic efficacy. However, one of the current challenges in developing theranostic nanoparticles is enhancing the tumor-specific targeting of both imaging probes and anticancer agents. Herein, we report the development of tumor-homing echogenic glycol chitosan-based nanoparticles (Echo-CNPs) that concurrently execute cancer-targeted ultrasound (US) imaging and US-triggered drug delivery. To construct this novel Echo-CNPs, an anticancer drug and bioinert perfluoropentane (PFP), a US gas precursor, were simultaneously encapsulated into glycol chitosan nanoparticles using the oil in water (O/W) emulsion method. The resulting Echo-CNPs had a nano-sized particle structure, composing of hydrophobic anticancer drug/PFP inner cores and a hydrophilic glycol chitosan polymer outer shell. The Echo-CNPs had a favorable hydrodynamic size of 432 nm, which is entirely different from the micro-sized core-empty conventional microbubbles (1-10 μm). Furthermore, Echo-CNPs showed the prolonged echogenicity via the sustained microbubble formation process of liquid-phase PFP at the body temperature and they also presented a US-triggered drug release profile through the external US irradiation. Interestingly, Echo-CNPs exhibited significantly increased tumor-homing ability with lower non-specific uptake by other tissues in tumor-bearing mice through the nanoparticle's enhanced permeation and retention (EPR) effect. Conclusively, theranostic Echo-CNPs are highly useful for simultaneous cancer-targeting US imaging and US-triggered delivery in cancer theranostics.

Abstract 1110: Colorectal liver metastases induce a systemic phase shift of the biological clock

Abstract Circadian clock genes drive cyclic expression of 10-15% of the transcriptome. Disruption of the circadian clock may deregulate the cell's normal functions and can lead to carcinogenesis, indicating that clock genes also act as tumor suppressor genes. In previous work, we have shown that circadian rhythmicity is disrupted in C26 colon carcinoma cell-derived hepatic metastases, as compared to the non-cancerous tissue of the liver. It has been suggested that neoplastic tissue can influence the circadian clock of adjacent tissues, causing a tissue specific phase shift. A better understanding of how tumors affect the circadian system may help elucidate the role of the clock in cancer patients suffering from fatigue and sleep disorders. Therefore, we have examined the circadian clock in liver and kidney tissue of mice carrying hepatic C26 colon carcinoma cell metastases. Male BALB/c mice were divided into two groups. In group 1, hepatic metastases were induced by injecting C26 colorectal carcinoma cells into the spleen. Group 2 was sham operated. Three weeks after (sham) surgery, animals were sacrificed over a 24-hour period (6 time points, n=4 per time point) and liver and kidneys were collected to compare circadian rhythmicity in control and tumor bearing animals. RNA was isolated and qRT-PCR was performed to determine the expression levels of 5 clock genes (Rev-Erbα, Per1, Per2, Bmal1, Cry1) and 3 clock controlled genes (Dbp, p21 and Wee1). Liver and kidney tissue of healthy control mice showed normal 24-hour oscillations of all clock genes, consistent with normal circadian rhythmicity. Liver and kidney tissue of tumor-bearing mice, as compared to control mice, showed a marked phase difference in clock rhythms. In the liver we observed a 4-hour phase advance for Bmal1, Rev-Erbα and Dbp. In contrast in the kidney, there was a 4-hour phase delay for Bmal1, Rev-Erbα, Cry1 and DBP, and a 8 hour delay for Per2. These data show that colorectal liver metastases induce a phase advance in the liver, and a phase delay in the kidney . This suggests a systemic effect of the tumor on peripheral clocks. This difference in clock phase between tumor-bearing mice and healthy controls may provide new clues to further understand the role of the clock for fatigue and sleep problems in cancer patients, but may also give opportunities to administer chemotherapeutic treatment in a chronotherapeutical setting . Citation Format: Sander A. Huisman, F. Tamanini, A. Ahmadi, J.N.M. IJzermans, G.T.J. van der Horst, R.W.F. de Bruin. Colorectal liver metastases induce a systemic phase shift of the biological clock. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2014-1110

Novel nitric oxide generating compound glycidyl nitrate enhances the therapeutic efficacy of chemotherapy and radiotherapy

Selective release of nitric oxide (NO) in tumors could improve the tumor blood flow and drug delivery for chemotherapeutic agents and radiotherapy, thereby increasing the therapeutic index. Glycidyl nitrate (GLYN) is a NO generating small molecule, and has ability to release NO on bioactivation in SCC VII tumor cells. GLYN-induced intracellular NO generation was significantly attenuated by NO scavenger carboxy-PTIO (cPTIO) and NAC. GLYN significantly increases tumor blood flow, but has no effect on the blood flow of normal tissues in tumor-bearing mice. When used with cisplatin, GLYN significantly increased the tumor growth inhibition effect of cisplatin. GLYN also had a modest radiosensitizing effect in vitro and in vivo. GLYN was well tolerated and there were no acute toxicities found at its effective therapeutic doses in preclinical studies. These results suggest that GLYN is a promising new drug for use with chemotherapy and radiotherapy, and provide a compelling rationale for future studies of GLYN and related compounds.

Changes in tumor microenvironments in high‐fat diet‐fed mice: role of macrophages and adipocytes (37.5)

Using several mouse tumor models, we have shown that the chronic consumption of a high‐fat diet (HFD) stimulates solid tumor growth and metastasis and thereby reduces the survival rate. The present study examined the mechanisms by which HFD stimulates tumor promotion. Angiogenesis and lymphangiogenesis in tumor tissues and lymph node (LN)s are markedly increased in HFD‐fed mice. HFD feeding increases adipocytes and M2‐macrophage (M2‐MΦ)s in tumor tissues and M2‐MΦs in adipose tissues of tumor‐bearing mice. Results from in vivo and in vitro co‐culture studies revealed that the crosstalk between adipocytes, tumor cells, M2‐MΦs, and/or lymphatic endothelial cell (LEC)s stimulates the production of several factors which stimulate tumor cell/monocyte migration, angiogenesis, and lymphangiogenesis via endocrine and paracrine mechanisms. In HFD‐fed mice, increased chemokine ligands in LECs recruit tumor cells expressing high levels of their receptors, thereby increasing LN metastasis. Our results indicate that increased adipocytes in HFD‐fed mice play important roles in the recruitment of M2‐MΦs in tumor tissues and lymphangiogenesis as well as tumor cell migration, thereby stimulating tumor metastasis.