Quantitative examination of the inhibitory activation of molecular targeting agents in hepatocellular carcinoma patient‐derived cell invasion via a novel in vivo tumor model

Abstract Background The outcomes for patients with advanced hepatocellular carcinoma (HCC) receiving sorafenib are far from satisfactory because of treatment resistance to sorafenib. However, the exact mechanism of resistance to sorafenib remains unclear and it is valuable to establish a novel mouse model to quantitatively analyze the inhibition rates of sorafenib on the invasive growth of HCC cells in the liver. Methods HCC tissue microblocks derived from patients were cultured and mixed with hydrogel drops. Then, hydrogel drops containing microblocks of HCC tissue were attached onto the surface of the livers of nude mice to form lesions or nodules of HCC. The mice received molecular targeting agents through oral administration. Livers with tumor nodules were harvested for H&E staining (hematoxylin‐eosin staining) analysis and H&E staining images were quantitatively analyzed using image J software. The invasive growth of HCC cells into the liver was calculated using the depth of the lesions compared with the total thickness of the liver. Results Microblocks containing cells derived from HCC patients can form lesions in the liver of nude mice. Oral administration of molecular targeting agents inhibited the invasive growth of HCC cells in the liver of nude mice. Conclusions The model established in this study involves the invasive growth of HCC cells in the liver of nude mice, and the model allows for the quantitative analysis of the inhibitory effect of molecular targeting agents on the invasion of HCC cells in vivo.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is still one of the foremost public health threats in Asia and the Pacific region, and specifically in China, because of high rates of hepatitis virus, such as hepatitis B virus (HBV). 1,2 Moreover, a large proportion of patients are diagnosed at the advanced stage of HCC at initial diagnosis and cannot receive certain treatments, such as surgery. 3 Advanced HCC is also known for its multi-drug resistance (MDR) features, whereby it is not sensitive to cytotoxic chemotherapies. 4 Therefore, current antitumor agents for advanced HCC are molecular targeting agents, represented by sorafenib. 5 However, during clinical treatment, some patients are initially resistant to sorafenib (initial resistance) and other patients who are initially sensitive to sorafenib can acquire resistance to sorafenib. 6 Therefore, it is valuable to determine whether a patient is sensitive to molecularly targeted drugs or if they are suitable to receive molecular targeting agents.
The patient-derived tumor xenograft (PDX) animal model involves inoculating patient-derived tumor cells into nude mice for an investigation of drug antitumor activity. 7 The PDX model is also an in vivo study model that can be used to detect the antitumor activity of drugs in animals. However, patient-derived tumor cells can reflect the sensitivity of cells in patients' tumor tissues to antitumor drugs. 8 In this work, patient-derived HCC tissues were prepared as tumor tissue microblocks, and a medical hydrogel was used to adhere the tissue microblocks to the liver surface in nude mice. A quantitative study of the invasive growth of HCC cells was performed by measuring the invasive depth of the cells in nude mice, and the effect of the oral administration of molecularly targeted drugs on HCC was measured by calculating the inhibition rate of HCC cell invasive growth.
The sensitivity of the cells to molecularly targeted drugs was also assessed.

| Tumor tissues
The collection of the clinical tissues and the study protocol were approved by the Ethics Committee of the fifth Medical Center of the Chinese People's Liberation Army General Hospital (original name: the 302nd Hospital), and written consent was obtained from all patients. The clinical tissues were obtained through a puncture biopsy using a coaxial needle (cat. no.: MCXS1815BP, RITA Company) immediately before radiofrequency ablation treatment, following a method described in our previous work. 9 The obtained tissues were preserved using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), and tumor tissue microblocks were prepared on a clean bench. The weights of the tumor tissue microblocks were accurately measured using a precision balance, and the size of the tumor microblocks was adjusted to make the weight of the microblocks uniform, at approximately 1 mg per microblock.

| MicroPET screening (living-imaging analysis of nude mice)
All animal experiments in thise present work wereh been re- Firstly, inhalation anesthesia was performed on the nude mice using isoflurane as an anesthetic agent (the dose for the first anesthesia was 1.5% [volume/volume]; the dose for continuous anesthesia was 0.5% [volume/volume]). Then, the mice received a 200 μCi

| In vivo antitumor activation of molecular targeting agents
For subcutaneous tumor formation, inhalation anesthesia was performed on nude mice using isoflurane as an anesthetic agent (the dose for the first anesthesia was 1.5% [volume/volume]; the dose for continuous anesthesia was 0.5% [volume/volume]). Next, microblocks of HCC tissues were directly injected subcutaneously. After injection (4-5 days), the mice received molecular targeting agents via oral administration. Mice were treated once every 2 days for a total

| Statistical analysis
The statistical analysis was performed using GraphPad Software (version 6.0, San Diego, CA. USA). The difference between the two groups was assessed using two-way analysis of variance (ANOVA) with Bonferroni correction method. A P-value of less than .05 was considered to indicate statistical significance.

| Molecular targeting agents inhibit the subcutaneous growth of HCC cells
The effect of molecular targeting agents on the subcutaneous growth of patient-derived HCC cells was examined. As shown in Figure 2 and Figure Table 1 (tumor volumes) and   of the molecular targeting agents, sorafenib, regorafenib, lenvatinib, anlotinib, or apatinib in the five PDCs are shown in Table 3 (images) and

| Molecular targeting agents inhibit the invasion of HCC cells into the liver
To further examine the effect of the molecular targeting agents, the invasion of HCC cells into the liver was examined. The    Tables 5 and 6. Among these molecularly targeted agents, lenvatinib had significantly higher antitumor activity against the subcutaneous growth of the HCC PDCs than the other four agents.
As shown in Figure 6, the invasion of the HCC cells in the liver was examined using H&E staining. Oral administration of the molecular targeting agents inhibited the subcutaneous growth of the HCC cells in nude mice ( Figure 6, Tables 5 and 6). The effect of sorafenib, a typical molecular targeting agent, is shown in Figure 6 as a representative result and PDC No.

Hepatitis viruses, such as HBV, have a very high infection rate in
China and related East-Asian regions. 1,16 Recently, more than 80 million people in China were reported to be infected with HBV or to suffer with various chronic liver diseases related to HBV. 1,2,16,17 Although many advances have been achieved in terms of antiviral treatments for HBV, patients with HBV-related chronic liver disease may still have disease progression and eventually suffer TA B L E 4 Inhibition rates of agents on lesions formed by HCC cells in liver organs  from HCC. [16][17][18] This means that HCC is a serious health threat and it also poses a great challenge for the public health system.
Unfortunately, because of current clinical diagnostic and treatment strategies, most HCC patients have an advanced stage of HCC at initial diagnosis and are unsuitable for surgery or liver transplantation. 19 Because advanced HCC has MDR characteristics for traditional cytotoxic chemotherapeutic drugs, existing antitumor treatment strategies mainly include various molecularly targeted drugs, such as sorafenib. 4 Sorafenib was approved by the U.S. food and drug administration (FDA) in 2007 for the treatment of advanced HCC, and sorafenib drug resistance has been reported. 6 To solve this problem, it is necessary to elucidate the molecular mechanism of sorafenib resistance. Despite the progress made in related research, the molecular mechanism of sorafenib resistance is still not very clear, and there is no convincing indicator for the clinical outcomes of patients undergoing treatment with sorafenib. 6 Therefore, using a variety of pharmacological and experimental techniques to examine the sensitivity of tumor cells to molecularly targeted drugs in patient tissues is important to determine whether a patient is eligible for treatment before receiving molecular targeted therapy. 20 In this study, patient-derived HCC tumor tissues were collected and prepared into tissue microblocks which were then mixed with hydrogel droplets. Next, the hydrogel droplets containing the HCC tissue microblocks were attached to   21,22 This study examined the in vivo invasion (intrahepatic invasion) of HCC cells in nude mice, which not only allows a determination of the antitumor activity of molecular targeting agents, but also has important implications for related research. Sorafenib has been widely used in clinical treatment and regorafenib or lenvatinib have been newly approved by the FDA. [23][24][25][26] In addition to these three agents, anlotinib and apatinib have been developed by Chinese manufacturers. [27][28][29][30][31] Anlotinib has been used for NSCLC (nonsmall cell lung cancer) treatment and apatinib is used TA B L E 6 Inhibition rates of agents on the intrahepatic invasion of HCC cells into nude mice's liver organs

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
All the listed authors meet the requirements for authorship. JH and RSL conceived and designed the experiments; HWS and FF performed the experiments and wrote the main manuscript text; HX, YTC and XJL performed the animal experiments; QTJ, RCY and ZJW performed the molecular biology experiment and analyzed the data.
All authors have read and approved the manuscript.