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Supplementary Figure Legends 1-9

Supplemental Fig. 6. Effect of over-expression of AKT on [pIC]PEI-induced apoptosis

Supplementary Figures 1-9. Sabutoclax has greater efficacy than ABT-737. (S1) Sabutoclax causes a G1-S phase cell cycle arrest. (S2) Pancreatic cancer cells exhibit varying levels of sensitivity to Minocycline. (S3) Sabutoclax and Minocycline induce cytotoxicity in PANC-1 cells that is reversed with zVAD. (S4) Sabutoclax and Minocycline show synergy. Combination index (CI) values for the combination of Sabutoclax and Minocycline in MIA PaCa-2 cells. (S5) The cytotoxicity induced by Sabutoclax and Minocycline is caspase-dependent and dependent upon loss of Stat3 activation. (S6) Sabutoclax and Minocycline reduce tumor growth in a subcutaneous xenograft model of pancreatic cancer. (S7) Tumors from Pdx-1-Cre/K-rasLSL-G12D/p53flox/wt and Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox mice overexpress Mcl-1 and show sensitivity to Sabutoclax and Minocycline. (S8) Tumors from KPC (Pdx-1-Cre/K-rasLSL-G12D/p53flox/flox )mice treated with Sabutoclax and Minocycline show decreased Stat3 activation. (S9)

Supplemental Materials. Supplemental methods and figure legend

Supplemental Fig. 3. [pIC]PEI does not change XIAP and survivin at the mRNA level

Supplemental Fig. 1. [pIC]PEI treatment increases the Sub G1 population in pancreatic cancer cells

Supplemental Fig. 8. In vivo cytoplasmic delivery of [pIC] using invivo jetPEI induces apoptosis in pancreatic tumors

Supplemental Fig. 2. Inhibition of autophagy by 3-MA or caspase-mediated-apoptosis by z-VAD rescues pancreatic cancer cells from [pIC]PEI-induced cell death

Supplemental Fig. 4. Inhibition of XIAP degradation does not rescue pancreatic cancer cells from [pIC]PEI-induced apoptosis

Supplemental Fig. 7. [pIC]PEI treatment inhibits mTOR and PKC-É› activation in pancreatic cancer cells

Supplemental Fig. 5. Effect of expression of XIAP alone, survivin alone and the combination on [pIC]PEI-induced apoptosis

Polyinosine–polycytidylic acid [pIC] is a synthetic dsRNA that acts as an immune agonist of TLR3 and RLR to activate dendritic and natural killer cells that can kill tumor cells. pIC can also trigger apoptosis in pancreatic ductal adenocarcinoma cells (PDAC) but its mechanism of action is obscure. In this study, we investigated the potential therapeutic activity of a formulation of pIC with polyethylenimine ([pIC]PEI) in PDAC and investigated its mechanism of action. [pIC]PEI stimulated apoptosis in PDAC cells without affecting normal pancreatic epithelial cells. Mechanistically, [pIC]PEI repressed XIAP and survivin expression and activated an immune response by inducing MDA-5, RIG-I, and NOXA. Phosphorylation of AKT was inhibited by [pIC]PEI in PDAC, and this event was critical for stimulating apoptosis through XIAP and survivin degradation. In vivo administration of [pIC]PEI inhibited tumor growth via AKT-mediated XIAP degradation in both subcutaneous and quasi-orthotopic models of PDAC. Taken together, these results offer a preclinical proof-of-concept for the evaluation of [pIC]PEI as an immunochemotherapy to treat pancreatic cancer. Cancer Res; 74(21); 6224–35. ©2014 AACR.

OBJECTIVE Hallux sesamoid injuries are well described and can be debilitating and chronically disabling. The role of orthobiologics such as platelet-rich plasma (PRP) in sesamoid injuries has not been reported. This study describes three cases of recalcitrant hallux sesamoid injuries in teenage athletes who returned to impact activities, pain free, following one treatment of PRP. METHODS This is a case-series study describing three teenage athletes presenting to a tertiary level pediatric sports medicine practice with chronic hallux sesamoid injuries. RESULTS The three patients (two female, one male) described in this case series were 13-, 16-, and 17-year-old athletes. Their primary sports were ballet, basketball, and Irish step dance, respectively. All three athletes received PRP: two received unilateral treatment (one tibial sesamoid, one fibular sesamoid) and one received treatment to bilateral tibial sesamoids. The average duration of symptoms prior to PRP was 52.5 weeks (14-128 weeks). The average time out of their primary sport was 48.7 weeks (20-78 weeks). Three of the 4 sesamoids treated with PRP were tibial sesamoids. Each site of injury was treated with one treatment of leukocyte-rich PRP. All three athletes were cleared to return to impact activities such as running and jumping at 6-9 weeks following PRP, specifically 9 weeks after the final PRP injection for the patient who underwent bilateral treatments. CONCLUSION In the three cases provided of sesamoid injuries treated with PRP, the time to return to impact activities was less than reported for athletes not treated with PRP. Acknowledging that other management factors likely contributed to return to impact activities, this case series sets the groundwork for future research investigating the role of PRP with needle fenestration in the treatment of sesamoid injuries.

We live in a time of accelerated environmental change. Links between ecosystem health and human health and well-being are increasingly recognized with ever more awareness of impending environmental...

Objective To investigate CrossFit-related injuries presenting to a pediatric sports medicine clinic. Design Retrospective review of pediatric CrossFit-related injuries from between January 1, 2003, and June 31, 2016. Setting Pediatric sports medicine clinic at a tertiary-level academic medical center. Patients Patients with injury related to CrossFit participation. Independent variables Sex, age, injury site, diagnosis, diagnostic imaging, and treatment. Main outcome measures Annual CrossFit-related injury proportion (%) over time. Results One hundred fifteen medical identified (N = 55 female; mean age, 25.2 ± 10.4 years). Proportion of CrossFit-related injuries presenting to clinic relative to overall clinic volume consistently increased over time (Pearson r = 0.825; P = 0.022). Injury location included head (0.08%), trunk/spine (25.2%), upper extremity (27.0%), and lower extremity (47.0%). Common injured joints included knee (27%), spine (24.3%), and shoulder (16.5%). Nearly half of patients had a single diagnostic imaging (49.6%; 57 of 115). Most common diagnostics included magnetic resonance imaging (60.0%; 69 of 115), plain radiographs (51.3%; 59 of 115), ultrasound (10.4%; 12 of 115), and computerized tomographic scan (9.6%; 11 of 115). Most commonly prescribed treatments included physical/occupational therapy (38.3%; 44 of 115), activity modification (19.1%; 22 of 115), crutches/brace/splinting/compression sleeve (13.0%; 15 of 115), and non-steroidal anti-inflammatory medications (10.4%; 12 of 115). Conclusions CrossFit-related injury proportion presenting to a pediatric sports medicine clinic increased over time. A notable proportion of injuries occurred to the trunk and spine. Advanced imaging was obtained in approximately half of these youth athletes. Further research in youth CrossFit athletes is required surrounding mechanism of injury to prevent future injury in this mode of training for youth athletes.

Purpose The purpose of the study was to investigate the impact on dose distribution and radiobiological metrics of common high-dose-rate vaginal brachytherapy treatment parameters and to analyze multiinstitutional data for clinically significant impact on outcomes in early-stage endometrial cancer. Methods and Materials Treatment plans were created for all combinations of prescription parameters and used to quantify the dosimetric impact of each parameter and to estimate the dose delivered using common voxel-integrated radiobiological metrics. A rating system, based on risk grouping from GOG and PORTEC trials, was used to consolidate staging information into a cancer “aggressiveness” measure. Correlations between the rating, toxicity, disease recurrence, and plan parameters were investigated. Results When prescribing to 5 mm depth, the variation caused by the diameter was very large across all dose metrics, ranging from 51% to 175% increase with the most divergence in BEDmax. For surface prescription, changing the cylinder diameter from 4 cm to 2 cm caused the dose metrics of BEDmin, Dmin, and gBEUD (a = −3) to increase by 117%, 67%, and 52%, respectively. Prescription to 5-mm depth caused changes across all dose metrics of 260% compared with surface prescription for a 2-cm cylinder. Deeper prescription point (p = 0.005) and longer treatment length (p = 0.01) were correlated with increased stenosis rates. No correlation between recurrence and any plan parameter was found. Conclusions Dramatic differences in dose distributions arise by small variations of plan parameters, with large impact on rates of vaginal stenosis, but no clear relation with local recurrence. To help radiation oncologists interpret the magnitude of these effects for their patients, we created a tool that allows comparison between dose and fractionation parameters.

BACKGROUND Adolescent ballet dancers have a higher incidence of injury than adult professional dancers. It is unclear which factors, including biomechanical changes related to intense dance training and/or the growth process itself, contribute to increased injury risk in this population. OBJECTIVE To assess changes in strength, flexibility, and functional performance in adolescent ballet dancers during a summer intensive dance program. DESIGN Prospective cohort study. SETTING Performing arts school in a suburb of Massachusetts. PARTICIPANTS A convenience sample of 58 male and female ballet dancers, 12 to 17 years old, was enrolled. One dancer did not have functional testing due to injury, but strength and range of motion results were included. METHODS Paired sample t-test was used to evaluate changes in (1) strength: lower abdominal muscle strength evaluated by the Kendall double leg lowering test; (2) flexibility: passive range of motion in hip extensibility, hip internal rotation, hip external rotation, hip turnout, and ankle dorsiflexion; and (3) functional athletic and dance assessment: the star excursion balance test (SEBT), vertical jump test, and dance technique performing demi-plie and passe dance positions. MAIN OUTCOME MEASUREMENTS Change in strength, flexibility, and functional dance measurements pre- and postsummer intensive dance training. RESULTS Lower abdominal strength testing improved 11° on the Kendall test (P

Objective To investigate clinical diagnoses, sports participation, and return to sport timeline associated with hallux sesamoid injuries with sex comparisons. Design Descriptive epidemiology study. Setting Sports medicine clinics at a tertiary-level pediatric medical center. Patients Six hundred eighty-three young athletes (546 women and 137 men). Independent variables Sex (women vs men). Main outcome measures Clinical diagnoses, participating sports, and injury timeline. Results The most common diagnosis was sesamoiditis (62.6%). The top 3 primary sports were dance (34.6%), running (13.7%), and soccer (11.7%). When stratified by sex, dance (40.1%), running (13.6%), and soccer (10.7%) were the top primary sports for women while running (19.4%), soccer (18.5%), and basketball (11.3%) were the leading diagnoses for male athletes. The mean time between injury occurrence and first clinic visit was 135.5 ± 229.3 days. The mean time between the first clinic visit and return to sport was 104.3 ± 128.2 days. Comparison by sex showed that women had a longer mean time than men (women: 111.5 ± 132.5 days, men: 67.2 ± 96.3 days, P = 0.001). The mean time from injury occurrence to return to sport was 235.2 ± 281.0 days. Women showed a longer mean timeline for return to sport compared with men (women: 245.2 ± 288.2 days, men: 179.3 ± 231.9 days, P = 0.014). Conclusion Sesamoiditis was the most common diagnosis, and dance, running, and soccer were top 3 sports. The most salient finding was that women taking almost twice as long to return the sport or activity compared with men, which likely stems from delay of reporting symptom onset to clinics.

Background Femoral neck stress fractures are rare in healthy children, with only 9 cases previously reported. The present article reviews our institutional experience with femoral neck stress fractures in children younger than 10 years of age, to highlight the unique features of this condition. Methods We undertook a retrospective review of clinical records of patients who had been treated at our institution for an idiopathic femoral neck stress fracture between 2000 and 2014. To focus on children rather than adolescents, the World Health Organization's definition of adolescent as a person between 10 and 19 years of age was used; we thereby limited our analysis to patients younger than 10 years of age. Results The study included 6 patients (3 males, 3 females) treated for an idiopathic femoral neck stress fracture, with a mean age at diagnosis of 7.7 years (range, 5.2 to 8.9 y). All patients presented with a limp, which worsened with activity and had persisted for a mean of 5 weeks (range, 2 to 9 wk). None of the patients had experienced an increase in activity level or sporting volume before symptom onset. On examination, 3 patients experienced pain with terminal hip flexion and 3 patients demonstrated pain-free hip range of motion. Plain radiography demonstrated inferior femoral neck cortical disruption, suggesting a compression-type stress fracture mechanism. The diagnosis was confirmed by cross-sectional imaging in all cases. All patients were initially treated with 6 to 8 weeks of non-weight-bearing followed by 4 to 6 weeks of partial weight-bearing, leading to complete healing in 4 patients. Two patients demonstrated incomplete healing and were managed with spica casting for an additional 6 weeks. Conclusions Our case series illustrates the unique features of this rare condition in children, with a history and examination profile distinct from those of adolescents and adults. Compliance with weight-bearing restrictions is difficult in this population and hip spica casting may be required to permit complete healing. Level of evidence Level IV-case series.

Objective To examine CrossFit-related injuries based on sex and age. Design Retrospective case series. Setting A tertiary-level pediatric sports medicine clinic. Participants CrossFit athletes. Main outcome measures CrossFit-related injuries by sex (males vs females) and age groups (≤19 years vs >19 years) using a χ analysis with P = 0.05, odds ratio (OR), and 95% confidence interval (95% CI). Results Among injured CrossFit athletes, female athletes sustained lower extremity injuries more frequently than male athletes (P = 0.011; OR, 2.65; 95% CI, 1.25-5.65). In observed CrossFit injuries, shoulder injuries were more frequently observed in male athletes compared with female athletes (P = 0.049; OR, 2.79; 95% CI, 0.98-7.95). Additionally, a greater proportion of CrossFit athletes aged 19 years and younger suffered trunk/spine injuries than those older than 19 years (P = 0.027; OR, 2.61; 95% CI, 1.10-6.21) in injured CrossFit athletes. Conclusions The current results indicated sex- and age-specific susceptibility to CrossFit-related injuries based on body parts and diagnoses. The presented information may be useful to develop a safer exercise program, especially for pediatric and adolescent CrossFit participants.

Background Sesamoid injuries of the first metatarsal phalangeal joint in athletes occur with sports that place repetitive stress on the plantar aspect of the great toe. Performing artist athletes are particularly at risk for injury given the load placed on the hallucal sesamoid bone often inherent in the activity. Risk factors may include choice of sport, volume of training, sex, bone density, BMI and biomechanical profile of the lower extremity. Hallucal sesamoid evaluation and treatment remains poorly defined in the literature. The aim of this study is to analyze all sesamoid injuries presenting to a sports medicine clinic. The goal of the study is to increase understanding of the injury profile, diagnostic evaluation, treatment regime, and return to sport of athletes with hallucal sesamoid injuries. The long-term goal is to develop evaluation and treatment algorithms that serve to guide clinical decision-making, and improve time to return to sport. Methods A comprehensive retrospective chart review was conducted of athletes presenting to a tertiary level sports medicine clinic located within a pediatric medical center. Electronic medical records were searched using the search term sesamoid. To be included in the study, the injury had to definitively involve the hallucal sesamoid and be related to sports participation. Exclusion criteria included patients with a chronic disease or condition that might affect bone healing or confuse the diagnosis of sesamoid injury, prior history of surgery to the foot, and insufficient management records. Descriptive statistics were used to analyze outcome variables including specific diagnosis, clinical prognoses, diagnostic imaging tools and treatment types. Additionally, a correlation analysis was performed for time from pain onset to first clinic visit, and time to return to participation. Little or no correlation was considered 0.00-0.25, weak correlation was considered 0.25-0.50, moderate correlation was considered 0.50-0.75 and strong correlation was considered 0.75 -1.00. Results 326 athletes with 359 hallucal sesamoid injuries were identified. The mean age of the cohort was 15.8 ± 3.8 years (median: 15.3, 95% CIs: 15.46 – 16.24); 86% (n=309) were female and 14% (n=50) of the injuries were male. The mean BMI of the cohort was 21.28 ± 3.5 mg/kg2. Table 1 presents the sports for the athletes in the cohort. The leading sports included 40% (n=144) dance, 13% (n=48) running, and 13% (n=47) soccer. Activities that top the list for females include dance 44% (n=137) and running 13% (n=39). In comparison, male athletes participated in soccer (20%, n=10), running (18%, n=9), and football (10%, n=5) as well as other diverse sports. The most common injuries across both sexes were sesamoiditis (30%, n=107), followed by sesamoid stress fracture (13%, n=46). Table 2 Where self-reported data on dance/sport practice time was recorded, 31% (n=65) reported practicing 10-15 hours per week. Figure 1 The average reported time between injury or the onset of pain to the first clinic visit was 143 days (median: 42, 95% CIs:116.87-169.15). The mean time between pain onset and first clinic visit was greater for female athletes as compared to male athletes (146 days and 119 days). The average time from first presentation to clinic to returning to participation was 115 days (median: 72, CIs:100.7-129.49). Spearman’s rho demonstrated a strong correlation between time from pain onset to first clinic visit and the time to return to participation in both males (? (rho) = 0.82, p < 0.001) and females (? = 0.79, p < 0.001). Males experienced a shorter duration from the first clinic visit to return to participation (mean: 72 days, median 33), than females (mean: 121 days, median 77). The most common diagnostic imaging modalities used were radiographs (72.14%, n=259) and MRI (56.55%, n=203). In both males and females the most common initial treatments included a combination of: walking boot (51.53%, n= 185), physical therapy (38.72%, n=139), and activity modification (34.82%, n=125). These remained the most popularly prescribed treatments in the second and third treatments as well. Conclusions/significance Female athletes participating in dance and running, and male soccer, running and football athletes lead the list for injury to the hallucal sesamoid. Sesamoiditis and sesamoid stress fracture were the leading diagnoses in this cohort. Athletes who presented to clinical attention sooner also returned to sport/dance sooner when compared to athletes who delayed seeking medical attention. Continued research will serve to support anticipatory guidance and education surrounding hallucal sesamoid clinical presentation and need for timely evaluation and treatment in order to minimize time loss from sport/performing artist activity. [Table: see text][Table: see text][Figure: see text]

First line treatment for pancreatic cancer consists of surgical resection, if possible, and a subsequent course of chemotherapy using the nucleoside analogue gemcitabine. In some patients, an active transport mechanism allows gemcitabine to enter efficiently into the tumor cells, resulting in a significant clinical benefit. However, in most patients, low expression of gemcitabine transporters limits the efficacy of the drug to marginal levels, and patients need frequent administration of the drug at high doses, significantly increasing systemic drug toxicity. In this article we focus on a novel targeted delivery approach for gemcitabine consisting of conjugating the drug with an EphA2 targeting agent. We show that the EphA2 receptor is highly expressed in pancreatic cancers, and accordingly, the drug-conjugate is more effective than gemcitabine alone in targeting pancreatic tumors. Our preliminary observations suggest that this approach may provide a general benefit to pancreatic cancer patients and offers a comprehensive strategy for enhancing delivery of diverse therapeutic agents to a wide range of cancers overexpressing EphA2, thereby potentially reducing toxicity while enhancing therapeutic efficacy.

Background: Physical rest after a concussion has been described as a key component in the management of the injury. Evidence supporting this recommendation, however, is limited. Purpose: To examine the association between physical activity and symptom duration in a cohort of patients after a concussion. Study Design: Cohort study; Level of evidence, 2. Methods: This study included 364 patients who were diagnosed with a concussion, were seen by a physician within 3 weeks of injury, and completed a questionnaire at the initial clinic visit. The questionnaire assessed the postconcussion symptom scale (PCSS) score, previous number of concussions, presence of the loss of consciousness or amnesia at the time of injury, and prior treatment for headaches. During each follow-up clinic visit, physical activity level was self-reported. A Cox proportional hazard model was constructed to determine the association between symptom duration, initial clinic visit responses, and self-reported physical activity level after the injury. Results: Study participants ranged in age from 8 to 27 years (mean age, 15.0 years) and had sustained a mean of 0.8 prior concussions; 222 patients (61%) were male. On initial examination, the mean PCSS score was 34.7. The mean symptom duration was 48.9 days after the injury. Among the variables included in the model, initial PCSS score and female sex were independently associated with symptom duration, while physical activity level after the injury was not. For participants aged between 13 and 18 years, however, higher levels of physical activity after the injury were associated with a shorter symptom duration. Conclusion: Results from this study indicate that physical activity after the injury may not be universally detrimental to the recovery of concussion symptoms.

A 26-year-old female professional dancer sustained an acute injury to her mid-foot during a performance of The Nutcracker. An intra-articular, comminuted, minimally displaced fracture of the cuboid was found. The patient was treated non-operatively with cast and boot immobilization, modified weightbearing, and progressive rehabilitation. She was able to return to professional dance at 6 months post-injury and continues to dance professionally over 1 year out from injury without issue. The unique demands of classical ballet, especially dancing en pointe, increase the risk for mid-foot fractures, and clinicians should have a high-index of suspicion in dancers suffering an acute injury to the foot and ankle with greater than expected pain or swelling. Multiple imaging modalities can be used to make the diagnosis, to include plain film radiographs, MRI, and CT scan. Fracture characteristics and patient-specific factors should be taken into account when deciding on a treatment plan.

SummaryThe development of novel, targeted delivery agents for anti-cancer therapies requires the design and optimization of potent and selective tumor-targeting agents that are stable and amenable to conjugation with chemotherapeutic drugs. While short peptides represent potentially an excellent platform for these purposes, they often get degraded and are eliminated too rapidly in vivo. In this study, we used a combination of nuclear magnetic resonance-guided structure-activity relationships along with biochemical and cellular studies to derive a novel tumor-homing agent, named 123B9, targeting the EphA2 tyrosine kinase receptor ligand-binding domain. Conjugating 123B9 to the chemotherapeutic drug paclitaxel (PTX) via a stable linker results in an agent that is significantly more effective than the unconjugated drug in both a pancreatic cancer xenograft model and a melanoma lung colonization and metastases model. Hence, 123B9 could represent a promising strategy for the development of novel targeted therapies for cancer.

Oral and oropharyngeal cancers are the sixth most common cancers worldwide. Despite intensive investigation, oral squamous cell carcinomas (OSCC) represent a clinical challenge resulting in significant morbidity and mortality. Resistance to cell death is common in OSCC and is often mediated by the Bcl-2 family proteins. Among all anti-apoptotic Bcl-2 family members, Mcl-1 functions as a major survival factor, particularly in solid cancers. Despite the confirmed importance of Mcl-1 in several neoplasms, the role of Mcl-1 in OSCC survival has yet to be explored. In this study, we found that knocking down Mcl-1 sensitized OSCC cells to ABT-737, which binds to Bcl-2/Bcl-xL but not Mcl-1. We report for the first time that a BH3 mimetic, Sabutoclax, which functions as an inhibitor of all anti-apoptotic Bcl-2 proteins, induced cancer-specific cell death in an Mcl-1-dependent manner through both apoptosis and toxic mitophagy. In vivo studies demonstrated that Sabutoclax alone decreased tumor growth in a carcinogen-induced tongue OSCC mouse model. In a combination regimen, Sabutoclax and COX-2 inhibitor, Celecoxib, synergistically inhibited the growth of OSCC in vitro and also significantly reduced OSCC tumor growth in vivo. Overall, these results identify Mcl-1 as a therapeutic prospective target in OSCC.

// Siddik Sarkar 1 , Bridget A. Quinn 1 , Xue-Ning Shen 1 , Rupesh Dash 2 , Swadesh K. Das 1,3,4 , Luni Emdad 1,3,4 , Alexander L. Klibanov 5 , Xiang-Yang Wang 1,3,4 , Maurizio Pellecchia 6 , Devanand Sarkar 1,3,4 and Paul B. Fisher 1,3,4 1 Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA 2 Institute of Life Sciences, Chandrasekharpur, Bhubaneswar, Orissa, India 3 VCU Institute of Molecular Medicine, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA 4 VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA 5 Division of Cardiovascular Medicine and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA 6 Infectious and Inflammatory Disease Center, Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Correspondence to: Paul B. Fisher, email: // Keywords : BH3 mimetic, cancer terminator virus (CTV, prostate cancer (CaP), truncated CCN1 (tCCN1)-Prom, PEG-Prom Received : January 02, 2015 Accepted : February 16, 2015 Published : March 12, 2015 Abstract Despite recent advances, treatment options for advanced prostate cancer (CaP) remain limited. We are pioneering approaches to treat advanced CaP that employ conditionally replication-competent oncolytic adenoviruses that simultaneously produce a systemically active cancer-specific therapeutic cytokine, mda- 7/IL-24, Cancer Terminator Viruses ( CTV ). A truncated version of the CCN1/CYR61 gene promoter, tCCN1-Prom, was more active than progression elevated gene-3 promoter (PEG-Prom) in regulating transformation-selective transgene expression in CaP and oncogene-transformed rat embryo cells. Accordingly, we developed a new CTV , Ad.tCCN1- CTV-m 7, which displayed dose-dependent killing of CaP without harming normal prostate epithelial cells in vitro with significant anti-cancer activity in vivo in both nude mouse CaP xenograft and transgenic Hi- Myc mice (using ultrasound-targeted microbubble (MB)-destruction, UTMD, with decorated MBs). Resistance to mda- 7/IL-24 - induced cell deathcorrelated with overexpression of Bcl-2 family proteins. Inhibiting Mcl-1 using an enhanced BH3 mimetic, BI-97D6, sensitized CaP cell lines to mda- 7/IL-24 - induced apoptosis. Combining BI-97D6 with Ads expressing mda- 7/IL-24promoted ER stress, decreased anti-apoptotic Mcl-1 expression and enhanced mda -7/IL-24expression through mRNA stabilization selectively in CaP cells. In Hi- myc mice, the combination induced enhanced apoptosis and tumor growth suppression. These studies highlight therapeutic efficacy of combining a BH3 mimetic with a novel CTV , supporting potential clinical applications for treating advanced CaP.

Pancreatic cancer is an aggressive disease with limited therapeutic options. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a potent antitumor cytokine, shows cancer-specific toxicity in a vast array of human cancers, inducing endoplasmic reticulum stress and apoptosis, toxic autophagy, an antitumor immune response, an antiangiogenic effect, and a significant "bystander" anticancer effect that leads to enhanced production of this cytokine through autocrine and paracrine loops. Unfortunately, mda-7/IL-24 application in pancreatic cancer has been restricted because of a "translational block" occurring after Ad.5-mda-7 gene delivery. Our previous research focused on developing approaches to overcome this block and increase the translation of the MDA-7/IL-24 protein, thereby promoting its subsequent toxic effects in pancreatic cancer cells. We demonstrated that inducing reactive oxygen species (ROS) after adenoviral infection of mda-7/IL-24 leads to greater translation into MDA-7/IL-24 protein and results in toxicity in pancreatic cancer cells. In this study we demonstrate that a novel chimeric serotype adenovirus, Ad.5/3-mda-7, displays greater efficacy in delivering mda-7/IL-24 compared with Ad.5-mda-7, although overall translation of the protein still remains low. We additionally show that d-limonene, a dietary monoterpene known to induce ROS, is capable of overcoming the translational block when used in combination with adenoviral gene delivery. This novel combination results in increased polysome association of mda-7/IL-24 mRNA, activation of the preinitiation complex of the translational machinery in pancreatic cancer cells, and culminates in mda-7/IL-24-mediated toxicity.

Objectives: To compare the demographics, metabolic bone health, radiologic features, treatment approaches and recurrence rates of pediatric versus young adult athletes with femoral neck stress fractures. Methods: A retrospective review was performed on all patients Results: Forty-nine patients (mean age 21.4 years, range 5-44, 78% females) met study inclusion criteria, including 28 pediatric patients (mean age 14.4 years, range 5-19 years, 71% females) and 21 young adults (mean age 30.8 years, range 20-44 years, 86% females). A higher percentage of females was seen with each increasing decade of age, with 50% of pediatric patients under 11 years-old being male. Mean BMI was lower (p=0.04) in the pediatric group (20.6 kg/m2 +/-3.42) than the adult group (21.8 kg/m2 +/-2.04). Pain was the presenting complaint in all patients, with pain localized to the groin in 80% of cases. Participation in running sports was higher for the young adult cohort (86%) than the pediatric cohort (50%), while multiple sports were played more by pediatric patients (29%) than young adults (5%). History of previous acute fractures (2%) and previous stress fractures (14%) was identical between groups. Delayed menarche was recorded in 6% of pediatric patients, and menstrual irregularity was reported in 29% and 33% of pediatric and adult females, respectively. The base of the femoral neck was most common location for fracture in both pediatric (67%) and adult (81%) groups, while transcervical fractures were more likely to occur in pediatric (29%) than adult patients (6%). More significant treatment interventions were pursued in the pediatric group (spica casting: n=2, operative screw fixation, n=4) than the adult group, all of whom demonstrated healing with activity modification, with varying degrees of weight bearing protection and medical optimization of metabolic bone health. There was no difference in the mean time to healing (13.3 weeks), or in the mean time to return to sports (Peds: 16wks, Adults: 13wks) between groups. There was a significant correlation between time to RTS and the extent of the femoral neck edema (p=0.048). Conclusion: Pediatric caregivers should be aware of femoral neck stress fractures in young athletes, an entity historically described almost exclusively in adults. Stress fractures in pediatric and adolescent patients are more likely to occur higher on the neck than adult patients, and both sexes in children may be affected to a greater degree than in adult counterparts, in whom females are affected much more commonly. Groin pain and participation in running sports are common in both groups, while multi-sport pediatric athlete patients may be more likely to be affected than in the adult population. More significant treatment interventions may be warranted in children. To avoid the catastrophic sequella of a displaced femoral neck fracture, proactive diagnostic workup and consideration of interventions such as spica casting or surgical screw fixation should be exercised given concerns related to non-compliance in this population.

Questionnaires were distributed to investigate body mass index (BMI) and menstrual patterns in female dancers aged 12 to 17 years. The study cohort consisted of 105 dancers, mean age 14.8 ± 1.1 years, and mean BMI 19.5 ± 2.3 kg/m2. In all, 92% were healthy weight for height. First menses age ranged from 10 to 15 years (mean 12.9 ± 1.1 years). A total of 44% reported irregular menses; of those, 14% described irregularity as “every other month,” 37% as “every 3 months,” and 49% as “skips a month occasionally.” A total of 36% of the dancers stop getting their menses during times of increased activity/dance, and 30% have gone >3 months at any time without getting their menses. A significant negative correlation between BMI and age of first menses was found with lower BMI associated with increased age of first menses (linear regression, β = -0.49, P = .021). This study supports an association between BMI and age of menarche among young female dancers. Given bone health reliance on hormonal milieu in female dancers, future research is warranted.

Structure-based modeling combined with rational drug design, and high throughput screening approaches offer significant potential for identifying and developing lead compounds with therapeutic potential. The present review focuses on these two approaches using explicit examples based on specific derivatives of Gossypol generated through rational design and applications of a cancer-specific-promoter derived from Progression Elevated Gene-3. The Gossypol derivative Sabutoclax (BI-97C1) displays potent anti-tumor activity against a diverse spectrum of human tumors. The model of the docked structure of Gossypol bound to Bcl-XL provided a virtual structure-activity-relationship where appropriate modifications were predicted on a rational basis. These structure-based studies led to the isolation of Sabutoclax, an optically pure isomer of Apogossypol displaying superior efficacy and reduced toxicity. These studies illustrate the power of combining structure-based modeling with rational design to predict appropriate derivatives of lead compounds to be empirically tested and evaluated for bioactivity. Another approach to cancer drug discovery utilizes a cancer-specific promoter as readouts of the transformed state. The promoter region of Progression Elevated Gene-3 is such a promoter with cancer-specific activity. The specificity of this promoter has been exploited as a means of constructing cancer terminator viruses that selectively kill cancer cells and as a systemic imaging modality that specifically visualizes in vivo cancer growth with no background from normal tissues. Screening of small molecule inhibitors that suppress the Progression Elevated Gene-3-promoter may provide relevant lead compounds for cancer therapy that can be combined with further structure-based approaches leading to the development of novel compounds for cancer therapy.

Adenovirus (Ad)-based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie-adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5-based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR-independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an effective wide-spectrum cancer-selective therapeutic. In low CAR human colorectal cancer cells RKO, wild-type Ad.5 virus expressing mda-7/IL-24 (Ad.5-mda-7) failed to infect efficiently resulting in lack of expression of MDA-7/IL-24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda-7/IL-24 (Ad.5/3-mda-7) efficiently infected RKO cells resulting in higher MDA-7/IL-24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl-2 family pharmacological inhibitor Apogossypol derivative BI-97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3-mda-7. A combination regimen of suboptimal doses of Ad.5/3-mda-7 and BI-97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5-mda-7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3-mda-7 alone or in combination with BI-97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients.

Human cancers are genetically and epigenetically heterogeneous and have the capacity to commandeer a variety of cellular processes to aid in their survival, growth and resistance to therapy. One strategy is to overexpress proteins that suppress apoptosis, such as the Bcl-2 family protein Mcl-1. The Mcl-1 protein plays a pivotal role in protecting cells from apoptosis and is overexpressed in a variety of human cancers.Targeting Mcl-1 for extinction in these cancers, using genetic and pharmacological approaches, represents a potentially effectual means of developing new efficacious cancer therapeutics. Here we review the multiple strategies that have been employed in targeting this fundamental protein, as well as the significant potential these targeting agents provide in not only suppressing cancer growth, but also in reversing resistance to conventional cancer treatments.We discuss the potential issues that arise in targeting Mcl-1 and other Bcl-2 anti-apoptotic proteins, as well problems with acquired resistance. The application of combinatorial approaches that involve inhibiting Mcl-1 and manipulation of additional signaling pathways to enhance therapeutic outcomes is also highlighted. The ability to specifically inhibit key genetic/epigenetic elements and biochemical pathways that maintain the tumor state represent a viable approach for developing rationally based, effective cancer therapies.

Limited options are available for treating patients with advanced prostate cancer (PC). Melanoma differentiation associated gene-7/interleukin-24 ( mda -7/IL-24), an IL-10 family cytokine, exhibits pleiotropic anticancer activities without adversely affecting normal cells. We previously demonstrated that suppression of the prosurvival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), is required for mda -7/IL-24–mediated apoptosis of prostate carcinomas. Here we demonstrate that pharmacological inhibition of Mcl-1 expression with the unique Apogossypol derivative BI-97C1, also called Sabutoclax, is sufficient to sensitize prostate tumors to mda -7/IL-24–induced apoptosis, whereas ABT-737, which lacks efficacy in inhibiting Mcl-1, does not sensitize mda -7/IL-24–mediated cytotoxicity. A combination regimen of tropism-modified adenovirus delivered mda -7/IL-24 (Ad.5/3- mda -7) and BI-97C1 enhances cytotoxicity in human PC cells, including those resistant to mda -7/IL-24 or BI-97C1 alone. The combination regimen causes autophagy that facilitates NOXA- and Bim-induced and Bak/Bax-mediated mitochondrial apoptosis. Treatment with Ad.5/3- mda -7 and BI-97C1 significantly inhibits the growth of human PC xenografts in nude mice and spontaneously induced PC in Hi-myc transgenic mice. Tumor growth inhibition correlated with increased TUNEL staining and decreased Ki-67 expression in both PC xenografts and prostates of Hi-myc mice. These findings demonstrate that pharmacological inhibition of Mcl-1 with the Apogossypol derivative, BI-97C1, sensitizes human PCs to mda -7/IL-24–mediated cytotoxicity, thus potentially augmenting the therapeutic benefit of this combinatorial approach toward PC.

Dance is as much a sport as an art form. Sports medicine clinicians seeing dancers in their practice will need to be familiar with the unique characteristics of dance in order to provide proper care. Dance encompasses different forms, which vary in equipment and terminology. The epidemiology of dance injuries has historically focused on ballet, but there is increasing research on other dance forms. Lower extremity and back injuries predominate. Injury prevention, both primary and secondary, is at the heart of dance medicine. Primary prevention includes preseason conditioning, identifying risk factors for injury, and recognizing the female athlete triad. Secondary prevention includes a comprehensive approach to injury rehabilitation, an appreciation for the unique demands of dance, and an understanding of the particulars of the injury being treated. Dancers may have difficulty accessing medical care or following prescribed advice; the proactive clinician will anticipate these situations.

A 26-year-old female professional dancer sustained an acute injury to her mid-foot during a performance of The Nutcracker. An intra-articular, comminuted, minimally displaced fracture of the cuboid was found. The patient was treated non-operatively with cast and boot immobilization, modified weightbearing, and progressive rehabilitation. She was able to return to professional dance at 6 months post-injury and continues to dance professionally over 1 year out from injury without issue. The unique demands of classical ballet, especially dancing en pointe, increase the risk for mid-foot fractures, and clinicians should have a high-index of suspicion in dancers suffering an acute injury to the foot and ankle with greater than expected pain or swelling. Multiple imaging modalities can be used to make the diagnosis, to include plain film radiographs, MRI, and CT scan. Fracture characteristics and patient-specific factors should be taken into account when deciding on a treatment plan.

Improved treatments for pancreatic cancer remain a clinical imperative. Sabutoclax, a small-molecule BH3 mimetic, inhibits the function of antiapoptotic Bcl-2 proteins. Minocycline, a synthetic tetracycline, displays antitumor activity. Here, we offer evidence of the combinatorial antitumor potency of these agents in several preclinical models of pancreatic cancer. Sabutoclax induced growth arrest and apoptosis in pancreatic cancer cells and synergized with minocycline to yield a robust mitochondria-mediated caspase-dependent cytotoxicity. This combinatorial property relied upon loss of phosphorylated Stat3 insofar as reintroduction of activated Stat3-rescued cells from toxicity. Tumor growth was inhibited potently in both immune-deficient and immune-competent models with evidence of extended survival. Overall, our results showed that the combination of sabutoclax and minocycline was highly cytotoxic to pancreatic cancer cells and safely efficacious in vivo. Cancer Res; 75(11); 2305–15. ©2015 AACR.

Polyinosine–polycytidylic acid [pIC] is a synthetic dsRNA that acts as an immune agonist of TLR3 and RLR to activate dendritic and natural killer cells that can kill tumor cells. pIC can also trigger apoptosis in pancreatic ductal adenocarcinoma cells (PDAC) but its mechanism of action is obscure. In this study, we investigated the potential therapeutic activity of a formulation of pIC with polyethylenimine ([pIC]PEI) in PDAC and investigated its mechanism of action. [pIC]PEI stimulated apoptosis in PDAC cells without affecting normal pancreatic epithelial cells. Mechanistically, [pIC]PEI repressed XIAP and survivin expression and activated an immune response by inducing MDA-5, RIG-I, and NOXA. Phosphorylation of AKT was inhibited by [pIC]PEI in PDAC, and this event was critical for stimulating apoptosis through XIAP and survivin degradation. In vivo administration of [pIC]PEI inhibited tumor growth via AKT-mediated XIAP degradation in both subcutaneous and quasi-orthotopic models of PDAC. Taken together, these results offer a preclinical proof-of-concept for the evaluation of [pIC]PEI as an immunochemotherapy to treat pancreatic cancer. Cancer Res; 74(21); 6224–35. ©2014 AACR.

The aesthetic athlete is a unique combination of athleticism and artistry. Their training begins at a young age with increased demands and high-level performance by their adolescence. They are susceptible to spine injuries based on the unique physical and aesthetic demands of their art/sport and errors in technique and training. The biomechanics of dance, gymnastics, and figure skating is explored as it applies to risk for spinal injury. This is followed by a discussion on spinal injuries in the aesthetic athlete. Injuries reviewed include spondylolysis, posterior element overuse syndrome, sacroiliac joint dysfunction, disc injury, Schuermann’s and atypical Schuermann’s, scoliosis, and mechanical back pain. Injury diagnosis, rehabilitation, and prevention are addressed.

Prostate cancer (PC) is the most frequently diagnosed cancer and is the second leading cause of cancer death among men in the United States (Damber and Aus, 2008; Siegel et al., 2012). It is estimated that 238,590 new PC cases will be diagnosed in 2013 and 29,720 men will die of PC. Patients with localized disease may be treated with surgery or radiation, whereas the treatment options for patients with metastatic disease is purely palliative. Current therapies include hormonal therapy, radiotherapy, and cytotoxic chemotherapeutic agents (Sternberg, 2002; Siegel et al., 2012). Although existing approaches are beneficial in men with various stages of PC, the complications frequently associated with these conventional treatment options diminish positive clinical outcomes. Consequently, more efficient and innovative treatments are mandatory, and genetic therapies represent promising approaches for the treatment of this neoplasm. Using subtraction hybridization combined with induction of cancer cell terminal differentiation, our laboratory cloned melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) (Jiang et al., 1995, 1996), a novel member of the IL-10-related cytokine gene family (Sarkar et al., 2002a; Wolk et al., 2002; Sauane et al., 2003; Dash et al., 2010a). Subsequent studies documented that mda-7/IL-24 displays almost ubiquitous anti-tumor properties in vitro and in vivo, leading to its rapid entry into the clinic, where its safety and clinical efficacy, when administered by adenovirus (Ad.mda-7; INGN 241), was observed in a phase I clinical trial in humans with advanced carcinomas and melanomas (Jiang et al., 1996; Fisher et al., 2003, 2007; Cunningham et al., 2005; Tong et al., 2005; Lebedeva et al., 2007a,c). mda-7/IL-24 preferentially induces apoptosis in cancer cells while exerting no discernible toxic effects toward normal cells (Sarkar et al., 2002b; Sauane et al., 2008; Dash et al., 2010a, 2011a) and it also elicits potent “anti-tumor bystander activity” in distant cancer cells as a consequence of autocrine and paracrine secretion of MDA-7/IL-24 (Su et al., 2001a, 2005a; Fisher, 2005; Lebedeva et al., 2007a,b; Sauane et al., 2008; Dash et al., 2010b). Since PC is generally a relatively slow-growing disease, it may require repeated gene therapy treatments, with single or multiple genes, over the lifespan of the patient. Conditionally replication-competent adenoviruses (CRCAs) provide a potentially valuable reagent for gene therapy (Curiel and Fisher, 2012). Using subtraction hybridization we cloned a novel rodent gene, progression elevated gene-3 (PEG-3), in the context of tumor progression in transformed rat embryo cells (Su et al., 1997). PEG-3: (i) displays elevated expression as a function of oncogenic transformation (by diverse oncogenes) (Su et al., 1997, 2002); (ii) induces an aggressive cancer phenotype without promoting transformation when expressed in normal cells (Su et al., 1999, 2002); and (iii) the gene promoter (PEG-Prom) has been isolated and shown to display elevated expression in both rodent and human tumors (including PC), with negligible expression in normal cells (including human prostate epithelium) (Su et al., 2001b, 2005b; Sarkar et al., 2005a,b, 2006a,b, 2007b, 2008; Bhang et al., 2011; Das et al., 2012). Considering the cancer-specific expression aspects of the PEG-Prom, we constructed a bipartite serotype 5 CRCA (called a cancer terminator virus, Ad.5-CTV) in which the expression of E1A and E1B genes of Ad, necessary for replication, is controlled by the PEG-Prom (Sarkar et al., 2005a, 2006, 2007b, 2008). This novel Ad.5-CTV also expressed mda-7/IL-24 from the E3 region (Ad.PEG-E1A-mda-7). The ability of Ad.5-CTV to infect and express MDA-7/IL-24 in PC cells depends on the presence of Coxsackie-Adenovirus Receptors (CARs) on their surface. Ad.5-CTV is capable of efficiently infecting high CAR cells (such as DU-145) and expressing robust levels of mda-7/IL-24, whereas infection is restricted and expression of MDA-7/IL-24 is minimal in low CAR cells, such as PC-3 (Dash et al., 2010b, 2011b). An approach to circumvent the low efficiency of Ad.5 infection of tumor cells involves “tropism modification” in which virus capsid proteins that normally associate with CAR are modified, permitting both CAR-dependent and CAR-independent infectivity of tumor cells. Studies using various tumor cell types have shown that inclusion of the infective type 3 Ad sequence within the Ad type 5 virus knob (Ad.5/3 recombinant virus) promotes viral infectivity in tumor cells displaying reduced or no CAR expression (Hamed et al., 2010; Dash et al., 2010b; Eulitt et al., 2011; Park et al., 2011; Azab et al., 2012). It is worth noting that Ad.5/3 also retains high infectivity in CAR-expressing tumor cells showing equal efficacy when compared with Ad.5, thereby providing an expanded range of utility for Ad.5/3, in both low and high CAR-expressing tumor cells. In the present study, we constructed and evaluated the in vitro and in vivo efficacy in low and high CAR PCs of a novel tropism-modified CTV in which the virus capsid proteins that normally associate with CAR were modified, Ad.5/3-CTV, permitting CAR-independent infectivity of tumor cells. In low CAR PC-3 cells, Ad.5/3-CTV is more efficient than Ad.5-CTV in infecting tumor cells, delivering a transgene (mda-7/IL-24), expressing MDA-7/IL-24 protein and inducing cancer-specific apoptosis. In an in vivo context, Ad.5/3-CTV is superior to the Ad.5-CTV in inhibiting in vivo tumor growth and exerting an anti-tumor “bystander” effect in nude mouse human PC xenografts and Ad.5/3-CTV potently suppresses PC development in an immunocompetent Hi-Myc transgenic mouse model of PC.

Programmed cell death is well-orchestrated process regulated by multiple pro-apoptotic and anti-apoptotic genes, particularly those of the Bcl-2 gene family. These genes are well documented in cancer with aberrant expression being strongly associated with resistance to chemotherapy and radiation.This review focuses on the resistance induced by the Bcl-2 family of anti-apoptotic proteins and current therapeutic interventions currently in preclinical or clinical trials that target this pathway. Major resistance mechanisms that are regulated by Bcl-2 family proteins and potential strategies to circumvent resistance are also examined. Although antisense and gene therapy strategies are used to nullify Bcl-2 family proteins, recent approaches use small molecule inhibitors (SMIs) and peptides. Structural similarity of the Bcl-2 family of proteins greatly favors development of inhibitors that target the BH3 domain, called BH3 mimetics.Strategies to specifically identify and inhibit critical determinants that promote therapy resistance and tumor progression represent viable approaches for developing effective cancer therapies. From a clinical perspective, pretreatment with novel, potent Bcl-2 inhibitors either alone or in combination with conventional therapies hold significant promise for providing beneficial clinical outcomes. Identifying SMIs with broader and higher affinities for inhibiting all of the Bcl-2 pro-survival proteins will facilitate development of superior cancer therapies.

Background Most cases of ovarian cancer are epithelial in origin and diagnosed at advanced stage when the cancer is widely disseminated in the peritoneal cavity. The objective of this study was to establish an immunocompetent syngeneic mouse model of disseminated epithelial ovarian cancer (EOC) to facilitate laboratory-based studies of ovarian tumor biology and preclinical therapeutic strategies. Methods Individual lines of TgMISIIR-TAg transgenic mice were phenotypically characterized and backcrossed to inbred C57BL/6 mice. In addition to a previously described line of EOC-prone mice, two lines (TgMISIIR-TAg-Low) were isolated that express the oncogenic transgene, but have little or no susceptibility to tumor development. Independent murine ovarian carcinoma (MOVCAR) cell lines were established from the ascites of tumor-bearing C57BL/6 TgMISIIR-TAg transgenic mice, characterized and tested for engraftment in the following recipient mice: 1) severe immunocompromised immunodeficient (SCID), 2) wild type C57BL/6, 3) oophorectomized tumor-prone C57BL/6 TgMISIIR-TAg transgenic and 4) non-tumor prone C57BL/6 TgMISIIR-TAg-Low transgenic. Lastly, MOVCAR cells transduced with a luciferase reporter were implanted in TgMISIIR-TAg-Low mice and in vivo tumor growth monitored by non-invasive optical imaging. Results Engraftment of MOVCAR cells by i.p. injection resulted in the development of disseminated peritoneal carcinomatosis in SCID, but not wild type C57BL/6 mice. Oophorectomized tumor-prone TgMISIIR-TAg mice developed peritoneal carcinomas with high frequency, rendering them unsuitable as allograft recipients. Orthotopic or pseudo-orthotopic implantation of MOVCAR cells in TgMISIIR-TAg-Low mice resulted in the development of disseminated peritoneal tumors, frequently accompanied by the production of malignant ascites. Tumors arising in the engrafted mice bore histopathological resemblance to human high-grade serous EOC and exhibited a similar pattern of peritoneal disease spread. Conclusions A syngeneic mouse model of human EOC was created by pseudo-orthotopic and orthotopic implantation of MOVCAR cells in a susceptible inbred transgenic host. This immunocompetent syngeneic mouse model presents a flexible system that can be used to study the consequences of altered gene expression (e.g., by ectopic expression or RNA interference strategies) in an established MOVCAR tumor cell line within the ovarian tumor microenvironment and for the development and analysis of preclinical therapeutic agents including EOC vaccines and immunotherapeutic agents.

Our laboratory developed a transgenic mouse model of spontaneous epithelial ovarian cancer (EOC) in which tumors are initiated by expression of the early region of the Simian Virus 40 (SV40) under transcriptional control of the 5' upstream regulatory region of the Müllerian inhibiting substance type II receptor (MISIIR) gene. Female TgMISIIR-Tag-DR26 transgenic mice develop bilateral ovarian tumors with variable latency and survive an average of 152 days. In the absence of reliable methods for disease detection and evaluation of therapeutic response, preclinical studies of this transgenic mouse model of EOC would be limited to longitudinal experiments involving large numbers of animals with euthanasia as the endpoint. Therefore, a non-invasive method for detecting tumors, measuring tumor volume and calculating parameters relevant to the evaluation of therapeutic or preventive interventions (i.e., tumor growth rates, tumor initiation, tumor regression and the time for tumors to reach a given size) is required. We developed and optimized a non-invasive Magnetic Resonance Imaging (MRI) scanning protocol to obtain high resolution abdominal images that is well tolerated by mice. Superior contrast and contrast to noise ratio (CNR) was found with Gd-DTPA contrast enhanced T(1)-weighted sequences. Image sets in both the axial and coronal orientations for redundant measurements of normal ovary and ovarian tumor volume can be acquired in approximately 20 minutes. Accuracy of MRI-based ovary and tumor volume determinations was verified by standard volume measurements at necropsy. Serial imaging studies were performed on 41 ovarian cancer bearing TgMISIIR-Tag-DR26 transgenic mice to quantitate tumor progression over time in this model. A chemotherapy study was conducted on TgMISIIR-Tag-DR26 transgenic mice using a standard combination therapy consisting of cisplatin and paclitaxel. Our results demonstrate that MRI is well tolerated and can be repeated in serial imaging studies, permitting quantitative analysis of tumor growth and progression and response to therapeutic interventions.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer is an aggressive cancer without currently effective treatment options. To develop effective therapies for pancreatic and other cancers, we developed bipartite Adenoviruses (Ads) that conditionally replicate in cancer cells and simultaneously express the cancer-specific apoptosis-inducing cytokine melanoma differentiation associated gene-7/interleukin-24 ( mda- 7/IL-24), termed Cancer Terminator Virus ( CTV-M7 ). To enhance transduction of CTV-M7 in cancer cells in a Coxsackie-Adenovirus receptor (CAR) independent manner, a chimeric tropism-modified CTV - M7 was generated in which the Ad.5 fiber knob was replaced by the Ad.3 fiber knob (Ad.5/3- CTV-M7 ) . This Ad displays enhanced infectivity in cancer cells with low as well as high CAR. Although mda -7 displays broad-spectrum anticancer properties, pancreatic ductal adenocarcinoma (PDAC) cells are intrinsically resistant to mda -7-mediated killing due to an mda -7 mRNA translational block. However, when Ad.5- mda -7 is combined with reactive oxygen species (ROS) inducers, there is a conversion of mda -7 mRNA into protein resulting in pancreatic cancer cell death. Hence, we employed perillyl alcohol (POH), which induces ROS and when combined with mda- 7 results in profound killing of PDAC cells, a chemoprevention gene therapy (CGT) approach. ROS induced by POH results in phosphorylation of p70S6-Kinase, 4EBP-1 and eIF4E leading to formation of the pre-initiation complex of protein translation machinery causing an association of polysomes with weakly translated mda -7 mRNA, subsequently enhancing MDA-7 translation. We presently evaluated Ad.5/3- CTV-M7 plus POH as a therapy for PDAC by MTT assays and western blotting. Cell viability was reduced in pancreatic cancer cell lines irrespective of K-ras status following treatment with POH and infection with CTV-M7 . This combination synergistically induced mda -7-mediated cancer-specific apoptosis by inhibiting anti-apoptotic Bcl-xL and Bcl-2 protein expression and inducing an endoplasmic reticulum stress response through induction of BiP/GRP-78, which was most evident in chimeric-modified CTV-M7 -infected PDAC cells. Moreover, it was found that Ad.5/3- CTV-M7 in combination with POH sensitized MIA PaCa-2 cells over-expressing either Bcl-2 or Bcl-xL to mda- 7-mediated apoptosis, demonstrating that CGT can overcome therapy resistance frequently seen in PDAC with elevated expression of these anti-apoptotic proteins. Treatment of MIA PaCa-2 overexpressing Bcl-xL cells established in both flanks of nude mice with POH and systemic administration of Ad.5/3. CTV-M7 using a ultrasound-targeted microbubble-destruction technique in tumors in one flank, resulted in tumor elimination on both flanks. These exciting studies support the potential of the CGT approach using POH and Ad.5/3- CTV-M7, for the therapy of currently intractable, therapy-resistant pancreatic cancers. Citation Format: Siddik Sarkar, Belal Azab, Bridget A. Quinn, Xuening Shen, Paul Dent, Alexander L. Klibanov, Luni Emdad, Swadesh K. Das, Devanand Sarkar, Paul B. Fisher. Chemoprevention gene therapy (CGT) approach for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-315. doi:10.1158/1538-7445.AM2013-LB-315