A novel chemotherapeutic sensitivity-testing system based on collagen gel droplet embedded 3D–culture methods for hepatocellular carcinoma

Background Patients suffering from advanced stage hepatocellular carcinoma (HCC) often exhibit a poor prognosis or dismal clinical outcomes due to ineffective chemotherapy or a multi-drug resistance (MDR) process. Thus, it is urgent to develop a new chemotherapeutic sensitivity testing system for HCC treatment. The presence study investigated the potential application of a novel chemotherapeutic sensitivity-testing system based on a collagen gel droplet embedded 3D–culture system (CD-DST). Methods Primary cells were separating from surgical resection specimens and then tested by CD-DST. To identify whether HCC cell lines or cells separating from clinical specimens contain MDR features, the cells were treated with an IC 50 (half maximal inhibitory concentration) or IC max (maximal inhibitory concentration) concentration of antitumor agents, e.g., 5-furuolouracil (5-FU), paclitaxel (PAC), cisplatin (CDDP), epirubicin (EPI), or oxaliplatin (L-OHP), and the inhibitory rates (IRs) were calculated. Results HepG2 cells were sensitive to 5-FU, PAC, CDDP, EPI, or L-OHP; the IC 50 value is 0.83 ± 0.45 μg/ml, 0.03 ± 0.02 μg/ml, 1.15 ± 0.75 μg/ml, 0.09 ± 0.03 μg/ml, or 1.76 ± 0.44 μg/ml, respectively. Only eight (8/26), nine (9/26), or five (5/26) patients were sensitive to the IC max concentration of CDDP, EPI, or L-OHP; whereas only three (3/26), four (4/26), or two (2/26) patients were sensitive to the IC 50 concentration of CDDP, EPI, or L-OHP. No patients were sensitive to 5-FU or PAC. Conclusions The in vitro drug sensitivity exanimation revealed the MDR features of HCC and examined the sensitivity of HCC cells from clinical specimens to anti-tumor agents. CD-DST may be a useful method to predict the potential clinical benefits of anticancer agents for HCC patients.


Background
HCC is currently one of the most common causes of cancer-related deaths in China and the Asian-Pacific region [1][2][3]. In China, there are 466,100 new cases, and 422,100 deaths occur per year [2][3][4][5]. Although hepatic resection may be the first choice of treatment, only a small proportion (10-15%) of patients are suitable [6,7]. Patients often suffer from advanced stage HCC upon their initial diagnosis and exhibit poor prognosis due to multi-drug resistance (MDR) features [8][9][10]. Thus, it is urgent and significant to develop a new chemotherapeutic sensitivity testing system for HCC treatment. Previously, the sensitivity of HCC cells to anti-tumor agents is often tested via MTT (3-(4, 5dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) assays or SRB (Sulforhodamine B) assays based on the two-dimensional (2-D) monolayer culture [11]. However, cancer cells in solid tumors grow in a three-dimensional (3-D) pattern [12]. In this respect, an in vitro 2-D cell cultural model is limited in evaluating the clinical efficacy of chemotherapies [13]. Therefore, it is urgent to establish an in vitro model reflecting the 3-D culture of tumor cells.
Applications of CD-DST in chemotherapeutic sensitivity examination have been demonstrated in human cancers, e.g., lung cancer, gastric cancer, breast cancer, or colorectal cancer [11][12][13][14][15][16]. However, reports focused on potential applications and efficacy of CD-DST in HCC remain rare [17,18]. This work aims to identify potential applications of CD-DST in HCC.

Cell lines and reagents
HepG2 (an HCC cell line) cells (Cat. 3111C0 001CCC000035) or HepG2/ADR (an HCC cell line resistant to Adriamycin), which were purchased from Cell Culture Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, or descripted previously [8], were cultured in complete DMEM (Invitrogen, Carlsbad, CA, USA) in a sterile incubator maintained at 37°C, 5% CO 2 . Anti-tumor agents, 5-furuolouracil      Table 2) for 24 h. After treatment with the anticancer agents, cells were incubated in a serum-free medium (4 ml PCM-2 medium to inhibit fibroblast proliferation) for 5-7 days to form colonies. Living cells were stained with neutral red, fixed with neutral formalin buffer, and directly examined/quantified by the cultured tests. An IR value >25% indicated that cells were sensitive to the compound.

Statistical analyses
Data are expressed as the mean ± SD. Statistical analyses were performed using the paired t-test or Fisher's exact probability test. The IC 50 and IC max values were calculated using Origin 8.5 software.

HepG2 cell cultures in collagen gel droplets
To establish the CD-DST methods, HepG2 cells were cultured in collagen gel droplets. The O.D. 540 nm values were  Table 3, similar results were obtained from each experiment, and the growth rates of HepG2 were from 4.1 to 4.62-fold (Table 3). These results were further confirmed by representative photographs. As shown in Fig. 1, after 5-7 days' growth, the colony sizes were much larger than that at baseline.

Sensitivity of primary cells separated from HCC clinical specimens
Next, the sensitivity of primary cells separated from HCC clinical specimens to anti-tumor agents was detected by CD-DST methods (Fig. 5). Cells were treated with an IC max or IC 50 concentration of antitumor agents. As shown in Table 4 Fig. 6. The characteristics of the tumor (histological grade, BCLC grade number of nodules) in relation to the sensitivity to chemotherapeutic agents are shown in Table 5. Taken together, patients suffering from HCC were not sensitive to PAC or 5-Fu, and only a few obtained clinical benefits from CDDP, EPI, or L-OHP.

2-D cultured HepG2 cells were more sensitive to anti-tumor agents
Moreover, to confirm the sensitivity of HCC cells to anti-tumor agents, the effect of agents on HepG2 or HepG2/ADR, an MDR cell line, was examined by MTT assays and the IC 50 /IC max concentrations were calculated. The results are shown in Table 6. The IC 50 value of agents on HpeG2 cells obtained from the MTT assays were much lower than those obtained from CD-DST. Moreover, the IC 50 value of agents on HpeG2/ADR cells obtained from the MTT assays were similar with those obtained from CD-DST. These results indicated that sensitivity of 2-D cultured HepG2 cells to anti-tumor agents was much higher than that in 3-D-cultured HepG2 cells.

Discussion
HCC is currently one of the most common malignancies, especially in China and the Asia-Pacific region.
Although an oral multi-targeted kinase inhibitor, sorafenib, has been used as a curative approach, cytotoxicity chemotherapeutic agents are still treatment choices, e.g., oral medication or hepatic artery chemotherapy and transcatheter arterial chemoembolization (TACE) [21][22][23][24][25]. 5-FU, CDDP, EPI, and L-OHP can induce double DNA strand breaking (DSB) and block DNA replication via multi-mechanisms [26]. PAC targets microtubules and disrupts cell division [27]. It is well known that HCC may be resistant to almost all kinds of cytotoxicity chemotherapeutic agents due to its multi-drug resistance (MDR) features. During the MDR process, breast cancer  L-OHP 26 5 resistance protein (BCRP), multi-drug resistance relative protein2/3 (MRP2/3) or multi-drug resistance 1 (MDR-1) may participate in the un-anticipated efficacy loss of chemotherapeutic agents via the phase I or phase II drug metabolizing process [28]. Some other signaling pathways, e.g. JAK/STAT (janus kinase / signal transducers and activators of transcription), PI3K/mTOR (phosphatidylinositol 3-kinase / mammalian target of rapamycin) or hypoxiarelated pathways, would also participate in the MDR of HCC [29,30]. Moreover, a higher level of these resistance genes has been confirmed in HCC/liver clinical specimens than that in HCC cell lines. Tumor cells in solid tumors grow in a 3-D pattern. Therefore, it is urgent to establish an in vitro model reflecting the 3-D culture of tumor cells. To date, there is no effective or rapid testing system for screening sensitive drugs for HCC patients to guide the clinical individual chemotherapy. For the first time, our presence work establishes the application of CD-DST in HCC chemotherapeuticefficacy examination. The sensitivity of HepG2 or primary cells separated from HCC clinical specimens to 5-FU, PAC, CDDP, EPI, and L-OHP was identified. No patients were sensitive to PAC or 5-FU. As shown in Table 5, the characteristics of the tumors (histological grade, BCLC grade number of nodules) in relation to their sensitivity to chemotherapeutic agents were identified. Patients with BCLC A or well-differentiated HCC would more sensitive to CDDP or L-OHP, whereas patients with BCLC B or moderate-differentiation HCC would be more sensitive to EPI than CDDP or L-OHP. Moreover, patients with poordifferentiation HCC would not be sensitive to anti-tumor agents, and the tumor numbers seemed unrelated to antitumor agent sensitivity.
Traditionally, the biological behavior of cancer cells was determined using 2-D methods, e.g., MTT or colony formation. However, 2-D methods may be insufficient to reflect the in vivo behavior of cancer cells in solid tumor tissues [7]. Thus, it is valuable to develop and validate a rapid method to examine the biological behavior of cancer cells in a 3-D manner. Tissue engineering research, e.g., 3-D spatial array, investigated the involvement of the extracellular matrix (ECM) in solid tissues [7]. The interaction between cells and the ECM modulates the behavior and features of solid tumors [8][9][10]. Collagen is a main constituent of the ECM and supports the formation of the microenvironment in solid tumors [8][9][10][11][12]. CD-DST is a mature test in which primary-culture cells of HCC are separated from surgical resection specimens embedded in collagen droplets [7][8][9][10][11][12]. Applications of CD-DST in chemotherapeutic sensitivity examination have been demonstrated in some kinds of human cancers, e.g., lung cancer, gastric cancer, breast cancer, and colorectal cancer [8][9][10][11][12]. Higashiyama et al. (2010) predicted the chemotherapeutic effect on postoperative recurrence in non-small cell lung cancer patients [9]. Sun et al. (2013) established a 3-D gastric tumor culture model [10]. Lin et al. (2015) revealed the effect of pyruvate kinase M2 on Epirubicin and 5-Fluorouracil in breast cancer by CD-DST [11]. Takahashi et al. (2015) examined the anti-tumor activity of fucoxanthin and fucoxanthinol on cell lines or primary cells from colorectal cancer clinical specimens [12]. In the present work, for the first time, we developed and validated an effective method to predict the resistance of HCC patients to anti-tumor agents. The result showed that 2-D cultured HepG2 cells were more sensitive to anti-tumor agents than cells cultured in a 3-D manner but similar to a 2-D cultured resistance cell line, HepG2/ADR. Moreover, primary cells separated from clinical specimens were much more resistant to anti-tumor agents. Therefore, CT-DST, a 3-D tumor model, is valuable for evaluating and mimicking the in vivo efficacy of antitumor agents.

Conclusions
The in vitro drug sensitivity exanimation validated the MDR features of HCC. CD-DST may be a useful method to predict the potential clinical benefits of anticancer agents for HCC patients. We thank Dr. Juan-juan Zhao for her advice and excellent technical assistances to this study.

Funding
This work was supported by direct grants from the Chinese Government: the Dean Innovation Fund (YNKT2014039) from the 302nd Hospital of Chinese PLA, Beijing 100,039, People's Republic of China. These funding sources or funding bodies had no role in the design of this study and will not have any role during its execution, analyses, interpretation of the data, or decision to submit the results.

Availability of data and materials
Additional data and materials may be requested from the corresponding author on reasonable request.

Open access
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Authors' contributions ZXH, JFB and ZZ made substantial contributions to conception and design. JH, ZXH, FF, YTC. YKZ QYJ, SQW, and YH made substantial contributions to acquisition of data, or analysis and interpretation of data. JH, FF and ZZ have been involved in drafting the manuscript or revising it critically for important intellectual content. YSW, XNG, QC and YW have given final approval of the version to be published. Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.

Ethics approval and consent to participate
Our studies are in compliance with the Helsinki Declaration. The collection of tissues and the study protocol were approved by the Ethics Committee of No. 302 Military Hospital of China, with informed written and verbal consent from 26 patients. The methods did not relate to the clinical trial or methods.

Competing interests
The authors declare that they have no competing interests. Author Xunian Gao works at/is employed by Guangzhou Darui Biotechnology as the supplier of the chemosensitivity assay for this research.

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