2.20 Development of Theranostic Mesoporous Silica Nanoparticles for Pancreatic Cancer

Posted on December 22, 2014

D. S. Pender1, A. Khanal1, S. Hudson1, L. McNally1  1University Of Louisville,Louisville, KY, USA

Introduction: Modern methods of pancreatic cancer diagnosis and treatment are severely lacking and have failed to provide effectual treatment options for patients. The root cause of this inadequacy stems from the hypovascularized nature of pancreatic cancer, making traditional chemotherapeutics and cancer detecting contrast agents nearly obsolete. A potential solution for tumor-targeting difficulties is through the implementation of nanotechnology, specifically targeting ligand capped, theranostic nanoparticles. We hypothesize that pH-responsive chitosan-capped mesoporous silica nanoparticles (MSNs) with the targeting ligand, urokinase plasminogen activator (UPA) will serve as theranostic agents for treatment and diagnosis of pancreatic cancer.

Methods: MSNs were synthesized by employing cetyl trimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS) and chitosan through the sol-gel method. The synthesized MSNs were characterized by transmission electron microscopy (TEM) and zeta-potential measurements. Afterwards, gemzar chemotherapeutic drug was encapsulated into these nanoparticles to observe the pH dependent release profiles in vitro. Furthermore, MSNs were tagged with UPA to increase the binding efficiency of these nanoparticles towards the pancreatic tumor cells (S2CP9 and S2VP10). The binding efficiency of both tagged and non-tagged MSNs was observed at various pHs (7.4 to 6.5) by employing fluorescence microscopy, Odyssey infrared imaging and tissue phantoms. For that, various types of dyes were used, such as, rhodamine B and indocyanine green (ICG). Finally, UPA-tagged MSNs with ICG were injected into mice infected with S2CP9 tumors cells to observe the distribution of these nanoparticles in-vivo through multispectral photoacoustic Tomography system (MSOT).

Results: TEM pictures showed that the synthesized MSN had a size around 120 nm.  Zeta-potential measurements revealed that charge density of MSN was dependent on pH.  The release experiments showed that these nanoparticles were pH-sensitive because the release of gemazar depended on the pH. Gemzar released ~2x the quantity from MSNs at pH 6.5 in comparison to pH 7.4.  Fluorescence microscopy, Odyssey infrared imaging and tissue phantoms showed that uptake of MSNs by pancreatic tumor cells depended on the pH and tagging of UPA. Lowering pH and tagging a ligand drastically increased the uptake of MSNs in pancreatic tumor cell in vitro. Specifically in tissue phantoms, UPA-ICG loaded MSNs at pH 6.5 demonstrated 20X and 7X more cell signal than without ligand or at pH 7.4, respectively.  Furthermore, UPA-ICG loaded MSNs were successfully detected in orthotopic pancreatic tumor of mice within 6 hours of imaging time by MSOT.

Conclusion: UPA tagged, pH sensitive MSNs demonstrate potential as a theranostic nanoparticle for pancreatic cancer.

 

19.04 Deficiency of the Immunostimulatory Cytokine IL-21 Promotes Intestinal Neoplasia

Posted on December 22, 2014

M. M. Shapiro1,2, B. Nandi1,2, G. Gonzalez1, R. Prabhala1,2,3, Q. Huang1,4, N. C. Munshi1,2,3, N. Y. Frank1,2,4, J. S. Gold1,2,3  4Brigham And Women’s Hospital,Boston, MA, USA 1VA Boston Healthcare System,West Roxbury, MA, USA 2Harvard School Of Medicine,Brookline, MA, USA 3Dana Farber Cancer Insititute,Boston, MA, USA

Introduction: Interest in the use of the cytokine IL-21 as an immunotherapeutic agent grew out of the initial success with interferon gamma and IL-2 in the treatment of melanoma. IL-21 functions by increasing proliferation and survival of B and T cells. It also increases granzyme B-mediated cytotoxicity of NK and T cells. Due to these properties, IL-21 has been investigated in the treatment of both renal cell carcinoma and melanoma. The role of IL-21 in spontaneous intestinal carcinogenesis has not been fully explored.

Methods: Mice with a targeted knockout of IL-21 (KO) were bred with APCMIN/+ (MIN) mice. MIN mice spontaneously develop numerous intestinal adenomas. Wild-type C57/Bl6 mice (WT) were used as a control. Mouse small intestines were harvested at 15 weeks. Polyps were measured and counted under a dissecting microscope. Mouse ileum was also either preserved for paraffin embedding and immunohistochemical staining or snap frozen for cDNA preparation and q-RT-PCR.

Results: Polyp-bearing ileum from MIN mice had a five-fold increase in IL-21 expression by q-RT-PCR as compared to the non-polyp bearing ileum of WT mice (p=0.03). MIN mice lacking IL-21 had increased intestinal polyp number and tumor load as compared to MIN mice with functional IL-21 (55 vs. 40 polyps, p=0.007; Figure panel A; tumor load 88 vs. 68, p=0.02). The differences in polyp number and tumor load were significant in the jejunum and ileum but not in the duodenum (duodenum 5.4 vs. 4.1 polyps, p=0.3 and tumor load 14 vs. 7.8, p=0.5; jejunum 21 vs. 14, p=0.02 and 31 vs. 22, p=0.04; ileum 29 vs. 22, p=0.01 and 44 vs. 36, p=0.03). KO-MIN mice had fewer CD3+ cells (T cells) and B220+ cells (B cells) in the polyp-bearing ileum than MIN mice (50 vs. 78 cells/0.07mm2, p<0.001 for T cells; 13 vs. 18, p<0.001 for B cells). Similarly, the number of granzyme B+ cells was much lower in polyp-bearing ileum of KO-MIN mice when compared to that of MIN mice (20 vs. 41 cells/0.07 mm2, p<0.001; Figure panel B).

Conclusion: Adenoma development is associated with upregulation of IL-21 in the intestine in a mouse model of spontaneous intestinal neoplasia. Deficiency of IL-21 leads to accelerated tumor development in this model. Loss of IL-21 is also associated with a decrease in B and T cell populations in the polyp-prone intestine as well as a concomitant decrease in granzyme B-releasing cells. These data support the hypothesis that IL-21 is involved in mediating spontaneous anti-tumor immunity controlling adenoma development. The use of IL-21 for the treatment of colorectal cancer warrants further investigation.

19.05 Histone Deacetylase Inhibitors Induce a Pro-inflammatory Phenotype in Pancreatic Cancer Fibroblasts

Posted on December 22, 2014

A. H. Nguyen1, S. Patel1, M. Vogelauer2, P. A. Toste1, N. Wu1, J. Williams3, L. Li1, D. W. Dawson4, S. Kurdistani2, T. R. Donahue1  1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Department Of Biological Chemistry,Los Angeles, CA, USA 3Harbor-UCLA Medical Center,Department Of Surgery,Torrance, CA, USA 4University Of California – Los Angeles,Department Of Pathology And Laboratory Medicine,Los Angeles, CA, USA

Introduction:
Histone deacetylase inhibitors (HDACi) are currently being investigated in early phase clinical trials for patients with pancreatic ductal adenocarcinoma (PDAC). Although there have been measurable responses in patients with hematologic malignancies treated with HDACi, similar results have not been demonstrated for solid organ tumors. We hypothesize that tumor associated fibroblasts (TAFs), the predominant cell type in the PDAC stoma, may contribute HDACi therapy resistance.

Methods:
Primary TAFs were isolated from human PDAC tumor samples. MTT assay was used to determine cell viability. Western blot was performed of whole cell lysate and acid extracted histone-enriched samples. Gene expression was determined by qRT-PCR. A modified Boyden chamber was utilized to assess tumor cell (TC) invasion. ChIP-seq was performed for HDAC2 and was overlaid with publically available H3K4me1 and H3K27ac ChIP data from the NIH Roadmap Epigonics Mapping Consortium website.

Results:
As has been described, PDAC TC (PANC-1, MIA PaCa-2) viability was significantly decreased (p<0.01) by all tested class I and II HDACi's (vorinostat, entinostat, panobinostat). However, slowly dividing primary TAFs showed neither growth arrest nor an upregulation of CDKN1A, despite an appropriate increase in global levels of acetylated histones H3 and H4. HDACi treatment of TAFs induced a tumor supportive secretory milieu, as conditioned media from treated cells increased TC invasion (p<0.001) and viability (p=0.05). HDACi treatment on primary TAFs increased secretion of a series of pro-inflammatory proteins (p=0.05) including CXCL1, IL-6, IL-8, and SPP1, by upregulating NFκB target genes (p<0.01). By western blot, we identified markers suggesting the cytosolic activation of NFκB, STAT3, and p38 MAPK pathways, yet, inhibition of STAT3 and p38 MAPK failed to completely abrogate the HDACi-induced inflammatory response and NFκB inhibition proved to be lethal to HDACi-treated fibroblasts. To elucidate how HDAC inhibition may directly affect gene regulation, pro-inflammatory gene enhancers were identified from published H3K4me1 ChIP-seq on normal fibroblasts. We overlaid our HDAC2 ChIP-seq from naïve primary PDAC TAFs to these poised enhancer regions. Our ChIP-seq shows HDAC2 binds these enhancer regions, and importantly, in cells that normally express the active enhancer mark H3K27ac. The promoter regions for these genes both show motif and ChIP-seq data supporting expression from STAT3 and NFκB mediated factors, suggesting HDAC inhibition provides a permissive chromatin landscape for the expression of inflammatory genes in primary PDAC TAFs.

Conclusion:
HDACi's are effective against PDAC TCs in culture, but induce a tumor supportive phenotype in primary PDAC TAFs, which may explain their disappointing results in solid organ tumors. These studies are beginning to uncover the mechanism of this detrimental response in PDAC TAFs.

18.08 Palliative Care Training in Surgical Oncology and Hepatobiliary Fellowship: National Fellows Survey

Posted on December 22, 2014

G. Larrieux1, J. T. Miura1, K. J. Brasel1, D. E. Weissman2, A. B. Nattinger3, T. C. Gamblin1, K. T. Turaga1, F. M. Johnston1  1Medical College Of Wisconsin,Surgery,Milwaukee, WI, USA 2Medical College Of Wisconsin,Palliative Care,Milwaukee, WI, USA 3Medical College Of Wisconsin,Medicine,Milwaukee, WI, USA

Introduction: Surgical Oncologists (SO) and Hepatobiliary (HPB) Surgeons frequently care for patients with advanced disease stages who are near the end of life, yet little is known about their training, comfort and readiness in the provision of palliative care.  This study sought to assess the quality, adequacy and extent of palliative care training and readiness of SO and HPB Fellows in delivering palliative care.   

Methods: A self-administered survey was distributed to all fellows enrolled in Society of Surgical Oncology (SSO) and HPB fellowships during the 2013-2014 academic years.  The survey assessed attitudes, training, experience, and readiness of fellows in caring for patients at the end of life.  Descriptive analysis was performed and Chi-square, Student’s t-test as well as Mann-Whitney U test were used to compare mean or median values as appropriate.

Results:The response rate was 47.2%. 50.9% of fellows reported exposure to a palliative care specialty service during their fellowship. 75% of our participants observed their faculty discussing the side effects of surgery compared to 54% observation of faculty’s communication regarding end of life goals with patients (p<0.01). 40% of fellows were never observed by faculty discussing symptoms management, goals of care, or hospice referral with patients and 56.7% never received feedback on their palliative skills. Fellows consistently rated their quality of teaching and managment of surgical disease at better compared to palliative and end of life topics (Figure 1).

Conclusion:Fellows rated the quality of palliative care education as poor compared to other aspects of fellowship training, implying the need and lack of palliative care teaching.  Surgical oncology and HPB fellows and ultimately patients may benefit from increased clinical and didactic palliative care training.
 

18.18 Robotic Simulator Curriculum Validation Study

Posted on December 22, 2014

J. L. Miller1, S. M. Novak1, D. L. Bartlett1, A. H. Zureikat1, H. J. Zeh1, M. E. Hogg1  1University Of Pittsburgh,Surgical Oncology/Surgery/Medicine,Pittsburgh, PA, USA

Introduction:

Robotic surgery is becoming widely used by general surgery and its specialties such as surgical oncology. Our institution has performed over 1,000 robotic surgical oncology cases and has identified the learning curve for several complex robotic resections. Critical to successful dissemination of the platform is a better understanding of how new surgeons learn the technology. This study aims to evaluate content and predictive validity of robotic simulation in surgery.  We hypothesize mastery-based simulation is a valid tool to train residents and fellows toward operative proficiency.

Methods:

A mastery-based simulation curriculum was performed in a virtual reality environment. Mastery was defined as 90% proficiency on each module. A pretest/posttest experimental design utilized virtual reality M score tasks (Match Board 3, Ring & Rail 2, Tubes and Continuous Suture) and inanimate environments using video analysis (Ring Rollercoaster 4, Around the World and Interrupted Suture) to evaluate technical improvement. Prior robotic training experience and curriculum assessment was self-reported in a survey; fellows were asked to rate modules based on difficulty and utility using a Likert scale of 5 (5 being greatest).

Results:

11 fellows enrolled in the curriculum. Prior robotic simulator experience showed: min=0, max=31, median=0.375 and mode=0 hours. Prior robotic case experience demonstrated: min=0, max=50, median=12 and mode=4 cases. 9 fellows (82%) completed the mandatory curriculum. 7 fellows (77.8%) achieved mastery on all 24 modules (one deficient on 4, one deficient on 9). Individual test scores improved; overall time and errors decreased (Table). Of the 24 modules, frequency to mastery demonstrated: min=1, max=17, median=2. Simulator hours spent completing curriculum showed: min=1.1, max=6.6 and med=4.2. 9 (100%) fellows continued modules beyond mastery. Fellows rated modules between 1 and 5 for difficulty and 3 and 5 for utility. Needle driving and Endowrist 2 modules were perceived as most difficult; needle driving modules were most useful. 8 (89%) fellows perceived improvement in robotic skills after completing the curriculum.

Conclusion:

This pilot study is limited by sample size; however, these preliminary results show overall score improvement, decrease in errors and decrease in total time. Time to complete the curriculum is manageable.To increase power for statistical comparison, the study is ongoing to include incoming fellows, senior general surgery residents and other fellowship programs. Ultimately, the study will assess correlation between performance on simulator curriculum with inanimate biotissue curriculum and operative improvement to assess content and predictive validity.

15.16 Readmissions Following Major Cancer Surgery in Older Adults Within a Large Multihospital System

Posted on December 22, 2014

R. C. Langan1,2, C. Huang3, K. Harris1,2,3, S. Colton1, A. L. Potosky2,3,4, L. B. Johnson1,2,3,4, N. M. Shara2,3,5, W. B. Al-Refaie1,2,3,4  1Georgetown University Hospital,Department Of Surgery,Washington, DC, USA 2MedStar-Georgetown Surgical Outcomes Research Center,Washington, DC, USA 3MedStar Health Research Institute,Washington, DC, USA 4Lombardi Comprehensive Cancer Center,Washington, DC, USA 5Georgetown-Howard Universities Center For Clinical And Translational Sciences,Washington, DC, USA

Introduction:  Readmissions are a focus of emerging efforts to improve the quality and affordability of healthcare. Yet, little is known about reasons for readmissions after major cancer surgery in the expanding elderly population (≥ 65 years) who are also at increased risk of adverse operative events. We sought to identify 1) the extent to which older age impacts readmissions and 2) factors predictive of 30- and 90-day readmissions after major cancer surgery among older adults. 

Methods:  We identified 2,797 older adults who underwent seven types of major thoracic or abdomino-pelvic cancer surgery within a large multihospital system from 2003-2012.  Multivariate logistic regression analyses were conducted to identify predictors of 30- and 90-day hospital readmission. 

Results: Overall 30-day and 90-day readmission rates were 16% and 24% with the majority of readmissions occuring within 15-days of discharge. Principal diagnoses of 30-day readmissions included gastrointestinal, pulmonary and infections complications. 30-day readmissions were associated with > 2 comorbid conditions and ≥ 2 postoperative complications. Readmissions significanctly varied according to cancer surgery type and across treating hospitals. Readmissions did not vary by increasing age. Factors associated with 90-day readmission were comparable to those observed at 30-days (Table 1). 

Conclusion: In this large multi-hospital study of older adults, multi-morbidities, procedure type, increased complications and the treating hospital predicted 30- and 90-day readmissions. These findings point toward the potential impact of hospital-level factors behind these readmissions. Our results also point towards the importance of assessing the influence of readmission on other important cancer care metrics; patient reported outcomes and the completion of adjuvant systemic therapies.

 

16.01 The Role of Breast MRI in Ductal Carcinoma in situ: Has it Improved Clinical Outcomes?

Posted on December 22, 2014

L. S. Sparber1, R. S. Chamberlain1,2,3  1Saint Barnabas Medical Center,Deptarment Of Surgery,Livingston, NJ, USA 2Saint George’s University,School Of Medicine,St. George’s, , Grenada 3New Jersey Medical School,Rutgers University – Department Of Surgery,Newark, NJ, USA

Introduction: For over three decades, screening mammography has played a central role in the early detection of in situ breast tumors in the United States.  More recently, breast magnetic resonance imaging (MRI) has emerged as a potentially more sensitive imaging modality than traditional mammography, but whether its use should be limited to adjunct screening for those at high risk or be universally utilized remains controversial.  While breast MRI undoubtedly detects subtler breast abnormalities, it is unclear whether this has resulted in an improvement in treatment decisions for patients with Ductal Carcinoma in situ (DCIS).

Methods: A comprehensive search for all published clinical studies on the use of MRI and its impact on DCIS management (2010-2014) was conducted using PubMed and Google Scholar.  The search focused on the value of MRI to guide treatment strategies, including mastectomy rates, re-excision rates and the overall benefit of this added imaging modality.  Keywords searched included: “breast MRI”, “mastectomy”, “DCIS”, “Ductal Carcinoma in situ”, and “surgical planning” in all possible combinations.

Results:  Six studies involving 3,296 patients have been published (Table 1). Pilewskie et al (2014) reported the largest study involving 2,321 DCIS patients (596 DCIS patients in MRI group; 1,725 patients in non-MRI group).  Within this group, 904 DCIS patients underwent radiation therapy [RT]; and 1,391 patients did not.  In the non-RT subgroup there was no association with the performance of an MRI and lower loco regional recurrence rates (p = 0.28). Three additional studies analyzed the impact of MRI on DCIS mastectomy rates, with Allen et al reporting no significant difference in mastectomy rates if an MRI was performed (p = .62). In contrast, Itakura et al reported increased mastectomy rates in patients undergoing preoperative MRI (p < .001). Re-excision rates were investigated in three studies, and preoperative performance of an MRI did not statistically impact these rates favorably or negatively. Pilewskie et al, demonstrated that breast conserving surgery was more successful in the non-MRI group (p = .06), whereas Allen et al and Kropcho et al found the results to not be statistically significant (p = .41 and p = .414, respectively). Across all studies, preoperative MRI was judged not routinely beneficial in DCIS patients.    

Conclusion: Breast MRI is associated with an increased sensitivity compared to other breast imaging technologies; however, it does not appear to improve clinical outcomes in patients with DCIS when added to conventional breast assessment.  Moreover, routine breast MRI in DCIS may contribute to an increase in unnecessary mastectomies.

 

14.16 Knowledge of Colorectal Carcinoma screening Among General Population in Western Region of Nepal

Posted on December 22, 2014

S. Nepal1, A. Shrestha2, J. Parajuli2, S. Sharma1, M. Acharya3, S. Baral2  1Manipal Teaching Hopital,Department Of Surgery,Pokhara, KASKI, Nepal 2Manipal Teaching Hopital,Medicine,Pokhara, KASKI, Nepal 3Manipal Teaching Hopital,Emergency,Pokhara, KASKI, Nepal

Introduction: Colorectal Carcinoma has emerged as third most common malignant tumor, second leading cause of death among cancer patients in the world and has been increasing in developing countries. In this study our objective was to determine the knowledge and attitude of CRC and to understand the factors that contribute to low screening rates in our region.

Methods: We interviewed 800 participants aged 40 years and above with 200 participants each from Kaski, Baglung, Parbat and Syangja district which are in Western region of Nepal. We used questionnaires to determine the socio-demographic characteristic, knowledge about CRC, screening, as well as screening test.

Results:The majority participants were illiterate with monthly income less than Nrs 10,000 ($100).Regarding lifestyle practices most of them were smokers (68%) and consumed alcohol (48%).Among the participants, 20% of them said there exists no cancer as Colorectal Carcinoma. The rest of them who knew CRC exists the knowledge about it and is screening were very poor. Only 25% and 10% of them knew about FOBT and Colonoscopy but none of them had idea about barium enema and flexible sigmoidoscopy .Majority of them (55%) agreed to do screening tests even if they did not have any symptom and 40% of the participants said the disease had good prognosis if diagnosed early.

Conclusion:The result of the current study provide information about the need for education campaigns about CRC and its screening to reduce the incidence of deaths due to CRC.

 

1.04 The Novel PARP Inhibitor ABT-888 Enhances Dacarbazine Induced Cytotoxicity in Carcinoids in vitro

Posted on December 22, 2014

Y. R. Somnay1, S. Lubner2, H. Gill1, B. Matsumura1, H. Chen1  1University Of Wisconsin,Endocrine Surgery Research Laboratories, Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Human Oncology,Madison, WI, USA

Introduction:  Carcinoids are slow-growing neuroendocrine tumors that often present insidiously with few options for medical management. Monoagent DNA-alkylating chemotherapies such as Dacarbazine (DTIC) are among a paucity of therapies for these tumors, but are limited by host toxicity and intrinsic chemoresistance through the base excision repair (BER) pathway via the activity of poly (ADP-ribose) polymerase (PARP). Inhibitors of PARP are thought to potentiate DNA-damaging chemotherapies by blocking cancer cells’ ability to repair DNA double strand breaks following base excision repair. Here we show that the PARP inhibitor ABT-888 (veliparib) enhances the cytotoxic effects of DTIC in a pancreatic carcinoid cell line and potentiates DNA damage, cell cycle arrest and apoptosis. 

Methods:  Human pancreatic carcinoid cells (BON) were incubated in ABT-888 (0-20µM), DTIC (0-400µM) or both, for 96 hours. Cell growth was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay. Western analysis showed expression levels of Chromogranin A (CgA) a well characterized marker for carcinoid malignancy, in addition to poly(ADP)-ribose polymerase (PARP), cleaved caspase 3, cyclin family kinases, and phosphorylated and total ataxia and telangiectasia mutated kinase (ATM), H2A histone family member X (H2AX), p53 and retinoblastoma gene (Rb). Flow cytometry using propidium iodide staining was used to assess cell cycle kinetics.  

Results: Cell survival after treatment with ABT-888 and DTIC was reduced synergistically as combination indices (CI) fell below 1 on the Chou-Talalay scale, as we've previously reported. Notably, ABT-888 alone was non-toxic at therapeutic doses. ABT-888 administered prior to varying DTIC doses reduced CgA expression beyond the combined effect of either drug alone. Of note, the degree of ATM and H2AX phosphorylation, indicative of double strand DNA breaks, was enhanced by ABT-888 prior to DTIC treatment, suggesting BER pathway attenuation via PARP inhibition. This was corroborated by an increase in phospho-p53, a tumor suppressor activated by DNA damage, alongside reductions in cyclin D and downstream inactive phospho-Rb. ABT-888 treatment prior to DTIC decreased levels of cyclin A expression, and accordingly augmented G2 and G0-G1 phase arrest, indicating an enhanced transition into apoptosis. Finally, ABT-888 potentiated DTIC-induced cleavage of terminal apoptotic markers caspase 3 and PARP relative to the effect exerted by either monotherapy.  

Conclusion: ABT-888 synergizes with DTIC to suppress carcinoid growth and phenotype. By targeting PARP activity, ABT-888 may impair BER pathway response to DTIC, inducing a futile cycle of persistent DNA strand breakage and potentiating cell cycle arrest and apoptosis through p53 by enhancing Rb hypophosphorylation. By targeting DNA repair pathways that confer cancer cell resistance, ABT-888 may help treat carcinoids that remain refractory to mainstay therapies.

1.05 MicroRNA-21 Regulates Melanoma Invasion via Inhibition of Tissue Inhibitor of Metalloproteinases-3

Posted on December 22, 2014

N. Latchana3, S. Martin Del Campo3, V. Grignol3, K. Levine3, E. Fairchild6, A. Ganju3, C. Jaime-Ramirez3, T. Dao5, V. Karpa5, M. Carson3, A. Chan5, W. Carson3  3Ohio State University,Columbus, OH, USA 5Wright State University,Dayton, OH, USA 6Nationwide Children’s Hospital,Columbus, OH, USA

Introduction: Melanoma accounts for the highest number of skin cancer associated deaths annually yet the progression towards a metastatic phenotype is not completely understood.  Increased cellular invasion through the extracellular matrix is a prerequisite for metastasis and is enhanced by matrix metalloproteinases (MMPs), MMPs are negatively regulated by the tissue inhibitor of metalloproteinases (TIMP) protein family such as TIMP3.  MicroRNAs (miRs) are small, noncoding RNAs that inhibit gene expression and regulate many cellular processes.  It was previously shown by our group that miR-21, a potential regulator of TIMP3, is over-expressed in cutaneous melanoma.  It was therefore hypothesized that increased levels of miR-21 expression would lead to decreased expression of TIMP3 and thereby promote the invasiveness of melanoma cells.

Methods: WM1552c, WM793b, A375 and MEL39 melanoma cell lines were transfected with control miR, pre-miR-21, or anti-TIMP3.  Oligonucleotide uptake was assessed by Real-Time PCR and transfection efficiency was determined using fluorescent microscopy with a FAM-miR construct during transfection.  Transfected cells were used for invasion, proliferation, and migration assays.  Immunoblot analysis was performed on transfected cells for targets of miR-21 including programmed cell death protein (PDCD4), tropomyosin-1 (TM1), phosphatase and tensin homolog (PTEN).  A375 cells were transfected with oligonucleotides against miR-21 or a control miR before injection into the flanks of 01B74 Athymic NCr-nu/nu mice.  Tumor growth was analyzed over three weeks.  Another group of mice were injected with untransfected A375 cells in the flank and monitored until tumor growth reached an average of 100mm3 before injections with PBS alone (control) or anti-miR 21 every three days for four cycles.  Tumor growth was monitored and tumor specimens were analyzed for TIMP3 expression by immunohistochemistry using a goat anti-TIMP3.

Results: Fluoroscopic evaluation revealed >90% transfection efficiency of miRs into melanoma cells with oligonucleotide uptake also confirmed by PCR.  Immunoblot analysis of miR-21 overexpressing cells revealed reduced expression of TIMP3.  This in turn led to an increase in the invasiveness of the 4 melanoma cell lines as wells as an additional melanoma cell line, 1174 MEL.  miR-21 transfection did not change the proliferation or migration capacity of melanoma cells.  Reduced expression of TIMP3 was achieved by siRNA knockdown and significantly enhanced invasion of melanoma cells, mimicking the effects of miR-21 over-expression.  Treatment of tumor cells in vivo with an antagomir to miR-21 inhibited tumor growth with a corresponding increased expression of TIMP3 on immunohistochemistry.  Intra-tumoral injections of anti-miR-21 in vivo produced similar effects.

Conclusion: Increased expression of miR-21 enhanced the invasive potential of melanoma cell lines through TIMP3 inhibition. Therefore, inhibition of miR-21 in melanoma may reduce melanoma invasiveness.

 

1.06 Suppression of CXCL10/CXCR3 Switches Polymetastatic Phenotype to Oligo- in a Melanoma Mouse Model

Posted on December 22, 2014

S. C. Wightman1, A. Uppal1, G. Oshima1, X. Huang1, S. Ganai2, N. N. Khodarev1, M. C. Posner1, R. R. Weichselbaum1  1University Of Chicago,Chicago, ILLINOIS, USA 2Southern Illinois University,Carbondale, ILLINOIS, USA

Introduction:   Oligometastasis is a state of limited metastasis with the potential for long-term disease free control by surgery or radiotherapy. We designed syngeneic mouse melanoma models with oligo-  and polymetastatic pulmonary disease. We found CXCL10, an interferon inducible chemokine that acts on the receptor CXCR3, consistently elevated in polymetastatic tumor clones both in vivo and in vitro.

Methods:   Two stable CXCL10 KDs (knockdowns) and two stable CXCR3 KDs of both oligo- and polymetastatic B16F1 clones (P2M5B and P2M3C respectively) were generated by lentiviral transfection and suppression confirmed by Western blot.  Lung metastases were counted 2.5-3.5 weeks after tail-vein injection of the KD clones and non-targeting controls (NTCs). Flank primary tumors were generated via injection of the KD clones or NTCs and measured serially over 19 days. 

Results:  The P2M3C CXCL10 KD #1 and KD #2 had decreased metastases, 27.7+/-21.1 and 26.7+/-16.9, compared to P2M3C-NTC with 89.7+/-14.3 metastases (p<0.001).  Similarly, the P2M5B CXCL10 KD #1 and KD #2 had decreased metastases, 9.4+/-6.2 and 2.8+/-1.6, compared to P2M5B-NTC with 25.4+/-11.0 metastases (p<0.01).  For CXCR3, the P2M3C CXCR3 KD #1 and KD #2 had decreased metastases, 86.1+/-32.3 and 77.3+/-19.8, compared to P2M3C-NTC with 134.4+/-39 metastases (p = 0.03 and 0.004 respectively).  The CXCR3 P2M5B KD #2 had decreased metastases to 1.7+/-0.8, compared to P2M5B-NTC with 31.3+/-25.5 metastases (p=0.02).  The CXCR3 P2M5B KD #1, while with a decreased number of metastases, was not statistically significant with 14.7+/-7.5 (p = 0.12). After experiments were done with KDs, primary tumors were grown in the flanks of mice to identify differences in the CXCL10 KDs outside of tumor metastases.  At day 19, volume in both of the P2M3C CXCL10 KDs were decreased with KD #1 having an average volume of 0.31+/-0.29 cm3 and KD #2 having an average volume of 0.15+/-0.15 cm3 compared to the P2M3C-NTC with a volume of 4.94+/-1.63 cm3 (p<0.001 for both).  The P2M5B CXCL10 KDs also both decreased with KD #1 having an average volume of 2.10+/-0.73 cm3 and KD #2 0.60+/-0.07 cm3 compared to the P2M5B-NTC with a volume of 7.81+/-1.80 cm3 (p = 0.03 and 0.001 respectively).

Conclusion:  Our experiments stress the vital role of the CXCL10/CXCR3 axis in metastases generation and primary tumor growth in our murine melanoma model.  As noted above, knocking down the CXCL10/CXCR3 axis is able to change the phenotype from polymetastatic to oligometastatic effecting the ability for metastatic tumor colonization. In primary tumors, decreasing the CXCL10 levels decrease tumor growth.  This potentially offers a gateway for therapeutic intervention in patients with melanoma.  These findings stress the importance of CXCL10 in tumor biology for both metastases and primary tumors in murine melanoma.  Further experiments are needed to dissect mechanisms through which CXCL10 regulates pulmonary metastases.

 

1.07 CDK4/6 inhibitor, PD-0332991 Synchronizes Sarcoma Cells for Synergistic Demise with WEE1 Inhibitor, MK-1775

Posted on December 22, 2014

A. M. Francis1, A. Alexander2, J. P. Carey2, V. Ravi3, K. Keyomarsi2, K. K. Hunt1  1University Of Texas MD Anderson Cancer Center,Surgical Oncology,Houston, TX, USA 2University Of Texas MD Anderson Cancer Center,Experimental Radiation Oncology,Houston, TX, USA 3University Of Texas MD Anderson Cancer Center,Sarcoma Medical Oncology,Houston, TX, USA

Introduction:
The retinoblastoma (RB) gene pathway is one of the most frequently altered pathways in human cancer. This pathway is regarded as one of the central regulators of cell proliferation through the coordination of the RB protein, cyclin-dependent protein kinases (CDK4, CDK6), D-type cyclins, the INK4 family of cyclin-dependent kinase inhibitors and the E2F-family of transcription factors. CDK4/6 inhibitors such as PD-03329991 (PD-991) have been shown to cause a G1 arrest in RB-positive cells while WEE1 inhibitors such as MK-1775 can cause premature mitotic entry of S-phase cells. We hypothesized that sarcoma cells could be synchronized in S-phase using PD-991 followed by a period of recovery to facilitate synergistic demise with MK-1775.

Methods:
Sarcoma cell lines (HT-1080, SK-LMS-1, SaOS-2) were evaluated for RB-pathway alterations using western blot analysis. Cells were treated with PD-991 for 6 days and MK-1775 for 2 days to determine IC50 values. FACS analysis was used to determine the optimal recovery timepoint for synchronization in S-phase after treatment with PD-991. Combination experiments involved application of PD-991 with or without a period of recovery followed by application of MK-1775. CalcuSyn 2.0 was used to yield a combination index (CI) to determine if the combination was synergistic (CI<1), additive (CI=1) or antagonistic (CI>1).

Results:
HT-1080 and SK-LMS-1 were RB-positive while SaOS-2 was RB-negative. PD-991 was more effective as single agent therapy in RB-positive cells (1.5-2.4uM) than RB-negative cells (7.0uM). MK-1775 had similar efficacy across all cell lines tested (1.4uM [HT-1080], 0.7uM [SK-LMS-1], 1.0uM [SaOS-2]). The greatest proportion of S-phase cells was observed after 6-9 hours of recovery from PD-991 (HT-1080: 21-25% vs 16% [vehicle], SK-LMS-1: 30-43% vs 21% [vehicle], SaOS-2: not obtainable due to its RB-negativity). PD-991 followed by MK-1775 was strongly synergistic in RB-positive cells (Figure). A period of recovery after treatment with PD-991 enhances this synergism. In RB-negative SaOS-2, the combination of PD-991 and MK-1775 was additive to antagonistic (CI 1.0-1.5).

Conclusion:
Synchronization of cells in S-phase using PD-991 followed by treatment with MK-1775 causes synergistic demise of RB-positive sarcoma cells. In vivo studies are warranted to test this novel treatment strategy and are currently underway.
 

1.08 GPR40 as a therapeutic target in melanoma and neural crest-derived tumors

Posted on December 22, 2014

M. I. Chang1,2, P. Nandivada1,2, S. J. Carlson1,2, A. Pan1,2, M. Puder1,2  1Boston Children’s Hospital,Surgery,Boston, MA, USA 2Boston Children’s Hospital,Vascular Biology Program,Boston, MA, USA

Introduction: Melanoma is a neural crest derived tumor and is the most deadly form of skin 
cancer. In advanced stages, this and other neural crest tumors (e.g., medullary thyroid, 
malignant peripheral nerve sheath tumor, neuroblastoma, etc) have limited treatment options as 
they often manifest as aggressive disease. Recent studies have demonstrated cellular 
apoptosis in these tumors in the presence of the omega-3 fatty acid, docosahexaenoic acid 
(DHA). The purpose of this study is to determine if 1) this effect is mediated through the free 
fatty acid G-protein coupled receptor 40 (GPR40) and 2) determine if selective agonism of 
GPR40 produces cell death in vitro. 

Methods: The presence of the GPR40 receptor was confirmed by PCR and Western blot 
analysis on neural crest tumor cell lines including: neuroblastoma, melanoma, medullary thyroid 
carcinoma, and malignant peripheral nerve sheath tumor. These cell lines were treated with 
varying concentrations of DHA and TAK-875 (a specific, high affinity GPR40 agonist) for 3 and 6 
days while using a human fibroblast cell line as a control. Cell viability was determined using an 
absorbance-based cell viability assay. 

Results: DHA exhibited a minimal inhibitory affect against medullary thyroid carcinoma. 
However, cell death was observed in neuroblastoma, melanoma, malignant peripheral nerve 
sheath tumor, and fibroblasts at high DHA concentration. TAK-875 produced a profound 
selective inhibitory effect on cell proliferation and promoted cell death of all of the neural crest 
derived tumor cell lines without toxicity in the control fibroblast line at nanomolar concentrations. 
Interestingly, no GPR40 expression was noted in the malignant peripheral nerve sheath tumor. 

Conclusion: GPR40 agonist TAK875 produces profound selective cell growth inhibition and 
death in neural crest derived tumor cell lines, with reduced cellular toxicity. While the GPR40 
receptor may serve as a novel target in the treatment of neural crest-derived tumors, the affect 
of this drug on the malignant peripheral nerve sheath tumor may indicate that GPR40 alone may 
not be responsible for this dramatic response in cell viability in neural crest-derived tumor cell 
lines. 

1.09 Notch2 Has an Opposing Role to Other Notch Isoforms in Neuroendocrine Tumors

Posted on December 22, 2014

T. V. Do1, A. Dammalapati1, A. Hundal1, H. Jin1, R. Jaskula-Sztul1, H. Chen1  1Department Of Surgery,Madison, WI, USA

Introduction: Notch signaling involves in various aspects of mammalian biology such as cellular differentiation, cell-cycle regulation, metabolism and apoptosis. Among the four identified mammalian Notch receptors, the role of Notch1 and 3 as tumor suppressor in neuroendocrine tumor cells (NET) have been elucidated. Nevertheless, the function of Notch2 signaling still remains unclear in NETs. The aim of this study was to access the role of Notch2 in gastrointestinal (GI)NETs (carcinoids).

Methods: pcDNA4/V5-His plasmid ,containing constitutively expressed human active portion of Notch2 (NICD2), was transiently transfected into GI carcinoid (BON) cells. The same plasmid without NICD2 was used as a control. Transfection efficiency was assessed by co- transfection with plasmid expressing the green fluorescent protein (GFP). The expression of NICD2 was confirmed by quantitative RT-PCR and Western Blot. Next the functional activity of NICD2 was analyzed by measuring the degree of CBF-1 binding by luciferase reporter assay. Cell viability was then tested by MTT (3-(4, 5-Dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide) assay after 24, 48, 72 and 96 hours after transfection. To investigate the potential effects of NICD2 on BON cells proliferation, cell cycle and anti-apoptotic markers such as X-linked inhibitor of apoptosis (XIAP), cyclin D1, and c-Myc were examined by Western Blot analysis.

Results: The expression of NICD2 was detected over all time points of transfection on both mRNA and protein levels. CBF-1 binding assay showed increased luciferase activity indicating functional activation of NICD2. In contract to Notch1 and 3 which are tumor suppressive, NICD2 transfected cells did not reduce proliferation comparing to the cells transfected with control plasmid. Moreover, Western blot analysis showed an increase of anti- apoptotic markers XIAP, C-Myc, and Cyclin D1 with the overexpression of NICD2.

Conclusion: Carcinoid tumor cells have paucity Notch2.  For the first time, we demonstrate the growth – promoting function of Notch2 receptor in carcinoid cancer with concomitant upturn of anti-apoptotic markers XIAP, c-Myc and cyclin D1.  This opposing role of Notch2 isoform in NE cancer progression warrant further investigation.

 

1.10 Cdk5/p25 Regulates Notch1 and Notch2 Intracellular Domains in Medullary Thyroid Carcinoma

Posted on December 22, 2014

S. K. Odorico1, X. Yu1, A. Dammalapati1, A. Harrison1, A. Hundal1, J. A. Bibb2, H. Chen1  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Texas Southwestern Medical Center,Department Of Psychiatry,Dallas, TX, USA

Introduction:  Medullary thyroid cancer (MTC) is a neuroendocrine carcinoma that arises from C cells.  Recently, it has been demonstrated that Cdk5 and its cofactor p25 promote C cell proliferation, leading to the development of MTC, while repressing p25 overexpression causes C cell growth arrest.  In our previous studies, we have shown that activation of Notch intracellular domain inhibits MTC cell proliferation and alters the neuroendocrine phenotype.  However, little is known about the possible interaction between Cdk5/p25 and pan Notch signaling.  Therefore, the purpose of this study was to investigate the role of Notch isoforms including Notch1 and 2 in the p25–mediated inhibition of proliferation in MTC cells.

Methods:  A murine thyroid cancer cell line MTCp25, which retains TetOp promoter-controlled p25-GFP expression, was used in our study.  The exogenous p25-GFP is stably overexpressed under basal conditions and repressed by treatment with doxycycline.  We treated MTCp25 cells with 3 different concentrations of doxycycline (1, 2 and 5 μg/mL) for 2, 4 and 6 days.  Notch1, Notch 2, p25, GFP and beta actin expression were then evaluated by Western blot.  We also quantified the mRNA levels of Notch isoforms during different time points using quantitative real-time polymerase chain reaction (PCR).

Results: Both p25 and GFP expression were consistently reduced during different time points in MTCp25 cells culturing in the presence of doxycycline.  No significant changes were detected by Western blot for the intracellular domain of Notch 1 or Notch2 after 2-day treatment of doxycycline.  Interestingly, protein expression of Notch1 and Notch2 intracellular domains profoundly increased in a dose dependent manner after repressing p25 for 4 and 6 days.  Furthermore, we found that the mRNA levels of Notch 1 and Notch 2 did not change significantly with p25 suppression, suggesting that p25 may be involved in the regulation of protein cleavage or degradation of Notch1 and Notch2.

Conclusion: Cdk5/p25 signaling up-regulates the protein level of Notch1 and Notch2 intracellular domains.  This intriguing finding could suggest the function of p25 involved in the post-translational processing of Notch1 and Notch2 receptor.  These results call for further investigation into the mechanism on the crosstalk between Notch and Cdk5 signaling pathways, which may play an important role in medullary thyroid cancer development and growth.

 

1.11 Association of Rosacea and Angiosarcoma/Lymphangiosarcoma (AS/LAS)

Posted on December 22, 2014

S. P. Olsen1, M. C. Perez2, A. M. Priddy1, E. S. Armbrecht1, A. K. Behera1, S. W. Fosko1, N. G. Zeitouni3, D. E. Winstead4, F. E. Johnson1  1Saint Louis University School Of Medicine,St. Louis, MO, USA 2Moffitt Cancer Center And Research Institute,Surgery,Tampa, FL, USA 3Roswell Park Cancer Institute,Dermatology,Buffalo, NY, USA 4Sarcoma Foundation Of America,Burlington, NC, USA

Introduction: Rosacea-affected tissue has increased angiogenesis/lymphangiogenesis.  Isolated case reports have intimated that rosacea, and particularly rhinophyma (a late manifestation of rosacea), might cause angiosarcoma (AS) or lymphangiosarcoma (LAS) in rosacea-affected tissue.  As in Stewart-Treves syndrome, AS/LAS feature prominent varicose veins, lymphatic obstruction, edema, and other features of chronic inflammation.  No reports aimed at quantitatively establishing a causal relationship have been published, as far as we are aware.  Recent evidence indicates that an aberrant innate immune response, mediated by altered endogenous polypeptides known as cathelicidins, is a primary pathologic event in rosacea.  They are pro-inflammatory and vasoactive agents that cause the excessive inflammation in rosacea.  Thus it is plausible that rosacea may be a risk factor for AS/LAS.  We carried out an observational study to confirm or refute this possibility.

Methods: IRB approval was obtained.  We developed a simple one-page data sheet to abstract data from pathology reports of patients with head and neck sarcomas of all sub-types.  We excluded Kaposi sarcoma, Ewing sarcoma, carcinoma, and sarcoma not in the head and neck region.  There were entries for other possible causes of sarcoma (prior radiation exposure, genetic predisposition, etc.).  We obtained data from several large cancer centers and calculated the crude odds ratio in this classic retrospective case-control study.

Results: Data were analyzed in “waves” corresponding to the data sources.  Wave 1 had 228 cases from 3 centers, of which 198 met inclusion criteria.  The odds ratio for the association between rosacea and AS/LAS was 11.9 (95% CI: 1.05, 136.0).  This is statistically significant.  The confidence interval is wide.  Wave 2 included 53 cases from one cancer center, of which all met the inclusion criteria.  The odds ratio for the association between rosacea and AS/LAS was 0.56 (95% CI: 0.08, 3.72).  This is not statistically significant.  Combining wave 1 and wave 2, we had 251 evaluable cases.  The odds ratio for the association between rosacea and AS/LAS was 4.76 (95% CI:  1.11, 20.50).  This is statistically significant but the confidence interval is wide.

Conclusion: This is the first quantitative analysis designed to measure the association between rosacea and head and neck angiosarcoma/lymphangiosarcoma.  A statistically significant association was observed but the confidence interval surrounding the odds ratio is wide.  We seek access to other large data sets for additional analyses.

1.14 Targeting Sirtuins Blocks Neuroblastoma Cell Proliferation And Induces Differentiation

Posted on December 22, 2014

E. J. Rellinger1, H. Song2, S. Park2, P. Paul1, B. T. Craig1, J. Qiao1, V. Athanasios2, D. R. Gius2, D. H. Chung1  1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA 2Northwestern University,Radiation Oncology,Chicago, IL, USA

Introduction:  Neuroblastoma (NB) is a pediatric solid tumor that arises from failed differentiation of neural crest progenitors. Infants and children with high-risk NB have a poor prognosis despite our most aggressive therapies, highlighting the need for novel drug targets. Sirtuins are a family of seven NAD+-dependent histone deacetylases that have emerged as key regulators of cell fate, DNA damage repair, neuronal protection, and tumorigenesis. However, the role of Sirtuins in NB tumorigenicity is unknown. The purpose of this study was to evaluate the exact role of Sirtuins in regulating NB cell proliferation and differentiation. 

Methods:  Sirtuin inhibition in BE(2)-C human NB cells was performed using nicotinamide (NAM), a nonspecific Sirtuin inhibitor, or shRNA transfection (shSIRT1, shSIRT2, shSIRT3, shSIRT6, shSIRT7). Cell viability was determined using CCK-8 assays. Confocal microscopy and immunoblotting with neuron specific enolase (NSE) and neurofilament-M (NF-M) were employed to assess for neural differentiation. Rescue experiments in shSirt6 cells were completed with pCDH-SIRT6 transfection. Sirt6 expression in human tumor sections was assessed using immunohistochemistry and counterstained with H&E to score histologic grade. Cell cycle analyses were completed using flow cytometry. Anchorage-independent soft agar colony growth was used for in vitro representation of tumor invasiveness.

Results: NAM treatment decreased cell viability, induced morphologic differentiation, and increased protein expression of p21 (CDK inhibitor) and NSE, our respective markers of cell cycle arrest and neural differentiation. Only transfection with shSirt6 similarly decreased proliferation and induced cell morphology and neural marker expression patterns consistent with neural differentiation in BE(2)-C cells (Fig. A). Silencing Sirt6 induced NB cell arrest at G0/G1 cell cycle phase and inhibited DNA synthesis and soft agar colony formation. Rescue of cell proliferation, morphologic differentiation, cell cycle arrest, and protein expression was achieved using pCDH-SIRT6 transfection (Fig. B). Sirt6 expression was decreased in differentiated human NB sections. Retinoic acid treatment diminished Sirt6 expression and induced differentiation synergistically with Sirt6 silencing. 

Conclusion: Targeted inhibition of Sirt6 in NB downregulates tumor growth and promotes neural differentiation synergistically with retinoic acid treatment. These findings suggest that Sirt6 is a novel therapeutic target in NB and highlights clinical strategies to optimize its efficacy.

 

1.19 ALDH marks a population of canine cancer stem cells which are preferentially targeted by dog NK cells

Posted on December 22, 2014

R. J. Canter5,6, E. Ames6, S. Mac6, S. Grossenbacher6, M. Kent3, W. Culp3, M. Chen4, W. J. Murphy6  3UC Davis School Of Veterinary Medicine,Surgical And Radiological Sciences,Davis, CA, USA 4University Of California – Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA 5University Of California – Davis,Surgery/Surgical Oncology,Sacramento, CA, USA 6University Of California – Davis,Laboratory Of Cancer Immunology,Sacramento, CA, USA

Introduction: Aldehyde dehydrogenase (ALDH) is a common cancer stem cell (CSC) marker in diverse solid human tumors. We hypothesized that ALDHbright cells would demonstrate the CSC phenotype in dog soft tissue sarcomas (STS) and that these dog CSCs could be preferentially targeted by dog NK cells.  

Methods: ALDHbright cell populations from canine tumor lines and fresh canine primary STS were evaluated for long term colony outgrowth and their ability to form tumors in NOD-SCID IL2 receptor gamma chain null (NSG). STS tissue was obtained from primary dog STS samples and canine patient-derived xenografts (PDX) and evaluated by immunohistochemy (IHC) and flow cytometry for CSC markers including CD24, CD44, and ALDH.  Stained slides were reviewed by a blinded pathologist and scored for percentage and intensity of ALDH-positive cells. Flow cytometry was performed using a BD Fortessa cell sorter (BD Biosciences), and cell viability was analyzed using 7-Aminoactinomycin (7-AAD). Dog NK cells were isolated from leukocyte filters obtained from healthy donors at the School of Veterinary Medicine. NK cytotoxicity was assessed by chromium release and flow cytometry. Parametric and non-parametric statistical tests were performed as appropriate.
 

Results: ALDHbright canine tumor cells displayed properties of CSCs, including selective tumor formation in NSG mice after cell sorting into ALDHbright and ALDHdim populations and long term colony outgrowth in methylcellulose. Using positive selection with magnetic beads, we observed that canine NK cells are responsive to human cytokines, including IL-2, IL-12, and IL-18 with a 3 – 10-fold expansion in NK cells over 14 days. Ex vivo activated dog NK cells demonstrated 35 – 45% cytotoxicity and 57 – 62% cytotoxicity against dissociated dog STS tumors at effector-to-target ratios of 10:1 and 20:1, respectively. IHC staining of dog PDX specimens showed a marked reduction in ALDH score (P<0.05) after intra-tumoral injection of allogeneic dog NK cells compared to controls.

Conclusion: ALDHbright cells exhibit CSC properties in dog STS, and dog NK cells appear to possess an intrinsic ability to recognize and target them. Dog STS appear to be a valuable model to facilitate clinical translation of NK immunotherapy and targeting of CSCs.

1.20 Gene silencing of SphK1 with nanoparticles as an innovative approach against cancer progression

Posted on December 22, 2014

I. Woelfel1, K. P. Terracina3, S. Lima5, C. Oyeniran5, J. Newton5, H. Aoki3, D. Avni5, P. Mukhopadhyay3, N. Hait5, A. Raza3, X. Wu4, H. Yamamoto4, S. Spiegel5, K. Takabe2,3,5  1Virginia Commonwealth University,School Of Medicine,Richmond, VA, USA 2VCU Massey Cancer Center,Richmond, VA, USA 3Virginia Commonwealth University,Department Of Surgery,Richmond, VA, USA 4Osaka University,Suita, Osaka, Japan 5Virginia Commonwealth University,Department Of Biochemistry And Molecular Biology,Richmond, VA, USA

Introduction:  

Gene therapy as an effective treatment modality for cancer has been sought for decades. Its full therapeutic potential has not been realized due to many barriers, including efficiency and cost. Small interfering RNA (siRNA) has emerged as a successful technology for specifically silencing gene expression in vitro. However, due to the small size and delicate nature of these molecules, an effective delivery system that allows these molecules to reach cancer and thus be applicable in patient treatment is necessary. Recent innovations in nanoparticle technology have enabled the development of a new delivery system: super carbonate apatite (sCA). sCA system is extremely inexpensive, and has been reported to deliver genes specifically to cancer due to the characteristic size and leakiness of peritumoral vessels. Sphingosine-1-phosphate, generated by Sphingosine kinase 1 (SphK1), has been established as a key lipid signaling molecule in cancer progression and is integral to cell survival, proliferation, migration, angiogenesis, and lymphangiogenesis. We investigated the ability of this new nanoparticle delivery system (sCA) with SphK1 siRNA to effectively knockdown SphK1 and suppress its physiological functions in vitro.

Methods:

A carbonate apatite inorganic nanoparticle delivery solution was created using CaCl2 and NaCO3 ions. 4T1 murine breast cancer cells were treated with 2 ug/mL of SphK1 siRNA, Non-targeting siRNA or a control containing no siRNA using the inorganic solution with a 4-hour serum free incubation time. At 4 hours 1 mL of 10% FBS media was added. 24 hours after the initial application of inorganic solution the old media was removed and 2 mL of DMEM with 10% FBS was added.  mRNA was harvested at the 48 hour time for qPCR. Cell survival was measured using WST-8 assay. To study the effects of silencing on cell migration, a point scratch assay was conducted using established technique with images collected at 0, 15 and 24 hours. Analysis of the images was conducted using Image J. 

Results:

We successfully introduced SphK1 siRNA into the 4T1 cells using sCA system, demonstrated through qPCR with a statistically significant reduction of SphK1 message production (67%, p value 0.008). We found that sCA transfection of SphK1 siRNA significantly decreased 4T1 cell survival compared to vector treated cells. We also observed faster closure of scratch area in cells treated with a non-targeting vector, as opposed to cells treated with SphK1 siRNA. 

Conclusion:

We have shown that our new inorganic nanoparticle delivery system, sCA, was successful in gene silencing of SphK1 in vitro and suppressed 4T1 cell proliferation and migration. Considering the advantage of sCA in gene delivery to cancer, we have high expectations regarding the application of this approach in vivo, which has the potential to transform the treatment frontier and to positively impact patient outcomes in the future. 

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