Identification of hepatic fibrosis inhibitors through morphometry analysis of a hepatic multicellular spheroids model

A chronic, local inflammatory milieu can cause tissue fibrosis that results in epithelial-to-mesenchymal transition (EMT), endothelial-to-mesenchymal transition (EndMT), increased abundance of fibroblasts, and further acceleration of fibrosis. In this study, we aimed to identify potential mechanisms and inhibitors of fibrosis using 3D model-based phenotypic screening. We established liver fibrosis models using multicellular tumor spheroids (MCTSs) composed of hepatocellular carcinoma (HCC) and stromal cells such as fibroblasts (WI38), hepatic stellate cells (LX2), and endothelial cells (HUVEC) seeded at constant ratios. Through high-throughput screening of FDA-approved drugs, we identified retinoic acid and forskolin as candidates to attenuate the compactness of MCTSs as well as inhibit the expression of ECM-related proteins. Additionally, retinoic acid and forskolin induced reprogramming of fibroblast and cancer stem cells in the HCC microenvironment. Of interest, retinoic acid and forskolin had anti-fibrosis effects by decreasing expression of α-SMA and F-actin in LX2 cells and HUVEC cells. Moreover, when sorafenib was added along with retinoic acid and forskolin, apoptosis was increased, suggesting that anti-fibrosis drugs may improve tissue penetration to support the efficacy of anti-cancer drugs. Collectively, these findings support the potential utility of morphometric analyses of hepatic multicellular spheroid models in the development of new drugs with novel mechanisms for the treatment of hepatic fibrosis and HCCs.

Hepatic fibrosis results as a consequence of a pattern of severe inflammation that leads to the excessive accumulation of extracellular matrix (ECM) proteins. Advanced liver fibrosis results in cirrhosis and is directly related to the high mortality of cirrhosis 1 . Because liver transplantation is currently the only treatment option for patients with advanced liver fibrosis and cirrhosis, there is an urgent need for the development of effective anti-fibrotic agents for the treatment of hepatic fibrosis.
Indeed, despite the high prevalence of liver fibrosis, there are no approved therapies, potentially because liver fibrosis represents a diverse state with numerous potential causes and complications. The main causes of liver fibrosis are chronic hepatitis virus infection 2 , alcohol abuse 3 , drug-induced liver injury (DILI) 4 , cholestasis 5 , and non-alcoholic steatohepatitis (NASH) 6 . These causal pathways are related in that they each contribute to a sustained pattern of hepatic injury and inflammation, which eventually contributes to the development of fibrotic tissue.
There are four basic cell types that reside in the liver. The specialized parenchymal cells are the hepatocytes, and the non-parenchymal cell types are principally liver sinusoidal endothelial cells (ECs), kupffer cells, and hepatic stellate cells (HCSs) 7 . Liver tissues with severe fibrosis suffer from sustained hepatocyte damage and the resulting production of fibrogenic cytokines 8 (such as TGF-β1, angiotensin II), which induce the activation of non-parenchymal cells such as hepatic stellate cells 9 and ECs 10 . Activation of HSCs 11,12 and endothelial-tomesenchymal transition (EndMT) of ECs 10,13 leads to deposition of uncommonly large amounts of ECM that contributes to liver fibrosis. To slow or reverse fibrosis, prior work in the development of anti-fibrotic drugs has Microarray analysis. Global gene expression analysis was performed using Affymetrix GeneChip Human Gene 2.0 ST Arrays. Total RNA from HCC spheroids and MCTS was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA quality was assessed using an Agilent 2100 Bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies), and the quantity was determined using a Nanodrop-1000 Spectrophotometer (Thermo Fisher Scientific). We used 300 μg of each RNA sample as input for the Affymetrix procedure, as recommended in the manufacturer's protocol (http:// www. affym etrix. com). Briefly, 300 ng of total RNA from each sample was converted to double-stranded cDNA using a random hexamer incorporating a T7 promoter, and amplified RNA (cRNA) was generated from the double-stranded cDNA template though an in vitro transcription (IVT) reaction and purified using the Affymetrix sample cleanup module. cDNA was regenerated through randomly primed reverse transcription using a dNTP mix containing dUTP. The cDNA was then fragmented by uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease (APE1) restriction enzymes, and end-labeled via a terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented end-labeled cDNA was hybridized to the GeneChip Human Gene 2.0 ST array for 17 h at 45 °C and 60 rpm, as described in the Gene Chip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix). After hybridization, Subsequently, a 20 μL sample of each compound was dispensed into each well of a 96-well assay plate. The plates were then incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 7 days. Sorafenib was added as a positive control into each assay plate at its IC 50 concentration as a low control and 0.5% DMSO (v/v) was were as a high control. After 10 days, spheroid images were acquired using a high-content screening system. The size of spheroids was measured using a self-developed algorithm. Hit compounds were selected using a threshold based on 3σ (standard deviation) from the IC 50 of sorafenib.

Establishment of multicellular tumor spheroids (MCTSs) that recapitulate important elements of hepatic fibrosis for high-throughput screening of potential liver fibrosis inhibitors. In our
previous study, we found that the interaction between HCC cells and various non-parenchymal cells affected the compactness of the spheroids as well as cell migration through accumulation of collagen and EMT-related proteins 11 . In order to generate a fibrosis model in vitro, various HCC cell lines (Huh7 cells, SNU449 cells, and HepG2 cells) were grown together with fibroblasts (WI38), hepatic stellate cells (LX2), and endothelial cells (HUVEC) in MCTS models. Despite the fact that both SNU449 cells and HepG2 cells innately formed loose aggregates, these cells acquired the rigidness of the spheroids following co-culture with stromal cells in spheroids. Similarly, although Huh7 cells formed a relatively solid spheroids, co-culture with stromal cells enhanced rigidness in spheroids (Fig. 1A). This result showed that crosstalk between stromal cells and HCC cells in MCTS models was an important determinant of rigidness of spheroids, emphasizing the importance of culturing these cells as a system rather than as individual components. Next, gene expression profiling was performed on the MCTS model systems to compare against the expression profiles observed in tumor spheroids. In the MCTS models, genes that were involved in the production of ECM structural constituents were significantly enriched. In particular, we found increased relative expression of MMP1, COL6A1, COL6A3, and TGFB1 in MCTS relative to tumor spheroids [ Fig. 1B].
Because the process of EMT leads to organ fibrosis, we compared the expression of mesenchymal markers such as vimentin, α-smooth muscle actin (α-SMA), Snail, and N-cadherin between the MCTS models and tumor spheroids. Mesenchymal markers were generally upregulated in all MCTS models relative to tumor spheroids alone. In particular, Snail, Vimentin, α-SMA, Collagen I and p-Smad2 were significantly elevated in all MCTS model. Because TGF-β1, which is a critical regulator of fibrosis, stimulates the EMT and EndMT processes MCTSs also displayed upregulated p-Smad3 expression ( Fig. 1C and Supplementary Fig. 1).
The MCTSs provided a useful in vitro model of liver fibrosis, where increased size of spheroids, which results from the loss of tight cross-linking among cells, indicates decreased expression of fibrosis-related proteins and thus provides a reliable morphometric indication of the reversal of liver fibrosis (Fig. 1D). Next, we sought to establish a MCTS-based drug screening platform for the evaluation of potential liver fibrosis inhibitors. To obtain reproducible results, we plated single, homogenously sized and configured spheroids in 384-well plates for HTS screening.
A library comprised of 4,763 drug compounds with known molecular targets was tested for potentially promising candidates as inhibitors of fibrosis. All compounds were screened at an initial concentration of 10 µM in duplicate to confirm the reproducibility of the observed effects. A correlation coefficient of 0.89 for replicate screens indicated that the assay was reliable (Fig. 1E). In that screening, we identified 12 positive compounds (HITs) including four compounds involved in the cAMP/PKA pathway, five retinoic acid analogs, an anti-diabetic drug, a regulator of cholesterol, and a NMDA receptor modulator (Table 1).
Because nintedanib and pirfenidone were recently authorized for the treatment of idiopathic pulmonary fibrosis, we evaluated the effects of both drugs on the size of HCC-MCTSs. Surprisingly, nintedanib and pirfenidone did not alter the morphology of HCC-MCTSs relative to control solvent (2% DMSO) ( Supplementary  Fig. 2). On the other hand, treatment with 10 µM concentration of the 12 HITs significantly increased of size of HCC-MCTSs, according to morphometric analyses, suggesting inhibition of fibrosis.
Next, dose-response studies were performed to find the most effective compounds among the 12 HITs identified from HTS in Huh7.5 cell in MCTSs. We found that the retinoic acid analogs and modulators of cAMP/ PKA pathway, particularly retinoic acid and forskolin, led to the most significant increases of HCC-MCTSs at concentrations as low as 0.1 µM, in a dose-dependent manner ( Fig. 1F and Supplementary Fig. 3).

Retinoic acid and forskolin reversed EMT and EndMT in stromal cells in multicellular tumor spheroids (MCTSs), but not multicellular hepatocyte spheroids (MCHSs). Generally, hepatic
fibrosis is associated with upregulated expression of α-SMA via EMT and EndMT. To investigate the architectural changes observed in MCTSs following treatment with retinoic acid and forskolin, we used immunofluorescence assay to evaluate the expression of F-actin in spheroid structures. Interestingly, when 5 µM of retinoic acid and forskolin were added to MCTSs for 48 h, spheroid size was bigger than DMSO-treated MCTSs, without increasing of cell size, demonstrating loss of tight cell-cell interactions and decreasing F-actin intensity among the cells (Fig. 2A).
Western blot analysis also showed that elevation of α-SMA expression in MCTSs was sufficiently attenuated by treatment with 1 µM retinoic acid and forskolin, whereas expression of CD31 was increased under the same conditions. These results indicated that retinoic acid and forskolin inhibit the EndMT process as well as fibrotic properties in MCTSs (Fig. 2B, Supplementary Fig. 4). The retinoic acid analogs AM580 and TTNPB and the water-soluble forskolin derivative NKH477 also inhibited α-SMA expression in MCTSs, but did not alter expression of CD31, in contrast to retinoic acid and forskolin in MCTSs (Supplementary Fig. 5A).
Next, we were curious whether the replacement of HCC cells with normal hepatocytes in the MCHSs would result in the same phenotypic effects. Instead of Huh7, we used Fa2N-4, a well-known normal hepatocyte cell line, to generate MCHSs. As shown in Fig. 2D, when stromal cells were mixed together, they formed compact spheroids. Similar to MCTSs, MCHSs also showed increasing expression of vimentin, α-SMA, collagen I, and Snail as well as decreasing E-cadherin and CD31 as was observed in the MCHS models ( Fig. 2E and Supplementary Fig. 6). However, the hit compounds identified from HTS, retinoic acid and forskolin, did not change www.nature.com/scientificreports/ the size of spheroids created with normal hepatocytes. This suggests that MCTSs composed of HCCs exist in a severe inflammatory environment that is treatable with these compounds, making it a more suitable model for screening compounds than MCHSs with normal hepatocytes (Fig. 2F). Liver fibrosis is a complex phenomenon orchestrated by numerous cellular actors in tumor microenvironments. These results suggested that retinoic acid and forskolin may inhibit hepatic fibrosis through reversing EMT and EndMT processes of stromal cells in MCTSs and suggested that an MCTS model-based morphometric screening approach may be a good strategy for the screening of novel effective therapies for fibrosis.

Retinoic acid and forskolin depolarized hepatic stellate cells in a fibrotic environment.
To confirm the potential efficacy of retinoic acid and forskolin in reprogramming activated HSCs, which are the main collagen-producing cells in liver fibrogenesis, we conducted cellular phenotype-based assays. Increasing production of α-SMA 23,24 and F-actin stress fibers are associated with HSC activation when HSCs are stimulated with TGF-β1. To define distinctive morphometric signatures before and after TGF-β1 treatment, we focused on the expression pattern of F-actin and α-SMA after treatment with TGF-β1. Treatment with TGF-β1 increased the intense cytoplasmic α-SMA and F-actin of LX2 cells in a dose-dependent manner (Fig. 3A). When the intensity of α-SMA and F-actin were analyzed by Harmony 3.5.1 high-content imaging and analysis software, we found that treatment with 5 ng/ml TGF-β1 increased the intensity of α-SMA more than 1.5-fold compared to the control, whereas intensity of F-actin increased only slightly (Fig. 3B). Therefore, we selected α-SMA as a marker of fiberized hepatic stellate cells. Western blot analysis also displayed similar results in agreement with the cellular . All images were obtained using the Operetta CLS system. Data are expressed as means ± SD (n = 3). ** P < 0.01, and *** P < 0.001 compared to the control group. www.nature.com/scientificreports/ phenotype-based assays. Expression of fibroblast markers, α-SMA, fibroblast activation protein (FAP) and collagen I were increased after TGF-β1 treatment in LX2 cells ( Fig. 3C and Supplementary Fig. 7). Next, we measured the effects of retinoic acid and forskolin on TGF-β1-induced HSC activation using cellular phenotype-based assays. As expected, 1 µM retinoic acid and forskolin inhibited the expression of α-SMA after treatment with TGF-β1 in LX2 cells, with efficacy comparable to 10 µM pirfenidone, which served as our positive control. Particularly, Retinoic acid more efficiently induced reprogramming of activated HSCs activated than forskolin (Fig. 3D). Pirfenidone 25,26 and nintedanib 27 , which are FDA-approved anti-fibrotic drug, inhibit TGF-β1 -induced fibrogenesis. However, in our system, the intensity of α-SMA was not decreased as much as 2% FBS-treated control when pirfenidone and nintedanib were treated at various concentrations ( Supplementary  Fig. 8). When 1 µM retinoic acid (Fig. 3E) and forskolin (Fig. 3F) were added with TGF-β1 to LX2 cells, EMTrelated markers N-cadherin and Snail were inhibited, but E-cadherin was elevated, in contrast to EMT-related markers (Fig. 3E,F). Collectively, this phenotypic-based 2D assay system using LX2 cells appears to be an effective tool for validating anti-fibrosis compounds and suggested that retinoic acid and forskolin can reprogram activated hepatic stellate cells.

Retinoic acid and forskolin suppress the EndMT process in HUVEC.
In our previous studies, we established a visual phenomic screening platform to measure radiation-induced EndMT using HUVECs 28 . Herein, this technology was applied to measure TGFβ1-induced EndMT in HUVECs. HUVECs treated Expression of EMT-related protein (N-cadherin, E-cadherin, Snail) when LX2 cells were treated with 0.5 or 1 µM of retinoic acid or forskolin with or without 20 ng/ml TGF-β1 for 48 h by western blot assay. (F) The quantitative of western blot images was analyzed. All images were obtained using the Operetta CLS system. Data are expressed as means ± SD (n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to the control group, # P < 0.05, ## P < 0.01, and ### P < 0.001 compared to the TGF-β1 treatment group. www.nature.com/scientificreports/ with TGF-β1 expressed increasing amounts of F-actin and cytoplasmic α-SMA in a dose-dependent manner (Fig. 4A). When the intensity of α-SMA and F-actin were analyzed by Harmony 3.5.1 high-content imaging and analysis software, we found that treatment with 20 ng/ml TGF-β1 increased the intensity of α-SMA more than 1.8-fold compared to the control, and intensity of F-actin increased 1.6-fold compared to the control (Fig. 4B). Expression of fibroblast marker, α-SMA was increased after TGF-β1 treatment in HUVEC cells (Fig. 4C). Next, we examined the effects of retinoic acid and forskolin on TGFβ1-induced HUVEC activation using the cellular phenotype-based 2D assay system. In this experiment, we found that 1 µM retinoic acid and forskolin decreased the expression of α-SMA after TGF-β1 treatment in HUVECs relative to treatment with 10 µM pirfenidone (Fig. 4D). Of interest, expression of α-SMA were inhibited when 1 µM retinoic acid and forskolin were added with TGF-β1 to HUVEC cells, (Fig. 4E,F). Retinoic acid also more efficiently induced reprogramming of HUVEC activated than forskolin. These results suggested that anti-fibrotic compounds, such as retinoic acid and forskolin, suppress the EndMT process in HUVECs.

The combination of anti-cancer drugs and anti-fibrosis compounds improves responses by enhancing penetration of anti-cancer drugs.
Liver cancer patients typically experience fibrosis, cirrhosis, and liver-related disease. As shown in Fig. 2A,F, spheroids showed loose compactness after treatment with anti-fibrosis compounds, and cell-cell tight junction interactions were also weak compared to controls. In our previous study 11 , we compared the efficacy of drug penetration by detecting the distribution of doxorubicin using fluorescence microscopy in HepG2 spheroids and HepG2-MCTS grown with LX2 or WI38 cells. In this study, we sought to determine whether the anti-fibrosis compounds may increase the penetration of anti-cancer All images were obtained using the Operetta CLS system. Data are expressed as means ± SD (n = 3). * P < 0.05 and ** P < 0.01 compared to the control group, # P < 0.05 and ## P < 0.01 compared to the TGF-β1 treatment group. www.nature.com/scientificreports/ drugs in MCTSs by decreasing cell-cell interactions. When the MCTSs that were treated with 5 µM retinoic acid or forskolin were treated with 10 µM doxorubicin for 8 h, the distribution of doxorubicin in MCTSs was highly increased relative to spheroids that were not treated with retinoic acid or forskolin (Fig. 5A). Indeed, doxorubicin only penetrated the periphery of MCTSs after treatment with 0.5% DMSO. This result was not surprising in light of the observed decreased compactness of MCTSs after treatment with anti-fibrosis compounds. Based on these results, we expected that anti-fibrosis compounds may accelerate anti-cancer effects by enabling delivery of anti-cancer compounds to the center of tumor tissues. In general, apoptosis-inducing mechanism were investigated refer to evaluate the anti-cancer effects. Among the apoptosis markers, caspase 3 play a role of collaborating the distribution of cellular structure including degradation of DNA and cytoskeleton proteins. In this study, we found that spheroids treated with 1 µM retinoic acid or forskolin combined with 1 or 3 µM of sorafenib had high expression of cleaved caspase-3, as an apoptosis marker, in MCTS model. Caspase 3 significantly higher relative to spheroids treated with sorafenib alone (Fig. 5B). From these results, it appears that anti-fibrosis compounds, such as retinoic acid or forskolin, may improve the efficacy of anti-cancer drugs and attenuate tissue compactness and stiffness observed in liver fibrosis.

Discussion
Fibrosis has been identified as a key factor that influences survival in patients with non-alcoholic steatohepatitis (NASH) 29,30 . Hepatic fibrosis frequently progresses to cirrhosis and hepatocellular carcinoma, but it does not cause symptoms itself. Since there is currently no standard treatment for hepatic fibrosis, there is currently a strong incentive for pharmaceutical companies to develop safe and effective therapeutics 29 . Further, because there are no therapies currently approved for the treatment of hepatic fibrosis, this disease is designated by the FDA as a Fast Track Development indication 31 . In recent years, strategies of target-based approaches for screening smallmolecules have shifted the strategic landscape in the evaluation of drugs that may treat hepatic fibrosis 32 . FXR agonists (such as obeticholic acid) have demonstrated a dramatic reduction in progression and improvement in fibrosis in a phase 2 clinical study, but ultimately failed in phase 3 clinical study because of long-term toxicity 33 . ASK-1(MAP3 kinase 5) inhibition with selonsertib reduced hepatic fibrosis in mouse models, but phase 3 study of selonsertib failed to reprogram hepatic fibrosis 34 . Additionally, a C-C chemokine receptor type 2 (CCR2) and type 5 (CCR5) antagonist (cenicriviroc) provided anti-fibrotic activity in adult patients with hepatic fibrosis, but the anti-fibrotic effect did not meet the primary end point in a phase 2 clinical study. Hence, there remains a significant unmet need for safe and effective medications for the treatment of hepatic fibrosis in NASH 35 .
Investigations of the molecular mechanisms of hepatic fibrosis have presented several clear targets such as TGF-β1, PPAR, ASK-1, angiotensin, YAP-TEAD, various inflammatory cytokines, and ROS. However, there remains no validated target for novel anti-fibrotic compounds 36,37 .
To develop the novel compounds for hepatic fibrosis, we first need to understand the complexity of the molecular mechanisms that govern hepatic fibrogenesis and the local microenvironment. Hepatic fibrosis is caused by chronic inflammation, and the liver tissue becomes rigid due ECM accumulation. Further, this environment results in EMT or EndMT activation 38 . There have been numerous studies assessing novel drugs for fibrosis and molecular mechanisms using 2D culture systems for cells in monolayer on plastic culture dishes. However, www.nature.com/scientificreports/ recent evidence has suggested that 2D systems fail to capture several crucial elements of the 3D environment, and 3D culture systems may be a more effective culture method. Diverse phenotypic approaches for drug screening assays have become increasingly popular in drug discovery as an alternative strategy to target-based approaches for the assessment of potential treatments for hepatic fibrosis [39][40][41] . Particularly, 3D co-culture models represent a high-throughput phenotypic screening system to efficiently screen for new anti-fibrotic therapeutics [42][43][44][45][46] . In this study, we tested whether MCTS models may recapitulate the in vivo microenvironment in fibrosis to generate a phenotype-based model that could overcome the shortcomings seen with 2D systems. In addition to abnormal HSC activation, ECM deposition and stiffness are key phenotypes observed in hepatic fibrosis in vivo 38,47 . Thus, strategies to reverse HSC activation, ECM deposition, and stiffness in spheroid models are critical to develop effective therapeutic agents for fibrosis. We found that MCTSs possessed ECM structural constituents (Fig. 1). Currently,the most common agents that are prescribed off-label for hepatic fibrosis in NASH include vitamin E, ursodeoxycholic acid, pioglitazone, metformin, and lipid-modifying agents 48,49 . Retinoic acid, retinoic acid analogs, and cAMP activators such as forskolin can attenuate hepatic stellate cell activation and have been validated with animal studies [50][51][52][53] . Interestingly, a series of off-label agents for hepatic fibrosis such as ursocholanic acid, rosiglitazone, and retinoic acid analogs were included among the HIT compounds identified herein (Fig. 2, Table 1). Forskolin and forskolin derivatives (NKH 477) attenuate carbon tetrachloride-induced liver fibrosis in rats 54 and were also identified as HIT compounds. The anti-fibrotic effects of forskolin have already been shown in animal models of liver fibrosis 54,55 and intestinal organoids 46 . Hence, our MCTS-based screening system appears to represent an effective approach for the identification of future therapeutics of fibrosis, providing comparable results with animal experiments.
TGF-β plays a key role in the progression of liver fibrosis, and drugs that inhibit TGF-β have been shown to have anti-fibrotic effects in animal studies 56 . The MCTS model used in this study also represented the ECMrelated protein accumulation as well as p-smad activation (Fig. 1). We found that reducing the expression of CD133, a cancer stem cell marker associated with liver cancer, had anti-fibrotic effects and also regulates the surrounding environment, potentially influencing risk of HCC.
Moreover, when we developed spheroids using normal hepatocytes instead of HCC cells in MCHSs, they showed ECM accumulation or mesenchymal cell properties, but did not change the phenotypic properties after drug treatment. From this result, HCC cells with stromal cells appear to best represent pathological characteristics of hepatic fibrosis.
As mentioned earlier, 2D phenotypic assay systems have been adapted for selecting anti-fibrotic compounds through LX2 cell activation with TGF-β1. In Figs. 3 and 4, we also utilized this system for secondary validation of hit compounds from the MCTS-based screening. Hit compounds from the screen were also effective at inhibiting endothelial activity and hepatic stellate activation. The failure rate in clinical trials could possibly be reduced by deriving the first hit through MCTS-based screens and then verifying drug efficacy in a 2D assay that can verify inhibition of EMT and EndMT, followed by confirmation in animal models of disease.
Biopsies of tissues from HCC patients commonly show evidence of cirrhosis and NASH. It has been suggested that drug treatment efficacy in liver cancer patients is lower than other carcinomas due to the hepatic microenvironment 30 . Tissue rigidity due to the accumulation of ECM and excessive inflammatory reactions lowers drug permeability, which reduces the ability of therapeutic compounds to access target cells. We have previously reported that losartan reduced the robustness of MCTS and consequently increased the permeability to doxorubicin. Similarly, in this work, drug permeability was increased when the anti-fibrotic drugs retinoic acid and forskolin were used to treat MCTSs (Fig. 5). The anti-cancer effect of sorafenib, a common treatment for liver cancer, was improved after combined treatment with these anti-fibrotics identified in our model system.
In this study, an in vitro model that reflects the microenvironment observed in hepatic fibrosis in vivo was constructed, characterized, and tested as a model for screening drugs that may be effective treatments for liver fibrosis. We expect that this model offers an efficient, high-throughput strategy to identify new drugs and targets through phenotypic screening. We found that anti-fibrotic drugs are not only effective in the treatment of liver fibrosis, but can also enhance the anti-cancer activity of other therapeutics by increasing tissue permeability, allowing drug delivery to cancer cells of interest.