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Purpose: Tumor microenvironmental (TME) factors play important roles in cancer progression and chemoresistance by inducing epithelial-mesenchymal transition (EMT) and related mechanisms. Pancreatic stellate cell (PSC) is a highly-enriched cell type in TME of pancreatic ductal adenocarcinoma (PDAC). Three dimensional (3D) tumor spheroids (TS) are considered as an in vivo-like tumor model demonstrating its clinical relevancy. We previously developed a minipillar array histochip for 3D culture and immunohistological analysis of TS in an increased throughput manner. Here, we present a minipillar-based co-culture of TS and PSC as a novel in vitro microtumor model of pancreatic ductal adenocarcinoma. Methods: Minipillar chips were obtained from MBD Co. (Cellvitro™ 55Cryo). 3D TS of Panc-1, human PDAC cells and PSCs (Sciencell) were cultured on minipillars and at the bottom of 96 well plates, respectively, as embedded in type I collagen. Changes in expression of extracellular matrix (ECM) and EMT-related factors in TS were assessed by confocal microscopy following cyrosection preparation. Results: Co-culture condition was optimized for the growth of TS, activation of PSC, and their interaction. Expression of EMT markers such as TGF-β1 and vimentin increased in co-cultured TS than mono-cultured TS. ECM remodeling as indicated by increased matrix deposition (collagen I, fibronectin) and fiber organization was observed in TS co-cultured with PSC compared to mono-cultured TS. Upon drug exposure (gemcitabine and paclitaxel), survival advantage was prominent in TS co-cultured with activated PSC. Conclusions: We established 3D model of PDAC by co-culturing TS and PSC using minipillar histochips. EMT-related changes including ECM remodeling and drug resistance were recapitulated in the model. Overall our results indicate usefulness of the present model not only in studying the role of TME factors and their interactions towards tumor progression but also in the evaluation of therapeutic efficacy of drug candidates and combinations. Citation Format: Hyun Ju Hwang, Min-Suk Oh, Seul-Ki Kim, Hyo-Jeong Kuh. 3D co-culture of pancreatic tumor sphroids and stellate cells using minipillar array as a microtumor model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2114.

Interaction between cancer cells and stromal components in the tumor microenvironment is well known for their significant roles in tumor progression and subsequent treatment failure. Hepatic stellate cells (HSCs), as a predominant cell type in the microenvironment of hepatocellular carcinoma (HCC), are involved in creating desmoplastic and chemoresistance-inducing microenvironment. HSCs secrete various paracrine factors that modify the HCC tumor microenvironment leading to tumor growth, drug resistance and metastasis via promoting epithelial-to-mesenchymal transition (EMT). Although several studies using 2D co-culture system have shown bidirectional cross-talk between cancer cells and HSCs, data obtained remain limited in their clinical relevance due to lack of in vivo tumor-like characteristics. We developed a mixed-cell spheroid model which recapitulates direct 3D tumor-HSCs interactions in paracrine and contact-mediated manners. Huh-7, human HCC cells, were mixed co-cultured with LX-2 cells, immortalized human HSC at 1:3 ratio using liquid overlay technique in 96-well plates. Electron microscopy was used to examine subcellular structural changes. Cellular distribution within spheroids was observed by labeling LX-2 cells with fluorescent tracer. The expression of pro-fibrotic and EMT markers was detected by immunohistochemistry or immunofluorescence staining on paraffin embedded sections. Invasion ability was determined using migration assay into 3D matrigel matrix. Dose-response curve was obtained by using APH assay and Ki-67 detection. Mixed co-culture of Huh-7 and LX-2 cells showed a spontaneous self-organization forming highly compact mixed-cell spheroids with well-defined contour. Heterotypic cell-to-cell contact increased as shown by gap junction and desmosomes in the mixed-cell spheroids. LX-2 cells were uniformly distributed within the spheroids. Expression of fibroblast-associated factors such as α-SMA, collagen I, TGF-β and CTGF showed similar patterns of distribution to that of LX-2 cells, but at an elevated level in the mixed-cell spheroids compared to that of cancer cell-alone spheroids. An increased expression of EMT-related factors was observed along with enhanced invasion into 3D matrix. Differential drug sensitivity was shown; mixed-cell spheroids were sensitive to sorafenib but not to other agents including oxaliplatin, gemcitabine, and 5-FU. Based on these results, the mixed cell-spheroids of HCC cells-HSCs may be proposed as a useful 3D model for in vitro therapeutic screening of targets and agents as well as for the study of malignant progression in HCC. Citation Format: Hyo-Jeong Kuh, Iftikhar Ali Khawar. Characterization of mixed-cell spheroid as an in vitro model of hepatocellular carcinoma for epithelial-to-mesenchymal transition and chemoresistance [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 5789. doi:10.1158/1538-7445.AM2017-5789

Purpose. Chemotherapeutic failure of human solid tumor is attributed to tumor microenvironmental (TME) factors associated with drug resistance. Three dimensional (3D) tumor spheroid (TS) model is known to be in vivo mimic and has demonstrated its clinical relevancy. Fibroblasts in TME have been shown to play a significant role in drug resistance. The present study was to develop and characterize a co-culture of 3D TS with fibroblasts for human colorectal tumor model. Methods. TS of human colorectal cancer cells (HT-29) was prepared by using liquid overlay method and co-cultured in collagen-coated 24-well plates with colon fibroblasts (CCD-18co) grown in Transwell® inserts. Differentail expression of extracellular matrix (ECM), growth signaling molecules, and microRNAs was analyzed using IHC, ELISA, western blot and qRT-PCR. For miRNA transfection study, changes in motility and drug sensitivity were measured by wound healing assays and APH or MTS assay, respectively. Results. Significant resistance to 5-FU was shown in TS either with or without fibroblasts compared to monolayers. Compared to single culture of TS, co-culture with fibroblast showed 1.3-fold increased outgrowth on collagen gel, indicating increased motility. Cell-to-cell adhesion structures, expression of ECM and active form TGFβ1 were higher in TS-fibroblast co-culture. Expression of several growth- and TME-related factors, such as fibronectin, EGFR, β-catenin and CTGF, was higher in fibroblast co-culture up to the level of HT-29 xenograft tissues. Among miRNAs showing differential expressions, miR-96 showed down-regulation in TS. HT-29 cells transfected with miR-96 showed increase in cell motility, proliferation, and sensitivity to 5-FU, suggesting its potential role in regulation of genes associated with 5-FU resistance. Conclusions. Co-culture of 3D TS-fibroblasts was successfully established and demonstrated in vivo mimic characteristics with respect to presence of adhesion microstructures, expression of ECM and growth signaling molecules, and resistance to 5-FU. MiR-96 showed its potential role in 3D drug resistance which warrants further studies. Citation Format: Sun-Ah Kim, Hyo-Jeong Kuh. Co-culture of 3D tumor spheroids with fibroblasts as an in vitro model for drug resistance study. [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 1970. doi:10.1158/1538-7445.AM2014-1970

Compared to the conventional monolayers or suspension cultures, 3D culture models have drawn attention as a vivo-mimic model which can produce clinically relevant data. Among the several types of 3D culture models, multicellular spheroid (MCS) is a appropriate model for study of penetration and efficacy of anticancer agents. Purpose of this study was to establish MCS of human pancreatic cancer cell (Panc-1) and evaluate its usefulness as anticancer efficacy test. Panc-1 MCS was produced successfully by using poly-dimethylsiloxane(PDMS) microwell instead of agarose-coated 96-well system. MCS with average diameter of 375 μm was prepared in 700 μm microwells after 4 days of culture. Spherical morphology with rather rough surface and junctional structures in outer cell layers were observed with no notable necrotic area. Extracellular matrix proteins such as collagen I, fibronectin, and laminin were detected in the interstitial space of MCS. Stem cell population with CD44+/CD24+/ESA+ expression increased from 0.1% to 2.1% when cultured as MCS. miRNA expression profiling showed a difference between monolayers and MCS, i.e., 36 and 85 genes were up- and down-regulated in MCS, respectively. Imaging of drug penetration and viability indicators including MitoSOX, Edu incorporation, and Ki-67 expression were optimized for microwell format. Colorimetric assays of MTS and APH were performed after transferring MCS to 96-well plates. Overall, we demonstrated that PDMS microwell is an appropriate platform for preparation of MCS of Panc-1 cells and MCS showed different biological features from monolayers, suggesting that Panc-1 MCS prepared using PDMS microwell may be a useful 3D model for anticancer efficacy test. Citation Format: Sang-Eun Yeon, Sang-Hoon Lee, Hyo-Jeong Kuh. Characteristics of pancreatic cancer spheroids as a model for anticancer efficacy test. [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 2722. doi:10.1158/1538-7445.AM2013-2722 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The limited efficacy of protein drugs is related to their poor distribution in tumor tissue. We examined interstitial delivery of four model proteins of different molecular size and bioaffinity as formulated of non-formulated forms in multicellular layers (MCL) of human cancer cells. Model proteins were tumor necrosis factor-related apoptosis-including ligand (TRAIL), cetuximab, RNase A, and IgG. MCLs were cultured in Transwell inserts, exposed to drugs, then cryo-sectioned for image acquisition using fluorescence microscopy (fluorescent dye-labeled TRAIL, RNase A, IgG) or Immunohistochemistry (cetuximab). TRAIL and cetuximab showed partial penetration into MCLs, whereas RNase A and IgG showed little penetration. At 10-fold higher dose, a significant increase in penetration was observed for IgG only, while cetuximab showed an intense accumulation limited to the front layers. PEGylated TRAIL and a heparin-Pluronic nanogel formulation of RNase A showed significantly improved penetration that was attributed to increased stability and extracellular matrix binding, respectively. IgG penetration was significantly enhanced with PTX pretreatment as a penetration enhancer. MCL culture was successfully used for the evaluation of protein movement in the tumor interstitum. Four proteins showed limited interstitial penetration in MCL cultures. Bioaffinity, rather than molecular size, seems to have a positive effect on tissue penetration, although strong binding affinity may lead to sequestration in the front layers. Nanoformulations, such as PEGylation and heparin-Pluronic (HP) nanogel, or penetration enhancers are potential strategies to increase interstitial delivery of anticancer biologics. 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 5708. doi:1538-7445.AM2012-5708

Intralesional chemotherapy is suggested to improve local tumor control as well as systemic toxicity profile of antitumor agents against solid tumors. Thermosensitive poly-(organophosphazene) hydrogel is a novel injectable polymer that transforms from sol to gel at body temperature. In this study, we evaluated distribution and efficacy of paclitaxel (PTX) when given as intratumoral injection using the hydrogel or solution formulation in human SNU-601 tumor xenograft-bearing nude mice. Following intratumoral injection of 60 mg/kg of PTX, plasma drug concentrations were lower than 0.5 μg/ml(LOQ) for both hydrogel and solution. For PTX tumor concentration, Cmax was 1.2 folds higher and T1/2 3.7 folds longer with hydrogel compared to solution. Over 21d, AUCtumor was 1.5 folds greater in hydrogel compared to solution, indicating greater drug exposure and retention at target site. The antitumor activity of PTX (30 mg/kg) when given alone or in combination with doxorubicin (DOX, 15 mg/kg) was evaluated after intratumoral hydrogel injection in SNU-601 (Td= 21 d) and SNU-398 (Td= 5 d) xenograft models. The synergism between DOX and PTX, independent of dosage form, was observed in SNU-398, but not in SNU-601. No toxicity was observed in hydrogel group in neither single nor combination treatment. For slow-growing SNU-601 tumor, combination of PTX and DOX given in hyrogel mixture showed greater activity than that of solution. In conclusion, poly-(organophosphazene) polymer may be useful in intralesional administration of PTX to achieve greater drug exposure at target site. Also, combination of DOX and PTX, showed a potential for greater antitumor efficacy, which warrants further evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3591.