A Phase I Study of Acapatamab, a Half-life Extended, PSMA-Targeting Bispecific T-cell Engager for Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Safety and efficacy of acapatamab, a prostate-specific membrane antigen (PSMA) x CD3 bispecific T-cell engager were evaluated in a first-in-human study in metastatic castration-resistant prostate cancer (mCRPC). PATIENTS AND METHODS: Patients with mCRPC refractory to androgen receptor pathway inhibitor therapy and taxane-based chemotherapy received target acapatamab doses ranging from 0.003 to 0.9 mg in dose exploration (seven dose levels) and 0.3 mg (recommended phase II dose) in dose expansion intravenously every 2 weeks. Safety (primary objective), pharmacokinetics, and antitumor activity (secondary objectives) were assessed. RESULTS: In all, 133 patients (dose exploration, n = 77; dose expansion, n = 56) received acapatamab. Cytokine release syndrome (CRS) was the most common treatment-emergent adverse event seen in 97.4% and 98.2% of patients in dose exploration and dose expansion, respectively; grade ≥ 3 was seen in 23.4% and 16.1%, respectively. Most CRS events were seen in treatment cycle 1; incidence and severity decreased at/beyond cycle 2. In dose expansion, confirmed prostate-specific antigen (PSA) responses (PSA50) were seen in 30.4% of patients and radiographic partial responses in 7.4% (Response Evaluation Criteria in Solid Tumors 1.1). Median PSA progression-free survival (PFS) was 3.3 months [95% confidence interval (CI): 3.0-4.9], radiographic PFS per Prostate Cancer Clinical Trials Working Group 3 was 3.7 months (95% CI: 2.0-5.4). Acapatamab induced T-cell activation and increased cytokine production several-fold within 24 hours of initiation. Treatment-emergent antidrug antibodies were detected in 55% and impacted serum exposures in 36% of patients in dose expansion. CONCLUSIONS: Acapatamab was safe and tolerated and had a manageable CRS profile. Preliminary signs of efficacy with limited durable antitumor activity were observed. Acapatamab demonstrated pharmacokinetic and pharmacodynamic activity.

Inhibition of Immunosuppressive Tumors by Polymer-Assisted Inductions of Immunogenic Cell Death and Multivalent PD-L1 Crosslinking.

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.

ETV4 Is Necessary for Estrogen Signaling and Growth in 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. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1234/F1.large.jpg.

A tissue preparation to characterize uterine fibroid tissue properties for thermal therapies.

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 < 0.05). Finally, the developed perfusion protocol successfully maintained tissue viability in ex vivo tissues as evaluated through histological analysis. CONCLUSIONS: This study developed an MR-compatible extracorporeal perfusion technique that effectively maintains tissue viability, allowing for the direct measurement of patient-specific MR, thermal, and acoustic property values for both fibroid and myometrium tissues. These measured tissue property values will enable further development and validation of treatment planning models that can be utilized during MRgFUS uterine fibroid treatments.

Clinical Performance of SurePath™ Preservative Compared to PreservCyt® with Cobas® and Hybrid Capture® 2 HPV Tests in a Colposcopy Population.

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.

Clinical and Genomic Crosstalk between Glucocorticoid Receptor and Estrogen Receptor α In Endometrial Cancer.

Steroid hormone receptors are simultaneously active in many tissues and are capable of altering each other's function. Estrogen receptor α (ER) and glucocorticoid receptor (GR) are expressed in the uterus, and their ligands have opposing effects on uterine growth. In endometrial tumors with high ER expression, we surprisingly found that expression of GR is associated with poor prognosis. Dexamethasone reduced normal uterine growth in vivo; however, this growth inhibition was abolished in estrogen-induced endometrial hyperplasia. We observed low genomic-binding site overlap when ER and GR are induced with their respective ligands; however, upon simultaneous induction they co-occupy more sites. GR binding is altered significantly by estradiol with GR recruited to ER-bound loci that become more accessible upon estradiol induction. Gene expression responses to co-treatment were more similar to estradiol but with additional regulated genes. Our results suggest phenotypic and molecular interplay between ER and GR in endometrial cancer.

Endometrial Cancers Harboring Mutated Fibroblast Growth Factor Receptor 2 Protein Are Successfully Treated With a New Small Tyrosine Kinase Inhibitor in an Orthotopic Mouse Model.

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 (PDGFß), 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.

Use of a highly parallel microfluidic flow cell array to determine therapeutic drug dose response curves.

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.

Imiquimod Induces Apoptosis in Human Endometrial Cancer Cells In vitro and Prevents Tumor Progression In vivo.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Design and Characterization of Bioengineered Cancer-Like Stem Cells.

Cancer stem cells (CSCs) are a small subset of cancer cells responsible for maintenance and progression of several types of cancer. Isolation, propagation, and the differentiation of CSCs in the proper stem niches expose the intrinsic difficulties for further studies. Here we show that induced cancer like stem cells (iCLSCs) can be generated by in vitro oncogenic manipulation of mouse embryonic stem cells (mESCs) with well-defined oncogenic elements; SV40 LTg and HrasV12 by using a mouse stem virus long terminal repeat (MSCV-LTR)-based retroviral system. The reprogrammed mESCs using both oncogenes were characterized through their oncogenic gene expression, the enhancement of proliferation, and unhampered maintenance of stem properties in vitro and in vivo. In addition, these transformed cells resulted in the formation of malignant, immature ovarian teratomas in vivo. To successfully further expand these properties to other organs and species, more research needs to be done to fully understand the role of a tumor- favorable microenvironment. Our current study has provided a novel approach to generate induced cancer like stem cells through in vitro oncogenic reprogramming and successfully initiated organ-specific malignant tumor formation in an orthotopic small animal cancer model.

Enhanced thermogenic program by non-viral delivery of combinatory browning genes to treat diet-induced obesity in mice.

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.

Uterine perfusion model for analyzing barriers to transport in fibroids.

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.

Combinatorial gene construct and non-viral delivery for anti-obesity in diet-induced obese mice.

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.

An estrogen-induced endometrial hyperplasia mouse model recapitulating human disease progression and genetic aberrations.

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 ß-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.

Thermosensitive progesterone hydrogel: a safe and effective new formulation for vaginal application.

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.

An immunocompetent, orthotopic mouse model of epithelial ovarian cancer utilizing tissue engineered tumor cell sheets.

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.

Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

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.

Ultrasound-assisted siRNA delivery via arginine-grafted bioreducible polymer and microbubbles targeting VEGF for ovarian cancer treatment.

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.

Mucoadhesive hybrid gel improves intraperitoneal platinum delivery.

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.

Characterization and evaluation of pre-clinical suitability of a syngeneic orthotopic mouse ovarian cancer model.

BACKGROUND/AIM: To develop and characterize the pre-clinical suitability of a syngeneic mouse epithelial ovarian cancer model in immunocompetent hosts. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.