Yilin Zhang

Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.

Reversal of Chemoresistance in Ovarian Cancer by Co-Delivery of a P-Glycoprotein Inhibitor and Paclitaxel in a Liposomal Platform

The overexpression of permeability-glycoprotein (P-gp), an ABC transporter involved in the cellular exclusion of chemotherapeutic drugs, is a major factor in paclitaxel-resistant ovarian cancer. However, in clinical trials, co-administration of P-gp inhibitors and anticancer drugs has not resulted in the efficient reversal of drug resistance. To improve administration, we encapsulated the third-generation P-gp inhibitor tariquidar (XR-9576, XR), alone or in combination with paclitaxel (PCT) in liposomes (LP). After optimization, the liposomes demonstrated favorable physicochemical properties and the ability to reverse chemoresistance in experiments using chemosensitive/chemoresistant ovarian cancer cell line pairs. Analyzing publicly available datasets, we found that overexpression of P-gp in ovarian cancer is associated with a shorter progression-free and overall survival. In vitro, LP(XR) significantly increased the cellular retention of rhodamine 123, a P-gp substrate. LP(XR,PCT) synergistically inhibited cell viability, blocked proliferation, and caused G2–M arrest in paclitaxel-resistant SKOV3-TR and HeyA8-MDR cell lines overexpressing P-gp. Holographic imaging cytometry revealed that LP(XR,PCT) treatment of SKOV3-TR cells induced almost complete mitotic arrest, whereas laser scanning cytometry showed that the treatment induced apoptosis. In proof-of-concept preclinical studies, LP(XR,PCT), when compared with LP(PCT), significantly reduced tumor weight (43.2% vs. 16.9%, P = 0.0007) and number of metastases (44.4% vs. 2.8%, P = 0.012) in mice bearing orthotopic HeyA8-MDR ovarian tumors. In the xenografts, LP(XR,PCT) efficiently induced apoptosis and impaired proliferation. Our findings suggest that co-delivery of a P-gp inhibitor and paclitaxel using a liposomal platform can sensitize paclitaxel-resistant ovarian cancer cells to paclitaxel. LP(XR,PCT) should be considered for clinical testing in patients with P-gp–overexpressing tumors. Mol Cancer Ther; 15(10); 2282–93. ©2016 AACR.

Urokinase Plasminogen Activator System–Targeted Delivery of Nanobins as a Novel Ovarian Cancer Therapy

The urokinase system is overexpressed in epithelial ovarian cancer cells and is expressed at low levels in normal cells. To develop a platform for intracellular and targeted delivery of therapeutics in ovarian cancer, we conjugated urokinase plasminogen activator (uPA) antibodies to liposomal nanobins. The arsenic trioxide–loaded nanobins had favorable physicochemical properties and the ability to bind specifically to uPA. Confocal microscopy showed that the uPA-targeted nanobins were internalized by ovarian cancer cells, whereas both inductively coupled plasma optical mass spectrometry (ICP-MS) and fluorescence-activated cell sorting (FACS) analyses confirmed more than four-fold higher uptake of targeted nanobins when compared with untargeted nanobins. In a coculture assay, the targeted nanobins showed efficient uptake in ovarian cancer cells but not in the normal primary omental mesothelial cells. Moreover, this uptake could be blocked by either downregulating uPA receptor expression in the ovarian cancer cells using short-hairpin RNA (shRNA) or by competition with free uPA or uPA antibody. In proof-of-concept experiments, mice bearing orthotopic ovarian tumors showed a greater reduction in tumor burden when treated with targeted nanobins than with untargeted nanobins (47% vs. 27%; P < 0.001). The targeted nanobins more effectively inhibited tumor cell growth both in vitro and in vivo compared with untargeted nanobins, inducing caspase-mediated apoptosis and impairing stem cell marker, aldehyde dehydrogenase-1A1 (ALDH1A1), expression. Ex vivo fluorescence imaging of tumors and organs corroborated these results, showing preferential localization of the targeted nanobins to the tumor. These findings suggest that uPA-targeted nanobins capable of specifically and efficiently delivering payloads to cancer cells could serve as the foundation for a new targeted cancer therapy using protease receptors. Mol Cancer Ther; 12(12); 2628–39. ©2013 AACR.

Prolactin receptor-mediated internalization of imaging agents detects epithelial ovarian cancer with enhanced sensitivity and specificity

Poor prognosis of ovarian cancer, the deadliest of the gynecologic malignancies, reflects major limitations associated with detection and diagnosis. Current methods lack high sensitivity to detect small tumors and high specificity to distinguish malignant from benign tissue, both impeding diagnosis of early and metastatic cancer stages and leading to costly and invasive surgeries. Tissue microarray analysis revealed that >98% of ovarian cancers express the prolactin receptor (PRLR), forming the basis of a new molecular imaging strategy. We fused human placental lactogen (hPL), a specific and tight binding PRLR ligand, to magnetic resonance imaging (gadolinium) and near-infrared fluorescence imaging agents. Both in tissue culture and in mouse models, these imaging bioconjugates underwent selective internalization into ovarian cancer cells via PRLR-mediated endocytosis. Compared with current clinical MRI techniques, this targeted approach yielded both enhanced signal-to-noise ratio from accumulation of signal via selective internalization and improved specificity conferred by PRLR upregulation in malignant ovarian cancer. These features endow PRLR-targeted imaging with the potential to transform ovarian cancer detection. Cancer Res; 77(7); 1684-96. ©2017 AACR.

Erratum: Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy (Nature Communications (2015) 6:6220 doi: 10.1038/ncomms7220)

using a primary human three-dimensional organotypic culture predicts in vivo efficacy Hilary A. Kenny, Madhu Lal-Nag, Erin A. White, Min Shen, Chun-Yi Chiang, Anirban K. Mitra, Yilin Zhang, Marion Curtis, Elizabeth M. Schryver, Sam Bettis, Ajit Jadhav, Matthew B. Boxer, Zhuyin Li, Marc Ferrer & Ernst Lengyel Nature Communications 6:6220 doi: 10.1038/ncomms7220 (2015); Published 5 Feb 2015; Updated 3 Feb 2016 The data plotted in the graph on the right in Fig. 2b of this Article were inadvertently duplicated in the adjacent graph during the production process. The correct version of Fig. 2 appears below.

Pancreatic adenocarcinoma human organoids share structural and genetic features with primary tumors

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids, the fidelity of molecular features, genetic heterogeneity, and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge, we created primary tumor- and PDX-derived organoids, and 2D cultures for in-depth genomic and histopathological comparisons to the primary tumor. Histopathological features and PDAC representative protein markers showed strong concordance. DNA and RNA sequencing of single organoids revealed patient-specific genomic and transcriptomic consistency. Single-cell RNAseq demonstrated that organoids are primarily a clonal population. In drug response assays, organoids displayed patient-specific sensitivities. Additionally, we examined the in vivo PDX response to FOLFIRINOX and Gemcitabine/Abraxane treatments, which was recapitulated in vitro by organoids. The patient-specific molecular and histopathological fidelity of organoids indicate that they can be used to understand the etiology of the patient’s tumor and the differential response to therapies and suggests utility for predicting drug responses.

Abstract POSTER-THER-1440: Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system

The therapeutic efficacy of antitumor drugs is often limited by non-specific, systematic delivery. Here we report a novel targeted delivery platform designed by coupling liposomal nanobins (NB) with the urokinase plasminogen activator receptor (u-PAR) antibody (ATN-658). The urokinase system is overexpressed in epithelial ovarian cancer (OvCa) cells and is expressed only at low levels in normal cells. Doxorubicin (Dox), a FDA approved antitumor drug, had been loaded into the nanobins as a payload. The size and surface charge of the nanobins were optimized to facilitate specific binding to u-PAR expressing OvCa cells. Confocal and transmission electron microscopy showed that ATN-658-NB(Dox) was internalized in OvCa cells in a receptor-dependent manner and was released from endosomes as a function of time. This uptake could be blocked by stably down-regulating u-PAR expression in the OvCa cells using shRNA. In an orthotopic ovarian cancer model, athymic mice treated with ATN-658-NB(Dox) had a significantly greater reduction in tumor burden (0.06±0.01g versus 0.1±0.01g; p Citation Format: Yilin Zhang, Elden P. Swindell, Patrick L. Hankins, Hilary A. Kenny, Thomas V. O’Halloran, Andrew P. Mazar, Ernst Lengyel. Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1440.

Abstract POSTER-CTRL-1217: Human placental lactogen-functionalized bimodal imaging probe for human ovarian cancer detection

The early detection of ovarian cancer (OvCa) is a challenge because the postmenopausal ovaries are hidden in the peritoneal cavity and current imaging technologies lack the ability to differentiate between benign and malignant masses. Our goal is to improve the detection and imaging of OvCa utilizing cancer cell-targeted, bimodal fluorescent and paramagnetic micelles that effectively detect malignant tumors. The human prolactin receptor (PrlR) is expressed in 98% of malignant ovarian tumors, but is not expressed in benign ovarian tumors or fallopian tubes. Therefore, we conjugated the maleimide-functional micelles to a thiol group of human placental lactogen (hPL), a ligand of PrlR, to specifically target OvCa cells. The micelles were 20-nm in diameter with a favorable surface charge (-5.8mV) characterized by dynamic light scattering, zeta potential, and transmission electron microscopy (TEM). After conjugation to hPL, hPL-micelles displayed an increased molecular weight and particle size as assessed by SDS-PAGE, MALDI-TOF, and size exclusion chromatography. Microscopic and cytometric analyses demonstrated that hPL-micelles were more efficiently internalized in human primary OvCa cells and OvCa cell lines than unconjugated micelles. The knockdown of PrlR using shRNA in OvCa cell lines or the triple mutation of hPL significantly inhibited uptake of hPL-fluorophore conjugates, suggesting receptor-mediated endocytosis. Furthermore, the Xenogen IVIS small animal imaging system detected a greater accumulation of hPL-micelles than of unconjugated micelles in the human ovarian tumor xenograft, confirming the enhanced imaging of OvCa through targeted delivery. Given the low prevalence of OvCa and the current unsatisfactory imaging modalities, “receptor-dependent imaging” through hPL-decorated bimodal micelles has the potential to improve detection of OvCa through enhanced specificity. Citation Format: Yilin Zhang, Karthik M. Sundaram, Nan-Sheng Li, Joseph A. Piccirilli, Ernst Lengyel. Human placental lactogen-functionalized bimodal imaging probe for human ovarian cancer detection [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1217.

Abstract 4416: Reversal of chemoresistance in ovarian cancer cells by the liposomal co-delivery of MDR inhibitors and paclitaxel

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer is one of the most lethal gynaecologic malignancies. This can be attributed to the emergence of multidrug resistance in the clinic due to the over-expression of drug-efflux pumps such as P-gp. To overcome this, we have developed a liposomal platform for the the co-delivery of potent P-gp inhibitors with chemotherapeutic drugs. Purpose: To allow for the effective reversal of chemo-resistance in ovarian cancer cells using liposomes co-loaded with tariquidar and paclitaxel. Methods: PEGylated liposomes composed of eggphosphatidylcholine, cholesterol and DOTAP loaded with equimolar amounts of tariquidar (XR) and paclitaxel (PCT) were prepared by the thin film hydration method followed by extrusion. Evaluation of drug resistance of ovarian cancer lines SKOV3, HeyA8 and Tynku was carried out by quantifying MDR1 and MRP1 receptor expression followed by a rhodamine123 exclusion assay. The cytotoxicity of the formulations on the above cancer cell lines was evaluated using the Promega cell viability assay. The effects of the formulations were evaluated in fluorescently stained cells using the iCyte® imaging cytometer (Thorlabs, USA). Long-term kinetic analysis of unstained live cultures was perfomed by holographic imaging cytometer Holomonitor ® M4 (Phase Holographic Imaging, Sweden) and fluorescence microscopy. Results: The liposomes had a particle size distribution of about 200 nm with a zeta potential of 30 mV. As compared to MRP1, a 2.4 fold, 1.9-fold and 1.6-fold higher expression of MDR1 was seen in the HeyA8-MDR, SKOV3-TR and Tynku-R cells respectively over their drug-sensitive counterparts. Using the rhodamine exclusion assay, 14 nM of liposomal XR showed better rhodamine inclusion than 140 nM free XR. In vitro cytotoxicity experiments showed that the drug co-loaded formulation was able to effectively overcome resistance to PCT even at low PCT doses. Using live-cell imaging, paclitaxel-induced arrest of cell cycle progression and onset of apoptosis following this mitotic catastrophe was visible from 1.5 μM to 50 nM only with the co-loaded liposomes. The cell cycle arrest was further visualized using time-lapse Holomonitor M4 which showed the increasing incidence of cells arrested in mitosis over time. Using fluorescence microscopy, we were able to clearly visualize the presence of polymerized microtubules around the cell nucleus as a result of the paclitaxel-mediated microtubule dysfunction. Conclusions: Uniform liposomes co-loaded with tariquidar and paclitaxel were prepared and characterized. The co-loaded liposomes were able to effectively reverse chemoresistance and induce cytotoxicity in a variety of drug-resistant ovarian cancers. The formulations were shown to arrest cell cycle, preventing its progression to the G1 phase. This arrest of cell cycle was shown to be due to the paclitaxel-mediated micro-tubule dysfunction. Note: This abstract was not presented at the meeting. Citation Format: Shravan K. Sriraman, Yilin Zhang, Ed Luther, Ernst Lengyel, Vladimir Torchilin, Vladimir Torchilin. Reversal of chemoresistance in ovarian cancer cells by the liposomal co-delivery of MDR inhibitors and paclitaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4416. doi:10.1158/1538-7445.AM2015-4416

Abstract 2885: Urokinase plasminogen activator system targeted delivery of arsenic trioxide loaded nanobins as a novel ovarian cancer therapeutic

That the urokinase plasminogen activator receptor (uPAR) system is constitutively expressed in primary and metastatic ovarian cancer regardless of grade or stage, while it is only weakly expressed in normal tissues, identifies it as a possible therapeutic target. Taking advantage of the tumor-specific expression of uPAR, a nanoscale liposome (nanobin, NB) was coupled with an antibody (ATN 291) against the uPAR ligand, uPA, to facilitate receptor-mediated, targeted drug delivery. The nanobin was loaded with arsenic trioxide (As 2 O 3, in short As), since our previous results had demonstrated effective tumor uptake and induction of tumor cell apoptosis using As loaded nanobins in an orthotropic model of human triple negative breast cancer. We hypothesized that the ATN 291-conjugated nanobin (NB-uPA Ab) would allow specific cellular internalization and increase the therapeutic index of As in ovarian cancer cells. Using flow cytometry and fluorescence microscopy, we measured the cellular uptake of NB(Calcein)-uPA antibody (Ab) in HeyA8, ovarian cancer cells. The data showed a dose-dependent pattern of internalization with maximum internalization at 24 hours. Furthermore, NB-uPA Ab showed elevated cellular uptake when compared to non-targeted NB. More importantly, delivery of the targeted nanobins is uPA/uPAR dependent. High doses of ATN-291 or scuPA inhibited the cellular binding of NB-uPA Ab to HeyA8 cells. Moreover, down-regulation of uPAR expression via RNA interference in ovarian cancer cells inhibited the cellular uptake of the NB-uPA Ab. The cell viability of HeyA8 cells was significantly reduced following treatment of both non-targeted NB(As) and NB(As)-uPA Ab when compared to empty NB treatment as determined by a cytotoxicity assay. However, the cytotoxicity of NB(As)-uPA Ab is 2.1-fold more potent than the non-targeted NB(As). Taken together, these data indicate that the uPA Ab conjugated nanobins improved drug delivery and enhanced the anti-cancer effects of As, demonstrating the utility of targeting the uPA/uPAR system in ovarian cancer chemotherapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2885. doi:1538-7445.AM2012-2885