Abstract
Background/Aim: Patient-derived xenograft (PDX) and Patient-derived orthotopic xenograft (PDOX) models are considered to recapitulate the heterogeneity and biological characteristics of original tumors more faithfully than conventional models. This study aimed to evaluate the extent to which specific tumor markers are retained in PDOX models of triple-negative breast cancer (TNBC), in comparison with their corresponding patient tumors.
Materials and Methods: PDOX models were established by orthotopically implanting tumor tissues from 15 TNBC patients into the mammary fat pads of immunodeficient mice. Immunohistochemical analyses were performed for CK5/6, HER2, EGFR, androgen receptor (AR), and Trop-2 in both the patient tumors and their corresponding PDOX tissues to evaluate the concordance of molecular marker expression.
Results: Trop-2 expression was consistently preserved across all tumor PDOX models. EGFR expression was preserved. In contrast, AR expression was observed in two of the original tumors, but it was completely lost in the corresponding PDOX models. CK5/6 and HER2 showed variable preservation.
Conclusion: TNBC PDOX models are promising tools for preclinical drug evaluation; however, variability in the retention of molecular targets, such as AR, CK5/6, and HER2, emphasizes the importance of verifying marker expression prior to functional analysis. Tumor heterogeneity and clonal selection during engraftment may contribute to these discrepancies.
Introduction
Preclinical models are crucial in connecting laboratory discoveries with clinical applications in cancer research. Traditional cell line-derived xenograft (CDX) models have been extensively used to assess anticancer treatments, but their limited capacity to replicate the original tumor environment has raised questions about their translational significance. In contrast, patient-derived xenograft (PDX) models maintain the molecular heterogeneity, differentiation status, tumor microenvironment, and histological features of the original tumors more effectively, enabling a more precise assessment of drug efficacy and resistance. In 2015, the National Cancer Institute (NCI) recommended transitioning anticancer drug screening from cell line-based approaches to those using PDX models. The EuroPDX consortium also acknowledges PDX as a vital preclinical tool for developing cancer therapies, considering it to have greater clinical relevance than traditional cell lines and serving as a basis for personalized medicine (1, 2).
Patient-derived xenograft (PDX) and patient-derived orthotopic xenograft (PDOX) models of breast cancer are generally believed to accurately replicate patient tumors (3-7). However, some studies have reported that these models may not fully retain certain tumor-specific features or intratumoral heterogeneity (8).
Breast cancer treatment is commonly based on molecular subtypes. Targeted therapies have greatly improved outcomes in estrogen receptor (ER)-positive luminal tumors and HER2-positive tumors (9-12). However, triple-negative breast cancer (TNBC), which lacks expression of ER, progesterone receptor (PgR), and HER2, still has a poor prognosis. Among potential therapeutic targets, the androgen receptor (AR) has long been considered important in TNBC (13-17). More recently, Trop-2 has also emerged as a promising target in this subtype (18-22). Establishing PDX or PDOX models that accurately recapitulate the expression of these target molecules is essential for the development of new therapeutic strategies.
In this study, we aimed to establish PDOX models of TNBC as a preclinical platform for therapeutic development, given the poor prognosis associated with this subtype. To evaluate whether key features of the original tumors were maintained, we performed immunohistochemical (IHC) analyses using TNBC tissues from different patients, which are known for their high degree of intratumoral heterogeneity (23, 24), and their corresponding PDOX models.
Materials and Methods
Patients. Since August 2020, we have obtained tumor tissues from 15 patients who underwent surgery at the Kobe University Hospital or the Kobe University International Cancer Center and were diagnosed with TNBC by core needle biopsy prior to surgery. Only tumors measuring ≥2 cm in diameter were included in this study. ER negativity was defined as <10% positively stained tumor cells, and HER2 negativity was defined as IHC score of 0 or 1, or IHC score of 2 with FISH negativity. FISH negativity was defined as a HER2/CEP17 ratio <2.0, and an average HER2 copy number of <4.0 signals per cell (25). Based on these criteria, cases were included only when diagnosed as TNBC by the pathologist. This study was approved by the Ethics Board of Kobe University (Ethics Committee No. B200065 and B200065-1), and written informed consent was obtained from all participants. TNBC status was confirmed by postoperative pathological evaluation.
Establishment of TNBC PDOX models. PDOX models were established as previously described (26). Tumor tissues from 15 TNBC patients were orthotopically implanted into the dorsal mammary fat pads of 2 to 3 female BALB/c-nu mice (5-6 weeks old) per patient (27). The first five cases were used as preliminary attempts to optimize and standardize the implantation technique. All tumor samples were collected following appropriate ethical approval and written informed consent. Mice were housed in individually ventilated cages and all animal procedures were conducted in accordance with institutional animal welfare guidelines. The experimental protocol was approved by the Animal Care and Use Committee of Oriental BioService Inc. (approval no. ma-K1804_2024).
Immunohistochemical (IHC) analysis. Tumor sections from both the original patient tissues and the corresponding PDOX tumors were subjected to IHC staining. The primary antibodies used were as follows: cytokeratin 5/6 (CK5/6; ARG66814, Arigo Biolaboratories, Hsinchu City, Taiwan, ROC; dilution 1:100), HER2 (29D8, Cell Signaling Technology, Danvers, MA, USA; dilution 1:400), EGFR (D38B1, Cell Signaling Technology; dilution 1:100), androgen receptor (AR; SP107, Abcam, Cambridge, UK; dilution 1:200), and Trop-2 (ab214488, Abcam; dilution 1:1,000). Sections were incubated at room temperature for 1 h with horseradish peroxidase (HRP)-labeled secondary antibodies (anti-rabbit EnVision HRP-conjugate, Dako, Santa Clara, CA, USA). Staining procedures followed previously reported protocols (28). Slides were imaged using a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan).
Immunohistochemical evaluation criteria. All IHC images were evaluated by two independent researchers who were blinded to the sample identity and followed standardized scoring criteria. HER2 scoring was based on the ASCO/CAP guidelines as 0, 1+, 2+ or 3+. CK5/6 signal was considered positive if >0% of tumor cells showed cytoplasmic and/or membranous staining, EGFR if >0% of tumor cells exhibited membranous staining, and Trop-2 if >0% of tumor cells exhibited membranous staining. Androgen Receptor (AR) signal was considered positive if >10% of tumor cell nuclei showed staining.
Results
An overview of the IHC expression patterns for all tumor–PDOX pairs is presented in Table I.
Results of immunohistochemical (IHC) staining.
Trop-2 and EGFR expression were preserved in the PDOX models. Trop-2 expression was consistently positive in all six original patient tumors and their corresponding PDOX models (Figure 1). EGFR expression, observed in two patient tumors (K006 and K008), was also retained in the respective PDOX models (Figure 2).
Immunohistochemical staining of trophoblast cell-surface antigen 2 (Trop-2) in patient tumors and corresponding PDOX models. Images at 4× (scale bar=1,000 μm) and 40× (scale bar=100 μm) magnification are shown for each.
Immunohistochemical staining of epidermal growth factor receptor (EGFR) in patient tumors and corresponding PDOX models. Images at 4× (scale bar=1,000 μm) and 40× (scale bar=100 μm) magnification are shown for each case.
Discrepancies in CK5/6 and HER2 expression were observed. CK5/6 staining was positive in all six patient tumor samples; however, one PDOX model (K006) showed a loss of CK5/6 expression despite positivity in the corresponding original tumor (Figure 3). HER2 IHC results were consistent between the patient tumors and PDOX tumors in four out of six cases. Discrepancies were observed in two cases: in case K007, the IHC score changed from 0 in the original tumor to 2 in the PDOX tumor, and in case K012, the score increased from 1 in the original tumor to 2 in the PDOX tumor (Figure 4).
Immunohistochemical staining of cytokeratin 5/6 (CK5/6) in patient tumors and corresponding PDOX models. Images at 4× (scale bar=1,000 μm) and 40× (scale bar=100 μm) magnification are shown for each case.
Immunohistochemical staining of human epidermal growth factor receptor 2 (HER2) in patient tumors and corresponding PDOX models. Images at 4× (scale bar=1,000 μm) and 40× (scale bar=100 μm) magnification are shown for each case.
AR expression was not preserved. Although AR expression was detected in two patient tumors (K006 and K010), it was not preserved in the corresponding PDOX tumors (Figure 5).
Immunohistochemical staining of androgen receptor (AR) in patient tumors and corresponding PDOX models. Images at 4× (scale bar=1,000 μm) and 40× (scale bar=100 μm) magnification are shown for each case.
Discrepancies in protein expression were observed in the F1 tumor tissues of PDOX. In this study, histological slides were prepared from PDOX tumors ranging from the first to the fifth generation (F1-F5). For cases in which changes in protein expression were observed, we examined multiple generations including F1 and confirmed that the alterations were already present and consistently observed from the F1 stage onward.
Discussion
This study assessed the retention of therapeutic markers in TNBC-PDOX models. Trop-2 and EGFR were consistently preserved, whereas AR was not. CK5/6 and HER2 showed variable expression between patient tumors and PDOX models. These findings highlight the importance of confirming the expression of molecular targets in PDOX models prior to their use in functional studies.
Discrepancies in IHC staining between original tumors and PDX may be attributed to intratumoral heterogeneity or clonal selection during the transplantation process (29, 30). Previous studies have reported that AR expression can be maintained in xenografts when androgens are supplemented post-transplantation, which may explain the loss of AR observed in our models (31). Tumor characteristics are known to change even during early passages (32, 33). In our study, we observed alterations in the expression of molecular targets as early the F1 generation. Given the aggressive and heterogeneous nature of TNBC, successful engraftment may favor highly proliferative clones, potentially excluding AR-positive clones with lower proliferative capacity (34, 35).
Although some studies have reported successful replication of tumor characteristics in TNBC-PDX models, the low engraftment rate often observed may be partly explained by intertumoral heterogeneity and clonal selection (36).
Unlike conventional studies using PDX or PDOX models to evaluate the efficacy of cytotoxic agents (37-42), this study provides valuable insight into the utility of these models in the context of developing targeted therapies such as antibody–drug conjugates (ADCs). As ADC efficacy relies on specific target expression, even minor discrepancies between patient tumors and PDOX or PDX models can affect preclinical outcomes. This underscores the need for careful model selection and characterization to ensure translational relevance in targeted drug development.
Study limitations. First, the number of cases analyzed was relatively small, which may limit the generalizability of the findings. Further analyses with larger cohorts will be necessary to validate and expand upon these observations. Second, while we initially expected AR expression to be preserved, the lack of androgen supplementation in the host mice might have contributed to its loss. Future studies may benefit from establishing PDOX models under hormone-supplemented conditions to better preserve AR signaling. Third, although PDOX models are considered powerful tools for translational cancer research, they do not fully replicate the human tumor microenvironment, which may influence biomarker expression and therapeutic responses.
Conclusion
Protein expression profiles in PDOX models can differ from those of the original patient tumors, with certain biomarkers being lost or variably retained during engraftment and serial passaging. These findings emphasize the importance of verifying biomarker status across PDOX generations, particularly when these models are used for preclinical evaluation of targeted therapies such as antibody–drug conjugates (ADCs). Given the reliance of ADC efficacy on specific target expression, rigorous molecular validation is essential to ensure the translational relevance of PDOX models in precision oncology.
Acknowledgements
The Authors thank Robert M. Hoffman, Professor at the University of California, San Diego for his expert guidance, as well as K. Katano, and S. Onishi of Kobe University for their technical assistance.
Footnotes
Authors’ Contributions
SI and SI designed the study, conducted the experiment, collected, and analyzed the data and wrote the paper. SM, MM, MY and MB obtained informed consent from the patients and collected tumor samples. SI, HT and TK supervised the study.
Conflicts of Interest
The Authors have no conflicts of interest to declare.
Funding
This work was supported by JSPS KAKENHI (21K07176, 24K02688).
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.
- Received May 20, 2025.
- Revision received July 11, 2025.
- Accepted July 22, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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