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SF1: ETV4 expression levels in endometrial cancer. SF2: Expression levels of ETV4 in candidate knockout clones. SF3: Short-term knockdown of ETV4 reduces ER genomic binding. SF4: Validation of rescue and overexpression of ETV4. SF5: Estrogen induced fold changes are higher in wildtype cells than ETV4 knockout cells and genes that lose estrogen induction are closer to ETV4 bound sites. SF6: Global chromatin accessibility is similar between wildtype and ETV4 knockout lines. SF7: Introduction of ETV4 in T-47D breast cancer cell line. SF8: GR localization does not change due to ETV4 loss. SF9: Growth phenotype observed in 2D growth of ETV4 knockout lines. SF10: ETV4 knockouts increase expression of cytokeratins, a marker of normal endometrium.

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells.Significance:Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells.

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells. Significance: Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells.

Vaginal drug delivery represents an attractive strategy for local and systemic delivery of drugs otherwise poorly absorbed after oral administration. The rather dense vascular network, mucus permeability and the physiological phenomenon of the uterine first-pass effect can all be exploited for therapeutic benefit. However, several physiological factors such as an acidic pH, constant secretion, and turnover of mucus as well as varying thickness of the vaginal epithelium can impact sustained drug delivery. In recent years, polymers have been designed to tackle challenges mentioned above. In particular, thermosensitive hydrogels hold great promise due to their stability, biocompatibility, adhesion properties and adjustable drug release kinetics. Here, we discuss the physiological and anatomical uniqueness of the vaginal environment and how it impacts the safe and efficient vaginal delivery and also reviewed several thermosensitive hydrogels deemed suitable for vaginal drug delivery by addressing specific characteristics, which are essential to engage the vaginal environment successfully.

PURPOSE Treating uterine fibroids with less invasive therapies such as magnetic resonance-guided focused ultrasound (MRgFUS) is an attractive alternative to surgery. Treatment planning can improve MRgFUS procedures and reduce treatment times, but the tissue properties that currently inform treatment planning tools are not adequate. This study aims to develop an ex vivo uterine fibroid model that can emulate the in vivo environment allowing for characterization of the uterus and fibroid MR, acoustic, and thermal tissue properties while maintaining viability for the necessary postsurgical histopathological assessments. METHODS Women undergoing a hysterectomy due to fibroid-related symptoms were invited to undergo a preoperative pelvic MRI and to permit postoperative testing of their uterine specimen. Patients that declined or could not be scheduled for a pre-operative MRI were still able to allow post-operative testing of their excised tissue. Following surgical removal of the uterus, nonmorcellated tissues were reperfused with a Krebs-Henseleit buffer solution. An MR-compatible perfusion system was designed to maintain tissue viability inside the MR suite during scanning. MR imaging protocols utilized preoperatively were repeated on whole sample, reperfused ex vivo uterus specimens. Thermal properties including thermal diffusivity and thermal conductivity of the uterus and fibroids were determined using an invasive needle sensor device in 50% of the specimens. Acoustic property measurements (density, speed of sound and attenuation) were obtained for approximately 20% of the tissue samples using both through-transmission and radiation force balance techniques. Differences between fibroid and uterus and in vivo and ex vivo measurements were evaluated with a two-tailed Student t test. RESULTS Fourteen patients participated in the study and measurements were obtained from 22 unique fibroids. Of the 16 fibroids available for preoperative MRI testing, 69% demonstrated classic hypo-intensity relative to the myometrium, with the remainder presenting with iso- (25%) or hyper-intensity (6%). While thermal diffusivity was not significantly different between fibroid and myometrium tissues (0.217 ± 0.047 and 0.204 ± 0.039 mm2 /s, respectively), the acoustic attenuation in fibroid tissue was significantly higher than myometrium (0.092 ± 0.021 and 0.052 ± 0.023 Np/cm/MHz, respectively). When comparing in vivo with ex vivo MRI T1 and T2 measurements in fibroids and myometrium tissue, the only difference was found in the fibroid T2 property (P

Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression; however, the molecular mechanisms underlying ER’s regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways; ETV4 loss impacts chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture where hollow organoids were formed and in vivo in the context of estrogen dependent growth. Our results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells.

The increasing incidence of endometrial cancer (EC), in younger age at diagnosis, calls for new tissue-sparing treatment options. This work aims to evaluate the potential of imiquimod (IQ) in the treatment of low-grade EC. Effects of IQ on the viabilities of Ishikawa and HEC-1A cells were evaluated using MTT assay. The ability of IQ to induce apoptosis was evaluated by testing changes in caspase 3/7 levels and expression of cleaved caspase-3, using luminescence assay and western blot. Apoptosis was confirmed by flow cytometry and the expression of cleaved PARP. Western blot was used to evaluate the effect of IQ on expression levels of Bcl-2, Bcl-xL, and BAX. Finally, the in vivo efficacy of IQ was tested in an EC mouse model. There was a decrease in EC cell viability following IQ treatment as well as increased caspase 3/7 activities, cleaved caspase-3 expression, and Annexin-V/ 7AAD positive cell population. Western blot results showed the ability of IQ in cleaving PARP, decreasing Bcl-2 and Bcl-xL expressions, but not affecting BAX expression. In vivo study demonstrated IQ’s ability to inhibit EC tumor growth and progression without significant toxicity. IQ induces apoptosis in low-grade EC cells in vitro, probably through its direct effect on Bcl-2 family protein expression. In, vivo, IQ attenuates EC tumor growth and progression, without an obvious toxicity. Our study provides the first building block for the potential role of IQ in the non-surgical management of low-grades EC and encouraging further investigations.

Background Until recently, no HPV test had been US FDA-approved for SurePath preservative. Clinical performance remains incompletely understood. The clinical performances of the Cobas HPV Test (Cobas) and Hybrid Capture 2 High-Risk HPV DNA Test (HC2) with PreservCyt and SurePath preservatives were compared. Methods Cervical cytology samples were collected in both preservatives in random order from women age 21+ (n = 244) referred for colposcopy. Before cytology processing and pelleting, SurePath samples were tested by the Cobas test with and without buffered SDS heat pretreatment. SurePath pellets were tested by the HC2 test and by the Cobas test (with pretreatment). Performance characteristics were calculated in relation to cases of cervical in-traepithelial neoplasia grade 2 or higher (CIN2+) as the clinical target outcome. All HPV-positive samples were also genotyped with the Linear Array test. Results CIN2+ was detected in 42 patients (17.2%). For both HPV tests, there was a trend towards higher positivity and sensitivity for SurePath compared to PreservCyt preservative. The Cobas test had higher sensitivity than HC2 and the HC2 test had higher specificity than Cobas. Pretreated SurePath samples produced results similar to untreated ones, despite a two-fold dilution during pretreatment [sensitivity %: 95.1 (82.2 - 99.2) vs. 94.3 (79.5 - 99.0); specificity %: 33.0 (26.6 - 40.1) vs. 33.0 (26.4 - 40.3)]. Conclusions There was good agreement between the preservatives and HPV tests in detecting HPV and between the Cobas and Linear Array tests for genotyping HR-HPV. These trends were not statistically significant due to the limited number of CIN2+ cases. However, these data may help in evaluations of preservative selection for colposcopy samples. Pre-treatment for Cobas testing eliminated invalid results due to clots. The Cobas test has been FDA-approved for use with heat pretreated SurePath samples.

Thermogenic program (also known as browning) is a promising and attractive anti-obesity approach. Islet amyloid polypeptide (IAPP) and irisin have emerged as potential browning hormones that hold high potential to treat obesity. Here, we have constructed a dual browning gene system containing both IAPP and irisin (derived from fibronectin type III domain containing 5; FNDC5) combined with 2A and furin self-cleavage sites. Intraperitoneal administration of the construct complexed with a linear polyethylenimine into diet-induced obese mice demonstrated the elevation of anti-obesogenic effects characterized as the decreased body weight, adiposity, and levels of glucose and insulin. In addition, the construct delivery increased energy expenditure and the expression of core molecular determinants associated with browning. The additional advantages of the dual browning gene construct delivery compared to both single gene construct delivery and dual peptide delivery can be emphasized on efficacy and practicability. Hence, we have concluded that dual browning gene delivery makes it therapeutically attractive for diet-induced obesity treatment.

This project uses an ex vivo human perfusion model for studying transport in benign, fibrous tumors. The uterine arteries were cannulated to perfuse the organ with a buffer solution containing blood vessel stain and methylene blue to analyze intratumoral transport. Gross examination revealed tissue expansion effects and a visual lack of methylene blue in the fibroids. Some fibroids exhibited regions with partial methylene blue penetration into the tumor environment. Histological analysis comparing representative sections of fibroids and normal myometrium showed a smaller number of vessels with decreased diameters within the fibroid. Imaging of fluorescently stained vessels exposed a stark contrast between fluorescence within the myometrium and relatively little within the fibroid tissues. Imaging at higher magnification revealed that fibroid blood vessels were indeed perfused and stained with the lipophilic membrane dye; however, the vessels were only the size of small capillaries and the blood vessel coverage was only 12% that of the normal myometrium. The majority of sampled fibroids had a strong negative correlation (Pearson's r=-0.68 or beyond) between collagen and methylene blue staining. As methylene blue was able to passively diffuse into fibroid tissue, the true barrier to transport in these fibroids is likely high interstitial fluid pressure, correlating with high collagen content and solid stress observed in the fibroid tissue. Fibroids had an average elevated interstitial fluid pressure of 4mmHg compared to -1mmHg in normal myometrium. Our findings signify relationships between drug distribution in fibroids and between vasculature characteristics, collagen levels, and interstitial fluid pressure. Understanding these barriers to transport can lead to developments in drug delivery for the treatment of uterine fibroids and tumors of similar composition.

The combinatorial peptidergic therapy of islet amyloid polypeptide (IAPP) and leptin (LEP) analogues was once an optimistic option in treating obese animals and patients. However, the need for frequent administrations and its negative side effects prevent it from being a viable choice. Here, we developed a combinatorial gene therapy of IAPP and LEP, where two genes are inserted into a single plasmid with self-cleaving furin and 2A sites to treat diet-induced obese (DIO) mice. The developed plasmid DNA (pDNA) individually produced both IAPP and LEP peptides in vitro and in vivo. The pDNA was delivered with a non-viral polymeric carrier, and its once-a-week administrations demonstrated a synergistic loss of body weight and significant reductions of fat mass, blood glucose, and lipid levels in DIO mice. The results suggest that the combinatorial gene therapy would have higher potential than the peptidergic approach for future translation due to its improved practicability.

Endometrial hyperplasia (EH) is a condition originating from uterine endometrial glands undergoing disordered proliferation including the risk to progress to endometrial adenocarcinoma. In recent years, a steady increase in EH cases among younger women of reproductive age accentuates the demand of therapeutic alternatives, which emphasizes that an improved disease model for therapeutic agents evaluation is concurrently desired. Here, a new hormone-induced EH mouse model was developed using a subcutaneous estradiol (E2)-sustained releasing pellet, which elevates the serum E2 level in mice, closely mimicking the effect known as estrogen dominance with underlying, pathological E2 levels in patients. The onset and progression of EH generated within this model recapitulate a clinically relevant, pathological transformation, beginning with disordered proliferation developing to simple EH, advancing to atypical EH, and then progressing to precancerous stages, all following a chronologic manner. Although a general increase in nuclear progesterone receptor (PR) expression occurred after E2 expression, a total loss in PR was noted in some endometrial glands as disease advanced to simple EH. Furthermore, estrogen receptor (ER) expression in the nucleus of endometrial cells was reduced in disordered proliferation and increased when EH progressed to atypical EH and precancerous stages. This EH model also resembles other pathological patterns found in human disease such as leukocytic infiltration, genetic aberrations in β-catenin, and joint phosphatase and tensin homolog/paired box gene 2 (PTEN/PAX2) silencing. In summary, this new and comprehensively characterized EH model is cost-effective, easily reproducible, and may serve as a tool for preclinical testing of therapeutic agents and facilitate further investigation of EH.

Purpose The safe and functional delivery of progesterone through the vaginal route remains an unmet clinical need. The purpose of this work is to prepare a new progesterone (P4) gel for vaginal application using a thermosensitive mucoadhesive polymer, glycol chitin (GC). Method Thermogelling, mucoadhesive, mechanical, and viscoelastic properties of GC and the new formulation were evaluated using rheometry. In vitro release profile and the bioactivity of P4 were determined using vaginal fluid simulant (VFS) pH 4.2, and PR-reporter gene assay, respectively. In vitro safety of the formulations was tested using (VK2/E6E7) vaginal epithelial cell line and Lactobacillus Crispatus. Finally, in vivo safety and the efficacy of this formulation were evaluated using an endometrial hypoplasia mouse model. Results Results shows the aqueous solution of 5%; (w/v) GC loaded with 0.1%; (w/v) P4 prepared in pH 4.2, (GC-P4), forms a thermosensitive mucoadhesive hydrogel and can maintain stable physical properties at 37°C. GC-P4 gel release 50% of P4 in 4 h after exposure to VFS, and no significant decrease in % viability of VK2/E6E7 or Lactobacillus was found after exposure to 5% GC or GC-P4. GC-P4 does not exhibit obvious toxicities to vaginal tissue in vivo even after repeated application. Efficacy studies indicated that GC-P4 was capable of preventing the progression of simple endometrial hyperplasia (SEH) to complex atypical endometrial hyperplasia (CAEH) in vivo. Conclusions Results indicates that GC-P4 retains many characteristics for an effective vaginal delivery system for P4. Therefore we believe that GC-P4 formulation is a promising alternative to current vaginal P4 formulation. Electronic supplementary material The online version of this article (doi:10.1007/s11095-014-1616-8) contains supplementary material, which is available to authorized users.

Despite the development of a myriad of anticancer drugs that appeared promising in preclinical ovarian cancer animal models, they failed to predict efficacy in clinical testing. To improve the accuracy of preclinical testing of efficacy and toxicity, including pharmacokinetic and pharmacodynamic evaluations, a novel animal model was developed and characterized. In this study, murine ID8 (epithelial ovarian cancer [EOC]) cells as injected cell suspensions (ICS) and as intact cultured monolayer cell sheets (CS) were injected or surgically grafted, respectively, into the left ovarian bursa of 6-8 week-old, female C57BL/6 black mice and evaluated at 8 and 12 weeks after engraftment. Tumor volumes at 8 weeks were as follows: 30.712 ± 18.800 mm(3) versus 55.837 ± 10.711 mm(3) for ICS and CS, respectively, p = 0.0990 (n = 5). At 12 weeks, tumor volumes were 128.129 ± 44.018 mm(3) versus 283.953 ± 71.676 mm(3) for ICS and CS, respectively, p = 0.0112 (n = 5). The ovarian weights at 8 and 12 weeks were 0.02138 ± 0.01038 g versus 0.04954 ± 0.00667 g for ICS and CS, respectively (8 weeks), p = 0.00602 (n = 5); and 0.10594 ± 0.03043 g versus 0.39264 ± 0.09271 g for ICS and CS, respectively (12 weeks), p = 0.0008 (n = 5). These results confirm a significant accelerated tumorigenesis in CS-derived tumors compared with ICS-derived tumors when measured by tumor volume/time and ovarian weight/time. Furthermore, the CS-derived tumors closely replicated the metastatic spread found in human EOC and histopathological identity with the primary tumor of origin.

Supplemental digital content is available in the text., Objectives AL3818 (anlotinib) is a receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFR1, VEGFR2/KDR, and VEGFR3), stem cell factor receptor (C-kit), platelet-derived growth factor (PDGFβ), and fibroblast growth factor receptors (FGFR1, FGFR2, and FGFR3). This study evaluates the efficacy of AL3818 studying tumor regression in an orthotopic murine endometrial cancer model. Methods We tested the cytotoxicity of AL3818 on a panel of 7 human endometrial cancer cell lines expressing either wild-type or mutant FGFR2 and also assessed the in vivo antitumor efficacy in a murine, orthotopic AN3CA endometrial cancer model. AL3818 was administered daily per os either alone or in combination with carboplatin and paclitaxel, which represent the current standard of adjuvant care for endometrial cancer. Results AL3818 significantly reduces AN3CA cell number in vitro, characterized by high expression of a mutated FGFR2 protein. Daily oral administration of AL3818 (5 mg/kg) resulted in a complete response in 55% of animals treated and in a reduced tumor volume, as well as decreased tumor weights of AN3CA tumors by 94% and 96%, respectively, following a 29-day treatment cycle. Whereas carboplatin and paclitaxel failed to alter tumor growth, the combination with AL3818 did not seem to exhibit a superior effect when compared with AL3818 treatment alone. Conclusions AL3818 shows superior efficacy for the treatment of endometrial cancer irresponsive to conventional carboplatin and paclitaxel combination and warrants further investigation.

A high-throughput, microfluidic flow cell array (MFCA) system has been modified to enable drug screening against small-volume cell-, and tissue cultures. The MFCA is composed of a 3D channel network that simultaneously flows fluids through forty-eight 830 μm by 500 μm flow cells, which physically divide and fluidically seal an existing culture into multiple compartments when docked onto the surface of a cell or tissue culture dish. The modified system provides temperature (37 °C) and CO2/pH level controls, while continuously flowing solutions (media or other liquid such as drug suspensions) over the cells/tissues. These assays were enhanced and validated using inverted microscopy and fluorescent staining techniques which also allow real time viability and toxicity assessments. This work presents the results of this new generation in vitro drug testing assay performed using this modified MFCA system. This setup allows the testing of 48 drug combinations on 48 different cell-, tissue specimen at once under flow conditions. All 48 flow cells were utilized to test 5 different concentrations of cisplatin (CDDP). CDDP solutions in various concentrations were continually flowed over cultured human ovarian cancer cells for 48 h. Viability assessments were performed using red-orange calcein and SYTOX ® Green nucleic acid stains. Cells were imaged at the beginning and end of the experiment (48 h). In order to compare and validate MFCAs suitability as drug screening assay, MTT assays were performed on cells. We found that both, MTT and MFCA assays generated dose-response curves with similar profiles. Innovative advantages of the MFCA system include the ability of handling smaller amounts of solutions compared to conventional and current state of the art drug screening and cell viability/toxicity methods. It also provides the ability to continually deliver fresh solution to the cell samples, while eliminating wastes that are produced. Based on our here reported findings MFCA may have a strong potential of providing a more physiological model than current state of the art static MTT assays.

Checkpoint blockade immunotherapies harness the host’s own immune system to fight cancer, but only work against tumors infiltrated by swarms of pre-existing T cells. Unfortunately, most cancers to date are immune-deserted. Here, we report a polymer-assisted combination of immunogenic chemotherapy and PD-L1 degradation for efficacious treatment in originally non-immunogenic cancer. “Priming” tumors with backbone-degradable polymer-epirubicin conjugates elicits immunogenic cell death and fosters tumor-specific CD8+ T cell response. Sequential treatment with a multivalent polymer-peptide antagonist to PD-L1 overcomes adaptive PD-L1 enrichment following chemotherapy, biases the recycling of PD-L1 to lysosome degradation via surface receptor crosslinking, and produces prolonged elimination of PD-L1 rather than the transient blocking afforded by standard anti-PD-L1 antibodies. Together, these findings established the polymer-facilitated tumor targeting of immunogenic drugs and surface crosslinking of PD-L1 as a potential new therapeutic strategy to propagate a long-term antitumor immunity, which might broaden the application of immunotherapy to immunosuppressive cancers.

Inactivation of p53 pathway is reported in more than half of all human tumors and can be correlated to malignant development. Missense mutation in the DNA binding region (DBD) of p53 is the most common mechanism of p53 inactivation in cancer cells. The resulting tumor-derived p53 variants, similar to wild-type (wt) p53, retain their ability to oligomerize via the tetramerization domain (TD). Upon hetero-oligomerization, mutant p53 enforces a dominant negative effect over active wt-p53 in cancer cells. To overcome this barrier, we have previously designed a chimeric superactive p53 (p53-CC) with an alternative oligomerization domain capable of escaping transdominant inhibition by mutant p53 in vitro. In this report, we demonstrate the superior tumor suppressor activity of p53-CC and its ability to cause tumor regression of the MDA-MB-468 aggressive p53-dominant negative breast cancer tumor model in vivo. In addition, we illustrate the profound effects of the dominant negative effect of endogenous mutant p53 over wt-p53 in cancer cells. Finally, we investigate the underlying differential mechanisms of activity for p53-CC and wt-p53 delivered using viral-mediated gene therapy approach in the MDA-MB-468 tumor model.

A leading cause of death and suffering in patients with abdominal or pelvic malignancies is progression of peritoneal surface disease. Changes in the use of chemotherapy have shown significant survival benefits for intraperitoneal or combined intraperitoneal and intravenous treatment following optimal surgical cytoreduction. However, broader clinical use of intraperitoneal therapy has not reached its full potential due to limited efficacy, accessibility and nonspecific toxicity. To overcome these problems, we developed a mucoadhesive hybrid gel (HG) for a local, intraperitoneal drug delivery. In vivo studies confirmed reliable adherence and residence of the gel to the peritoneal sidewall for at least 72 h exhibiting no signs of tissue toxicity. Functionally active CDDP was released from HG within 2h and was equal to free CDDP in vitro. Moreover, intraperitoneal application of HG-CDDP significantly enhanced CDDP accumulation in the genomic DNA of peritoneal tissues compared to the same CDDP dose administered intravenously. These findings indicate the potential application of this hybrid gel as a mucoadhesive drug carrier amendable to use for intraperitoneal drug delivery and possible expansion for use on other mucosal surfaces of the female reproductive tract.

In this study, a new thermo-sensitive polymer, glycol chitin, was synthesized by controlled N-acetylation of glycol chitosan and evaluated as a thermogelling system. The physico-chemical properties of glycol chitins with different degrees of acetylation (DA) were investigated in terms of degradation, cytotoxicity, rheological properties, and in vitro and in vivo gel formation. Aqueous solutions of glycol chitins were flowable freely at room temperature but quickly became a durable gel at body temperature. Thermo-reversible sol-gel transition properties were observed with fast gelation kinetics. Glycol chitins with higher DA showed faster degradation in the presence of lysozyme. They exhibited no significant biological toxicity against human cell lines. An anti-cancer drug, doxorubicin, could be incorporated into the hydrogel by a simple mixing process and released in a sustained pattern over 13 days. Our findings suggest that glycol chitins could be useful as a new thermogelling biomaterial for drug delivery and injectable tissue engineering.

The purpose of this study was to model data from a head to head comparison of the in vivo fate of hyper-branched PAMAM dendrimers with linear HPMA copolymers in order to understand the influence of molecular weight (MW), hydrodynamic size (Rh) and polymer architecture on biodistribution in tumor-bearing mice using compartmental pharmacokinetic analysis. Plasma concentration data was modeled by two-compartment analysis using Winnonlin® to obtain elimination clearance (E.CL) and plasma exposure (AUCplasma). Renal clearance (CLR) was calculated from urine data collected over 1 week. A plasma-tumor link model was fitted to experimental plasma and tumor data by varying the tumor extravasation (K4, K6) and elimination (K5) rate constants using multivariable constrained optimization solver in Matlab®. Tumor exposures (AUCtumor) were computed from area under the tumor concentration time profile curve by the linear trapezoidal method. Along with MW and Rh, polymer architecture was critical in affecting the blood and tumor pharmacokinetics of the PAMAM-OH dendrimers and HPMA copolymers. Elimination clearance decreased more rapidly with increase in hydrodynamic size for PAMAM-OH dendrimers as compared to HPMA copolymers. HPMA copolymers were eliminated renally to a higher extent than PAMAM-OH dendrimers. These results are suggestive of a difference in extravasation of polymers of varying architecture through the glomerular basement membrane. While the linear HPMA copolymers can potentially reptate through a pore smaller in size than their hydrodynamic radii in a random coil conformation, PAMAM dendrimers have to deform in order to permeate across the pores. With increase in molecular weight or generation, the deforming capacity of PAMAM-OH dendrimers is known to decrease, making it harder for higher generation PAMAM-OH dendrimers to sieve through the glomerulus as compared to HPMA copolymers of comparable molecular weights. PAMAM-OH dendrimer had greater tumor extravsation rate constants and higher tumor to plasma exposure ratios than HPMA copolymers of comparable molecular weights which indicated that in the size range studied, when in circulation, PAMAM-OH dendrimers had a higher affinity to accumulate in the tumor than the HPMA copolymers.

Spherical and rod-shaped gold nanoparticles with surface poly (ethylene glycol) (PEG) chains were characterized for size, shape, charge, poly dispersity and surface plasmon resonance. The nanoparticles were injected intravenously to 6–8 weeks old female nu/nu mice bearing orthotopic ovarian tumors and their biodistribution in vital organs was compared. Gold nanorods were taken up to a lesser extent by the liver, had longer circulation time in the blood, and higher accumulation in the tumors, compared with their spherical counterparts. The cellular uptake of PEGylated gold nanoparticles by a murine macrophage-like cell line as a function of geometry was examined. Compared to nanospheres, PEGylated gold nanorods were taken up to a lesser extent by macrophages. These studies point to the importance of gold nanoparticle geometry and surface properties on transport across biological barriers.

In vitro fertilization has experienced phenomenal progress in the last thirty years and awaits the additional refinement and enhancement of medication delivery systems. Opportunity exists for the novel delivery of gonadotropins, progesterone and other adjuvants. This review highlights the rationale for various medications, present delivery methods and introduces the status of novel ideas and possibilities.

Biodegradable cationic polymers have become promising alternatives to traditional polycationic gene delivery systems in which the high charge densities of high molecular weight polymers contribute significantly to cellular toxicities. Previous research has shown that biodegradable, multiblock copolymers (MBC), PEG-PLL-g-16% His, are efficient gene carriers with negligible cellular toxicities. The present research was designed to characterize the polymer degradation as well as to determine the biodistribution of the MBC after systemic administration. Polymer degradation was performed in buffer as a function of pH, in serum and within polymer/pDNA complexes. The MBC exhibited exponential decay with a half-life (t1/2) of approximately 14 min at pH 9.0, approximately 5 h at pH 7.4 and approximately 2 h in serum. However, there was little or no degradation observed at pH 4.0 and the MBC within the complexes degraded between 4 and 8 h in serum. Biodistribution data performed with fluorescently labeled polymer and pDNA revealed that intact complexes remained in the blood up to 3 days, which was also reflected in the organs as a function of time. Therefore, the cumulative data suggest that PEG may be sterically stabilizing complexes in vivo via dysopsonization in which serum proteins mask the complexes from elements of the reticuloendothelial system (RES).

Development of improved gene transfer methods is needed for gene therapy to achieve its clinical potential. The use of biocompatible polymeric gene carriers has shown effectiveness in overcoming the current problems associated with viral vectors in safety, immunogenicity and mutagenesis. Previous work has demonstrated that repeated, local, non-viral interleukin-12 (IL-12) gene delivery successfully slows down tumor progression, while improving immunogenicity. Combining IL-12 gene delivery with systemic paclitaxel (PCT) chemotherapy as a treatment for various subcutaneous mouse mammary carcinomas, we used PCT with either a biodegradable polymeric solubilizer, HySolv or Cremophor EL for systemic treatment and injected water soluble lipopolymer (WSLP)/plasmid-encoding IL-12 gene (p2CMVmIL-12) complexes local once every week. The amount of lung metastases being essential for survival as well as subcutaneous tumor volume were compared against untreated controls. We showed inhibition of tumor growth and decreased lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the PCT only treated groups. Compared to Cremophor, HySolv performed better alone or in combination with IL-12. Using polymeric vectors as gene carrier systems in combination with improved systemic therapies provide evidence for the efficacy and feasibility of polymer-based drug delivery systems. Especially local cytokine gene delivery showed augmentation of systemic chemotherapy while reducing the hosts risk for further systemic toxicity.

Repeated, local, nonviral IL12 (interleukin-12) gene delivery decreased tumor progression and increased immunogenicity. We combined our IL12 gene delivery with systemic paclitaxel chemotherapy as a treatment for paclitaxel (PCT)-resistant 4T1 subcutaneous mouse mammary carcinomas and PCT-sensitive, immunogenic/nonimmunogenic tumors. We mixed PCT with either a biodegradable polymeric solubilizer, HySolv, or Cremophor EL for bimonthly systemic treatments and injected water-soluble lipopolymer (WSLP)/p2CMVmIL-12 (plasmid encoding IL12 gene) complexes locally every week. We compared treated subcutaneous tumor volume and lung metastasis with controls. HySolv alone performed better compared to Cremophor EL in combination with WSLP/p2CMVmIL-12. We showed inhibition of 4T1 tumor growth and lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the paclitaxel-only treated groups. In parallel experiments we also demonstrated additive responses for tumor growth and number of lung metastases within other PCT-sensitive mammary tumor models using this combination strategy. Our combination therapy provides evidence for the efficacy and feasibility of improved drug delivery systems. Local cytokine gene delivery can augment local and systemic chemotherapy without placing the host at risk for further systemic toxicity.

Endometrial cancer (EC) is the most common uterine malignancy in industrialized countries and is the most frequent gynecologic cancer in the United States with an estimated 60,050 new cases in 2016. Traditionally observed in peri-, postmenopausal women, the incidence rate of EC has steadily increased among younger women of reproductive age over the past two decades partially owing to a more sedentary lifestyle and rampant obesity epidemic. Surgery in combination with either radio- and or chemotherapy is commonly curative for the early stage of the disease. However, patients with recurrent or metastatic disease, who only respond poorly to cytotoxic chemotherapy are in desperate need of therapeutic alternatives as are patients of reproductive age. Thus, innovative, safe and more effective therapies are mandated. Several studies have demonstrated the increased expression of various tyrosine kinase receptors involved in the development of EC. We hypothesized that a targeted therapeutic approach using a pan-tyrosine kinase inhibitor would prevent the proliferation and progression of EC. Anlotinib (AL3818) is a multi-targeted receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors 1 to 3 (VEGFR1-3), stem cell factor receptor (C-kit), platelet-derived growth factor (PDGFβ), and fibroblast growth factor receptors 1 to 3 (FGFR1-3). We tested a panel of seven human endometrial cancer cell lines (AN3Ca, Ishikawa, HEC1A, HEC1B, KLE, MFE280 and MFE296) to assess the cytotoxicity of anlotinib in vitro. A lower IC50 value was observed in AN3Ca cells (25 nM), a cell line characterized by a high level of expression of an FGFR2 mutant protein, while cell lines expressing FGFR2 wild-type are less sensitive to anlotinib (HEC1A: 33 μM, or HEC1B:36 μM). Somatic mutations of FGFR2 have been observed in 12% of EC, indicating the potential for this drug in a subset of EC patients. In summary, these results suggest improved efficacy of anlotinib for the treatment of ECs expressing an increased level of FGFR2 mutant proteins. The safety and efficacy of anlotinib are currently being evaluated in an orthotopic AN3Ca mouse model of EC. Experimental groups include a tumor bearing control not subjected to any treatment, anlotinib alone, carboplatin/paclitaxel, a combination of anlotinib and carboplatin/paclitaxel. Primary study endpoints include tumor growth retardation, the delayed onset of local metastases as well as reduced toxicity as secondary endpoint. Citation Format: Sebastien Taurin, Chieh-Hsiang Yang, Maria Reyes, Sungpil Cho, Elke A. Jarboe, Theresa L. Werner, Demetrius M. Coombs, Paul Chen, Margit M. Janát-Amsbury. Treatment of endometrial cancer cells with a new small tyrosine kinase inhibitor targeting mutated fibroblast growth factor receptor-2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3244. doi:10.1158/1538-7445.AM2017-3244

Endometrial cancer (EC) is the most common gynecological malignancy among American women. Steadily increasing numbers of newly diagnosed cases and deaths emphasize the necessity of improving current management strategies. Rather than the commonly known two types of EC based on histological classification, the Cancer Genome Atlas (TCGA) Research Network recently reported a reclassification that categorizes EC into four subtypes based on genomic characterization. Molecular similarities between these subtypes and other cancers, including colorectal, ovarian, and breast cancers, were identified. But to date, no prospective validation has delivered an improved understanding on how to translate these findings to the clinic in the form of treatments adapting genomic guidance. Objectives: To evaluate drug response according to TCGA classification using a panel of twenty-seven established endometrial cancer patient-derived xenografts (EC-PDXs). Methods: Twenty-seven EC-PDXs representing various stages of disease and histological subtypes were orthotopically transplanted and propagated over multiple generations. Upon thorough characterization including histology, metastatic status, and molecular features, EC-PDXs were further categorized into the four TCGA molecular subtypes on the basis of somatic copy number variations (CNV), microsatellite instability (MSI), and POLE mutations. A PDX clinical trial (PCT) was performed to re-evaluate current standard treatments in comparison to therapeutic agents adopted from treatment regimens for other cancer types based on their molecular similarities. Results: EC-PDXs established from patients with recurrent disease exhibited minimal activity to first-line chemotherapeutic drugs, as expected, but remained sensitive to other second-line drugs. None of the tested drugs exhibited sufficient activity in POLE EC-PDXs. MSI EC-PDXs were found to be less sensitive to platinum-based treatment and resistant to 5-FU as reported for MSI colorectal cancer. CNV-high EC-PDXs were more sensitive to drugs adopted from current ovarian and breast cancer treatment regimens. Conclusions: This is the first study that demonstrates the utility of TCGA classification for treatment guidance based on a PCT. Patients with recurrent and persistent disease are in desperate need of new therapeutic options. Our findings open up the possibility to utilize drugs which are currently used in the treatment of other cancers for the treatment of EC based on molecular similarities. In addition, this is the first study that successfully demonstrated the use of molecular classification to enable the prediction of drug response, and thus may facilitate the refinement of current EC management in a more precise and personalized way. Citation Format: Chieh-Hsiang Yang, David K. Gaffney, Theresa L. Werner, Elke A. Jarboe, Jason Gertz, Katherine E. Varley, Matthew Peterson, Margit M. Janat-Amsbury. Genomic reclassification of endometrial carcinoma predicts drug response and facilitates refinement of current management based on PDX-guided efficacy outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2017-1756

Background Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE. Methods The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509. Results Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p = 0.034). Conclusions Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-752) contains supplementary material, which is available to authorized users.

The major drawback hampering siRNA therapies from being more widely accepted in clinical practice is its insufficient accumulation at the target site mainly due to poor cellular uptake and rapid degradation in serum. Therefore, we designed a novel polymeric siRNA carrier system, which would withstand serum-containing environments and tested its performance in vitro as well as in vivo. Delivering siRNA with a system combining an arginine-grafted bioreducible polymer (ABP), microbubbles (MBs), and ultrasound technology (US) we were able to synergize the advantages each delivery system owns individually, and created our innovative siRNA-ABP-MB (SAM) complexes. SAM complexes show significantly higher siRNA uptake and VEGF protein knockdown in vitro with serum-containing media when compared to naked siRNA, and 25k-branched-polyethylenimine (bPEI) representing the current standard in nonviral gene therapy. SAM complexes activated by US are also able to improve siRNA uptake in tumor tissue resulting in decelerating tumor growth in vivo.

Cancer nanotechnology, defined as nanotechnology applicable for diagnosis, prognosis, and therapy of cancer, offers exciting possibilities but also challenges. Although nanotechnology became fruitful in the field of diagnostics, to date the desire of improving current cancer treatments through nanotechnology has surpassed the research or development stage only in a handful of cases. However, efforts to clinically translate more of these treatments remain highly focused. Drug-bearing nanoparticles, which are usually injected directly into the bloodstream, are sent on a journey full of obstacles to overcome such as bypassing RES organs and crossing endothelial cells, as well as various membranes of cellular and extracellular compartments, before finally encountering tumor tissue. Therefore, a cornucopia of different nanoparticle designs varying in size, architecture, and surface properties has been designed to be deployed to tumor sites within the human body either passively or promoting the association with a particular cell or tissue type through targeting. Early clinical results with nanomedicines were indeed able to show reduced toxicity, improving patients’ quality of life, but are still in need of stronger coupling with superior clinical efficacy to help justify the often high costs associated with this new class of therapeutics and their complexity. Nanotechnology aiming for the development of novel cancer therapeutics must focus in on potential, unforeseen toxicities and also consider the emerging role of epigenetics. The following chapter introduces most of the basic nanoconstructs developed to date, which inevitably will continue to evolve and eventually find an important place in health care, though to become the cancer medicines of tomorrow, nanotechnology’s future success and translation must early on be connected to diagnostic and therapeutic potential for clinical applications. To be equally aware of potential benefits, risks and costs will be necessary to stimulate future research with emphases on how to improve therapeutic outcomes such as the extension of life in patients with cancer in times burdened with unsustainable healthcare costs.

To develop and characterize the pre-clinical suitability of a syngeneic mouse epithelial ovarian cancer model in immunocompetent hosts.ID8 mouse ovarian surface epithelium cells were implanted into the left ovarian bursa of C57BL/6 mice. Using conventional as well as ultrasound-based techniques and histopathological analysis, the tumor weights, volumes, metastases, ascites and vascularity were observed over a period of 16 weeks.Ovarian weights and volume increased 12- and 7-fold, respectively. Ultrasound measurements of ovarian ID8 tumors correlated with the actual size obtained following surgical excision. Ascites and metastasis were first observed at 12 weeks post-orthotopic implantation. Histopathological analysis indicated similarities between orthotopically-generated ovarian tumors and human ovarian tumors. However, there was less evidence of angiogenesis in this animal model.The development of this mouse model closely replicates characteristics seen in human ovarian cancer with feasibility of using ultrasound to assess tumor formation, progression and vascularization.

Endometrial cancer (EC) is the fourth most frequently diagnosed gynecological cancer among American Women. Steady increased numbers of newly diagnosed cases and deaths necessitates an added attention. Despite survival rates between 60-80% for localized, early stage disease, only marginal advances have been made in the treatment of distant (16%) and recurrent (15% of early stage) EC over the past two decades. New treatment strategies, guided by more predictive preclinical disease models incorporating molecular characteristics are crucial. Recent findings from The Cancer Genome Atlas (TCGA) Research Network suggested that the current histo-pathologically-guided classification of EC may not suffice to direct treatment recommendations, and introduced the potential adequacy of genomic profiling for reclassifying EC. However, these findings are hampered by the lack of appropriate in vivo models, which adequately facilitate obtaining the necessary preclinical evidence before translating findings to genome-guided clinical trials. Objectives: To establish patient derived orthotopic endometrial tumor grafts recapitulating histology, metastasis, and molecular characteristics of the original tumor specimen, and integrating TCGA genomic characterization to facilitate the development of genomic-guided treatment. Methods: Patient derived EC tissues were orthotopically transplanted to murine uteri and propagated over multiple generations. Generated tumor grafts were characterized for metastases, histopathology, and molecular profiles to ensure grafts resemblance of the original tumor samples. Tumor grafts were then further categorized into the four reported TCGA clusters on the basis of mutation frequency, somatic copy number alterations, microsatellite instability, and POLE mutation. Results: Twenty EC tumor grafts were successfully generated. Histological features, including tumor grade and hormone receptor status, as well as genomic profiles were maintained for up to 7 generations. Xenografts were capable to form metastasis closely parallel to clinical relevant sites. Results from a survival study demonstrated that the established tumor grafts reflected clinical stage-, and grade-based disease progression, and recapitulate TCGA cluster-based survival findings. Conclusions: This study reports the first successful generation of orthotopic EC patient-derived tumor grafts, which retain crucial histo-pathological characteristics, the capacity to form distant metastasis following known clinical patterns, as well as recapitulating molecular features of the original human tumor specimen. Additionally, our model can indeed be used as a tool to investigate the need for reclassifying EC into four clusters rather than two simplified disease subtypes and feasibility in directing genomic-guided treatment based on TCGA recommendations. Citation Format: Chieh-Hsiang Yang, Elke A. Jarboe, Jason Gertz, Katherine E. Varley, C. Matthew Peterson, Margit M. Janát-Amsbury. Integrated genomic characterization of endometrial cancer tumor grafts: a step toward genomic-guided treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 648.

The performance and safety of current antineoplastic agents, particularly water-insoluble drugs, are still far from satisfactory. For example, the currently widely used Cremophor EL®-based paclitaxel (PTX) formulation exhibits pharmacokinetic concerns and severe side effects. Thus, the concept of a biodegradable polymeric drug-delivery system, which can significantly improve therapeutic efficacy and reduce side effects is advocated. The present work aims to develop a new-generation of long-circulating, biodegradable carriers for effective delivery of PTX. First, a multiblock backbone biodegradable N-(2-hydroxypropyl)methacrylamide(HPMA) copolymer- PTX conjugate (mP-PTX) with molecular weight (Mw) of 335 kDa was synthesized by RAFT (reversible addition-fragmentation chain transfer) copolymerization, followed by chain extension. In vitro studies on human ovarian carcinoma A2780 cells were carried out to investigate the cytotoxicity of free PTX, HPMA copolymer-PTX conjugate with Mw of 48 kDa (P-PTX), and mP-PTX. The experiments demonstrated that mP-PTX has a similar cytotoxic effect against A2780 cells as free PTX and P-PTX. To further compare the behavior of this new biodegradable conjugate (mP-PTX) with free PTX and P-PTX in vivo evaluation was performed using female nu/nu mice bearing orthotopic A2780 ovarian tumors. Pharmacokinetics study showed that high Mw mP-PTX was cleared more slowly from the blood than commercial PTX formulation and low Mw P-PTX. SPECT/CT imaging and biodistribution studies demonstrated biodegradability as well as elimination of mP-PTX from the body. The tumors in the mP-PTX treated group grew more slowly than those treated with saline, free PTX, and P-PTX (single dose at 20 mg PTX/kg equivalent). Moreover, mice treated with mP-PTX had no obvious ascites and body-weight loss. Histological analysis indicated that mP-PTX had no toxicity in liver and spleen, but induced massive cell death in the tumor. In summary, this biodegradable drug delivery system has a great potential to improve performance and safety of current antineoplastic agents.

The biodistribution profile of a series of linear N-(2-hydroxylpropyl)methacrylamide (HPMA) copolymers was compared with that of branched poly(amido amine) dendrimers containing surface hydroxyl groups (PAMAM-OH) in orthotopic ovarian-tumor-bearing mice. Below an average molecular weight (MW) of 29 kDa, the HPMA copolymers were smaller than the PAMAM-OH dendrimers of comparable molecular weight. In addition to molecular weight, hydrodynamic size and polymer architecture affected the biodistribution of these constructs. Biodistribution studies were performed by dosing mice with (125)iodine-labeled polymers and collecting all major organ systems, carcass, and excreta at defined time points. Radiolabeled polymers were detected in organ systems by measuring gamma emission of the (125)iodine radiolabel. The hyperbranched PAMAM dendrimer, hydroxyl-terminated, generation 5 (G5.0-OH), was retained in the kidney over 1 week, whereas the linear HPMA copolymer of comparable molecular weight was excreted into the urine and did not show persistent renal accumulation. PAMAM dendrimer, hydroxyl-terminated, generation 6.0 (G6.0-OH), was taken up by the liver to a higher extent, whereas the HPMA copolymer of comparable molecular weight was observed to have a plasma exposure three times that of this dendrimer. Tumor accumulation and plasma exposure were correlated with the hydrodynamic sizes of the polymers. PAMAM dendrimer, hydroxyl-terminated, generation 7.0 (G7.0-OH), showed extended plasma circulation, enhanced tumor accumulation, and prolonged retention with the highest tumor/blood ratio for the polymers under study. Head-to-head comparative study of HPMA copolymers and PAMAM dendrimers can guide the rational design and development of carriers based on these systems for the delivery of bioactive and imaging agents.

Attempted surgical removal of advanced stage peritoneal cancers is mostly insufficient and often fails in areas affected by disseminated disease. But the successful elimination of peritoneal surface spread is known to have a significant impact on patient survival. However, standard therapies such as surgical cytoreduction and systemic chemotherapy combined with radiation have only shown limited-efficacy, accessibility, and nonspecific toxicity as well as frequent development of multidrug resistance (MDR). To overcome those limitations, we previously reported about the development of a mucoadhesive, chitosan-hybrid gel (CS(BCDDP)) embedded with cisplatin (CDDP) containing alginate beads for intraperitoneal administration (# 1948, AACR 2012). Here we report additional results illustrating CDDP release from CS(BCDDP) at different pH in vitro, CDDP accumulation to genomic DNA (gDNA) isolated from tissues as well as toxicity data in vivo. To determine the amount of CDDP released from CS (BCDDP) in vitro, B50μgCDDP, CDDP-chitosan (CS50μgCDDP) and CS (B50μgCDDP) pellets (8 mm ø, 1.5 mm thickness) were prepared and placed into a 6 well plate containing 2 ml/well PBS (pH 6, 7, 7.4 and 8). Multiple samples were collected during the 24 h incubation (37°C, 100rpm) period. The majority (75%) of CDDP was successfully released from CS(BCDDP) within the first 2 h at pH 7.4 showing the rate of drug release to be inversely correlated with pH yielding a more rapid release under acidic pH conditions. To assess CDDP accumulation to gDNA, inductively coupled plasma mass spectrometry (ICP-MS) was performed with tissue samples obtained from the left peritoneal sidewall of female nu/nu mice 24 hours after treatment with CS50μgCDDP, CS(B50μgCDDP), intravenously (i.v.) CDDP(IV50μgCDDP) and intraperitoneal (i.p) CDDP (IP50μgCDDP), respectively. The results demonstrated a greater than three-time enhancement of CDDP-accumulation to the gDNA from CS(B50μgCDDP) when compared to CS50μgCDDP and IV50μgCDDP administration. In addition, CS(B50μgCDDP) showed similar levels of CDDP adduction compared to direct IP50μgCDDP administration. Moreover, assessment of CDDP accumulation in kidneys and blood 24 h following treatment with either IP50μgCDDP or CS(B50μgCDDP), demonstrated significantly decreased kidney toxicity from CS(B50μgCDDP) when compared to IP50μgCDDP. Our results, therefore strongly suggest that administration of this hybrid gel directly to mucosal surfaces such as the peritoneal cavity is feasible making it an advantageous, safe and non-toxic intraperitoneal drug delivery system for the treatment of disseminated peritoneal cancers such as advanced or recurrent ovarian cancer. Currently ongoing experiments are evaluating the efficacy and safety of our hybrid gel/CDDP in an orthotopic peritoneal cancer animal model. Citation Format: Sungpil Cho, Yongen Sun, Elke A. Jarboe, Andrew P. Soisson, Mark K. Dodson, David K. Gaffney, C.Mattew Peterson, Margit M. Janat-Amsbury. Tissue accumulation and toxicity of platinum released from a novel chitosan hybrid gel for intraperitoneal drug delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5402. doi:10.1158/1538-7445.AM2014-5402

Ovarian cancer is the second most frequent invasive malignancy of the female genital tract and the most common cause of death among women with gynecologic malignancies. Although epithelial ovarian cancer (EOC) accounts for over 90% of all ovarian malignancies, the lack of proper animal models that mimic the development and consequences of human EOC limits the ability of current preclinical ovarian cancer models to predict treatment response. The orthotopic development of syngeneic EOC within its relevant tumor microenvironment is an experimental ideal. Here, we demonstrate the development and characterization of a syngeneic EOC mouse model in immunecompetent C57BL/6 mice by orthotopic implantation of ID 8 mouse ovarian cancer cells. Using conventional and ultrasound-based techniques to compare tumor weight, volume and vascularity we followed the development of primary tumors, metastases and ascites over 16 weeks. ID 8 cells were implanted directly underneath the left ovarian bursa. Normal saline was injected to the contralateral ovary as control. Ovarian tumors grew consistently throughout the study period; ovarian weight and volume increased twelve and seven fold, respectively, compared to the contralateral, non-cancerous ovary. Ultrasound measurements of primary tumors and surrounding tumor tissue correlated with the actual size after surgical tumor harvest. Abdominal ascites were first observed at 12 weeks post orthotopic ID8 implantation with volume changes correlating with changes in abdominal circumference. Metastatic lesions were identified by ultrasound at 12 weeks after orthotopic ID8 implantation; sites included peritoneum, liver, and intestine. Histopathological analysis of tumors and metastases indicated similarities between orthotopic ID8 ovarian tumors and human ovarian tumors, except a significantly lower formation of angiogenic vasculature within the ID8 tumors. This study confirms the successful development of a mouse model that closely replicates characteristics seen in human ovarian cancer patients. It shows the feasibility of using ultrasound to assess tumor formation, progression and vascularization. Furthermore, we demonstrate potential shortcomings, which could significantly limit the use of current animal models predicting therapeutic efficacy of novel agents, especially anti-angiogenic therapeutics, impacting the translation of preclinical information to actual clinical outcomes. Citation Format: Sungpil Cho, Yongen Sun, Andrew P. Soisson, Mark K. Dodson, C. Matthew Peterson, Elke A. Jarboe, Anne M. Kennedy, Margit M. Janat-Amsbury. Characterization and evaluation of a mouse ovarian cancer model for its preclinical suitability. [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 325. doi:10.1158/1538-7445.AM2013-325

Cervical cancer is one of the leading gynecological malignancies affecting women worldwide. Although surgical cytoreduction and systemic chemotherapy combined with radiation are standard therapies, accessibility to such treatment as well as nonspecific toxicity limit its efficacy. We hypothesize that the site-specific local application of anticancer drugs utilizing innovative delivery technologies will improve efficacy, reduce toxicity and provide cost-effective, accessible therapeutic options. To test this hypothesis, we developed a mucoadhesive, polymeric hybrid chitosan gel embedding drug containing alginate beads. An optimized gel was achieved by mixing 1% chitosan (w/v) with glycerol dissolved in acetic acid (AA) solution at a volume ratio of 1:2 or 1:3 (2% AA:glycerol). Alginate beads were formed by dropping alginate solution (2%, w/v) into various concentrations of CaCl2 solution. In testing swelling properties, dried alginate beads transforming into wet ones were capable of releasing the drug load. Beads obtained from 1% CaCl2 solution demonstrated optimal and stable swelling properties that were 40 times greater than other formulations. Such beads, loaded with cisplatin (CDDP) dissolved in 0.9% NaCl were prepared. The loaded CDDP concentration was confirmed by colorimetric assay with Sn2Cl2 after re-swelling of the dried beads. Drug loaded-beads contained an average of 0.2mg of CDDP per 10 mg of dried beads. There was a linear correlation (R2 = 0.92) between the swelling ratio of the alginate beads and the amount of CDDP released, allowing the ability to estimate CDDP concentration based on the swelling ratio. Drug-loaded dried alginate beads were placed into a chitosan solution resulting in a hybrid hydrogel. To test the feasibility of local application of the gel on a mucosal surface, we investigated the swelling and mucoadhesive properties on a 4cm2 section of mouse peritoneal sidewall. We confirmed the complete swelling of beads within the hybrid gel suggested sufficient release of CDDP from the beads onto a biological surface. Mucoadhesive properties and toxicity were evaluated in vivo by surgically placing a 1cm2 sized gel pellet into the left peritoneal sidewall of immune competent female mice. The hybrid gel remained in place for 72 hours exhibiting no signs of local inflammation, tissue damage or systemic toxicity. Our results strongly suggest the feasibility of application of this hybrid gel to various mucosal surfaces. Ongoing experiments will evaluate the efficacy and safety of our hybrid gel/CDDP as a radiation enhancer in the treatment of cervical cancer and for the treatment of cervical dysplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3232. doi:10.1158/1538-7445.AM2011-3232