Patient-Derived Models of Liver Cancer to Inform Clinical Treatment Paradigms: Recent Updates

Abstract

Primary liver cancer remains a global health challenge due to rising incidence, limited curative options, and poor overall survival. Poor outcomes stem from tumor heterogeneity, limited efficacy of current therapies, and comorbid chronic liver disease. Despite recent advances in immunotherapy and combination treatments, response rates remain low, and predictive biomarkers are lacking. As a result, there is an urgent need for preclinical models that capture the molecular, cellular, and immune landscape of primary liver cancer. This review discusses the strengths and limitations of patient-derived models of liver cancer, including two-dimensional patient-derived cell lines (PDCL), three-dimensional (3D) patient-derived tumor organoids (PDTOs), and patient-derived xenografts (PDXs). While PDCLs and PDTOs enable high throughput studies, they lack a representative tumor microenvironment. PDXs, including PDXs in animals with humanized immune systems, may more effectively mimic tumor–environment interactions but are costly, complex, and still contain mouse stromal cells. Ex vivo tissue culture preserves tissue structure and cell–cell interactions in an immunocompetent environment; however, short duration of viable culture limits broader application. Continued innovation in the development of multicellular three-dimensional culture systems and in vivo humanization strategies will play a critical role in enabling the development of more personalized and effective therapies for primary liver cancer.

Keywords

hepatocellular carcinoma - cholangiocarcinoma - patient-derived models - organoids - patient-derived xenograft

Introduction

Primary liver cancer is the third leading cause of cancer-related mortality worldwide with both incidence and associated mortality expected to increase by over 50% over the next 20 years.[1] [2] Hepatocellular carcinoma (HCC) accounts for approximately 80% of primary liver cancer cases in adults, whereas cholangiocarcinoma (CCA) and combined hepatocholangiocarcinoma (cHCC–CCA) comprise most of the remaining cases. Morbidity and mortality rates remain high for these cancers with overall expected survival less than 2 years.[1] [3] This poor prognosis stems from the late stage at presentation, the modest efficacy of locoregional and systemic treatment options, and the chronic liver disease that is heavily comorbid in these patients.

Significant heterogeneity among HCC patients has been a major hurdle in the development of effective treatments.[4] [5] [6] [7] HCC develops in the context of chronic liver disease with over 80% of HCC patients having underlying cirrhosis but from multiple different etiologies that each result in differential impacts on the tumor microenvironment and distinct genetic, epigenetic, and environmental alterations that contribute to the multistep process of oncogenesis.[8] This is evidenced by the mutational landscape of HCCs that demonstrate an average of 30 to 40 mutations per tumor.[9] [10] [11] Notably, no mutation is observed in more than half of patients except mutations in the TERT promoter, and only 15 to 25% of tumors harbor targetable driver mutations.[7] [9] [10] [11]

Over the past decade, considerable progress has been made in developing a growing repertoire of therapeutic options for patients with incurable/unresectable HCC (uHCC) that include both locoregional and systemic therapies.[12] [13] Locoregional therapies (LRTs) continue to be the most commonly used treatments for patients with uHCC; however, choice of modality is mostly driven by provider/center experience and resources, as there are no multicenter, randomized controlled trials comparing these approaches nor treatment-specific biomarkers used in clinical practice.[14] [15] [16] [17] Duration of response remains highly variable, and repeated treatments significantly impact liver function over time. Excitingly, there has been a significant expansion of systemic therapy options driven largely by success using immune checkpoint inhibitors (ICIs) in combination therapies.[18] [19] [20] [21] [22] [23] [24] However, clinical trials in this space have highlighted a tremendous unmet need. While some patients exhibit dramatic and durable responses, overall objective response rates remain ≤30%.[22] [23] [24] Biomarkers of response to ICIs that have been useful in other malignancies have not proven predictive of response in HCC.[25] In addition, each therapeutic combination requires optimization of the scheduling and dosing, as well as appropriate patient selection that takes into account the diversity of molecular alterations and underlying liver dysfunction. Addressing these challenges through human clinical trials alone is infeasible given the vast numbers of potential therapeutic combinations and the prohibitive costs required. Consequently, there is an urgent need for sophisticated translational models that recapitulate the complexity of tumor-intrinsic factors, the tumor immune microenvironment, and underlying chronic liver disease seen in HCC patients.

Patient-Derived Models of Liver Cancer

Historically, two-dimensional cancer cell lines have been the backbone of liver cancer research, as they are relatively low-cost, can be grown in vitro as well as in vivo in immunocompromised mice, and allow for high-throughput drug screening approaches. Notably, there are several important drawbacks to these models, including the incomplete recapitulation of genetic variants observed in patients, absence of intratumoral heterogeneity, genotypic and phenotypic drift in culture, and lack of tumor microenvironment in vitro ([Table 1]).[26] [27] [28] [29] [30] Models designed to address some of these challenges, including novel patient-derived cell lines (PDCL) with coculture systems, patient-derived tumor organoids (PDTO), and patient-derived xenografts (PDX), will be herein reviewed ([Fig. 1]).

Table 1 Comparison of advantages and limitations of patient-derived models

	Patient-derived cell lines	Patient-derived tumor organoids	Patient-derived xenografts
Advantages	(1) Low cost, straightforward protocols (2) Robust system for investigating molecular mechanisms. (3) Easy to scale for high throughput screens	(1) Model 3D structure of tumors including nutrient and oxygen gradients (2) Can culture directly from patient biopsies (3) Scalable for high-throughput screens (4) Maintain molecular and histologic features for at least 1 y in culture	(1) Preclinical drug testing for efficacy, toxicity, and pharmacokinetics (2) Large amount of tissue for experiments (3) Maintain molecular and histologic features through at least five passages (4) Allow for studies of locoregional therapies
Limitations	(1) Loss of intratumor heterogeneity (2) Loss of 3D structure, cell–cell and cell–ECM interactions (3) Lack of tumor microenvironment (4) Higher potential for genotypic/phenotypic drift (5) Low take rates (6) Poor representation of well differentiated tumors	(1) Require more technical expertise and resources that PDCLs (2) Lack of tumor microenvironment (3) Low take rates (4) Poor representation of well differentiated tumors	(1) Severely immunodeficient animals required (2) Stromal cells are rodent (3) Expensive and resource-intensive to scale (4) Low take rates (5) Poor representation of well-differentiated tumors
Recent advances and future direction	(1) Coculture systems with nontumor cells (2) Expanded number with LIMORE to over 81 PDCLs (3) Multiregional tumor sampling to capture intratumor heterogeneity	(1) Multicellular clusters rather than single cell suspension for increased take rates (2) Coculture systems with nontumor cells (3) Ex vivo culture systems of PCTS	(1) Rodent models with humanized immune system and humanized livers (2) Potential to combine locoregional and systemic therapies

Abbreviations: 3D, 3 dimensional; ECM, extracellular matrix; PDCLs, patient-derived cell lines; PCTS, precision cut tissue slices.

Fig. 1 Preclinical models of liver cancer using patient tissue. Schematic representation of different patient-derived model systems. Created in BioRender. Simon, C. (2026) https://BioRender.com/uh0pvpu .

Patient-Derived Cell Lines

PDCLs have been widely used in preclinical cancer biology research due to their utility in dissecting molecular mechanisms and testing drug sensitivity, as well as their relative ease of use and low cost. These lines can be used for high throughput screening to elucidate tumor-specific vulnerabilities to a rapidly escalating number of molecularly targeted cancer therapies. With increasing recognition of the enormous degree of genomic heterogeneity across human cancers, there has been considerable effort to generate large datasets that link the molecular and pharmacologic profiles of a large number of cell lines in order to identify biomarkers of response to therapies that would likely be missed when only testing a few tumor lines.[31] [32] [33] [34] However, these efforts in liver cancer have been limited by several key factors. First and foremost has been the difficulty in culturing primary liver cancer cells, and, therefore, the inability to represent the full diversity of patient tumors. Until recently, there were approximately 30 HCC cell lines available and only a handful of CCA lines. The majority of these PDCLs were established in the 20^th century without molecular profiling available for the primary tumor tissue; therefore, it is not clear how well these cell lines recapitulated the source tumors.[32] In addition, several commonly used lines are not HCC (SKHEP, HepG2), and others are known to be contaminated with other human cell lines, particularly HeLa cells.[35] [36]

To address some of these concerns, Caruso et al. performed genomic, transcriptomic, and proteomic profiling, as well as drug screening, on 34 publicly available HCC PDCLs and compared the findings to profiling done on 821 primary HCC tumors.[37] Their results demonstrated that HCC PDCLs had similar molecular signatures to HCC patient tumors with a few important caveats. First, HCC PDCLs were derived from mostly Asian patients with viral hepatitis (55% hepatitis B virus [HBV], 33% hepatitis C virus). Second, the PDCLs were enriched for molecular signatures of the “proliferative” HCC signature, including more frequent mutations in TP53, AXIN1, and FGF19/CCND1. These existing HCC PDCLs underrepresent tumors with CTNNB1 mutations and “nonproliferative” transcriptional profile, most likely due to the bottleneck created by in vitro culture. Lastly, the PDCLs had a higher median mutation rate compared to primary tumors, suggesting selection for cells with greater genetic instability or the occurrence of genetic drift in culture. Qiu et al. sought to more directly evaluate HCC PDCL fidelity to parent tumors by developing nine novel HCC PDCLs with molecular comparison to the parent tumor through early passages of these primary cells and established PDCLs after >20 passages.[38] Whole-genome sequencing demonstrated that HCC PDCLs shared greater than 80% of genetic alterations with the matched primary HCCs, including single nucleotide variants (SNVs), copy number alterations (CNAs), and HBV integration sites. While additional mutations were seen in PDCLs (established > early passages) compared with the primary tumor, the cell line-specific mutations were unique to each cell line with no common mutations seen among the cell lines and no mutations found in any reported HCC driver gene. Instead, these mutations were enriched for extracellular matrix- and cell cycle-associated pathways likely reflecting the nature of in vitro culture. Expanding on this work, the Liver Cancer Model Repository (LIMORE) was established, combining publicly available and newly generated HCC PDCLs.[39] Using a ROCK inhibitor to facilitate attachment of primary cells and a TGFb inhibitor to inhibits mesenchymal cells, success rate of primary HCC culture increased to over 40% allowing for the development of an additional 50 HCC PDCLs, for a total of 81 cell lines included in the repository. This expansion increased the diversity of available HCC cell lines with 85% of HCC driver mutations covered by at least one PDCL and over 50% of HCC driver mutations covered by at least three PDCLs. While these cell lines still overrepresent Asian patients with HBV and the “proliferative” phenotype (Hoshida S1 and S2, TP53 and FGF19 mutations), around 30% had mutations in the Wnt/B-catenin pathway (CTNNB1, AXIN1, APC) with a “nonproliferative” transcriptional signature (Hoshida S3).

To address challenges with inter- and intra-tumoral heterogeneity, Gao et al. demonstrated that culturing patient-derived cells from multiregional sampling of HCC tumors enabled increased representation of the genetic and phenotypic heterogeneity of HCC and provides a platform to determine how this intra-tumoral heterogeneity impacts sensitivity due different therapies.[40] Comparing 55 HCC PDCLs from 10 tumors, the mean percentage of heterogeneous mutations was around 40% with fewer than half of driver alterations occurring early in HCC evolution. Importantly, of the druggable alterations found in two tumors, all were later branch events leading to differential sensitivity of subclones within one tumor, highlighting the complexity of targeted therapy in HCC. However, a drug screen of 28 compounds from their in-house library was able to identify drugs that were effective against all subclones, suggesting that this strategy may be a powerful tool for precision medicine approaches though more costly and labor intensive. Notably, the HCC tumors used in this study were all from resection specimens of patients with HBV-driven HCC—a limitation of HCC PDCLs in general as these approaches are limited by tissue availability. Resection specimens are inherently biased to solitary lesions, early-stage disease, and patients without advanced cirrhosis, leading to dearth of PDCLs that represent intermediate and advanced HCC patients—the patients most in of need targeted therapies. Even with an expanding number of HCC PDCLs, cell lines are inherently limited in their ability to retain tumor cell three-dimensional (3D) structure, cell-to-cell interactions, and the tumor microenvironment. Novel culture systems have been developed to minimize each of these limitations with a major technical advance coming with the development 3D culture systems.

Patient-Derived Tumor Organoids

Over the past two decades, there has been an increased awareness of the importance of the 3D aspects of solid tumors in tumorigenesis, as well as in tumor cell proliferation, invasion, metastasis, and response to therapy.[41] [42] [43] [44] [45] [46] [47] While in vivo models are important tools for investigating these factors, they are limited by intrinsic differences between animal models and human biology, as well as increased cost and complexity of experimental design. Advances in 3D cell culture techniques, driven by a better understanding of extracellular matrix biology and regulation of stem cell niches/differentiation, have allowed for the development of PDTOs that preserve their histologic and molecular integrity over months in culture.[48] [49] [50] The 3D structure of the PDTOs enables modeling of in vivo conditions such as cell morphology, cell–cell interactions, and cell growth kinetics. Nutrient and oxygen conditions mimic cancer cell programs, including hypoxia and angiogenesis signaling activation, that are not seen in two-dimensional (2D) models.[46] [51] Similarly, drug sensitivities may be dependent on the culture type, especially with cytostatic drugs, and drug sensitivities observed in PDTOs have better correlated with molecular alterations than drug sensitivities in PDCLs.[46] [47] Importantly, PDTOs have demonstrated utility for high-throughput drug screening approaches that were previously only possible in 2D culture systems, although these approaches in PDTOs do require more technical expertise and resource investment.[52] [53]

Patient-Derived Liver Cancer Organoids

Huch et al. optimized conditions for the development of first mouse and then human liver organoids.[54] [55] [56] In brief, liver cells are isolated from liver tissue using collagenase–acutase digestion and then resuspended in a basement–membrane matrix (Matrigel or Basement Membrane Extract) as domes that are then submerged in culture media. The media is initially supplemented with a ROCK inhibitor to minimize apoptosis and promote proliferation, as well as Wnt and Noggin to prevent premature cell differentiation and promote the maintenance of stem/progenitor cells. Interestingly, these culture systems favor growth of primary human bile duct cells; however, these cells can be differentiated into functional hepatocytes. Building on this work, Broutier et al. optimized protocols that allowed for the establishment of primary liver cancer organoids from resection specimens and demonstrated that these models recapitulate the histologic architecture, genomic landscape, and transcriptomic and protein expression profiles of the parent tumor even after long-term expansion in vitro for up to 1 year.[57] Comparing whole-exome sequencing (WES) of the parent tumor tissue to early PDTOs (<2 months) and late PDTOs (>4 months) confirmed that tumoroid cultures retained >90 and >80%, respectively, of the genetic variants found in the parent tumor. Importantly, PDTOs from different patients showed differential drug sensitivities when treated with 29 different anticancer compounds. In addition, when PDTOs were implanted into immunocompromised mice, each recapitulated parent tissue histology, metastatic capability, and drug sensitivity. Notably, only eight tumoroid lines were generated during this initial study, limiting the generalizability of the results; however, it clearly established the broad range of translational potential for liver tumor organoids. Saito et al. further leveraged these techniques to establish additional organoid lines for 3/6 (50%) intrahepatic CCA and 1/5 (20%) gallbladder carcinomas.[58] Nuciforo et al. adapted the protocol to allow for development of PDTOs from liver cancer biopsies specimens with much more limited primary tissue, representing an important advance in the clinical utility of tumor organoids, as this allows inclusion of patients with unresectable tumors—the population most likely to receive systemic therapy and with the greatest unmet therapeutic needs.[59] In addition to confirming that PDTOs generated from patient biopsies recapitulate the histologic, genetic, and transcriptional profiles of the parent tissue, Nuciforo et al. also demonstrated that PDTOs are polyclonal and largely preserve the intratumor heterogeneity of patient biopsies. To further investigate the impact of tumor heterogeneity, Li et al. generated 27 PDTOs from 5 patients with primary liver cancer using multifocal tumor sampling of resection specimens.[60] The authors then tested drug response to 129 cancer drugs and highlighted the impact of inter- and intratumor heterogeneity on drug sensitivities by classifying drugs into four categories: (1) pan-effective; (2) intra-patient divergence; (3) inter-patient divergence; and (4) largely ineffective. Notably sorafenib and gemcitabine were two of the top interpatient divergent drugs and are clinically used in HCC and CCA, respectively, suggesting that better patient selection through functional testing could improve treatment outcomes. Overall, this approach would likely increase the chances of discovering clinically relevant phenotypes, though resection specimens or multifocal liver biopsies would be necessary. In addition, it remains unclear how much sampling per patient and/or tumor would be sufficient to capture clinically relevant heterogeneity.

Limitations of Patient-Derived Liver Cancer Organoids

Current liver cancer PDTOs remain limited in their utility for precision medicine approaches by their low derivation efficiency. Thus far, liver cancer PDTOs have been unable to overcome the low take rates also seen in PDCLs with only approximately 30% of primary tumors developing PDTOs regardless of tissue specimen source (resection or biopsy), noting that this rate is lower for HCC organoids than CCA organoids (26–50 vs. 50–75%, respectively).[57] [58] [59] [61] [62] [63] [64] Established HCC PDTOs are also enriched for poorly differentiated tumors with notably no HCC PDTOs generated from well differentiated (Edmondson Grade I) tumors using conventional protocols.[57] [59] [61] [62] [63] Dong et al. showed that using multicellular clusters that include tumor cells, stromal cells, and noncellular components increased take rates of HCC PDTOs to 65%, including one PDTO from a well differentiated tumor.[65] Maier et al. also found that using cell clusters improved establishment of CCA PDTOs.[66] While increasing expertise and standardization of tissue collection, preparation, and culture management have improved take rates, the heterogeneity in cancer stem cells, particularly in HCC, will likely require a deeper understanding of the drivers of tumor stem cell proliferation in order to represent the diversity of primary liver cancers. In addition, cell culture conditions still need to be optimize to allow for high throughput drug screening without impacting tumor cell biology.[52] [53] [65] [67] Finally, while genetic and phenotypic drift in culture is less with PDTOs than traditional 2D cell lines, genetic and transcriptional discordance increases with time in culture and number of passages, and it is not yet known at what point these discrepancies become clinically relevant.

Future Directions of Patient-Derived Liver Cancer Organoids

Current HCC PDTOs have focused on expanding the epithelial component of tumors and are notably lacking in the cell populations and spatial organization that constitute the tumor microenvironment, limiting their clinical utility especially for the study of immune and antiangiogenic therapies, which are currently the standard of care for advanced disease. Several emerging coculture model systems have demonstrated promise in dissecting tumor–stromal cross talk. Coculture of HCC cell line-derived organoids with endothelial cells from human umbilical veins demonstrated endothelial cell differentiation and tubule network formation in the absence of exogenous growth factors (VEGF, SDF-1) with a hypoxic tumor core and true gradient penetration of drugs comparable to PDX tumors.[68] [69] In addition, the endothelial-HCC interactions results in production of tumor necrosis factor signaling and proinflammatory cytokine production from HCC cells. Notably, vessel formation required a synthetic hydrogel specifically designed to have adhesion ligands and matrix metalloproteinase (MMP)-sensitive domains, and the vasculature network generally declined after 3 days of coculture due to absence of pericytes; however, this model was able to identify differences in the ability of antiangiogenic therapies to induce apoptosis of endothelial cells versus only prevent angiogenesis.[68] Zhou et al. demonstrated that coculture of HCC PDTOs with autologous tumor infiltrating lymphocytes (TILs) compared with autologous peripheral blood lymphocytes resulted in enhanced antitumor activity in a TIL coculture system, though only the TILs with the most cytolytic activity resulted in tumor regression in vivo.[61] Further studies are needed to dissect the clinical relevance of these in vitro findings in the in vivo setting. Similarly, cultures systems for CCA PDTOs have also focused on expanding the epithelial counterpart of the tumor despite knowledge that these tumors contain a high degree of stromal reaction and desmoplasia.[70] Interestingly, when CCA PDTOs were implanted into immunodeficient mice, the tumors displayed areas of desmoplastic stroma reaction, suggesting that tumor intrinsic features play a role in recapitulating this phenotype if the appropriate factors are present in the tumor microenvironment.[59] Notably, coculture with cancer-associated fibroblasts promoted liver cancer PDTO growth and conferred drug resistance via direct cell–cell contact and paracrine factors in both HCC and CCA PDTOs.[71]

Even with these advances, PDTOs are inherently limited in their ability to represent the complex interactions of the in vivo tumor microenvironment and its impact on therapeutic response. Ex vivo culture of precision cut tissue slices (PCTS) can preserve the tissue structure, extracellular matrix composition, and cellular heterogeneity and function of liver tissue and has been used to study drug metabolism and fibrosis. However, these models are significantly limited by a functional life span of 24 to 48 hours that is thought to be due to cell death from hypoxia.[72] Innovative strategies to improve oxygenation through air–liquid interfaces or proprietary bioreactors have increased liver tissue viability to 7 to 8 days.[73] [74] Jagatia et al. reported the first PCTS of human liver cancer and demonstrated that tumor morphology, stroma, TILs, and tumor immunophenotype could be maintained for at least 8 days in vitro.[75] Lastly, Collins et al. have shown that PCTS of cell line-derived HCC xenografts could be leveraged for scaled drug screening in the 96-well plate format.[76]

Patient-Derived Xenografts

While advances in 3D culture are improving our ability to mimic the tumor microenvironment in vitro, in vivo animal models remain the gold standard for investigation into tumor biology and response to therapy. Genetically engineered mouse models (GEMMs) have proven utility in studying of the impact of a single or combined genetic alteration(s) on tumorigenesis and tumor biology; however, these models lack the heterogeneity and complexity of the molecular alterations present in human HCC.[77] Chemotoxic models allow for autochthonous tumors that have greater genetic diversity than GEMMs with tumor vascularization that mirrors human tumors, but tumorigenesis is more variable with longer latency and by different mechanisms than those seen in patients.[78] [79] These syngeneic models utilize immunocompetent animals and allow for investigations into interactions between HCC tumors and the immune system with a few notable limitations. First, there are known differences in immunotherapy targets for human and murine homologs, and several FDA-approved immunotherapies fail to bind to equivalent mouse targets.[80] Second, GEMM tumors often have limited immunogenicity and often require additional strategies to produce the immunosurveillance and immunotherapy responses seen in patients.[81] [82] Lastly, there are notable recognized, and likely unrecognized, differences in both innate and adaptive immunity between the murine and human immune systems that can limit translatability of findings.[83]

PDXs, in which human tumors are implanted into immunodeficient mice, recapitulate the heterogeneity of human tumors and patient responses to therapy and are frequently used in preclinical therapeutic trials.[84] [85] Historically, liver cancer PDXs have largely been generated from patients with surgically cured, early-stage disease, likely representing less aggressive tumors that may never require the systemic therapies being investigated.[86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] Recent studies demonstrate the potential to generate liver cancer PDXs from biopsy samples, allowing for the development of PDXs from patients with all stages of disease.[103] [104] Historically, the lack of immune system in these models has limited their utility for informing pressing questions in immuno-oncology; however, advances in immunodeficient mice and our understanding of hematopoietic stem/progenitor cells has promoted the development of humanized mouse models that allow for the in vivo interaction of human immune cells and human tumors.

Patient-Derived Liver Cancer Xenografts in Immunocompromised Mice

The limitations of cell cultures systems, along with the increased availability of severely immunodeficient mice, have led to widespread use of PDXs for studying cancer biology and drug responsiveness. Patient-derived tumor cells or tissue can be implanted heterotopically in the flank using a subcutaneous injection or orthotopically in their organ of origin by surgical approaches ([Fig. 2]). Xenograft models of primary liver cancer have included implantation of liver cancer cell lines, organoids, and patient cells or tissue. The first HCC PDX was established in 1996 via orthotopic implantation of HCC resection specimens in nude mice, noting the development of a PDX in only 1/30 (3.3%) mice.[86] Engraftment rates improved with the use of more immunodeficient mouse strains and tissue fragments rather than cell suspension; however, overall engraftment rates still remain low at around 20 to 40% in large cohorts.[62] [91] [93] [98] [103] The highest take rates have been seen in Nod-scid and Nod-scid IL2Rγ^null (NSG) mice that lack functional B, T, and natural killer (NK) cells with impaired macrophages, dendritic cells, and complement response.[89] [95] [97] [98] [104] In addition, various mechanisms of liver injury, including partial hepatectomy or induction of cirrhosis, may improve PDX engraftment, underscoring the important role that the tumor microenvironment plays in regulating tumor cell proliferation.[91] [100] [102] [105] Importantly, several studies have reported human lymphoma formation during the initial establishment of PDX models due to outgrowth of human lymphocytes infected with Epstein-Barr virus from patient liver cancer resection or biopsy specimens, highlighting the importance of histologic confirmation for all PDX lines.[89] [103] [104] Tischfield et al. demonstrated that implanting HCC tissue fragments in Matrigel reduces the risk of lymphoma formation from 2/4 PDXs (50%) to 1/7 PDXs (14%).[104] Xian et al. directly compared success rates of developing primary liver cancer PDTOs and PDXs from the same tissue samples and found no significant difference (29 vs. 24%, respectively), though notably this study was done using nude mice rather than NSG mice.[62] Success rates for CCA and cHCC–CCA were much higher for both PDTOs (9/17, 53% and 5/5, 100%) and PDXs (7/17, 41% and 3/5, 60%), likely reflecting the generally more aggressive tumor biology of these liver cancer subtypes.

Fig. 2 In vivo patient-derived models of liver cancer. Schematic of animal model systems used to implant liver cancer cell lines, organoids or tissue to model liver cancer in vivo. HSCs, hematopoietic stem cells; IV/IO, intravenous/intraosseous; PBMCs, peripheral blood mononuclear cells; SQ/OT, subcutaneous/orthotopic, TACE, transarterial chemoembolization; TAE, transarterial embolization. Created in BioRender. Simon, C. (2026) https://BioRender.com/uh0pvpu .

Multiple studies have demonstrated the fidelity of liver cancer PDXs to their primary tumors in regard to histology, molecular profiles, clinical prognosis, and response to approved therapies.[62] [93] [94] [96] [98] [100] [103] [104] Analysis of the histology of these tumors demonstrates that liver cancer PDXs retain the features of their parent tumor and maintain intertumor heterogeneity as evaluated by differentiation status, growth patterns, cytological subtypes, and liver cancer and hepatocyte/cholangiocyte markers by immunohistochemistry.[93] [98] [103] Liver cancer PDXs retain the genotypes of their parent tumor as measured by WES, short-tandem repeat analyses, and single-nucleotide polymorphism (SNP) arrays with a median of 85% (range: 65–100%) of somatic mutations in the HCC parent tumor expressed in the respective HCC PDX.[103] Notably, all missense mutations in HCC driver mutations were expressed in the corresponding PDX tumors stably over at least six PDX generations. With regard to gene expression profiles, HCC PDX tumors show downregulation of inflammatory and angiogenesis pathways, consistent with the loss of human immune cells and replacement of human vasculature and stroma with mouse vessels and stromal cells.[103] Importantly, the PDX tumors maintained the tumor-specific transcriptomic profiles of their parent tumors, and these profiles remained remarkably stable between the first and sixth generation of PDX tumors. These findings were consistent with prior data suggesting HCC PDXs have the least amount of genome discordance from their primary tumors compared to PDXs from multiple solid tumors in a large scale analysis of CNAs assessed by SNP arrays and WES.[106] Further supporting the fidelity of PDX to parent tumor, HCC PDXs have also been shown to predict patient prognosis, specifically the risk of relapse after resection and patient response to tyrosine kinase inhibitors.[100]

Limitations of Patient-Derived Liver Cancer Xenografts in Immunocompromised Mice

A major limitation of PDX models is the limited scalability due the cost and resources required. In addition, the low derivation rate and underrepresentation of well-differentiated, “nonproliferative” subtype of HCC, similarly to liver cancer PDCLs and PDTOs, limits the generalizability of findings from liver cancer PDXs.[93] [96] [98] Interestingly, PDTOs are more enriched for aggressive cell types than PDXs based on expression of HCC stem cell markers in PDTOs and PDXs from the same parent tissue.[62] While PDXs better represent the interactions between tumor cells and the stromal and vasculature compartments, current models are still limited in their ability to recapitulate the phenotypes of chronic liver disease and cirrhosis seen in patients, as well as tumor–immune interactions given the lack of functional immune system.[78] Huang et al. demonstrated the impact of the tumor microenvironment on prognosis and therapeutic response by treating HCC PDXs in mice with and without cirrhosis.[100] Notably, metastases only developed in the cirrhotic mice and correlated with early recurrence postresection in patients. In addition, only the cirrhotic PDX model predicted patient responses to sorafenib and lenvatinib. Lastly, while PDXs have been leveraged to model ablation and external radiotherapy, HCC PDXs to date have been developed in mice, and their small size has prohibited investigation into endovascular LRTs.[107] [108] [109] Recent developments in immunodeficient rat strains have allowed for the implantation of PDX tumors into rats allowing for larger tumor development, more relevant pharmacokinetic and toxicology analysis, and the potential for investigation into endovascular LRTs.[110] [111] [112]

Patient-Derived Liver Cancer Xenografts in Humanized Mice

Advances in immunodeficient rodents strains have resulted in not only more efficient PDX engraftment rates, but also the development of various humanized models that have become indispensable tools in modeling human-specific biological processes across a wide array of disciplines.[113] [114] [115] These humanized rodent models can be engrafted with human hematopoietic cells, human tissues, and human pathogens and have been leveraged for investigation into cancer pathogenesis, progression, immune evasion, and response to therapies, including ICIs, bispecific antibodies, and chimeric antigen receptor (CAR) T cells.[115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126]

Most commonly “humanized mice” refers to immunodeficient mice xenotransplanted with human hematopoietic cells to generate mice with a humanized immune system (HIS). For immune–oncology applications, these HIS mice can then be implanted with PDX tumors to allow for human immune cell and human tumor cell interactions in vivo. There are several different HIS models mostly based on the source of human hematopoietic cells ([Fig. 2]). Human peripheral blood mononuclear cells (PBMCs) or isolated T cells can be injected intravenously in NSG mice leading to good engraftment of memory and effector T cells; however, B cells and myeloid cells do not engraft well, and these mice uniformly develop xenogeneic graft-versus-host disease (xGVHD) within 2 months of implantation.[127] This model can be useful for evaluating therapies that suppress human T cell responses, such as antibody- or regulatory cell-based therapies or cytokines, in highly proliferative PDX tumors. Alternatively, engraftment with human hematopoietic stem and progenitor cells (HSPCs, i.e., CD34⁺ cells) from fetal livers, cord blood, or bone marrow result in HIS mice that produce all lineages of hematopoietic cells, including both innate and adaptive immune cells, with delayed onset of xGVHD to over 6 months posthumanization.[128] [129] While lymphoid and myeloid progenitor cells engraft well in the bone marrow, there are low rates of mature innate immune cells in the periphery of these mice, including immune cell types important for tumor immune evasion such as macrophages, neutrophils, dendritic cells, and NK cells.[113] [114] [115] [130] In addition, these models are limited by the lack of HLA expression for HLA-restricted T cell maturation and the relative absence of secondary lymphoid structures.[115] [131] [132] [133]

Next-generation humanized mice have been engineered to express various human cytokines and/or HLA proteins in order to overcome many of these limitations, including models that minimize the development of xGVHD and support the expansion and maturation of specific immune cell populations and lymphoid structures.[115] [131] [133] [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] [145] [146] Each model has its own strengths and limitations that need to be considered in the context of the specific research question being asked.[115] Mouse strains genetically engineered to lack mouse major histocompatibility complex (MHC) class I and II molecules can be engrafted with PBMCs or isolated T cells with the onset of xGVHD delayed from 3 to 5 weeks to 8 to 11 weeks posthumanization without any deaths observed up to 14 weeks.[144] [145] [146] Building on this further, HUMAMICE were engineered to have deficient mouse MHC molecules and express human HLA molecules, reducing xGVHD and allowing for antigen-specific T and B cell responses when HLA-matched PBMCs were engrafted.[134] Alternatively, the coimplantation of human fetal liver HSPCs and fetal thymic tissue (BLT mice) also allows for T cell education on human rather than mouse MHC molecules.[137] [147] [148] BLT mice develop multilineage hematopoiesis, secondary lymphoid organs, and functional adaptive immune responses; however, this model is limited by the availability of human fetal liver and thymic tissue, limited development of the innate immune compartment, and earlier development of xGVHD than mice humanized with fetal liver cells alone. Similarly, two novel immunodeficient mice strains have been developed that restore the generation of lymphoid tissue inducer (LTi) cells and lymph node development through either the expression of IL-2R in LTi cells or expression of thymic stromal lymphopoietin in epithelial cells.[132] [133] While these updated models improve lymphoid cell maturation and function, they do not address the limited development of the myeloid compartment in humanized models. To that end, immunodeficient mice were genetically engineered to express M-CSF, IL-3/GM-CSF, TPO^+/− SIRPa (MITRG, MISTRG).[143] HIS MISTRG mice have high efficiency HSPC engraftment and support the development of functional and diverse innate immune cells, including monocytes, macrophages, and NK cells, with the caveat that increased phagocytosis of mouse erythroid cells leads to anemia in these models.

In the context of liver cancer, HCC PDXs have been used in several adopted cell transfer models using human PBMCs, T cells, and CAR T cells, though many investigators implant HCC cell lines rather than PDXs due to the rapid onset of xGVHD in these models.[121] [122] [123] Zhao et al. developed a HIS HCC PDX model that utilized human fetal liver-derived, partially HLA-matched HSPCs to humanize NSG mice and then HCC PDX tumors were implanted subcutaneously.[117] The model recapitulated key features of the HCC tumor immune microenvironment, specifically the upregulation of immune checkpoint markers on tumor cells, a T cell exhaustion phenotype in the TILs, and enrichment for tumor-associated macrophages (MØ2) and myeloid-derived suppressor cells. In addition, HCC PDX tumor growth was inhibited by ICIs in the HIS mice but not in the nonhumanized controls. Importantly, the humanized mice also exhibited immunotoxicity consistent with the immune-mediated adverse events that occur in patients. In a follow-up study, the HIS HCC PDX mouse model was successfully used to elucidate molecular and cellular pathways through which HCC tumor cells interacted with the immune system, as well as the effect of combination therapies.[118] Weinfurtner et al. demonstrated that utilizing an immunodeficient mouse strain that expresses human GM-CSF and IL-3 (NOG-EXL) increases overall tumor immune infiltration and the proportion of myeloid cells and regulatory immune cells, including MØ2 macrophages and regulatory T cells.[149] In addition, the model was further optimized for orthotopic rather than subcutaneous implantation of HCC PDX tumors and partial HLA-matching of HSPCs to the HCC PDX tumor using adult bone marrow-derived HSPCs. As this study and others demonstrated, using adult bone marrow-derived HSPCs requires more cells than HSPCs from fetal liver or cord blood, but these cells are more readily available in the quantities required for HLA-matching.[149] [150] Due to supraphysiological levels of human GM-CSF and IL-3, all mice in this model eventually develop macrophage activation syndrome (MAS), a fatal condition similar to hemophagocytic lymphohistiocytosis seen in patients.[151] [152] The onset and severity of MAS varies by mouse strain, degree of human chimerism, HSPC donor, and HCC PDX.[150] [152] [153] HIS MITRG/MISTRG mice do not develop MAS as the human cytokines are knocked-in to their respective mouse cytokine loci and are expressed at more physiologic levels; however, these strains are not commercially available and have not yet been implanted with HCC PDXs.[143]

Ongoing Limitations and Future Directions of Humanized Patient-Derived Xenograft Models

HIS PDX models represent significant progress in the evolution of patient-derived models and the ability to study tumor and immune interactions in vivo; however, there are several important limitations that currently hinder their scalability and broader application in translational research. The generation of these models is resource-intensive, requiring highly immunodeficient host strains, access to rare and ethically sensitive human tissues, and the use of specialized facilities with significant technical expertise. Given the logistical challenges of obtaining autologous bone marrow or mobilized peripheral blood from patients with cancer, most of these models are allografts rather than autografts with significant variability between donors in engraftment efficiency and immune reconstitution, which can reduce experimental consistency, throughput, and translatability. Importantly, there has been considerable effort to improve HSPC engraftment efficacy to reduce the amount of cells need and therefore cost of experiments, including new immunodeficient mouse strains, direct interosseus injections, and humanizing pups rather than adult mice.[153] [154] [155] Lastly, clinically relevant interventions such as locoregional therapies (e.g., endovascular LRTs) are not technically feasible in mice due to their small size and vascular anatomy. To overcome this limitation, there is growing interest in developing HIS models in larger animals, particularly in rats.[156]

Overall, substantial progress has been made in the ability to model human tumor immunology in rodents, and the continued evolution of HIS PDX models will be critical for advancing translational therapeutics for cancer patients. Future efforts in HIS PDX models of primary liver cancer will focus on improved representation of the heterogeneity of patient tumors and the complexity of the human tumor microenvironment. Humanizing other elements of the tumor microenvironment, such as hepatocytes, hepatic stellate cells, and endothelial cells, has been shown to improve human immune cell reconstitution in the liver, including tissue resident immune cells (i.e. Kupffer cells).[157] [158] In addition, HIS mice with humanized livers faithfully recapitulate features of chronic liver disease seen in patients, including alcohol-associated hepatitis, metabolic dysfunction-associated steatohepatitis, and viral hepatitis, allowing for a more accurate representation of the tumor microenvironment.[158] [159] [160] It is possible that mice with these liver microenvironments could improve engraftment rates for nonproliferative, well-differentiation subtypes of HCC that are underrepresented in current HCC PDXs. In addition, advances in gene editing using CRISPR/Cas9 could be leveraged to introduce specific driver mutations in human liver progenitor cells or PDX tumors prior to implantation in these humanized models to study the impact of specific mutations on tumor–immune cell interactions in the setting of varying causes of liver injury. Furthermore, the integration of HCC PDXs with autologous HSPCs could enable fully personalized humanized models for preclinical immunotherapy screening and assessment of individualized treatment regimens.

Concluding Remarks

As the incidence of and mortality from primary liver cancer continue to rise globally, there is an urgent need for preclinical models that can faithfully recapitulate the molecular, immunologic, and microenvironment complexity of human disease. The development and refinement of patient-derived models, including cell lines, organoids, and xenografts, have significantly advanced our ability to study liver cancer in preclinical settings. As discussed in this review, each model offers unique advantages and limitations. More recently, the advancements in humanized rodent models provide an in vivo model to study tumor–immune interactions, assess immunotherapy responses, and investigate mechanisms of resistance; however, the full translational potential of these HIS models is currently limited by high cost, limited scalability, and incomplete immune reconstitution. Simultaneously, innovative coculture systems for PDTOs have dramatically improved our ability to reproduce the tumor microenvironment in vitro and have the potential to revolutionize high throughput drug screening. Together, these novel patient-derived model systems promise to bridge the gap between experimental findings and clinical phenotypes, offering a path forward toward more effective and personalized treatment strategies for primary liver cancer.

Conflict of Interest

K.W. received research funding from Astra Zeneca through the Society of Interventional Oncology. T.P.G. is on scientific advisory board for Trisalus Life Sciences. D.E.K. receives research funding from Astra Zeneca, Roche Genetech, Exact Sciences, and Bausch. R.A. and N.S. have no conflict of interest to declare.

Contributors' Statement

K.W.: study concept and design; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscript for important intellectual content. R.A.: drafting of the manuscript. N.S.: drafting of the manuscript. T.P.G.: critical revision of the manuscript for important intellectual content. D.E.K.: critical revision of the manuscript for important intellectual content.

• References

• 1 Bray F, Laversanne M, Sung H. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024; 74 (03) 229-263
• 2 Rumgay H, Arnold M, Ferlay J. et al. Global burden of primary liver cancer in 2020 and predictions to 2040. J Hepatol 2022; 77 (06) 1598-1606
• 3 Brar G, Greten TF, Graubard BI. et al. Hepatocellular carcinoma survival by etiology: a SEER-Medicare database analysis. Hepatol Commun 2020; 4 (10) 1541-1551
• 4 Jovel J, Lin Z, O'keefe S. et al. A survey of molecular heterogeneity in hepatocellular carcinoma. Hepatol Commun 2018; 2 (08) 941-955
• 5 Scudellari M. Drug development: try and try again. Nature 2014; 516 (7529): S4-S6
• 6 Llovet JM, Hernandez-Gea V. Hepatocellular carcinoma: reasons for phase III failure and novel perspectives on trial design. Clin Cancer Res 2014; 20 (08) 2072-2079
• 7 Llovet JM, Villanueva A, Lachenmayer A, Finn RS. Advances in targeted therapies for hepatocellular carcinoma in the genomic era. Nat Rev Clin Oncol 2015; 12 (07) 408-424
• 8 Mittal S, El-Serag HB, Sada YH. et al. Hepatocellular carcinoma in the absence of cirrhosis in US Veterans is associated with non-alcoholic fatty liver disease. Clin Gastroenterol Hepatol 2016; 14 (01) 124-31.e1
• 9 Villanueva A, Llovet JM. Liver cancer in 2013: mutational landscape of HCC–the end of the beginning. Nat Rev Clin Oncol 2014; 11 (02) 73-74
• 10 Guichard C, Amaddeo G, Imbeaud S. et al. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma. Nat Genet 2012; 44 (06) 694-698
• 11 Schulze K, Imbeaud S, Letouzé E. et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet 2015; 47 (05) 505-511
• 12 Yang C, Zhang H, Zhang L. et al. Evolving therapeutic landscape of advanced hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2023; 20 (04) 203-222
• 13 Llovet JM, De Baere T, Kulik L. et al. Locoregional therapies in the era of molecular and immune treatments for hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 2021; 18 (05) 293-313
• 14 Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival. Hepatology 2003; 37 (02) 429-442
• 15 Salem R, Gordon AC, Mouli S. et al. Y90 radioembolization significantly prolongs time to progression compared with chemoembolization in patients with hepatocellular carcinoma. Gastroenterology 2016; 151 (06) 1155-1163.e2
• 16 Brown AM, Kassab I, Massani M. et al. TACE versus TARE for patients with hepatocellular carcinoma: Overall and individual patient level meta analysis. Cancer Med 2023; 12 (03) 2590-2599
• 17 Xu MJ, Feng M. Radiation therapy in HCC: what data exist and what data do we need to incorporate into guidelines?. Semin Liver Dis 2019; 39 (01) 43-52
• 18 Llovet JM, Ricci S, Mazzaferro V. et al; SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008; 359 (04) 378-390
• 19 Kudo M, Finn RS, Qin S. et al. Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial. Lancet 2018; 391 (10126): 1163-1173
• 20 Bruix J, Qin S, Merle P. et al; RESORCE Investigators. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2017; 389 (10064): 56-66
• 21 Abou-Alfa GK, Meyer T, Cheng AL. et al. Cabozantinib in patients with advanced and progressing hepatocellular carcinoma. N Engl J Med 2018; 379 (01) 54-63
• 22 Finn RS, Qin S, Ikeda M. et al; IMbrave150 Investigators. Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma. N Engl J Med 2020; 382 (20) 1894-1905
• 23 Abou-Alfa GK, Lau G, Kudo M. et al; HIMALAYA Investigators. Tremelimumab plus durvalumab in unresectable hepatoceullar carcinoma. NEJM Evid 2022; 1 (08) a2100070
• 24 Yau T, Galle PR, Decaens T. et al; CheckMate 9DW investigators. Nivolumab plus ipilimumab versus lenvatinib or sorafenib as first-line treatment for unresectable hepatocellular carcinoma (CheckMate 9DW): an open-label, randomised, phase 3 trial. Lancet 2025; 405 (10492): 1851-1864
• 25 Lin ZF, Qin LX, Chen JH. Biomarkers for response to immunotherapy in hepatobiliary malignancies. Hepatobiliary Pancreat Dis Int 2022; 21 (05) 413-419
• 26 Roschke AV, Tonon G, Gehlhaus KS. et al. Karyotypic complexity of the NCI-60 drug-screening panel. Cancer Res 2003; 63 (24) 8634-8647
• 27 Olivotto M, Dello Sbarba P. Environmental restrictions within tumor ecosystems select for a convergent, hypoxia-resistant phenotype of cancer stem cells. Cell Cycle 2008; 7 (02) 176-187
• 28 Daniel VC, Marchionni L, Hierman JS. et al. A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro . Cancer Res 2009; 69 (08) 3364-3373
• 29 Gillet JP, Calcagno AM, Varma S. et al. Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance. Proc Natl Acad Sci U S A 2011; 108 (46) 18708-18713
• 30 Januszyk M, Rennert RC, Sorkin M. et al. Evaluation the effect of cell culture on gene expression in primary tissue samples using microfluidic-based single cell transcriptional analysis. Microarrays (Basel) 2015; 4 (04) 540-550
• 31 Wilding JL, Bodmer WF. Cancer cell lines for drug discovery and development. Cancer Res 2014; 74 (09) 2377-2384
• 32 Barretina J, Caponigro G, Stransky N. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012; 483 (7391): 603-607
• 33 Garnett MJ, Edelman EJ, Heidorn SJ. et al. Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature 2012; 483 (7391): 570-575
• 34 Ghandi M, Huang FW, Jané-Valbuena J. et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature 2019; 569 (7757): 503-508
• 35 Rebouissou S, Zucman-Rossi J, Moreau R, Qiu Z, Hui L. Note of caution: Contaminations of hepatocellular cell lines. J Hepatol 2017; 67 (05) 896-897
• 36 Bian X, Yang Z, Feng H, Sun H, Liu Y. A combination of species identification and STR profiling identifies cross-contaminated cells from 482 human tumor cell lines. Sci Rep 2017; 7 (01) 9774
• 37 Caruso S, Calatayud AL, Pilet J. et al. Analysis of liver cancer cell lines identifies agents with likely efficacy against hepatocellular carcinoma and markers of response. Gastroenterology 2019; 157 (03) 760-776
• 38 Qiu Z, Zou K, Zhuang L. et al. Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers. Sci Rep 2016; 6: 27411
• 39 Qiu Z, Li H, Zhang Z. et al. A pharmacogenomic landscape in human liver cancers. Cancer Cell 2019; 36 (02) 179-193.e11
• 40 Gao Q, Wang ZC, Duan M. et al. Cell culture system for analysis of genetic heterogeneity within hepatocellular carcinomas and response to pharmacologic agents. Gastroenterology 2017; 152 (01) 232-242.e4
• 41 Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer 2001; 1 (01) 46-54
• 42 Bissell MJ, Kenny PA, Radisky DC. Microenvironmental regulators of tissue structure and function also regulate tumor induction and progression: the role of extracellular matrix and its degrading enzymes. Cold Spring Harb Symp Quant Biol 2005; 70: 343-356
• 43 Aranda V, Haire T, Nolan ME. et al. Par6-aPKC uncouples ErbB2 induced disruption of polarized epithelial organization from proliferation control. Nat Cell Biol 2006; 8 (11) 1235-1245
• 44 Leslie K, Gao SP, Berishaj M. et al. Differential interleukin-6/Stat3 signaling as a function of cellular context mediates Ras-induced transformation. Breast Cancer Res 2010; 12 (05) R80
• 45 Lee GY, Kenny PA, Lee EH, Bissell MJ. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 2007; 4 (04) 359-365
• 46 Imamura Y, Mukohara T, Shimono Y. et al. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncol Rep 2015; 33 (04) 1837-1843
• 47 Jabs J, Zickgraf FM, Park J. et al. Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations. Mol Syst Biol 2017; 13 (11) 955
• 48 Sato T, Vries RG, Snippert HJ. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009; 459 (7244): 262-265
• 49 Sato T, Stange DE, Ferrante M. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 2011; 141 (05) 1762-1772
• 50 Rock JR, Onaitis MW, Rawlins EL. et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A 2009; 106 (31) 12771-12775
• 51 Däster S, Amatruda N, Calabrese D. et al. Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment. Oncotarget 2017; 8 (01) 1725-1736
• 52 Kondo J, Ekawa T, Endo H. et al. High-throughput screening in colorectal cancer tissue-originated spheroids. Cancer Sci 2019; 110 (01) 345-355
• 53 Bergdorf K, Phifer C, Bharti V. et al. High-throughput drug screening of fine-needle aspiration-derived cancer organoids. STAR Protoc 2020; 1 (03) 100212
• 54 Huch M, Dorrell C, Boj SF. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 2013; 494 (7436): 247-250
• 55 Huch M, Gehart H, van Boxtel R. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 2015; 160 (1-2): 299-312
• 56 Broutier L, Andersson-Rolf A, Hindley CJ. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 2016; 11 (09) 1724-1743
• 57 Broutier L, Mastrogiovanni G, Verstegen MM. et al. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening. Nat Med 2017; 23 (12) 1424-1435
• 58 Saito Y, Muramatsu T, Kanai Y. et al. Establishment of patient-derived organoids and drug screening for biliary tract carcinoma. Cell Rep 2019; 27 (04) 1265-1276.e4
• 59 Nuciforo S, Fofana I, Matter MS. et al. Organoid models of human liver cancers derived from tumor needle biopsies. Cell Rep 2018; 24 (05) 1363-1376
• 60 Li L, Knutsdottir H, Hui K. et al. Human primary liver cancer organoids reveal intratumor and interpatient drug response heterogeneity. JCI Insight 2019; 4 (02) e121490
• 61 Zhou Z, Yan X, Shi W. et al. Evaluation of the tumoricidal efficacy of adoptive cell transfer using hepatocellular carcinoma-derived organoids. J Gastrointest Oncol 2022; 13 (02) 732-743
• 62 Xian L, Zhao P, Chen X. et al. Heterogeneity, inherent and acquired drug resistance in patient-derived organoid models of primary liver cancer. Cell Oncol (Dordr) 2022; 45 (05) 1019-1036
• 63 Wang S, Wang Y, Xun X. et al. Hedgehog signaling promotes sorafenib resistance in hepatocellular carcinoma patient-derived organoids. J Exp Clin Cancer Res 2020; 39 (01) 22
• 64 Ren X, Huang M, Weng W. et al. Personalized drug screening in patient-derived organoids of biliary tract cancer and its clinical application. Cell Rep Med 2023; 4 (11) 101277
• 65 Dong H, Li Z, Bian S. et al. Culture of patient-derived multicellular clusters in suspended hydrogel capsules for pre-clinical personalized drug screening. Bioact Mater 2022; 18: 164-177
• 66 Maier CF, Zhu L, Nanduri LK. et al. Patient-derived organoids of cholangiocarcinoma. Int J Mol Sci 2021; 22 (16) 8675
• 67 Ji S, Feng L, Fu Z. et al. Pharmaco-proteogenomic characterization of liver cancer organoids for precision oncology. Sci Transl Med 2023; 15 (706) eadg3358
• 68 Chiew GGY, Wei N, Sultania S, Lim S, Luo KQ. Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: a model system for dual analysis of tumor growth and angiogenesis. Biotechnol Bioeng 2017; 114 (08) 1865-1877
• 69 Lim JTC, Kwang LG, Ho NCW. et al. Hepatocellular carcinoma organoid co-cultures mimic angiocrine crosstalk to generate inflammatory tumor microenvironment. Biomaterials 2022; 284: 121527
• 70 Sirica AE, Gores GJ. Desmoplastic stroma and cholangiocarcinoma: clinical implications and therapeutic targeting. Hepatology 2014; 59 (06) 2397-2402
• 71 Liu J, Li P, Wang L. et al. Cancer-associated fibroblasts provide a stromal niche for liver cancer organoids that confer trophic effects and therapy resistance. Cell Mol Gastroenterol Hepatol 2021; 11 (02) 407-431
• 72 de Graaf IAM, Olinga P, de Jager MH. et al. Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat Protoc 2010; 5 (09) 1540-1551
• 73 Wu X, Roberto JB, Knupp A. et al. Precision-cut human liver slice cultures as an immunological platform. J Immunol Methods 2018; 455: 71-79
• 74 Paish HL, Reed LH, Brown H. et al. A bioreactor technology for modeling fibrosis in human and rodent precision-cut liver slices. Hepatology 2019; 70 (04) 1377-1391
• 75 Jagatia R, Doornebal EJ, Rastovic U. et al. Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing. EBioMedicine 2023; 97: 104826
• 76 Collins AL, Kirkness K, Ramon-Gil E. et al. Precision-cut tumor slices for modeling hepatocellular carcinoma enable at-scale drug screening. Hepatol Commun 2025; 9 (06) e0706
• 77 Olson B, Li Y, Lin Y, Liu ET, Patnaik A. Mouse models for cancer immunotherapy research. Cancer Discov 2018; 8 (11) 1358-1365
• 78 Nevzorova YA, Boyer-Diaz Z, Cubero FJ, Gracia-Sancho J. Animal models for liver disease - a practical approach for translational research. J Hepatol 2020; 73 (02) 423-440
• 79 Connor F, Rayner TF, Aitken SJ. et al. Mutational landscape of a chemically-induced mouse model of liver cancer. J Hepatol 2018; 69 (04) 840-850
• 80 Magiera-Mularz K, Kocik J, Musielak B. et al. Human and mouse PD-L1: similar molecular structure, but different druggability profiles. iScience 2020; 24 (01) 101960
• 81 Ruiz de Galarreta M, Bresnahan E, Molina-Sánchez P. et al. B-catenin activation promotes immune escape and resistance to anti-PD-1 in hepatocellular carcinoma. Cancer Discov 2019; 9 (08) 1124-1141
• 82 Wang J, Perry CJ, Meeth K. et al. UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model. Pigment Cell Melanoma Res 2017; 30 (04) 428-435
• 83 Mestas J, Hughes CCW. Of mice and not men: differences between mouse and human immunology. J Immunol 2004; 172 (05) 2731-2738
• 84 Gao H, Korn JM, Ferretti S. et al. High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response. Nat Med 2015; 21 (11) 1318-1325
• 85 Byrne AT, Alférez DG, Amant F. et al. Interrogating open issues in cancer precision medicine with patient-derived xenografts. Nat Rev Cancer 2017; 17 (04) 254-268
• 86 Sun FX, Tang ZY, Lui KD. et al. Establishment of a metastatic model of human hepatocellular carcinoma in nude mice via orthotopic implantation of histologically intact tissues. Int J Cancer 1996; 66 (02) 239-243
• 87 Armengol C, Tarafa G, Boix L. et al. Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line. Clin Cancer Res 2004; 10 (06) 2150-2157
• 88 Huynh H, Soo KC, Chow PKH, Panasci L, Tran E. Xenografts of human hepatocellular carcinoma: a useful model for testing drugs. Clin Cancer Res 2006; 12 (14 Pt 1): 4306-4314
• 89 Chen K, Ahmed S, Adeyi O, Dick JE, Ghanekar A. Human solid tumor xenografts in immunodeficient mice are vulnerable to lymphomagenesis associated with Epstein-Barr virus. PLoS One 2012; 7 (06) e39294
• 90 Yan M, Li H, Zhao F. et al. Establishment of NOD/SCID mouse models of human hepatocellular carcinoma via subcutaneous transplantation of histologically intact tumor tissue. Chin J Cancer Res 2013; 25 (03) 289-298
• 91 Ahmed SU, Zair M, Chen K. et al. Generation of subcutaneous and intrahepatic human hepatocellular carcinoma xenografts in immunodeficient mice. J Vis Exp 2013; 25 (79) e50544
• 92 Xin H, Wang K, Hu G. et al. Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts. PLoS One 2014; 9 (01) e85308
• 93 Gu Q, Zhang B, Sun H. et al. Genomic characterization of a large panel of patient-derived hepatocellular carcinoma xenograft tumor models for preclinical development. Oncotarget 2015; 6 (24) 20160-20176
• 94 Zhao Q, Zhou H, Liu Q. et al. Prognostic value of the expression of cancer stem cell-related markers CD133 and CD44 in hepatocellular carcinoma: From patients to patient-derived tumor xenograft models. Oncotarget 2016; 7 (30) 47431-47443
• 95 Jiang Z, Jiang X, Chen S. et al. Anti-GPC3-CAR T cells suppress the growth of tumor cells in patient-derived xenografts of hepatocellular carcinoma. Front Immunol 2017; 7: 690
• 96 He S, Hu B, Li C. et al. PDXliver: a database of liver cancer patient derived xenograft mouse models. BMC Cancer 2018; 18 (01) 550
• 97 Liu R, Li Y, Tian L. et al. Gankyrin drives metabolic reprogramming to promote tumorigenesis, metastasis and drug resistance through activating β-catenin/c-Myc signaling in human hepatocellular carcinoma. Cancer Lett 2019; 443: 34-46
• 98 Hu B, Li H, Guo W. et al. Establishment of a hepatocellular carcinoma patient-derived xenograft platform and its application in biomarker identification. Int J Cancer 2020; 146 (06) 1606-1617
• 99 Nazzal M, Sur S, Steele R. et al. Establishment of a patient-derived xenograft tumor from hepatitis C-associated liver cancer and evaluation of imatinib treatment efficacy. Hepatology 2020; 72 (02) 379-388
• 100 Huang DQ, Muthiah MD, Zhou L. et al. Predicting HCC response to multikinase inhibitors with in vivo cirrhotic mouse model for personalized therapy. Cell Mol Gastroenterol Hepatol 2021; 11 (05) 1313-1325
• 101 Wu Y, Wang J, Zheng X. et al. Establishment and preclinical therapy of patient-derived hepatocellular carcinoma xenograft model. Immunol Lett 2020; 223: 33-43
• 102 Zhu M, Li L, Lu T. et al. Uncovering biological factors that regulate hepatocellular carcinoma growth using patient-derived xenograft assays. Hepatology 2020; 72 (03) 1085-1101
• 103 Blumer T, Fofana I, Matter MS. et al. Hepatocellular carcinoma xenografts established from needle biopsies preserve the characteristics of the originating tumors. Hepatol Commun 2019; 3 (07) 971-986
• 104 Tischfield DJ, Ackerman D, Noji M. et al. Establishment of hepatocellular carcinoma patient-derived xenografts from image-guided percutaneous biopsies. Sci Rep 2019; 9 (01) 10546
• 105 Zou C, El Dika I, Vercauteren KOA. et al. Mouse characteristics that affect establishing xenografts from hepatocellular carcinoma patient biopsies in the United States. Cancer Med 2022; 11 (03) 602-617
• 106 Hoge ACH, Getz M, Zimmer A. et al. DNA-based copy number analysis confirms genomic evolution of PDX models. NPJ Precis Oncol 2022; 6 (01) 30
• 107 Su T, Liao J, Dai Z. et al. Stress-induced phosphoprotein 1 mediates hepatocellular carcinoma metastasis after insufficient radiofrequency ablation. Oncogene 2018; 37 (26) 3514-3527
• 108 Yeoh KW, Prawira A, Saad MZB. et al. Vinorelbine augments radiotherapy in hepatocellular carcinoma. Cancers (Basel) 2020; 12 (04) 872
• 109 Zhou K, Jiang Y, Feng S. et al. Establishment of image-guided radiotherapy of orthotopic hepatocellular carcinoma mouse model. Animal Model Exp Med 2023; 6 (05) 419-426
• 110 He D, Zhang J, Wu W. et al. A novel immunodeficient rat model supports human lung cancer xenografts. FASEB J 2019; 33 (01) 140-150
• 111 Noto FK, Sangodkar J, Adedeji BT. et al. The SRG rat, a Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies. PLoS One 2020; 15 (10) e0240169
• 112 Gade TPF, Hunt SJ, Harrison N. et al. Segmental TAE in a translational rat model of hepatocellular carcinoma. J Vasc Interv Radiol 2015; 26 (08) 1-9
• 113 Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol 2012; 12 (11) 786-798
• 114 Walsh NC, Kenney LL, Jangalwe S. et al. Humanized mouse models of clinical disease. Annu Rev Pathol 2017; 12: 187-215
• 115 Chuprin J, Buettner H, Seedhom MO. et al. Humanized mouse models for immuno-oncology research. Nat Rev Clin Oncol 2023; 20 (03) 192-206
• 116 Marín-Jiménez JA, Capasso A, Lewis MS. et al. Testing cancer immunotherapy in a human immune system mouse model: correlating treatment responses to human chimerism, therapeutic variables, and immune cell phenotypes. Front Immunol 2021; 12: 607282
• 117 Zhao Y, Shuen TWH, Toh TB. et al. Development of a new patient-derived xenograft humanised mouse model to study human-specific tumour microenvironment and immunotherapy. Gut 2018; 67 (10) 1845-1854
• 118 Zhao Y, Wang J, Liu WN. et al. Analysis and validation of human targets and treatments using a hepatocellular carcinoma immune humanized mouse model. Hepatology 2021; 74 (03) 1395-1410
• 119 Horowitz NB, Mohammad I, Moreno-Nieves UY, Koliesnik I, Tran Q, Sunwoo JB. Humanized mouse models for the advancement of innate lymphoid cell-based cancer immunotherapies. Front Immunol 2021; 12: 648580
• 120 Mhaidly R, Verhoeyen E. Humanized mice are precious tools for preclinical evaluation of CAR T and CAR NK cell therapies. Cancers (Basel) 2020; 12 (07) 1915
• 121 Yu L, Yang X, Huang N. et al. A novel targeted GPC3/CD3 bispecific antibody for the treatment hepatocellular carcinoma. Cancer Biol Ther 2020; 21 (07) 597-603
• 122 Du K, Li Y, Liu J. et al. A bispecific antibody targeting GPC3 and CD47 induced enhanced antitumor efficacy against dual antigen-expressing HCC. Mol Ther 2021; 29 (04) 1572-1584
• 123 Habibollahi P, Gurevich A, Hui JZ. et al. Integrated imaging probe and bispecific antibody development enables in vivo targeting of glypican-3-expressing hepatocellular carcinoma. Mol Cancer Ther 2024; 23 (12) 1815-1826
• 124 Barrett DM, Zhao Y, Liu X. et al. Treatment of advanced leukemia in mice with mRNA engineered T cells. Hum Gene Ther 2011; 22 (12) 1575-1586
• 125 Li D, Li N, Zhang YF. et al. Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas. Gastroenterology 2020; 158 (08) 2250-2265.e20
• 126 Lu LL, Xiao SX, Lin ZY. et al. GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy. Cell Biol Toxicol 2023; 39 (06) 3101-3119
• 127 King MA, Covassin L, Brehm MA. et al. Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex. Clin Exp Immunol 2009; 157 (01) 104-118
• 128 Shultz LD, Lyons BL, Burzenski LM. et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 2005; 174 (10) 6477-6489
• 129 Sonntag K, Eckert F, Welker C. et al. Chronic graft-versus-host-disease in CD34(+)-humanized NSG mice is associated with human susceptibility HLA haplotypes for autoimmune disease. J Autoimmun 2015; 62: 55-66
• 130 Tanaka S, Saito Y, Kunisawa J. et al. Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rγKO mice. J Immunol 2012; 188 (12) 6145-6155
• 131 Danner R, Chaudhari SN, Rosenberger J. et al. Expression of HLA class II molecules in humanized NOD.Rag1KO.IL2RgcKO mice is critical for development and function of human T and B cells. PLoS One 2011; 6 (05) e19826
• 132 Halkias J, Yen B, Taylor KT. et al. Conserved and divergent aspects of human T-cell development and migration in humanized mice. Immunol Cell Biol 2015; 93 (08) 716-726
• 133 Li Y, Masse-Ranson G, Garcia Z. et al. A human immune system mouse model with robust lymph node development. Nat Methods 2018; 15 (08) 623-630
• 134 Zeng Y, Liu B, Rubio MT. et al. Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells. PLoS One 2017; 12 (04) e0173754
• 135 Brehm MA, Kenney LL, Wiles MV. et al. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression. FASEB J 2019; 33 (03) 3137-3151
• 136 Shultz LD, Saito Y, Najima Y. et al. Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice. Proc Natl Acad Sci U S A 2010; 107 (29) 13022-13027
• 137 Lan P, Tonomura N, Shimizu A, Wang S, Yang YG. Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation. Blood 2006; 108 (02) 487-492
• 138 Li Y, Teteloshvili N, Tan S. et al. Humanized mice reveal new insights into the thymic selection of human autoreactive CD8+ T cells. Front Immunol 2019; 10: 63
• 139 Takahashi T, Katano I, Ito R. et al. Enhanced antibody response in a novel NOG transgenic mouse with restored lymph node organogenesis. Front Immunol 2018; 8: 2017
• 140 Billerbeck E, Barry WT, Mu K. et al. Development of human CD4+FoxP3+ regulatory T cells in SGM3 humanized mice. Blood 2011; 117 (11) 3076-3086
• 141 Maser IP, Hoves S, Bayer C. et al. The tumor milieu promotes functional human tumor-resident plasmacytoid dendritic cells in humanized mouse models. Front Immunol 2020; 11: 2082
• 142 Herndler-Brandstetter D, Shan L, Yao Y. et al. Humanized mouse model supports development, function, and tissue residency of human natural killer cells. Proc Natl Acad Sci U S A 2017; 114 (45) E9626-E9634
• 143 Rongvaux A, Willinger T, Martinek J. et al. Development and function of human innate immune cells in a humanized mouse model. Nat Biotechnol 2014; 32 (04) 364-372
• 144 Yaguchi T, Kobayashi A, Inozume T. et al. Human PBMC-transferred murine MHC class I/II-deficient NOG mice enable long-term evaluation of human immune responses. Cell Mol Immunol 2018; 15 (11) 953-962
• 145 Ashizawa T, Iizuka A, Nonomura C. et al. Antitumor effect of PD-1 blockade in humanized the NOG-MHC double knockout mouse. Clin Cancer Res 2017; 23 (01) 149-158
• 146 Ka Y, Katano I, Nishinaka E. et al. Improved engraftment of human PBMCs in NOG MHC double knockout mice generated using CRISPR/Cas9. Immunol Letters 2021; 229: 55-61
• 147 Lockridge JL, Zhou Y, Becker YA. et al. Mice engrafted with human fetal thymic tissue and hematopoietic stem cells develop pathology resembling chronic graft-versus-host disease. Biol Blood Marrow Transplant 2013; 19 (09) 1310-1322
• 148 Madley R, Nauman G, Danzl N. et al. Negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen in the human thymus. J Transl Autoimmun 2020; 3: 100061
• 149 Weinfurtner K, Tischfield D, McClung G. et al. Human GM-CSF/IL-3 enhance tumor immune infiltration in humanized HCC patient-derived xenografts. JHEP Rep Innov Hepatol 2024; 7 (03) 101264
• 150 Lepus CM, Gibson TF, Gerber SA. et al. Comparison of human fetal liver umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NSG, BRG, and C.B.-17-scid immunodeficient mice. Hum Immunol 2009; 70 (10) 790-802
• 151 Tarrant JC, Binder ZA, Bugatti M. et al. Pathology of macrophage activation syndrome in humanized NSGS mice. Res Vet Sci 2021; 134: 137-146
• 152 Willis E, Verrelle J, Banerjee E. et al. Humanization with CD34-positive hematopoietic stem cells in NOG-EXL mice results in improved long-term survival and less severe myeloid cell hyperactivation phenotype relative to NSG-SGM3 mice. Vet Pathol 2024; 61 (04) 664-674
• 153 McIntosh BE, Brown ME, Duffin BM. et al. Nonirradiated NOD,B6.SCID Il2rγ-/- Kit(W41/W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells. Stem Cell Reports 2015; 4 (02) 171-180
• 154 Yahata T, Ando K, Sato T. et al. A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow. Blood 2003; 101 (08) 2905-2913
• 155 Brehm MA, Cuthbert A, Yang C. et al. Parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rgamma(null) mutation. Clin Immunol 2010; 135 (01) 84-98
• 156 Ménoret S, Ouisse LH, Tesson L. et al. In vivo analysis of human immune response in immunodeficient rats. Transplantation 2020; 104 (04) 715-723
• 157 Guo J, Li Y, Shan Y. et al. Humanized mice reveal an essential role for human hepatocytes in the development of the liver immune system. Cell Death Dis 2018; 9 (06) 667
• 158 Kaffe E, Roulis M, Zhao J. et al; AlcHepNet. Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions. Cell 2023; 186 (18) 3793-3809.e26
• 159 Kaffe E, Mehal WZ. Generation and characterization of a humanized mouse model of alcohol-induced steatosis, inflammation, and fibrosis. AASLD Liver Meeting 2023. Abstract 46
• 160 Keng CT, Sze CW, Zheng D. et al. Characterisation of liver pathogenesis, human immune responses and drug testing in a humanised mouse model of HCV infection. Gut 2016; 65 (10) 1744-1753

Publication History

Received: 30 August 2025

Accepted: 09 December 2025

Accepted Manuscript online:
14 January 2026

Article published online:
24 February 2026