Cancer

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21728

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Fluorescence polarization based detection of hyaluronidase activity as a biomarker for skin cancer.
(2015-03) Chib, Rahul; Shah, Sunil; Mummert, Mark; Raut, Sangram; Bora, Ilkay; Pedro, Bobby; Gryczynski, Ignacy; Gryczynski, Zygmunt; Fudala, Rafal
Purpose: Malignant melanoma (MM) is a type of skin cancer that has a high potential to metastasize to distant organs and cause death. MM is the third most common skin cancer in the United States and has an incidence of 18 new cases per 100,000. It is more lethal compared to other type of skin cancers due to its higher rate of metastasis and has a 5 years survival rate of 7-18%. In MM, the levels of hyaluronidase are overexpressed. Hyaluronidase is an endoglycosidase that degrades glycosaminoglycan and the hyaluronic acid (HA). Therefore, monitoring the hyaluronidase activity can be used as a contrasting mechanism for its detection. A novel fluorescence polarization based detection of enzyme activity using a fluorophore with a long fluorescence lifetime can enable a simple wide field molecular analysis of the cancer activities at the cellular and tissue level. Hyaluronic acid is a large biopolymer (over 1MDa) that is cleaved by the hyaluronidase enzyme to smaller elements. Monitoring kinetics of HA degradation by fluorescence polarization will be a simple and precise tool reflecting hyaluronidase activity and can be used for the detection, diagnosis and monitoring of the malignant melanoma. Methods: A long lifetime fluorophore (ADOTA) with a fluorescence lifetime of ̴ 20 ns was used to label hyaluronic acid. The hyaluronidase activity was measured as a function of change in the steady state fluorescence intensity and fluorescence polarization. Results: An intact HA will rotate slowly and the fluorescence polarization will be high, on the other hand, the cleaved HA ( ̴300KDa) will depolarize within the fluorescence lifetime of ADOTA yielding low polarization. The change in the polarization directly reflects hyaluronidase activity. We found an increase in the fluorescence intensity with increasing time. This is due to release of HOMO-FRET. We also observed a decrease in the fluorescence correlation time as cleaved hyaluronic acid fragments needs smaller time to rotate. Conclusions: Thus, preliminary results show the ability of HA-ADOTA probe to efficiently detect hyaluronidase activity. This study will develop a new technology for the non-invasive detection of the molecular activity of tumor in situ. In future, this technology can be used to construct a devise which can be used in the primary care setting for the detection of melanoma and monitoring the therapy progress.
Assessment of Bladder Cancer Risk Factors: Central European Health Study
(2015-03) Shikdar, Sufana; Tao, Menghua
Purpose: Urinary bladder cancer is the ninth most commonly diagnosed cancer in the world and fifth most common malignancy in the United States. Because of the lifetime needs for surveillance, treatment and management of recurrent tumors, it poses a significant economic burden. A pilot case-control study was conducted to explore relationship between established and suspected risk factors and risk of different types of bladder cancer. We preliminarily described distributions of risk factors among bladder cancer cases. Method: A multi-center case-control study was conducted in three Central European countries during 2012-2013. Cases (n=83) were aged 30-79, diagnosed with primary, histologically or cytologically confirmed incident bladder cancer. Participants were in-person interviewed to collect information on tobacco and alcohol use, second-hand smoking (SHS), history of chemical exposures, medical history and habitual dietary history. We stratified the cases by gender to compare distributions of risk factors in men and women. Result: Mean age of cases was 62.7 years; there were more males (78.3%) than females (21.7%). 77% of all patients ever smoked, and 65% of the smokers have quitted smoking before diagnosis. Among smokers, men were more likely to be heavy smokers than women (p value is 66.7% for men and women respectively, p value≤0.05). More male patients ever exposed to potential chemical carcinogens than female patients (58.5% for men and 38.9 % for women, p value=0.20), and the major sources of chemical exposures were from petroleum/gasoline, vehicle exhaust, paining and wood dust. About 14% of female ever used hair dyes. Compared to female, men had higher body mass index (BMI) (27.2 for men and 26.9 for women, p value≤0.05), and men were more likely to have higher frequency of meat consumption everyday (14.3% for men and 5.6% for women, p value<0.05). Conclusion: The preliminary results showed different distributions of risk factors among male and female bladder cancer cases. Further case control comparisons are needed to investigate different risk factors associated with bladder cancer across gender and subtype of bladder cancer in this study.
Development of Docetaxel-Loaded Lipid Nanoparticles for Prostate Cancer Treatment
(2015-03) Tanaudommongkon, Asama; Tanaudommongkon, Irin; Prathipati, Priyanka; Dong, Xiaowei
Purpose: Using docetaxel-based therapy (DTX) for treatment for men with castration-resistant prostate cancer (CRPC) showed efficacy on improving overall survival. Despite this promising outcome, toxicities and adverse events of DTX limit the dose and dosage frequency. Moreover, patients develop DTX resistance eventually. Nanoparticle (NP) drug delivery systems offer alternative therapeutic options for the treatment of prostate cancer. The goal of this study is to develop novel DTX NPs to treat CRPC. Method: Lipid and surfactant were selected using the solubility test. DTX NPs were prepared by an emulsion method. The NPs were characterized in terms of particle size, polydispersity index, short-term stability, drug loading, drug entrapment efficiency, in vitro release study, and cytotoxicity studies in DU145 and PC3 prostate cancer cell lines. Results: DTX NPs were prepared using a proportionally amount of Migloyl 812 as the oil phase and TPGS as the surfactant phase. All tested NPs had particle size less than 150 nm with polydispersity index of less than 0.35. DTX NPs were physically stable at 4°C over five months and in PBS at 37°C over 96 hours as measured by particle size. DTX NPs had the drug entrapment efficiency over 90% with drug loading over 5%. The cytotoxicity studies demonstrated that there was no significant difference in IC50 values for the sensitive PC3 and DU145 cells between DTX NPs and free DTX. For the resistant PC3 and DU145 cells, DTX NPs significantly reduced IC50 values compared to free DTX. Conclusions: DTX NPs were successfully prepared and characterized. DTX NPs showed comparable cytotoxicity in sensitive prostate cancer cells, and superior cytotoxicity in resistant prostate cancer cells compared to free DTX. Therefore, DTX NPs have the potential to treat CRPC and overcome drug resistance.
Development of methylene blue-loaded nanoparticles for glioblastoma treatment
(2015-03) Castañeda-Gill, Jessica M.; Ranjan, Amalendu P.; Yang, Shaohua; Vishwanatha, Jamboor K.
Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults over 45, resulting in an average survival of 15 months post-diagnosis and treatment. While recent research has provided essential information to aid diagnosis and treatment, GBM is known to cause relapse following traditional combinatorial regimens, necessitating the development of more effective and less toxic therapies. Methylene blue (MB), a dye with noted medicinal applications, has received recent consideration as a potential neurotherapeutic due to its ability to infiltrate the blood-brain barrier (BBB) and improve cellular processes within distinct brain cell compartments and types; however, one drawback is an increased administration to produce desired therapeutic effects, leading to excessive brain deposition and potential neurotoxicity. A method commonly used to enhance drug delivery while reducing unwanted side effects is via encapsulation in nanoparticles (NPs) composed of the biodegradable/biocompatible co-polymer, poly(lactic-co-glycolic) acid (PLGA). Based on our previous studies, we are developing a MB-loaded PLGA NP capable of permeating the BBB to treat GBM, to test our hypothesis that MB encapsulation into PLGA NPs will enhance accumulation in cancerous brain regions, resulting in reduced tumor size and prolonged survival. In this study, we formulated and characterized MB-loaded PLGA NPs, with a 3:1 molar ratio of sodium oleate to methylene blue at 5mg, based on particle size, drug loading, encapsulation efficiency, and release kinetics. Currently, we are establishing their in vitro effects in two different commercially-available GBM cell lines, according to their responses to commonly-used chemotherapeutics. Following loading of 5mg MB and their comparison to blank NPs, we obtained NP preparations in the range of 120-145nm, with encapsulation efficiencies from 25-40% and drug loading between 1-2%. Additionally, we have found that 50% of the MB initially added is released at 24 hours, and stays constant up to two weeks, demonstrating sustained drug release. In conclusion, based on studies that have demonstrated in vitro effects with MB at a minimum of 1μM (~0.3mg) and 150nm particles, our formulation should elicit comparable, if not better, results when treating GBM.
Neutron-Activatable Holmium-Containing Nanoparticles for the Treatment of Non-small Cell Lung and Skin Cancers
(2015-03) Shi, Yi; Munaweera, Imalka; Koneru, Bhuvaneswari; Levesque-Bishop, Daniel; Aliev, Ali; Coyle, Russell; Saez, Ruben; Balkus, Kenneth J.; Di Pasqua, Anthony J.
Purpose: To improve therapeutic outcomes for patients with non-small cell lung and skin cancers using radiation therapy. Methods: Magnetic holmium-165 (Ho) nanoparticles (HoIG) and derivatives containing platinum-based chemotherapeutic drugs (HoIG-Pt) were prepared and then neutron-activated to radioactive holmium-166 (166Ho; Eβ-max = 1.84 MeV; t½ = 26.8 h) nanoparticles (166HoIG and 166HoIG-Pt, respectively). Cytotoxicities of 166HoIG and 166HoIG-Pt were tested against non-small cell lung cancer cells, and in vivo studies were performed. 165HoIG were also introduced into poly-acrylonitrile (PAN) polymer solutions; these solutions were electrospun to produce bandages that can be applied directly to tumor lesions after neutron-activation. Results: Ho materials were carefully characterized and successfully neutron-activated. HoIG contained approximately 56% w/w Ho. Two 166HoIG-Pt derivatives showed greater toxicity toward non-small cell lung cancer cells than blank 166HoIG and free platinum drug. The animal study data collected showed that the radiation and platinum drug act synergistically. HoIG was distributed homogenously throughout the radioactive bandages and the bandage was stable upon neutron-activation. Conclusion: Pt-based chemotherapeutic drugs and 166Ho have been incorporated together in a magnetic nanoparticle for the first time, and then used successfully in human cell culture and animal studies. A novel radiotherapeutic bandage was successfully prepared using PAN solutions and HoIG nanoparticles, and subsequently neutron-activated. The radiotherapeutic bandage is a more flexible strategy than that currently being used in the clinic for the treatment of skin cancer.
A Comparative Study of Rural Communities for Colorectal Cancer Screening by Means of Colonoscopy Provided by Family Physicians
(2015-03) Stanley, Russell F.; Deen, Briar; Luz Chiapa-Scifres, Ana; Bowling, John
Purpose: Colorectal cancer has a high incidence and prevalence in the United States and in particular, rural Texas. Some rural family physicians in Texas do colonoscopy procedures whereas other rural family physicians refer their patients to urban centers. This study will look at whether or not patients with access to family medicine physicians who perform colonoscopy exams within their own rural community are more likely to comply with colorectal cancer screening guidelines as opposed to rural patients in another community who are referred to urban centers. Methods: A 24 question survey pertaining to colon cancer and adapted from the Behavior Risk Factor Surveillance System (BRFSS) was given to patients at primary care clinics in Clifton and Haskell. Both clinics are located in small, rural towns in Texas. The family medicine physicians at the Clifton clinic provide colonoscopies within the community whereas the residents in Haskell drive to urban centers for colonoscopies. In order to take the survey, participants had to be patients of either the Clifton or Haskell clinic and be at least 50 years of age. Logistic regression was used to look at an association between the two clinics and patient colon cancer screening awareness, knowledge of what age a person should be screened for colon cancer and whether or not patients had ever had either a sigmoidoscopy or a colonoscopy. All statistics were done using SAS 9.3. Results: 92 surveys were collected at Clifton and 76 were collected at Haskell for a total of 168 surveys. Of the collected patient surveys, 80.12% patients were white and 62.05% were female with a mean age of 63.90. A higher odds (OR=3.61; CI = (1.11, 11.69)) was seen in Clifton compared to Haskell for patient colon cancer screening awareness after being adjusted for gender, race, age, employment status and family history of colon cancer. There was a higher odds (OR=2.50; CI = (1.13, 5.54) of knowing what age a person should be screened for colon cancer in Clifton compared to Haskell after being adjusted for gender, age, employment status, education level and family history of colon cancer. A higher odds (OR=3.61; CI = (1.42, 9.20)) was seen in Clifton compared to Haskell for patients ever having a colonoscopy or sigmoidoscopy after being adjusted for patient race, age, gender, education, employment status and for the patient not having insurance. Conclusion: This study supports the idea that having family medicine physicians perform colonoscopies within a rural community leads to a higher odds of patients following cancer screening guidelines. Therefore, it is beneficial to have colonoscopies performed locally in a rural community in order to better prevent colorectal cancer.
Therapeutic Leukapheresis in Pediatric Leukemia: The Cook Children’s Experience
(2015-03) Thapa, Namisha; Cole, Charles; Ray, Anish; Bowman, W. Paul
Purpose (a): In Pediatrics, acute leukemia is the most common cause of hyperleukocytosis, as defined by WBC above 100K. In children, hyperleukocytosis is associated with early morbidity and mortality due to leukastasis-related complications such as intracranial hemorrhage and pulmonary distress. Additionally, tumor lysis syndrome, a dreaded complication due to high rate of cell turnover, can especially occur if chemotherapy is initiated without leukoreduction. Currently, therapeutic leukapheresis (LK) serves as an adjunctive therapy for select population presenting with hyperleukocytosis and/or leukastasis-related symptoms. Although LK is commonly used for this purpose, specific guidelines regarding when to use LK are not well-established. The purpose of this study is to evaluate the efficacy of LK and to determine the specific patient population that will benefit the most from this procedure. To our knowledge, this is the largest study conducted on efficacy of LK and its clinical outcome in pediatric leukemia patients. Methods (b): After obtaining institutional IRB approval, a retrospective chart review was conducted on 20 pediatric leukemia patients (14 ALL, 5 AML, and 1 CML) who underwent LK at Cook Children’s Medical Center from 2000 to 2014. Data on white blood cell count (WBC), platelets, chemistry, complications due to leukastasis at presentation, complete remission (CR), and overall survival rate were collected. Results (c): At presentation, 15% children had CNS symptoms, 15% had respiratory symptoms, and 5% had both. First round of LK showed 61.6% reduction in WBC from median value of 474.2 (233 – 910 x 109/L) to 182.5 (99.2 – 845 x 109/L). Six patients underwent second LK that reduced WBC by another 28.9% with a final median WBC of 139.35 (27.1 – 725 x 109/L). Overall, 19 out of 20 patients were alive immediately post LK, and 15 patients achieved complete remission. Conclusions (d): LK significantly reduces WBC in pediatric leukemia in patients as young as 22 day old presenting with WBC > 250 9/L; 250 9/L and leukastasis-related complications. LK procedure itself has no significant complications and is concluded to be a safe adjunctive procedure in pediatric leukemia prior to initiation of induction chemotherapy.
Primary Esophageal Adenosquamous carcinoma presenting as a pancreatic tail mass
(2015-03) Reed, Kristina M.; Yurvati, Albert
This is a case report of a case of primary esophageal adenosquamous carcinoma. This report highlights the rarity of this subtype of cancer as well as the difficulty establishing an accurate diagnosis. This difficulty in this case was due to the cancer presenting as a pancreatic tail mass. All information was gathered while on a core surgery rotation with a hospital affiliated with UNTHSC (Dallas Methodist). Results are that this is an aggressive subtype of esophageal cancer that is lacking in the literature due to its rarity. There is an immense need for further research to establish more targeted and effective post surgical care in order to increase prognosis for these patients. There is also a lack of understanding of the pathophysiology in adenosquamous carcinoma, determining if it arises first as adenocarcinoma, squamous cell carcinoma, or independently as both could help determine risk factors and chemotherapy strategies. Known risk factors include diet, like the consumption of hot beverages and foods.
Overcoming Chemoresistance in Neuroblastoma Cells
(2015-03) Panchoo, Marlyn; Sabnis, Nirupama; Lacko, Andras G.
Background: The development of multi-drug resistance (MDR) in tumor cells continues to be a major challenge to effective cancer therapeutics. Regarding neuroblastoma (NB), a large proportion of the cases (~40%), designated as high risk NB (HRNB), present with a poor prognosis, with death occurring within 2-3 years of diagnosis. Most of these HRNB tumors become resistant and thus unresponsive during extended treatment, leading to the exceedingly poor prognosis. MDR is often due to the efflux (pumping out) of the drugs from the malignant cells via the ATP Binding Cassette (ABC) transporters. Hypothesis: The design of this study is based on the concept that the incorporation of the drug doxorubicin (Dox) into reconstituted high density lipoprotein (rHDL) nanoparticles will overcome MDR during treatment. This hypothesis also proposes a mechanism where delivery of Dox via the scavenger receptor type B1 (SR-B1), directly into the cytosol, will allow the drug to bypass the membrane MDR efflux pump and thus limit or eliminate drug resistance. Methods and Results: Drug resistance was induced in SMS-KCNR neuroblastoma cells by incubating the cells with 50 ng/ml of interleukin-6 (IL- 6) for five days. The IL-6 treated cells showed a 5 fold increase in resistance compared to the untreated cells. To evaluate the Dox encapsulated in rHDL (rHDL-Dox nanoparticles) for its ability to overcome MDR, the cytotoxicity of the free Dox vs. the encapsulated Dox was compared against resistant cells. The results showed that the rHDL-Dox formulation was more effective in killing the drug resistant cells than the free Dox (IC50 = 0.08 µg/ml vs. 0.52 µg/ml). These findings show that the rHDL-Dox formulation is indeed effective in limiting drug resistance. Conclusion: We anticipate that the increased sensitivity of MDR cells to rHDL-Dox nanoparticles, shown by these studies, could be extended to other drugs. Consequently, we also anticipate that these rHDL nanoparticle formulations could provide a safe and effective treatment for HRNB patients that otherwise would be resistant to therapy. Future plans will include screening of drug resistant NB cells for the expression of the SR-B1 receptor, monitoring of downstream events such as apoptosis, cell migration, and localization of the drug to document cytosolic delivery, in addition to studies with tumor carrying mice.
Small Molecule, Tolfenamic Acid Induces the Apoptotic Response of Cis-Retinoic Acid in High-Risk Neuroblastoma Cells
(2015-03) Shelake, Sagar; Sankpal, Umesh; Wadhwani, Anmol; Tabor-Simecka, Leslie; Bowman, W. Paul; Basha, Riyaz
Neuroblastoma (NB) is an aggressive and highly heterogeneous extra-cranial solid tumor found in children. NB accounts for 15% deaths among pediatric cancer patients. 13-cis-retinoic acid (RA) is commonly used as adjuvant therapy during the remission maintenance phase of NB treatment. There is, however, a greater than 50% risk of relapse in high-risk neuroblastoma (HRNB) that necessitates the development of an alternative therapeutic strategy. Specificity protein 1 (Sp1) is a transcription factor that is involved in the regulation of various cellular functions including cell growth, differentiation, and apoptosis. The present study is aimed at investigating the effect of a small molecule and Sp1 inhibitor, Tolfenamic Acid (TA) for enhancing the anti-proliferative effect of RA in HRNB cell lines, LA1-55n and SH-SY5Y. The optimized doses obtained from dose/time-dependent response of individual agents were used for the combination treatment experiments. LA1-55n and SH-SY5Y cells were treated with TA (30 µM) or RA (20 µM) or both for 48 h and tested to assess the effect on cell viability, apoptosis and selected molecular markers- Sp1, survivin, AKT and ERK1/2. Cell viability and caspase activity were measured using the CellTiter-Glo and Caspase-Glo kits. The apoptotic cell population was determined by flow cytometry with Annexin-V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c-PARP were evaluated by western blot analysis. Treatment with TA and RA resulted in significant inhibition of cell viability in dose/time- dependent manner in tested cell lines. Even though the individual agents showed anti-proliferative response, the combination of both agents increased cell growth inhibition in LA1-55n (91.8%), SH-SY5Y (78.2%) as compared to TA alone (LA1-55n: 65%; SH-SY5Y: 51%) and RA alone (LA1-55n: 50%; SH-SY5Y: 33.5%). This anti-proliferative effect is accompanied by a decline in Sp1 and survivin expression. Furthermore, TA and RA combination treatment resulted in a significant increase in apoptotic (Annexin-V positive) cells, caspase 3/7 activation (LA1-55n: 12 fold and SH-SY5Y: 9 fold; p
Bone Targeted Polymeric Nanoparticles for Metastatic Prostate Cancer
(2015-03) Gdowski, Andrew; Ranjan, Amalendu; Vishwanatha, Jamboor
Purpose: Bone is the most frequent site of metastasis in several types of cancers including breast, prostate, and lung. The majority of patients that develop bone metastasis will experience complications that include pathological fractures and severe bone pain. Current treatment options for bone metastasis often cause many serious off target side effects and are ineffective. We hypothesize that delivery of cabazitaxel encapsulated bone-targeted biodegradable nanoparticles is an effective therapy for bone metastatic prostate cancer. Materials & Methods: Bone targeted nanoparticles were made using a modified water in oil in water double emulsion solvent evaporation technique. Cabazitaxel was encapsulated within PLGA nanoparticles and alendronate was used to coat the nanoparticles. Nanoparticles were characterized with mass spectroscopy, fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and dynamic light scattering (DLS). Functionally, the nanoparticles were tested in 3D prostate tumor spheroids and ex vivo bone affinity experiments. Results: Successful encapsulation of cabazitaxel within poly (lactic-co-glycolic acid) (PLGA) nanoparticles yielded an encapsulation efficiency of 58% and a drug loading of 3.74%. Release kinetics demonstrated a controlled release of the drug with 60% of cabazitaxel released at 1 hour and 98% at 72 hours. Surface characterization with FTIR confirmed alendronate attachment to the surface of the nanoparticle. SEM and DLS showed an acceptable degree of size dispersity with spherical nanoparticles. Targeted nanoparticles had a 4-fold increased affinity to bone compared to non-targeted nanoparticles at 6 hours and a 8-fold increased affinity to bone at 72 hours in bone affinity experiments. 3D tumor spheroid assay indicated that spheroids treated with equivalent doses of free drug and drug loaded nanoparticles resulted in similar cytotoxic performance. Conclusion: We have engineered bone targeted PLGA nanoparticles for treating metastatic prostate cancer. Future studies will assess the in vivo bone targeting capabilities of the nanoparticles as well as therapeutic effects in an intraosseous tumor model.
Targeted Therapy for Non-Small Cell Lung Cancer Using Antibody-Coated Gold Nanoparticles
(2015-03) Koneru, Bhuvaneswari; Shi, Yi; Moron, Sebastian; Di Pasqua, Anthony
Purpose:Lung cancer is the leading cause of cancer-related death in the United States among both men and women. Approximately 85 to 90% of all lung cancers are non-small cell lung cancer (NSCLC). Unfortunately, NSCLC is extremely difficult to treat and survival rates are low (the five year survival rate is Methods: AuNPs with pendant –COOH and –OH groups are synthesized by the reduction of gold ions in the presence of a mixture of alkanethiols. A targeting ligand (mAb) is attached to the AuNPs by EDC-coupling. The size and zeta potential of the AuNPs is determined using dynamic light scattering and TEM, and the amount of antibody conjugation quantified via colorimetry. Uptake of AuNP and AuNP-mAb in various NSCLC cell lines with various levels of expression of the adhesion molecule was then performed Results: AuNP-mAb was synthesized, characterized and uptake tested in cell culture. We anticipate next to attach a platinum prodrug and test for use in targeted therapy for NSCLC.
Exosomal AnnexinA2 promotes angiogenesis and breast cancer metastasis
(2015-03) Maji, Sayantan; Akopova, Irina; Nguyen, Phung M.; Hare, Richard J.; Gryczynski, Ignacy; Vishwanatha, Jamboor K.
Purpose: Early detection of cancer using circulating biomarkers is a realistic possibility with the discovery of exosomes. Cells under both physiological and pathological conditions secrete a wide array of membranous vesicles containing specific protein and RNA signatures. Exosomes, which are 40-100nm in size, comprise a major portion of these vesicles. Annexin A2 (AnxA2) is a 36 KD Ca+2 dependent phospholipid-binding protein up-regulated in many cancer types and implicated in promoting tumorigenesis and angiogenesis. Cancer cells have been shown to secrete more AnxA2 and it is also highly expressed in the exosomes; but the function of exosomal AnxA2 (exo-AnxA2) has never been studied. We hypothesize that exo-AnxA2 promotes angiogenesis and breast cancer metastasis and we propose to explore this in our study. Methods: Exosomes were isolated from MCF10A progression model for progression studies. For the metastasis studies MDA-MB-231 and its organ specific metastatic variants MDA-MB-831 (brain met) and MDA-MB-4175 (lung met) were used. They were characterized via Western blotting, particle size analyzer, transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). In vitro and in vivo angiogenesis assays were performed to study the role of exo-AnxA2 in angiogenesis. Exo-AnxA2 was either inhibited by use of LCKLSL inhibitory peptide or knocked down in these studies. Tail vein and intracardiac injection metastasis models were used after priming the animals with exosomes to study the role of exo-AnxA2 in breast cancer metastasis. Results: Characterization of exo-AnxA2 by Western blot and AFM revealed that it is highly expressed in cancer exosomes than exosomes from normal cells. In vitro and in vivo angiogenesis studies revealed exo-AnxA2 to be a potent inducer of angiogenesis and inhibition of exo-AnxA2 significantly inhibits angiogenesis (~3 fold and ~5 fold decrease with LCKLSL vs. control in endothelial assay and matrigel plug assay respectively). Tail vein and intracardiac metastasis models showed that exo-AnxA2 promotes lung and brain metastasis. Knockdown of exo-AnxA2 showed ~2 fold decrease in lung and brain metastasis vs. control respectively, as evident from bioluminescence imaging. Conclusion: We found that exo-AnxA2 correlates positively with breast cancer progression. Furthermore, we found that exo-AnxA2 is a potent inducer of angiogenesis and breast cancer metastasis indicating a possible role of exo-AnxA2 in tumor- microenvironment signaling and cancer progression.
Bacteriomimetic Nanoparticles for Immunotherapy against Breast Cancer
(2015-03) Kokate, Rutika; Thamake, Sanjay; Chaudhary, Pankaj; Raut, Sangram; Mott, Brittney; Vishwanatha, Jamboor K.
Short description: Immunotherapy represents a potential and innovative means to combat cancer. It essentially harnesses the body’s immune system to fight against cancer. Previous literature suggests that cancer vaccines designed against a specific tumor antigen have been efficiently utilized to trigger immune responses against tumor cells. Despite the preliminary evidence in animal models, low immunogenicity is one of the major hurdles in the development of vaccines in humans. In order to surmount this obstacle, several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored. Purpose: The purpose of this study was to develop “bacteriomimetic nanoparticles” to enhance adaptive cell-mediated immune responses (CD4+ and CD8+ T cell responses) against tumor antigen as a therapeutic option for cancer treatment. Materials and Methods: NPs were prepared by modified solid/oil/water solvent evaporation method using an ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany). We used membrane preparations of the 4T1 mouse mammary cancer cell line as a tumor antigen and CpG ODN’s as a “bacteriomimetic” stimulant. Fourteen days before tumor challenge BALB/c female mice (6-8 weeks) were pre-immunized with CpG followed by secondary immunization using respective NPs encapsulated with the membrane antigen preparation. Subsequently, mice (n=5) were challenged subcutaneously (SC) with 105 tumor cells and the primary tumor size was monitored using vernier caliper and bioluminiscence imaging (Caliper Life Sciences Inc., MA, USA). Mice were sacrificed on day fourteen after tumor challenge; spleen cells were used for flow cytometric analysis and primary tumor tissue was used to evaluate effect of NP immunization on tumor growth, survival as well as the immune response (CD4+ and CD8+ T cell) via immunohistochemistry. Results: We found significant reduction in progression of tumor growth in mice immunized with CpG coated NPs containing tumor antigen (CpG-NP-Tag). Histological analysis confirmed that tumors in CpG-NP-Tag mice were relatively well differentiated and of lower grade in contrast to CpG-Blank tumors. Immunofluorescence (IF) data further revealed that CpG-NP-Tag tumors had lesser proliferation and higher apoptotic activity. Tumor CD4+ and CD8+T cell infiltration as well as T cell response in spleen was found be higher in CpG-NP-Tag NP immunized mice as compared to the controls (CpG-NP-Blank and NP-Tag). Conclusions: Primary tumor size, IHC, IF and flow cytometry analysis indicate that CpG-NP-Tag NPs were successfully employed to boost the immune response against tumor cells.
Reproductive Health in Female Members of Cook Children's Life After Cancer Program
(2015-03) Chitturi, Kalyan R.; Prendergast, Christina; Bashore, Lisa; Shah, Deep; Paxton, Raheem; Bowman, W. Paul
Reproductive Health in Female Members of Cook Children’s Life After Cancer Program Purpose: Late effects from cancer treatment have been a topic of growing interest in pediatric oncology. In this study, we assessed the relationship between cancer treatment modalities (radiation therapy and chemotherapeutic drugs) on spontaneous primary ovarian insufficiency (POI) and hormone replacement therapy (HRT) among female pediatric cancer survivors enrolled in the Life After Cancer Program (LACP) at Cook Children’s Medical Center. Reproductive ability is an important quality of life issue in pediatric cancer survivors. Oncologists can utilize this information to minimize risks during treatment and recommend fertility-preserving steps such as removal and cryopreservation of oocytes and ovarian tissues prior to treatment. Female pediatric patients benefit from being more informed of reproductive late effects as fertility-preserving measures can be pursued prior to initiation of cancer treatment. Methods: Chart review (n = 194) was conducted of LACP cancer survivors from 1/1/2011 to 6/30/2013. POI was defined clinically as females <40 years old with metrorrhagia or amenorrhea in association with elevated serum FSH levels as determined by individual lab assay method. Bivariate and stepwise logistic regression models assessed associations between treatment-related factors and both POI and HRT. Two-sided statistical tests (significance = 0.10) were performed in the model. Inclusion criteria as follows: Females who are currently ≥12 years of age, Females who completed last cancer treatment ≥2 years ago, Females who were seen at least once in LACP clinic between 1/1/2011 to 6/30/2013. Results: Mean age of diagnosis = 6.69 years. 25 subjects required HRT. Age at diagnosis, busulfan, ifosfamide, carboplatin, radiation therapy affecting ovaries/uterus, and total body irradiation (TBI) were found to be significantly associated (p < .05) with HRT in stepwise model. Twenty-three patients developed POI. Age at diagnosis, busulfan, carboplatin, radiation therapy affecting ovaries/uterus, and total body irradiation (TBI) were found to be significantly associated (p < .05) with POI in stepwise model Conclusions: Findings show certain alkylating agents (busulfan, ifosfamide), heavy metals (carboplatin), and radiation therapy increased odds of HRT and POI among LACP pediatric and young adult cancer survivors. Future analyses are ongoing with an expanded cohort (n = 449).
Formulating adjuvant therapy of rHDL nanoparticles with saquinavir to combat high-risk neuroblastoma.
(2015-03) Sheikh, Irtiza; Sabnis, Nirupama; Lacko, Andras G.
Purpose: Despite major advances in pediatric cancer research, there has been only modest progress in the survival of children with high risk neuroblastoma (HRNB). Current chemotherapy regimens have a serious limitation due to off target toxicity. The purpose of our project is to evaluate the effectiveness of a drug delivery platform with reconstituted/synthetic high density lipoprotein nanoparticles (rHDL) using rHDL-saquinavir formulation for the treatment of HRNB. It is anticipated that upon establishing an improved chemotherapeutic regimen for HRNB, the rHDL technology could be extended to enhance the chemotherapy for other pediatric cancers. Materials and Methods: The rHDL-Saquinavir nanoparticles were prepared by cholate dialysis method. The entrapment efficiency of Saquinavir was determined by Fluorimetric measurements. The chemical composition of rHDL/Saquinavir particles was estimated by standard kits. The average size of the particles was measured by DeLsa Nano particle size analyzer. The stability of particles was estimated by dialyzing the particles at 37°C, for 48 hours at pH 7.4. The cytotoxic effectiveness of the formulation was tested against two HRNB cell lines (SJNKP and IMR-5 obtained from Dr F. Temius, Regina Margherita Children’s Hospital, Turin, Italy)) as compared to that of the free Saquinavir using CCK-8 kit. The Inhibitory concentration to kill 50% of the cells (IC50) was determined. Results: The entrapment efficiency of the rHDL-SAQ particles was determined to be 70%. The chemical composition study indicated that the rHDL-SAQ nanoparticles were composed of 60% phospholipids, 24% protein, 9% cholesterol, and 7% of Saquinavir. The average diameter of the particle was 7.3 nm. The stability of the nanoparticle formulation measured as retention of the drug under experimental conditions indicated that 71% of the drug was preserved. When testing the survival of the IMR-5 cell lines in presence of Free and rHDL-Saquinavir, it was found that the rHDL particles were 10 times more effective than free Saquinavir. The effect on the SJNKP cells was observed to be 2 fold greater when using the rHDL particles compared to the free drug. Moreover, the rHDL-SAQ particles were both able to achieve 100% killing while the free SAQ did not achieve 100% killing effect in the given range. Conclusions: The rHDL-Saquinavir nanoparticles were successfully formulated. The particles appeared to be small, stable and non-leaking. In vitro survival studies suggested that rHDL-Saquinavir formulation is more effective than the free saquinavir. Thus, these studies support the potential of this novel drug delivery platform for treating HRNB. These studies could be extended to other types of cancers as well.
Development of Curcumin Loaded Nanoparticles and Evaluation of Antitumor Effects on Prostate Cancer Cell Lines
(2015-03) Tanaudommongkon, Irin; Tanaudommongkon, Asama; Prathipati, Priyanka; Dong, Xiaowei
Purpose (a): Curcumin (CUR) is a low molecular weight, lipophilic, yellow polyphenolic compound of Indian spice turmeric. In recent years, CUR has been shown as an effective antiproliferative agent in many cancer cell lines such as breast cancer and prostate cancer. Due to low water solubility and instability, the formulation of CUR is challenging and this prevents the usage of CUR in anticancer application. Lipid based nanoparticle (NP) delivery system is a promising approach to formulate CUR, in terms of its abilities in formulating lipophilic drugs, improving the drug’s pharmacokinetics and biodistribution, and reducing drug toxicity. The main objective of this study is to develop CUR NPs by optimizing NPs with varying compositions of lipids and surfactants. Methods (b): The preparation of nanoparticles was performed by a warm o/w microemulsion system. The drug loading and entrapment efficiency of CUR NPs were measured by using HPLC. Particle size was determined by using photon correlation spectroscopy. The in-vitro cytotoxicity of CUR NPs were performed by using MTT assays in PC3 and DU145 prostate cancer cell lines. Results (c): We were able to load CUR into the NPs made of Migloyl 812 and TPGS (1:1, w/w). Particle size was less than 150 nm with polydispersity index95% and drug loading was >5%. CUR NPs were stable for up to 5 months at 4°C and up to 96 hours at 37°C in PBS buffer (pH 7.4) without significant changes in particle sizes. For both sensitive and resistant PC3 and DU145 cell lines, CUR NPs significantly reduced IC50 values over free drug. Conclusions (d): We successfully prepare CUR NPs using lipid-based NPs. CUR NPs significantly improved cytotoxicity of CUR in sensitive and resistant prostate cancer cells compared to free CUR.
Tolfenamic Acid Induces Therapeutic Efficacy of Chemotherapeutic Agents in Medulloblastoma Cells
(2015-03) Jones, Michelle; Shelake, Sagar; Hernandez, Yazmin; Wadhwani, Anmol; Sankpal, Umesh; Bowman, W. Paul; Murray, Jeffrey; Basha, Riyaz
Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Currently, MB is treated using a multimodal approach consisting of surgery, craniospinal irradiation, and chemotherapy. Among the most commonly used chemotherapeutic agents are vincristine (Vinc), etoposide (Etop) and Cisplatin (Cis), while doxorubicin (Dox) is also used rarely. All of these agents carry significant delayed consequences for the patients including cognitive deficits. It is imperative that the strategies to improve the therapeutic efficacy of standard chemotherapy will include reduction of side effects in order to have a significant impact for treating MB patients. The primary objective of this study is to determine the effectiveness of a combination treatment to enhance the therapeutic efficacy of chemotherapeutic agents using MB cell lines. We have tested the combination of Tolfenamic Acid (TA), a small molecule and non-steroidal anti-inflammatory drug along with Vinc or Etop or Cis or Dox using MB cell lines, DAOY and D283. These cell lines were purchased from American Type Culture Collection (ATCC), Manassas, VA. TA has been shown to inhibit cell proliferation, induce apoptosis, and decrease the expression of Sp1 and Survivin which play roles in growth and cell survival in MB cells. Our results show that each drug together with TA causes a time and dose dependent decrease of MB cell viability which is more than that of single drug treatment. The cell growth inhibition is accompanied by an induction of apoptotic markers and the decrease in expression of Sp1 and survivin. The preliminary results of this preclinical model are in favor of combining TA with Vinc or Etop or Cis or Dox to achieve maximum therapeutic benefit while limiting the duration of treatment. Further studies are under investigation to precisely understand the underlying mechanisms and to confirm these results via in vivo assays. Addition of TA to current chemotherapy regimen for MB may reduce the dose and amount of time necessary for chemotherapy and therefore potentially reduce the toxicity and side-effects in children.
AnxA2 contributes to TNBC progression
(2015-03) Gibbs, Lee D.; Vishwanatha, Jamboor
Purpose: The National Cancer Institute (NCI) estimates that in 2015, approximately 232,340 women will be diagnosed with new cases of invasive breast cancer and 39,620 will succumb to the disease. Advances in breast cancer research have led to the identification of four molecular subtypes; Luminal A, Luminal B, Triple negative/basal-like, and HER2 (Human Epidermal Growth Factor [Erbb2]) type. Triple-negative breast cancers (TNBC) are identified by the absence of three major receptors that drive most breast cancer: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2); and constitutes 15 to 20% of diagnosed breast cancers. Given the heterogeneity and lack of standard molecular targets, patients with TNBC do not benefit from currently available targeted therapy, such as endocrine or anti-HER2 agents. Therefore, the only systemic treatment option available for these patients is surgery (+/- radiation) and chemotherapy with standard cytotoxic agents. Overall, TNBC is associated with poor prognosis, high mortality rate, and shorter median time to relapse (due to its aggressive tumor phenotype(s)), high recurrence rate, and visceral metastatic spread to the brain and lungs. This presents an urgent clinical need to identify new biomarker(s) that can be used for diagnosis and/ or as potential therapeutic targets. Our previous studies have demonstrated that AnxA2 is abundantly expressed in TNBC and has a reciprocal relationship with HER2 (Human Epidermal Growth Factor Receptor 2/ErBb2) at mRNA and protein levels. We have also demonstrated that AnxA2 and EGFR interact at the cell surface and this association is essential for EGFR mediated downstream signaling events that lead to cancer cell proliferation and migration. Furthermore, we have found that addition of growth factors, like EGF, stimulate the expression of AnxA2 at the mRNA and protein level in cancer cells expressing EGFR and basal levels of AnxA2. Based on our previous published studies and new preliminary data we hypothesize that AnxA2 expression in TNBC provides a selective advantage for TNBC fueling cancer progression. Our investigation will allow us to identify the AnxA2-EGFR relationship as a molecular contributor to TNBC progression. Materials and Methods: We serum starved MCF-7 cells overnight and treated with EGF (100ng/mL) at different time intervals. Increased expression of AnxA2 was observed at 48 and 72 hours at the protein level and mRNA. Membrane and cytosolic extraction of metastatic TNBC cell lines was performed for analysis of AnxA2 protein expression. TCGA(The Cancer Genome Atlas) analysis of breast cancer subtypes from clinical samples will also performed for analysis of AnxA2 and EGFR expression in cancer progression. Results: Our data indicate that increased expression of AnxA2 was observed at 48 and 72 hours at the protein level and mRNA fold change increased exponentially with EGF treatment. TCGA analysis unveils a significant increase in AnxA2 mRNA expression in comparison with other breast cancer subtypes. Conclusions: Our results suggest that AnxA2 increased expression during EGF stimulation of cancer cells with low expression of AnxA2 may be a potential mechanism of the AnxA2-EGFR interaction in TNBC. Increased expression of AnxA2 in TNBC in comparison with other breast cancer subtypes from the TCGA portal demonstrates AnxA2 as a potential biomarker for TNBC.

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