DSTYK predicts Chemoresistance in Triple-Negative Breast Cancer Patient-Derived Xenograft Models

DSTYK predicts Chemoresistance in Triple-Negative Breast Cancer Patient-Derived Xenograft Models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article DSTYK predicts Chemoresistance in Triple-Negative Breast Cancer Patient-Derived Xenograft Models Samuel Rojas, Jinyu Zhang, Brianna M Elam, Genevieve Schwarz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9321136/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Patient-derived xenograft (PDX) models are widely regarded as robust preclinical platforms because they preserve the histopathological and molecular features of primary tumors. In this study, we established twenty triple-negative breast cancer (TNBC) PDX models using freshly resected patient tumors, including ten with high DSTYK expression and ten with low DSTYK expression. Tumor fragments were orthotopically implanted into the fourth mammary fat pads of NSG mice. DSTYK expression was validated by immunohistochemistry and western blotting. Histological evaluation across three serial passages demonstrated that PDX tumors retained the cellular morphology, stromal architecture, and lineage characteristics of their corresponding primary tumors. Using these models, we assessed in vivo responses to combination chemotherapy with doxorubicin and docetaxel. DSTYK-low PDX tumors exhibited significantly greater chemosensitivity, whereas DSTYK-high tumors showed marked resistance, indicating a critical role for DSTYK in mediating chemotherapy resistance. Consistent with these findings, analyses from The Human Protein Atlas and Kaplan-Meier Plotter databases revealed that high DSTYK expression is associated with poor survival outcomes in TNBC patients. Collectively, our results from clinical specimens, PDX models, and public datasets identify DSTYK as a promising prognostic biomarker and predictor of chemotherapeutic response in TNBC, with potential implications for patient stratification and treatment optimization. Breast cancer Orthotopic PDX mouse model PDX expansion Subcutaneous PDX mouse model TNBC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression 1 . TNBC represents the most aggressive subtype of breast cancer, with high rates of recurrence, pronounced molecular heterogeneity, and limited targeted therapeutic options 2 , 3 . Currently, chemotherapy remains the primary neoadjuvant treatment for TNBC. However, the frequent development of chemoresistance is a major contributor to treatment failure and poor clinical outcomes 4 – 6 . Despite extensive investigation, the molecular mechanisms underlying chemoresistance in TNBC remain incompletely understood, underscoring an urgent need to identify novel biomarkers and therapeutic targets to improve patient survival 7 , 8 . Among commonly used regimens, the combination of doxorubicin (DOX) and docetaxel (DXL) has demonstrated substantial efficacy in breast cancer and has been widely applied in clinical trials for TNBC treatment 9 – 11 . A deeper understanding of how chemotherapy-induced cell death is regulated will provide new strategies to overcome chemoresistance in TNBC 12 , 13 . Cancer progression is fundamentally driven by an imbalance between cell proliferation and programmed cell death, particularly apoptosis 14 , 15 . Under normal physiological conditions, apoptosis serves as a critical safeguard to eliminate damaged or transformed cells; however, cancer cells frequently evade apoptosis by inactivating key regulatory pathways 16 . Consequently, successful chemotherapeutic agents exert their antitumor effects largely by reactivating apoptotic signaling in cancer cells 17 – 19 . In our previous studies, we demonstrated that dual serine/threonine and tyrosine protein kinase (DSTYK) suppresses chemotherapy-induced apoptosis in TNBC cells⁷. Building upon these findings, the present study aims to further elucidate the role of DSTYK in regulating apoptosis and chemotherapeutic response using PDX models. Recently, trophoblast cell-surface antigen 2 (Trop-2) has emerged as an important biomarker for TNBC 20 – 22 . Trop-2, encoded by the TACSTD2 gene, is overexpressed in multiple malignancies and strongly associated with chemoresistance in lung, pancreatic, colorectal, hepatic cancers, and TNBC 11 – 15 . Functionally, Trop-2 promotes tumor cell survival, proliferation, and metastasis 23 . Notably, Trop-2-targeting antibody–drug conjugates have shown remarkable clinical efficacy, significantly improving progression-free and overall survival in patients with metastatic TNBC 24 . In this study, we observed a positive correlation between DSTYK and Trop-2 expression in clinical TNBC specimens, suggesting a potential functional link between these two molecules. Previous work from our group and others has shown that DSTYK promotes transforming growth factor-β (TGF-β)–induced epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells 16 and enhances chemoresistance in TNBC using cellular and orthotopic mouse models 7 . Mechanistically, DSTYK activates ERK signaling 17 , 18 , downregulates the pro-apoptotic protein Bax 7 , promotes STING pathway activation 19 , and inhibits apoptosis induced by UV irradiation in skin cells 20 . In addition, inhibition of DSTYK sensitizes cancer cells to taxane-based chemotherapy and enhances T cell–mediated cytotoxicity by suppressing autophagy-dependent survival pathways 21 . However, these studies have largely relied on in vitro systems or conventional xenograft models. The role of DSTYK in chemoresistance has not yet been investigated in TNBC PDX models. PDX models are widely recognized as superior preclinical platforms because they can faithfully recapitulate the histological architecture, molecular heterogeneity, and tumor microenvironment of primary human tumors 2 , 25 , 26 . In the present study, we employed DSTYK-high and DSTYK-low TNBC PDX models to evaluate responses to combined DOX/DXL chemotherapy. We demonstrate that DSTYK-high PDX tumors exhibit elevated Trop-2 expression and profound resistance to chemotherapy, whereas DSTYK-low tumors display reduced Trop-2 levels and significantly enhanced chemosensitivity. Collectively, our findings establish DSTYK as a critical determinant of chemotherapeutic response in TNBC and highlight its potential as a prognostic biomarker and therapeutic target for overcoming chemoresistance. Materials and Methods Establish PDX mouse model and chemotherapeutic assays All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Toledo (UT). De-identified, fresh, treatment-naïve triple-negative breast cancer (TNBC) specimens were obtained from TNBC patients for the establishment of patient-derived xenograft (PDX) models. Based on DSTYK expression levels, the top ten TNBC specimens with relatively high DSTYK expression and the bottom ten specimens with relatively low DSTYK expression were selected for chemoresistance analyses using PDX models. NOD-SCID-IL2Rγ null (NSG) mice were housed and maintained in the UT animal facilities. Fresh tumor specimens were sectioned into small fragments (~ 3–5 mm³) and aseptically implanted into the fourth mammary fat pad of 6-7-week-old female NSG mice, with one tumor fragment implanted per mouse). Tumor engraftment and growth were monitored regularly. Successfully engrafted tumors were serially passaged to generate P0, P1 (first passage), and P2 (second passage) PDX tumors via subcutaneous expansion. To minimize inter-experimental variability, only tumors from the same generation (P2) were used for all treatment studies. Once P2 tumors reached an approximate volume of 50 mm³, mice were randomized into treatment groups and administered a combination of doxorubicin (DOX) and docetaxel (DXL) via intraperitoneal injection. Treatment was initiated at doses of 4.0 mg/kg DOX and 1.0 mg/kg DXL, and delivered twice weekly for 3–4 weeks. Tumor growth and treatment responses were monitored throughout the treatment period. Immunohistochemistry (IHC) Staining Tumor specimens derived from patients were collected and processed according to our previously described immunohistochemistry protocol 27 . Paraffin-embedded tissue sections (5 µm thick) were incubated overnight at 4°C with the following primary antibodies: anti-DSTYK (OriGene Technologies), anti-Trop2 (Abcam), anti-Ki67 (Abcam), anti-Ku80 (R&D System), anti-CD45, and anti-Bax (Cell Signaling Technology). Immunoreactive signals were visualized using the VECTASTAIN® Elite ABC Kit (Vector Laboratories, Burlingame, CA) in accordance with the manufacturer’s instructions. Hematoxylin was used for nuclear counterstaining. Quantitative analysis of IHC staining intensity was performed using ImageJ software. Western Blotting Western blot (WB) analyses were performed as previously described 27 , 28 . Briefly, total proteins were extracted from whole-cell lysates, and equal amounts of protein were separated by electrophoresis on 8–10% SDS–polyacrylamide gels. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk and incubated with primary antibodies against Trop2, Bax, DSTYK, and GAPDH (Santa Cruz Biotechnology). After incubation with horseradish peroxidase–conjugated secondary antibodies (Thermo Fisher Scientific), protein signals were detected using enhanced chemiluminescence (ECL; Bio-Rad) and visualized by chemiluminescence imaging. Each Western blot experiment was performed at least three times independently. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay The TUNEL assay was performed as previously described 27 , 28 , following the manufacturer’s instructions using an apoptosis detection kit (ABP Biosciences, catalog no. A050). Briefly, tissue sections were fixed in 4% paraformaldehyde for 10 min and subsequently washed with phosphate-buffered saline (PBS). Sections were permeabilized with proteinase K (20 µg/mL in PBS). The terminal deoxynucleotidyl transferase (TdT) reaction mixture was freshly prepared and incubated with the sections for 1 h at 37°C in a humidified chamber. Afterward, the slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Fluorescence signals were examined and images were captured using a Leica fluorescence microscope. RNA isolation and quantitative real-time PCR Total RNA was extracted from the cell pellets by the RNeasy Mini kit (Cat# 74104; QIAGEN), and 1 µg RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Cat# 43-688-14; Thermo Fisher). Quantitative real-time PCR (real-time-qPCR) was performed using iTaq Universal SYBR Green Supermix (Cat# 1725124, Bio-Rad). DSTYK Forward, 5′-ATTGCCAACCGAAAGCAGGAGG-3′, Reverse, 5′-GGTGCCTACTGGTTCTCCATTC-3′; β-actin Forward 5′-CACCAACTGGGACGACAT-3′, Reverse 5′-ACAGCCTGGATAGCAACG-3′. The gene expression levels were determined by the 2 −ΔΔ C q method and values were normalized to β-actin expression as an internal control. Statistical Analysis Data were analyzed using Prism 7 and are expressed as the mean ± standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test, with a confidence level of 95%. Comparisons between two groups were made using a parametric paired t-test for normally distributed data or non-parametric Wilcoxon paired t-test for non-normally distributed data. Unless otherwise indicated, experiments included more than five mice or samples per group ( n > 5). Data were analyzed using the unpaired student t-test, and differences were considered statistically significant at P-values of < 0.05 (*), < 0.01 (**), < 0.001 (***) and < 0.0001 (****) that were considered statistically significant or very significant, respectively. Results Determination of DSTYK expression levels in human TNBC tissues First, histopathological analyses of human triple-negative breast cancer (TNBC) tissues were performed using hematoxylin and eosin (H&E) staining to confirm the characteristic features of breast cancer ( Fig. 1 A, left panel ). We then assessed DSTYK protein expression in TNBC tissues by immunohistochemical (IHC) staining (Fig. 1 A, right panel ). Based on IHC staining intensity, we selected the top 10 TNBC specimens with the high DSTYK expression (DSTYK High ) and the bottom 10 specimens with the low DSTYK expression (DSTYK Low ). Quantitative analysis was conducted using ImageJ software (Fig. 1 B). Consistently, DSTYK mRNA expression was significantly higher in DSTYK High tissues compared with DSTYK Low tissues (Fig. 1 C). Together, these results validate the classification of DSTYK High and DSTYK Low TNBC patient specimens. DSTYK expression correlates with the expression of cancer stem cell (CSC) markers The association between DSTYK expression and breast cancer stem cell (CSC) markers was systematically evaluated based on The Human Atlas database. DSTYK expression showed a modest but significant positive correlation with CD133 (r = 0.0621, p = 0.0474; Fig. 2 A). Further analyses revealed that DSTYK was positively correlated with multiple established CSC markers, including OCT4 (r = 0.2352, p < 0.0001; Fig. 2 B), NANOG (r = 0.3933, p < 0.001; Fig. 2 C), LGR5 (r = 0.2399, p < 0.0001; Fig. 2 D), CD44 (r = 0.3104, p < 0.0001; Fig. 2 E), and EPCAM (r = 0.1189, p < 0.0001; Fig. 2 F). In addition, DSTYK expression was negatively correlated with the pro-apoptotic marker Bax (r = − 0.3446, p < 0.0001; Fig. 2 G), while a positive association was observed between DSTYK and the proliferation marker Ki67 (r = 0.1300, p < 0.0001; Fig. 2 H). Collectively, these findings suggest that elevated DSTYK expression is associated with enhanced cancer stemness, reduced apoptotic potential, and increased proliferative capacity, indicating that DSTYK may contribute to apoptosis inhibition and decreased chemosensitivity in breast cancer cells. Identification of DSTYK as a Potential Prognostic Biomarker in TNBC To assess the clinical relevance of DSTYK in breast cancer, we analyzed patient data from the GDC TCGA Breast Cancer (BRCA) cohort. Our statistical analysis revealed that elevated DSTYK expression is significantly associated with an increased risk of mortality in breast cancer patients (Fig. 3 A), indicating that DSTYK may serve as a negative prognostic factor. These findings further imply a potential role for DSTYK in promoting chemoresistance in breast cancer, including triple-negative breast cancer (TNBC). Given that Trop2 is highly expressed on the surface of most TNBC cells 21 , 29 , we next examined the relationship between DSTYK and Trop2 expression. Immunoblotting analyses were performed using randomly selected human TNBC tissue specimens. The results revealed a positive correlation between DSTYK and Trop2 protein levels (Fig. 3 B). Quantification of immunoblot band intensities using ImageJ, followed by statistical analysis, confirmed a significant positive correlation between DSTYK and Trop2 expression (Fig. 3 C). Based on these immunoblotting results, TNBC tissues were stratified into relative DSTYK High and DSTYK Low groups. Subsequent statistical analysis demonstrated that the pro-apoptotic factor Bax was negatively correlated with DSTYK expression (Fig. 3 B & 3 D). Collectively, these data suggest that DSTYK may function as a tumor-promoting element in TNBC by modulating the expression of Trop2 and Bax, thereby contributing to tumor progression and resistance to chemotherapeutic drug-induced apoptosis. Establishment of TNBC PDX Models in NSG Mice To establish PDX models, NOD-SCID-IL2Rγ null (NSG) mice were used as illustrated in Fig. 4 . For each tumor specimen, ten mice were employed. Fresh TNBC tumor tissues were aseptically sectioned into small fragments (~ 5 mm³) and orthotopically implanted into the fourth mammary fat pad of 6-7-week-old female NSG mice, with one tumor fragment implanted per mouse (Fig. 4 ). The first passage (P0) of subcutaneous TNBC PDX tumors required approximately 6–8 weeks for expansion, whereas the second (P1) and third (P2) passages required approximately 4 weeks and 3–4 weeks, respectively. All subcutaneous PDX tumors were expanded to a volume of ~ 300–400 mm³ prior to subsequent transplantation. Following orthotopic implantation, P1 and P2 PDX tumors demonstrated significantly accelerated in vivo growth compared with P0 tumors, with growth rates significantly increased (Fig. 4 E). To assess whether key characteristics of the original tumors were preserved during passaging, randomly selected PDX models derived from two patients with triple-negative breast cancer (TNBC) were subjected to histopathological evaluation. Hematoxylin and eosin (H&E) staining and Ki67 immunohistochemistry of P2 tumors confirmed retention of original TNBC histological features and high proliferative activity (Fig. 5 A). Immunohistochemical analysis of estrogen receptor (ER), progesterone receptor (PR), and HER2 further verified maintenance of the triple-negative phenotype (Fig. 5 B). Moreover, Ku80 and CD45 staining confirmed the human origin of the tumor cells and revealed minimal murine hematopoietic cell contamination, supporting the fidelity of the PDX models (Fig. 5 B). DSTYK expression correlates with Trop2 upregulation and Bax suppression in clinical TNBC. Our previous studies demonstrated that chemoresistant TNBC cells regain chemosensitivity upon DSTYK knockout (KO) following chemotherapeutic treatment 9 . To explore the mechanisms by which DSTYK promotes chemoresistance in TNBC, we performed immunohistochemical (IHC) analyses to evaluate the expression of DSTYK, Trop2, and Bax in paired primary TNBC specimens and corresponding residual tumors obtained after neoadjuvant chemotherapy. IHC staining revealed that DSTYK protein expression was significantly increased in residual TNBC tissues compared with their matched pre-treatment DSTYK Low tumors (Fig. 6 A, B). Consistently, Trop2 expression was markedly upregulated in residual tumors following chemotherapy. In contrast, expression of the pro-apoptotic protein Bax was significantly reduced in residual TNBC tissues relative to untreated DSTYK Low tumors (Fig. 6 A, C). Correlation analyses further demonstrated a positive association between DSTYK and Trop2 expression and an inverse relationship between DSTYK and Bax expression. Specifically, TNBC tissues with low DSTYK expression exhibited reduced Trop2 levels and elevated Bax expression, whereas tumors with high DSTYK expression showed increased Trop2 expression and concomitant suppression of Bax (Fig. 6 B, C). Collectively, these findings suggest that Trop2 and Bax may serve as downstream effectors of DSTYK signaling. Our data identify Trop2 and Bax as novel components of a DSTYK-associated regulatory network that contributes to chemoresistance in TNBC and supports DSTYK as a potential prognostic biomarker in the clinical setting. DSTYK Promotes Chemoresistance in In Vivo PDX Models To provide clinically relevant evidence that DSTYK contributes to chemoresistance, we employed patient-derived xenograft (PDX) mouse models. The experiment procedure is depicted in Fig. 7 A. Following orthotopic implantation, tumor volume, tumor weight, and body weight were monitored twice a week, and tumor growth curves were generated to compare therapeutic responses between tumors with high versus low DSTYK expression, no significant differences in tumor growth were observed between DSTYK high and DSTYK low PDX tumors (Fig. 7 B), consistent with our previous findings that DSTYK does not significantly influence cancer cell proliferation 28 . In contrast, after DOX + DXL treatment, tumors derived from the DSTYK low group exhibited marked regression compared with those from the DSTYK high group (Fig. 7 B-D). Consistently, TUNEL staining revealed a significantly higher level of apoptosis in DSTYK Low tumors following chemotherapy compared with DSTYK High tumors (Fig. 7 E & F ). These findings indicate that elevated DSTYK expression attenuates chemosensitivity by suppressing chemotherapy-induced tumor cell death. Taken together, these results demonstrate that DSTYK plays a pivotal role in promoting chemoresistance in TNBC. Targeting DSTYK in combination with standard chemotherapy may therefore represent a promising strategy to enhance therapeutic efficacy in TNBC patients. Discussion This study demonstrates that primary TNBC tumors with elevated DSTYK expression exhibit significantly greater chemoresistance than those with lower DSTYK levels. DSTYK expression is positively correlated with the newly identified TNBC marker Trop2 and negatively correlated with the pro-apoptotic protein Bax. Together with DSTYK and Trop2, Bax may therefore also serve as a potential biomarker for assessing chemoresistance in TNBC, consistent with several previous reports 18 , 19 , 30 . Furthermore, in our PDX mouse models, tumors characterized by high DSTYK and low Bax expression displayed markedly reduced sensitivity to drug treatment compared with tumors expressing lower DSTYK and higher Bax levels. Our previous studies revealed that DSTYK suppresses chemotherapy-induced apoptosis in TNBC cells and destabilizes Bax in colorectal cancer cells 27 . In the present study, we extend these findings by providing clinical evidence that DSTYK and Bax protein levels are inversely correlated in TNBC specimens. Combined with our functional PDX data showing that DSTYK inhibits apoptosis and attenuates chemosensitivity, these results strongly suggest that DSTYK may similarly downregulate Bax stability in TNBC. Previously, we demonstrated that chemoresistant cancer cells regain chemosensitivity following DSTYK knockout 28 , here in, immunohistochemical analysis of clinical TNBC samples reveals that DSTYK protein levels are significantly elevated in residual tumors after neoadjuvant chemotherapy compared with treatment-naïve tumors. This observation suggests that DSTYK may be a key determinant driving the chemoresistant phenotype of residual TNBC. Collectively, these findings reinforce our earlier discovery and establish DSTYK as a potential prognostic biomarker and therapeutic target for chemoresistant TNBC. To further characterize the role of DSTYK in chemoresistance, we employed patient-derived xenograft (PDX) mouse models generated from clinical TNBC specimens. PDX models are highly valued in cancer research 12 , 25 , 31 , particularly for TNBC, as they better mirror the characteristics of human disease compared with conventional cancer cell lines 32 . By engrafting freshly resected patient tumor tissues into immunodeficient mice, PDX models preserve tumor architecture, histopathological features, and intratumoral heterogeneity, and these factors that are critical for accurately assessing therapeutic response. In our PDX studies, tumors with low DSTYK expression exhibited significantly greater regression following chemotherapy than those with high DSTYK expression. These findings further support the conclusion that elevated DSTYK levels confer increased chemoresistance. To enhance the translational relevance of our work, future studies should evaluate the impact of DSTYK on resistance to additional chemotherapeutic agents beyond doxorubicin and docetaxel. Statistical analysis of the patient dataset indicates that a high DSTYK level is correlated with an increased risk of mortality in breast cancer patients, TNBC patients, and TNBC patients treated only with chemotherapy 28 . However, the molecular mechanisms by which DSTYK mediates chemoresistance remain poorly understood 33 . Notably, we identified a strong positive correlation between DSTYK and Trop2 expression. Trop2 is a well-established therapeutic target in TNBC, as it is highly expressed on the surface of most TNBC cells while exhibiting minimal expression in normal tissues. Trop2 is a transmembrane glycoprotein that functions as an intracellular calcium signal transducer and plays a critical role in tumor progression by regulating oncogenic signaling pathways, including AKT, ERK, and JAK/STAT. Trop2 overexpression is consistently associated with poor prognosis and aggressive TNBC phenotypes 32 , 34 , 35 . Trop2 antibody drug conjugates sacituzumab govitecan (Trodelvy) and datopotomab deruxtecan (Datroway) have been approved by FDA not long ago for the clinical treatment of TNBC patients, which is a groundbreaking achievement to significantly increase both progression-free survival (PFS) and overall survival (OS) probability of metastatic TNBC patients according to a statistical analysis 24 , 36 . In our study, TNBC specimens with high DSTYK expression consistently exhibited elevated Trop2 levels, whereas tumors with low DSTYK expression showed correspondingly low Trop2 expression. This statistically significant correlation suggests a previously unrecognized link between DSTYK and Trop2 and highlights a compelling direction for future mechanistic studies aimed at elucidating how DSTYK promotes chemoresistance. Given that DSTYK is a protein kinase, it represents a highly druggable target. The development of cell-permeable DSTYK inhibitors may therefore offer a novel therapeutic strategy to overcome chemoresistance and enhance the efficacy of existing treatments, including Trop2-targeted therapies, ultimately improving clinical outcomes for patients with TNBC. Conclusion In summary, our findings provide important advances in therapeutic strategies for the ongoing challenge of chemotherapy resistance in TNBC. By further validating, we identify DSTYK as a promising druggable target for targeting and eliminating chemoresistant TNBC in clinical patients. Declarations Ethics approval and consent to participate All animal experiments were performed in accordance with institutional regulations after protocol review and approval by the University of Toledo's Institutional Animal Care and Use Committee. The authors complied with the ARRIVE guidelines Consent for publication Not applicable. Availability of supporting data Not applicable. Competing interests The authors declare that they have no competing interests. Author contributions J.Z., S.R., and Y.J. wrote the manuscript; Y.J. revised the manuscript according to the comments and suggestions of G.S. and Y.Z. All authors have read and agreed to the published version of the manuscript. Conflict of interests The authors declared no conflict of interest. Funding The author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by National Institutes of Health grants R01CA272760 and startup funding from the University of Toledo. Clinical trial number Not applicable. Acknowledgements Not applicable. References Lai, R. C. et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 4 , 214-222 (2010). https://doi.org/10.1016/j.scr.2009.12.003 Abdolahi, S. et al. Patient-derived xenograft (PDX) models, applications and challenges in cancer research. J Transl Med 20 , 206 (2022). https://doi.org/10.1186/s12967-022-03405-8 Agelidis, A., Ter-Zakarian, A. & Jaloudi, M. Triple-Negative Breast Cancer on the Rise: Breakthroughs and Beyond. Breast Cancer (Dove Med Press) 17 , 523-529 (2025). https://doi.org/10.2147/BCTT.S516125 Kim, C. et al. Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing. Cell 173 , 879-893 e813 (2018). https://doi.org/10.1016/j.cell.2018.03.041 Lusby, R., Zhang, Z., Mahesh, A. & Tiwari, V. K. Decoding gene regulatory circuitry underlying TNBC chemoresistance reveals biomarkers for therapy response and therapeutic targets. NPJ Precis Oncol 8 , 64 (2024). https://doi.org/10.1038/s41698-024-00529-6 Dhara, A. et al. Enhanced ETS1 stability by DNAPKcs orchestrates transcriptional changes during chemoresistance in triple negative breast cancer. NPJ Breast Cancer 11 , 114 (2025). https://doi.org/10.1038/s41523-025-00782-3 Trnkova, L. et al. Molecular profiling of chemotherapy-resistant breast cancer reveals DNA methylation remodeling associated with the acquisition of paclitaxel resistance. Drug Resist Updat 85 , 101350 (2026). https://doi.org/10.1016/j.drup.2026.101350 Fleisher, B., Clarke, C. & Ait-Oudhia, S. Current advances in biomarkers for targeted therapy in triple-negative breast cancer. Breast Cancer (Dove Med Press) 8 , 183-197 (2016). https://doi.org/10.2147/BCTT.S114659 Baltali, E., Ozisik, Y., Guler, N., Firat, D. & Altundag, K. Combination of docetaxel and doxorubicin as first-line chemotherapy in metastatic breast cancer. Tumori 87 , 18-19 (2001). https://doi.org/10.1177/030089160108700104 Ricci, M. S. & Zong, W. X. Chemotherapeutic approaches for targeting cell death pathways. Oncologist 11 , 342-357 (2006). https://doi.org/10.1634/theoncologist.11-4-342 Zafar, A., Khatoon, S., Khan, M. J., Abu, J. & Naeem, A. Advancements and limitations in traditional anti-cancer therapies: a comprehensive review of surgery, chemotherapy, radiation therapy, and hormonal therapy. Discov Oncol 16 , 607 (2025). https://doi.org/10.1007/s12672-025-02198-8 Liu, Y. et al. Programmed cell death in triple-negative breast cancer. Cell Mol Biol Lett 30 , 111 (2025). https://doi.org/10.1186/s11658-025-00789-5 Nedeljkovic, M. & Damjanovic, A. Mechanisms of Chemotherapy Resistance in Triple-Negative Breast Cancer-How We Can Rise to the Challenge. Cells 8 (2019). https://doi.org/10.3390/cells8090957 Loftus, L. V., Amend, S. R. & Pienta, K. J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. Int J Mol Sci 23 (2022). https://doi.org/10.3390/ijms23094723 Wei, Y., Hankey, W., Xu, D. & Yuan, F. Programmed Cell Death in Cancer. MedComm (2020) 6 , e70357 (2025). https://doi.org/10.1002/mco2.70357 Gielecinska, A. et al. Apoptosis, necroptosis, and pyroptosis as alternative cell death pathways induced by chemotherapeutic agents? Biochim Biophys Acta Rev Cancer 1878 , 189024 (2023). https://doi.org/10.1016/j.bbcan.2023.189024 Mantri, S. & Doshi, G. M. Reactivating apoptotic pathways in cancer: A review of novel therapeutic approaches. Eur J Pharmacol 1003 , 177965 (2025). https://doi.org/10.1016/j.ejphar.2025.177965 Krajewski, S. et al. Reduced expression of proapoptotic gene BAX is associated with poor response rates to combination chemotherapy and shorter survival in women with metastatic breast adenocarcinoma. Cancer Res 55 , 4471-4478 (1995). McCurrach, M. E., Connor, T. M., Knudson, C. M., Korsmeyer, S. J. & Lowe, S. W. bax-deficiency promotes drug resistance and oncogenic transformation by attenuating p53-dependent apoptosis. Proc Natl Acad Sci U S A 94 , 2345-2349 (1997). https://doi.org/10.1073/pnas.94.6.2345 Jeon, Y., Jo, U., Hong, J., Gong, G. & Lee, H. J. Trophoblast cell-surface antigen 2 (TROP2) expression in triple-negative breast cancer. BMC Cancer 22 , 1014 (2022). https://doi.org/10.1186/s12885-022-10076-7 Liao, Q. et al. TROP2 is highly expressed in triple-negative breast cancer CTCs and is a potential marker for epithelial mesenchymal CTCs. Mol Ther Oncol 32 , 200762 (2024). https://doi.org/10.1016/j.omton.2024.200762 Alhamed, A. S. et al. Modulation of Chemotherapy Sensitivity of Breast Cancer Cells through Transforming Growth Factor-beta Pathway-mediated Alterations in DNA Damage Response. Int J Med Sci 22 , 2031-2039 (2025). https://doi.org/10.7150/ijms.111217 Sakach, E., Sacks, R. & Kalinsky, K. Trop-2 as a Therapeutic Target in Breast Cancer. Cancers (Basel) 14 (2022). https://doi.org/10.3390/cancers14235936 Alhushki, S., Deshmukh, S., Levy, B. & Desai, A. A tale of two TROP-2 antibody-drug conjugates: a comparative saga of datopotamab deruxtecan and sacituzumab govitecan. Oncologist 30 (2025). https://doi.org/10.1093/oncolo/oyaf251 Liu, Z. et al. A fast, simple, and cost-effective method of expanding patient-derived xenograft mouse models of pancreatic ductal adenocarcinoma. J Transl Med 18 , 255 (2020). https://doi.org/10.1186/s12967-020-02414-9 Gu, A., Li, J., Li, M. Y. & Liu, Y. Patient-derived xenograft model in cancer: establishment and applications. MedComm (2020) 6 , e70059 (2025). https://doi.org/10.1002/mco2.70059 Zhang, J. et al. DSTYK Promotes Metastasis and Chemoresistance via EMT in Colorectal Cancer. Front Pharmacol 11 , 1250 (2020). https://doi.org/10.3389/fphar.2020.01250 Ogbu, S. C. et al. DSTYK Enhances Chemoresistance in Triple-Negative Breast Cancer Cells. Cells 11 (2021). https://doi.org/10.3390/cells11010097 Ebrahim, N. A. A., Hussein, M. A., Sobeih, M. E. & Amin, N. H. From biomarker to targeted therapy: investigating trophoblast cell-surface antigen 2 expression in triple-negative breast cancer- insights from the national cancer institute. BMC Cancer 25 , 1008 (2025). https://doi.org/10.1186/s12885-025-14402-7 Lopez-Gonzalez, L. et al. Exploring Biomarkers in Breast Cancer: Hallmarks of Diagnosis, Treatment, and Follow-Up in Clinical Practice. Medicina (Kaunas) 60 (2024). https://doi.org/10.3390/medicina60010168 Blanchard, Z., Brown, E. A., Ghazaryan, A. & Welm, A. L. PDX models for functional precision oncology and discovery science. Nat Rev Cancer 25 , 153-166 (2025). https://doi.org/10.1038/s41568-024-00779-3 El-Sahli, S. et al. Nanoparticle-Mediated mRNA Delivery to Triple-Negative Breast Cancer (TNBC) Patient-Derived Xenograft (PDX) Tumors. ACS Pharmacol Transl Sci 8 , 460-469 (2025). https://doi.org/10.1021/acsptsci.4c00597 Echepare, M. et al. DSTYK Inhibition Sensitizes NSCLC to Taxane-Based Chemotherapy. J Thorac Oncol 20 , 345-365 (2025). https://doi.org/10.1016/j.jtho.2024.11.003 Goldenberg, D. M., Stein, R. & Sharkey, R. M. The emergence of trophoblast cell-surface antigen 2 (TROP-2) as a novel cancer target. Oncotarget 9 , 28989-29006 (2018). https://doi.org/10.18632/oncotarget.25615 Aslan, M. et al. Oncogene-mediated metabolic gene signature predicts breast cancer outcome. NPJ Breast Cancer 7 , 141 (2021). https://doi.org/10.1038/s41523-021-00341-6 Huang, J., Huang, T., Guo, J., Hu, K. & Zhou, H. The efficacy and safety of datopotamab deruxtecan (Dato-DXd) in advanced solid tumors: a systematic review and meta-analysis. Eur J Med Res 30 , 1265 (2025). https://doi.org/10.1186/s40001-025-03538-8 Additional Declarations No competing interests reported. Supplementary Files S1.jpg S2.jpg S3.jpg S4.jpg Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 23 Apr, 2026 Editor assigned by journal 14 Apr, 2026 Submission checks completed at journal 14 Apr, 2026 First submitted to journal 04 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9321136","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633560979,"identity":"cf2c30ee-ccac-4c37-bac0-b253fa205a20","order_by":0,"name":"Samuel Rojas","email":"","orcid":"","institution":"University of Toledo","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Rojas","suffix":""},{"id":633560980,"identity":"ddefe4fb-9f70-486a-87ee-58151472b1cc","order_by":1,"name":"Jinyu Zhang","email":"","orcid":"","institution":"The University of Toledo College of Medicine and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jinyu","middleName":"","lastName":"Zhang","suffix":""},{"id":633560981,"identity":"e79a7994-7621-40ec-928b-8622a7198e3c","order_by":2,"name":"Brianna M Elam","email":"","orcid":"","institution":"East Tennessee State University","correspondingAuthor":false,"prefix":"","firstName":"Brianna","middleName":"M","lastName":"Elam","suffix":""},{"id":633560982,"identity":"5febb001-db1d-4e87-ac73-6d8f125909b8","order_by":3,"name":"Genevieve Schwarz","email":"","orcid":"","institution":"The University of Toledo College of Medicine and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Genevieve","middleName":"","lastName":"Schwarz","suffix":""},{"id":633560983,"identity":"a9f7cfdc-5d66-48b5-bc8d-83e8e686bab4","order_by":4,"name":"Yue Zou","email":"","orcid":"","institution":"The University of Toledo College of Medicine and Life Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zou","suffix":""},{"id":633560984,"identity":"09656a63-9d32-4734-a9ae-f4b0fd7261a4","order_by":5,"name":"Yong Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYNACHgkgwXwASFgwMIAoHuK0sCUAVUsQqwWiz4A4LQbHew+/YJCxkDfnX/NN+kOFhBzf8QbGB2/b8Gg5cy7NAugww50z3m6TOHBGwljyzAFmw7n4tNzIMTMAamHccOPsNomDbRKJG24ksEnzEqHFfsONM89AWuqBWth/E9Bi/ACoJXHD+R42kJYEA6AtzPi0SJ45Y8aQwCORvOEGm7HFmTMShjPPHGyWnHMOtxa+4z3GHz721NluOH/44Y2KCht5vuPNBz+8KcOtReEAA5tEYg+QJZEAE2NswK0eCOQbGJg/MPwAsvgP4FU4CkbBKBgFIxgAAEQCWGBunUxQAAAAAElFTkSuQmCC","orcid":"","institution":"The University of Toledo College of Medicine and Life Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2026-04-04 14:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9321136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9321136/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108807234,"identity":"77d5a66b-e673-4ad6-88f0-7f3e4df997ef","added_by":"auto","created_at":"2026-05-08 15:30:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":424366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of DSTYK expression in patient-derived TNBC tumors.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) Representative H\u0026amp;E staining showing the histological morphology of DSTYK-high and DSTYK-low TNBC tumors. Immunohistochemical (IHC) staining specific for DSTYK demonstrates differential protein expression in TNBC tumor samples. Scale bar: 100 mm.\u003cbr\u003e\n(\u003cstrong\u003eB\u003c/strong\u003e) Quantitative real-time PCR (RT–qPCR) analysis of DSTYK mRNA expression in tumor specimens from DSTYK-high and DSTYK-low TNBC patients. Data are presented as mean ± SD. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/815b5464826a44126742216a.png"},{"id":108807071,"identity":"e81a25f0-2a38-421e-8aed-90a3b569b2e4","added_by":"auto","created_at":"2026-05-08 15:30:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between DSTYK expression and cancer stem cell-related markers in breast cancer based on data from The Human Protein Atlas (HPA).\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) Correlation between DSTYK and CD133. (\u003cstrong\u003eB\u003c/strong\u003e) Correlation between DSTYK and OCT4. (\u003cstrong\u003eC\u003c/strong\u003e) Correlation between DSTYK and NANOG. (\u003cstrong\u003eD\u003c/strong\u003e) Correlation between DSTYK and LGR5. (\u003cstrong\u003eE\u003c/strong\u003e) Correlation between DSTYK and CD44. (\u003cstrong\u003eF\u003c/strong\u003e) Correlation between DSTYK and EPCAM. (\u003cstrong\u003eG\u003c/strong\u003e) Correlation between DSTYK and Ki67. (\u003cstrong\u003eH\u003c/strong\u003e) Correlation between DSTYK and BAX. Data are presented as mean ± standard deviation (S.D.). Statistical significance is indicated as the P\u003cem\u003e \u003c/em\u003evalue. P \u0026lt; 0.05 means a significant difference.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/ca8a7fad18f0ad9a18157dff.png"},{"id":108735211,"identity":"6d416090-13e2-451a-8226-fe50df13ce25","added_by":"auto","created_at":"2026-05-07 20:01:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":314767,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpdated statistical analysis of DSTYK-associated survival probability in breast cancer patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(\u003cstrong\u003eA\u003c/strong\u003e) Survival curves comparing overall survival probability in breast cancer patients with high versus low DSTYK expression, based on data from the GDC TCGA Breast Cancer (BRCA) cohort. (\u003cstrong\u003eB\u003c/strong\u003e) Western blot analysis of DSTYK protein levels in human triple-negative breast cancer (TNBC) tumor tissues, categorized into DSTYK\u003csup\u003ehigh\u003c/sup\u003e and DSTYK\u003csup\u003elow\u003c/sup\u003e groups. (\u003cstrong\u003eC\u003c/strong\u003e) Correlation analysis between DSTYK and Trop2 protein expressions in breast cancer patient samples. (\u003cstrong\u003eD\u003c/strong\u003e) Correlation analysis between DSTYK and Bax protein expression in breast cancer patient samples.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/78af9d0700d21c9c8c1e6aa5.png"},{"id":108735213,"identity":"706e1321-d013-46b3-be74-5ed1bba00ea4","added_by":"auto","created_at":"2026-05-07 20:01:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":350668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of patient-derived xenograft (PDX) mouse models.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) NOD-SCID-IL2Rγ\u003csup\u003enull\u003c/sup\u003e (NSG) mice (The Jackson Laboratory) were used for tumor implantation. Fresh patient tumor specimens were cut into small fragments (~5 mm³) and aseptically implanted into the fourth mammary fat pad of 6-7-week-old female mice (one fragment per mouse) to generate P0 tumors. (\u003cstrong\u003eB\u003c/strong\u003e) P0 tumors were harvested, sectioned into ~5 mm³ fragments, and used for subsequent transplantation. (\u003cstrong\u003eC\u003c/strong\u003e) Tumor fragments from P0 tumors were aseptically implanted into the fourth mammary fat pad of 6-7-week-old female mice (one fragment per mouse) to generate P1 tumors; the same procedure was repeated to generate P2 tumors. (\u003cstrong\u003eD\u003c/strong\u003e) Representative P2 tumors derived from P1 tumors. (\u003cstrong\u003eE\u003c/strong\u003e) Schematic timeline and workflow of PDX model establishment.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/1c6742fa0369894c445cc127.png"},{"id":108735216,"identity":"bea43820-63e7-4b7e-9eba-4d249121b6b0","added_by":"auto","created_at":"2026-05-07 20:01:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1231260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP2 tumors preserve original human tumor features.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eH\u0026amp;E\u003c/em\u003e staining and Ki67 staining in representative P2 PDX tumors compared to original human TNBC tumors. Scale bar: 100 mm. (\u003cstrong\u003eB\u003c/strong\u003e) IHCstaining for Ku80 and CD45 was performed to confirm the human origin of the P2 tumors. ER, PR, and HER2 staining further verified the maintenance of the triple-negative phenotype.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/0833bc7488591b866ea40994.png"},{"id":108735218,"identity":"5bf30fa0-caca-4902-aa7e-78e90f3e69fd","added_by":"auto","created_at":"2026-05-07 20:01:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":339016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDSTYK serves as a prognostic biomarker in clinical TNBC.\u003c/strong\u003e\u003cbr\u003e\n(\u003cstrong\u003eA\u003c/strong\u003e) Representative IHC staining images showing the expression of DSTYK, Trop2, and Bax in DSTYK\u003csup\u003eLow\u003c/sup\u003e TNBC tissues and the corresponding residual TNBC tissues. (\u003cstrong\u003eB\u003c/strong\u003e) Representative IHC images comparing the expression levels of DSTYK, Trop2, and Bax in TNBC tissues with high versus low DSTYK expression. (\u003cstrong\u003eC\u003c/strong\u003e) Quantitative analysis of IHC staining from panels (A) and (B).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/fd6724d64a3e02c28f91daba.png"},{"id":108806701,"identity":"6fc7edce-4239-49dd-b2b6-b58a2bf214ef","added_by":"auto","created_at":"2026-05-08 15:29:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":188317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced DSTYK expression enhances chemosensitivity in PDX models.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Schematic illustration of the treatment regimen used in PDX mouse models receiving combined doxorubicin (DOX) and docetaxel (DXL) therapy. (\u003cstrong\u003eB\u003c/strong\u003e) Tumor growth curves of PDX models stratified by DSTYK expression (DSTYK\u003csup\u003eHigh\u003c/sup\u003e and DSTYK\u003csup\u003eLow\u003c/sup\u003e) with or without drug treatment, as indicated. Data are presented as mean ± S.D. (\u003cstrong\u003eC\u003c/strong\u003e) Representative images of excised tumors from DSTYK\u003csup\u003eHigh\u003c/sup\u003e and DSTYK\u003csup\u003eLow\u003c/sup\u003e PDX groups with and without drug treatment.\u003cbr\u003e\n(\u003cstrong\u003eD\u003c/strong\u003e) Quantification of tumor weights at the experimental endpoint. Data are shown as mean ± S.D. (\u003cstrong\u003eE\u003c/strong\u003e) TUNEL assays assessing cell death in tumor tissues from DSTYK\u003csup\u003eHigh\u003c/sup\u003e and DSTYK\u003csup\u003eLow\u003c/sup\u003e groups with and without drug treatment. (\u003cstrong\u003eF\u003c/strong\u003e) Quantification of TUNEL-positive cells from panel (E). Data are presented as mean ± S.D. Statistical significance is indicated as follows: *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/702b208f4d46467c4bb74f4e.png"},{"id":108979684,"identity":"1a69e98f-5815-4a69-8208-404c431986d9","added_by":"auto","created_at":"2026-05-11 12:00:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3187720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/2a7c633e-1b69-4be9-be99-a4fe35b53e38.pdf"},{"id":108735208,"identity":"f0a809bb-60d0-4b7d-9e11-4b4a9381ee50","added_by":"auto","created_at":"2026-05-07 20:01:54","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":351918,"visible":true,"origin":"","legend":"","description":"","filename":"S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/3ea0d4fddf5d21916d8de1f3.jpg"},{"id":108807182,"identity":"39eaba6f-be8f-4ba6-a528-b68876726e26","added_by":"auto","created_at":"2026-05-08 15:30:17","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":552611,"visible":true,"origin":"","legend":"","description":"","filename":"S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/71916e064b44138b32b22aa6.jpg"},{"id":108976777,"identity":"0e2ab401-3eb9-4955-bcc9-85ffbfdd84fa","added_by":"auto","created_at":"2026-05-11 11:28:28","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":515960,"visible":true,"origin":"","legend":"","description":"","filename":"S3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/01008afcdbf868268946985f.jpg"},{"id":108806811,"identity":"63e9fd72-af17-46e3-8bb8-dc590fe8695d","added_by":"auto","created_at":"2026-05-08 15:29:31","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":351276,"visible":true,"origin":"","legend":"","description":"","filename":"S4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9321136/v1/f9223df41a34dfc4517821c1.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"DSTYK predicts Chemoresistance in Triple-Negative Breast Cancer Patient-Derived Xenograft Models","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTriple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. TNBC represents the most aggressive subtype of breast cancer, with high rates of recurrence, pronounced molecular heterogeneity, and limited targeted therapeutic options\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Currently, chemotherapy remains the primary neoadjuvant treatment for TNBC. However, the frequent development of chemoresistance is a major contributor to treatment failure and poor clinical outcomes\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Despite extensive investigation, the molecular mechanisms underlying chemoresistance in TNBC remain incompletely understood, underscoring an urgent need to identify novel biomarkers and therapeutic targets to improve patient survival\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong commonly used regimens, the combination of doxorubicin (DOX) and docetaxel (DXL) has demonstrated substantial efficacy in breast cancer and has been widely applied in clinical trials for TNBC treatment\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A deeper understanding of how chemotherapy-induced cell death is regulated will provide new strategies to overcome chemoresistance in TNBC\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Cancer progression is fundamentally driven by an imbalance between cell proliferation and programmed cell death, particularly apoptosis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Under normal physiological conditions, apoptosis serves as a critical safeguard to eliminate damaged or transformed cells; however, cancer cells frequently evade apoptosis by inactivating key regulatory pathways\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Consequently, successful chemotherapeutic agents exert their antitumor effects largely by reactivating apoptotic signaling in cancer cells\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In our previous studies, we demonstrated that dual serine/threonine and tyrosine protein kinase (DSTYK) suppresses chemotherapy-induced apoptosis in TNBC cells⁷. Building upon these findings, the present study aims to further elucidate the role of DSTYK in regulating apoptosis and chemotherapeutic response using PDX models.\u003c/p\u003e \u003cp\u003eRecently, trophoblast cell-surface antigen 2 (Trop-2) has emerged as an important biomarker for TNBC \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Trop-2, encoded by the TACSTD2 gene, is overexpressed in multiple malignancies and strongly associated with chemoresistance in lung, pancreatic, colorectal, hepatic cancers, and TNBC\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Functionally, Trop-2 promotes tumor cell survival, proliferation, and metastasis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Notably, Trop-2-targeting antibody\u0026ndash;drug conjugates have shown remarkable clinical efficacy, significantly improving progression-free and overall survival in patients with metastatic TNBC\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this study, we observed a positive correlation between DSTYK and Trop-2 expression in clinical TNBC specimens, suggesting a potential functional link between these two molecules.\u003c/p\u003e \u003cp\u003ePrevious work from our group and others has shown that DSTYK promotes transforming growth factor-β (TGF-β)\u0026ndash;induced epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and enhances chemoresistance in TNBC using cellular and orthotopic mouse models\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Mechanistically, DSTYK activates ERK signaling\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, downregulates the pro-apoptotic protein Bax\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, promotes STING pathway activation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and inhibits apoptosis induced by UV irradiation in skin cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In addition, inhibition of DSTYK sensitizes cancer cells to taxane-based chemotherapy and enhances T cell\u0026ndash;mediated cytotoxicity by suppressing autophagy-dependent survival pathways\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, these studies have largely relied on in vitro systems or conventional xenograft models. The role of DSTYK in chemoresistance has not yet been investigated in TNBC PDX models.\u003c/p\u003e \u003cp\u003ePDX models are widely recognized as superior preclinical platforms because they can faithfully recapitulate the histological architecture, molecular heterogeneity, and tumor microenvironment of primary human tumors\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In the present study, we employed DSTYK-high and DSTYK-low TNBC PDX models to evaluate responses to combined DOX/DXL chemotherapy. We demonstrate that DSTYK-high PDX tumors exhibit elevated Trop-2 expression and profound resistance to chemotherapy, whereas DSTYK-low tumors display reduced Trop-2 levels and significantly enhanced chemosensitivity. Collectively, our findings establish DSTYK as a critical determinant of chemotherapeutic response in TNBC and highlight its potential as a prognostic biomarker and therapeutic target for overcoming chemoresistance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstablish PDX mouse model and chemotherapeutic assays\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Toledo (UT). De-identified, fresh, treatment-na\u0026iuml;ve triple-negative breast cancer (TNBC) specimens were obtained from TNBC patients for the establishment of patient-derived xenograft (PDX) models. Based on DSTYK expression levels, the top ten TNBC specimens with relatively high DSTYK expression and the bottom ten specimens with relatively low DSTYK expression were selected for chemoresistance analyses using PDX models.\u003c/p\u003e \u003cp\u003eNOD-SCID-IL2Rγ\u003csup\u003enull\u003c/sup\u003e (NSG) mice were housed and maintained in the UT animal facilities. Fresh tumor specimens were sectioned into small fragments (~\u0026thinsp;3\u0026ndash;5 mm\u0026sup3;) and aseptically implanted into the fourth mammary fat pad of 6-7-week-old female NSG mice, with one tumor fragment implanted per mouse). Tumor engraftment and growth were monitored regularly. Successfully engrafted tumors were serially passaged to generate P0, P1 (first passage), and P2 (second passage) PDX tumors via subcutaneous expansion. To minimize inter-experimental variability, only tumors from the same generation (P2) were used for all treatment studies.\u003c/p\u003e \u003cp\u003eOnce P2 tumors reached an approximate volume of 50 mm\u0026sup3;, mice were randomized into treatment groups and administered a combination of doxorubicin (DOX) and docetaxel (DXL) via intraperitoneal injection. Treatment was initiated at doses of 4.0 mg/kg DOX and 1.0 mg/kg DXL, and delivered twice weekly for 3\u0026ndash;4 weeks. Tumor growth and treatment responses were monitored throughout the treatment period.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry (IHC) Staining\u003c/h3\u003e\n\u003cp\u003eTumor specimens derived from patients were collected and processed according to our previously described immunohistochemistry protocol\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Paraffin-embedded tissue sections (5 \u0026micro;m thick) were incubated overnight at 4\u0026deg;C with the following primary antibodies: anti-DSTYK (OriGene Technologies), anti-Trop2 (Abcam), anti-Ki67 (Abcam), anti-Ku80 (R\u0026amp;D System), anti-CD45, and anti-Bax (Cell Signaling Technology). Immunoreactive signals were visualized using the VECTASTAIN\u0026reg; Elite ABC Kit (Vector Laboratories, Burlingame, CA) in accordance with the manufacturer\u0026rsquo;s instructions. Hematoxylin was used for nuclear counterstaining. Quantitative analysis of IHC staining intensity was performed using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eWestern blot (WB) analyses were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Briefly, total proteins were extracted from whole-cell lysates, and equal amounts of protein were separated by electrophoresis on 8\u0026ndash;10% SDS\u0026ndash;polyacrylamide gels. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk and incubated with primary antibodies against Trop2, Bax, DSTYK, and GAPDH (Santa Cruz Biotechnology). After incubation with horseradish peroxidase\u0026ndash;conjugated secondary antibodies (Thermo Fisher Scientific), protein signals were detected using enhanced chemiluminescence (ECL; Bio-Rad) and visualized by chemiluminescence imaging. Each Western blot experiment was performed at least three times independently.\u003c/p\u003e\n\u003ch3\u003eTerminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay\u003c/h3\u003e\n\u003cp\u003eThe TUNEL assay was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, following the manufacturer\u0026rsquo;s instructions using an apoptosis detection kit (ABP Biosciences, catalog no. A050). Briefly, tissue sections were fixed in 4% paraformaldehyde for 10 min and subsequently washed with phosphate-buffered saline (PBS). Sections were permeabilized with proteinase K (20 \u0026micro;g/mL in PBS). The terminal deoxynucleotidyl transferase (TdT) reaction mixture was freshly prepared and incubated with the sections for 1 h at 37\u0026deg;C in a humidified chamber. Afterward, the slides were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) for 5 min. Fluorescence signals were examined and images were captured using a Leica fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eRNA isolation and quantitative real-time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from the cell pellets by the RNeasy Mini kit (Cat# 74104; QIAGEN), and 1 \u0026micro;g RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Cat# 43-688-14; Thermo Fisher). Quantitative real-time PCR (real-time-qPCR) was performed using iTaq Universal SYBR Green Supermix (Cat# 1725124, Bio-Rad). DSTYK Forward, 5\u0026prime;-ATTGCCAACCGAAAGCAGGAGG-3\u0026prime;, Reverse, 5\u0026prime;-GGTGCCTACTGGTTCTCCATTC-3\u0026prime;; β-actin Forward 5\u0026prime;-CACCAACTGGGACGACAT-3\u0026prime;, Reverse 5\u0026prime;-ACAGCCTGGATAGCAACG-3\u0026prime;. The gene expression levels were determined by the 2\u003csup\u003e\u0026minus;ΔΔ\u003c/sup\u003e\u003cem\u003eC\u003c/em\u003eq method and values were normalized to β-actin expression as an internal control.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using Prism 7 and are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s honestly significant difference post hoc test, with a confidence level of 95%. Comparisons between two groups were made using a parametric paired t-test for normally distributed data or non-parametric Wilcoxon paired t-test for non-normally distributed data. Unless otherwise indicated, experiments included more than five mice or samples per group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;5). Data were analyzed using the unpaired student t-test, and differences were considered statistically significant at P-values of \u0026lt;\u0026thinsp;0.05 (*), \u0026lt;\u0026thinsp;0.01 (**), \u0026lt;\u0026thinsp;0.001 (***) and \u0026lt;\u0026thinsp;0.0001 (****) that were considered statistically significant or very significant, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of DSTYK expression levels in human TNBC tissues\u003c/h2\u003e \u003cp\u003eFirst, histopathological analyses of human triple-negative breast cancer (TNBC) tissues were performed using hematoxylin and eosin (H\u0026amp;E) staining to confirm the characteristic features of breast cancer \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eleft panel\u003c/b\u003e). We then assessed DSTYK protein expression in TNBC tissues by immunohistochemical (IHC) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cb\u003eright panel\u003c/b\u003e). Based on IHC staining intensity, we selected the top 10 TNBC specimens with the high DSTYK expression (DSTYK\u003csup\u003eHigh\u003c/sup\u003e) and the bottom 10 specimens with the low DSTYK expression (DSTYK\u003csup\u003eLow\u003c/sup\u003e). Quantitative analysis was conducted using ImageJ software (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistently, DSTYK mRNA expression was significantly higher in DSTYK\u003csup\u003eHigh\u003c/sup\u003e tissues compared with DSTYK\u003csup\u003eLow\u003c/sup\u003e tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Together, these results validate the classification of DSTYK\u003csup\u003eHigh\u003c/sup\u003e and DSTYK\u003csup\u003eLow\u003c/sup\u003e TNBC patient specimens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDSTYK expression correlates with the expression of cancer stem cell (CSC) markers\u003c/h2\u003e \u003cp\u003eThe association between DSTYK expression and breast cancer stem cell (CSC) markers was systematically evaluated based on The Human Atlas database. DSTYK expression showed a modest but significant positive correlation with CD133 (r\u0026thinsp;=\u0026thinsp;0.0621, p\u0026thinsp;=\u0026thinsp;0.0474; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Further analyses revealed that DSTYK was positively correlated with multiple established CSC markers, including OCT4 (r\u0026thinsp;=\u0026thinsp;0.2352, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), NANOG (r\u0026thinsp;=\u0026thinsp;0.3933, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), LGR5 (r\u0026thinsp;=\u0026thinsp;0.2399, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), CD44 (r\u0026thinsp;=\u0026thinsp;0.3104, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), and EPCAM (r\u0026thinsp;=\u0026thinsp;0.1189, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eIn addition, DSTYK expression was negatively correlated with the pro-apoptotic marker Bax (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.3446, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), while a positive association was observed between DSTYK and the proliferation marker Ki67 (r\u0026thinsp;=\u0026thinsp;0.1300, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eCollectively, these findings suggest that elevated DSTYK expression is associated with enhanced cancer stemness, reduced apoptotic potential, and increased proliferative capacity, indicating that DSTYK may contribute to apoptosis inhibition and decreased chemosensitivity in breast cancer cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of DSTYK as a Potential Prognostic Biomarker in TNBC\u003c/h2\u003e \u003cp\u003eTo assess the clinical relevance of DSTYK in breast cancer, we analyzed patient data from the GDC TCGA Breast Cancer (BRCA) cohort. Our statistical analysis revealed that elevated DSTYK expression is significantly associated with an increased risk of mortality in breast cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that DSTYK may serve as a negative prognostic factor. These findings further imply a potential role for DSTYK in promoting chemoresistance in breast cancer, including triple-negative breast cancer (TNBC).\u003c/p\u003e \u003cp\u003eGiven that Trop2 is highly expressed on the surface of most TNBC cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we next examined the relationship between DSTYK and Trop2 expression. Immunoblotting analyses were performed using randomly selected human TNBC tissue specimens. The results revealed a positive correlation between DSTYK and Trop2 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Quantification of immunoblot band intensities using ImageJ, followed by statistical analysis, confirmed a significant positive correlation between DSTYK and Trop2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Based on these immunoblotting results, TNBC tissues were stratified into relative DSTYK\u003csup\u003eHigh\u003c/sup\u003e and DSTYK\u003csup\u003eLow\u003c/sup\u003e groups. Subsequent statistical analysis demonstrated that the pro-apoptotic factor Bax was negatively correlated with DSTYK expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Collectively, these data suggest that DSTYK may function as a tumor-promoting element in TNBC by modulating the expression of Trop2 and Bax, thereby contributing to tumor progression and resistance to chemotherapeutic drug-induced apoptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of TNBC PDX Models in NSG Mice\u003c/h2\u003e \u003cp\u003eTo establish PDX models, NOD-SCID-IL2Rγ\u003csup\u003enull\u003c/sup\u003e (NSG) mice were used as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. For each tumor specimen, ten mice were employed. Fresh TNBC tumor tissues were aseptically sectioned into small fragments (~\u0026thinsp;5 mm\u0026sup3;) and orthotopically implanted into the fourth mammary fat pad of 6-7-week-old female NSG mice, with one tumor fragment implanted per mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe first passage (P0) of subcutaneous TNBC PDX tumors required approximately 6\u0026ndash;8 weeks for expansion, whereas the second (P1) and third (P2) passages required approximately 4 weeks and 3\u0026ndash;4 weeks, respectively. All subcutaneous PDX tumors were expanded to a volume of ~\u0026thinsp;300\u0026ndash;400 mm\u0026sup3; prior to subsequent transplantation.\u003c/p\u003e \u003cp\u003eFollowing orthotopic implantation, P1 and P2 PDX tumors demonstrated significantly accelerated in vivo growth compared with P0 tumors, with growth rates significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To assess whether key characteristics of the original tumors were preserved during passaging, randomly selected PDX models derived from two patients with triple-negative breast cancer (TNBC) were subjected to histopathological evaluation. Hematoxylin and eosin (H\u0026amp;E) staining and Ki67 immunohistochemistry of P2 tumors confirmed retention of original TNBC histological features and high proliferative activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Immunohistochemical analysis of estrogen receptor (ER), progesterone receptor (PR), and HER2 further verified maintenance of the triple-negative phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, Ku80 and CD45 staining confirmed the human origin of the tumor cells and revealed minimal murine hematopoietic cell contamination, supporting the fidelity of the PDX models (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDSTYK expression correlates with Trop2 upregulation and Bax suppression in clinical TNBC.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous studies demonstrated that chemoresistant TNBC cells regain chemosensitivity upon DSTYK knockout (KO) following chemotherapeutic treatment\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To explore the mechanisms by which DSTYK promotes chemoresistance in TNBC, we performed immunohistochemical (IHC) analyses to evaluate the expression of DSTYK, Trop2, and Bax in paired primary TNBC specimens and corresponding residual tumors obtained after neoadjuvant chemotherapy.\u003c/p\u003e \u003cp\u003eIHC staining revealed that DSTYK protein expression was significantly increased in residual TNBC tissues compared with their matched pre-treatment DSTYK\u003csup\u003eLow\u003c/sup\u003e tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Consistently, Trop2 expression was markedly upregulated in residual tumors following chemotherapy. In contrast, expression of the pro-apoptotic protein Bax was significantly reduced in residual TNBC tissues relative to untreated DSTYK\u003csup\u003eLow\u003c/sup\u003e tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, C).\u003c/p\u003e \u003cp\u003eCorrelation analyses further demonstrated a positive association between DSTYK and Trop2 expression and an inverse relationship between DSTYK and Bax expression. Specifically, TNBC tissues with low DSTYK expression exhibited reduced Trop2 levels and elevated Bax expression, whereas tumors with high DSTYK expression showed increased Trop2 expression and concomitant suppression of Bax (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003eCollectively, these findings suggest that Trop2 and Bax may serve as downstream effectors of DSTYK signaling. Our data identify Trop2 and Bax as novel components of a DSTYK-associated regulatory network that contributes to chemoresistance in TNBC and supports DSTYK as a potential prognostic biomarker in the clinical setting.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDSTYK Promotes Chemoresistance in\u003c/b\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003ePDX Models\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo provide clinically relevant evidence that DSTYK contributes to chemoresistance, we employed patient-derived xenograft (PDX) mouse models. The experiment procedure is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eFollowing orthotopic implantation, tumor volume, tumor weight, and body weight were monitored twice a week, and tumor growth curves were generated to compare therapeutic responses between tumors with high versus low DSTYK expression, no significant differences in tumor growth were observed between DSTYK\u003csup\u003ehigh\u003c/sup\u003e and DSTYK\u003csup\u003elow\u003c/sup\u003e PDX tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), consistent with our previous findings that DSTYK does not significantly influence cancer cell proliferation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In contrast, after DOX\u0026thinsp;+\u0026thinsp;DXL treatment, tumors derived from the DSTYK\u003csup\u003elow\u003c/sup\u003e group exhibited marked regression compared with those from the DSTYK\u003csup\u003ehigh\u003c/sup\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eConsistently, TUNEL staining revealed a significantly higher level of apoptosis in DSTYK\u003csup\u003eLow\u003c/sup\u003e tumors following chemotherapy compared with DSTYK\u003csup\u003eHigh\u003c/sup\u003e tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE \u003cb\u003e\u0026amp; F\u003c/b\u003e). These findings indicate that elevated DSTYK expression attenuates chemosensitivity by suppressing chemotherapy-induced tumor cell death.\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that DSTYK plays a pivotal role in promoting chemoresistance in TNBC. Targeting DSTYK in combination with standard chemotherapy may therefore represent a promising strategy to enhance therapeutic efficacy in TNBC patients.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that primary TNBC tumors with elevated DSTYK expression exhibit significantly greater chemoresistance than those with lower DSTYK levels. DSTYK expression is positively correlated with the newly identified TNBC marker Trop2 and negatively correlated with the pro-apoptotic protein Bax. Together with DSTYK and Trop2, Bax may therefore also serve as a potential biomarker for assessing chemoresistance in TNBC, consistent with several previous reports\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Furthermore, in our PDX mouse models, tumors characterized by high DSTYK and low Bax expression displayed markedly reduced sensitivity to drug treatment compared with tumors expressing lower DSTYK and higher Bax levels.\u003c/p\u003e \u003cp\u003eOur previous studies revealed that DSTYK suppresses chemotherapy-induced apoptosis in TNBC cells and destabilizes Bax in colorectal cancer cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In the present study, we extend these findings by providing clinical evidence that DSTYK and Bax protein levels are inversely correlated in TNBC specimens. Combined with our functional PDX data showing that DSTYK inhibits apoptosis and attenuates chemosensitivity, these results strongly suggest that DSTYK may similarly downregulate Bax stability in TNBC. Previously, we demonstrated that chemoresistant cancer cells regain chemosensitivity following DSTYK knockout\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, here in, immunohistochemical analysis of clinical TNBC samples reveals that DSTYK protein levels are significantly elevated in residual tumors after neoadjuvant chemotherapy compared with treatment-na\u0026iuml;ve tumors. This observation suggests that DSTYK may be a key determinant driving the chemoresistant phenotype of residual TNBC. Collectively, these findings reinforce our earlier discovery and establish DSTYK as a potential prognostic biomarker and therapeutic target for chemoresistant TNBC.\u003c/p\u003e \u003cp\u003eTo further characterize the role of DSTYK in chemoresistance, we employed patient-derived xenograft (PDX) mouse models generated from clinical TNBC specimens. PDX models are highly valued in cancer research\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, particularly for TNBC, as they better mirror the characteristics of human disease compared with conventional cancer cell lines\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. By engrafting freshly resected patient tumor tissues into immunodeficient mice, PDX models preserve tumor architecture, histopathological features, and intratumoral heterogeneity, and these factors that are critical for accurately assessing therapeutic response. In our PDX studies, tumors with low DSTYK expression exhibited significantly greater regression following chemotherapy than those with high DSTYK expression. These findings further support the conclusion that elevated DSTYK levels confer increased chemoresistance. To enhance the translational relevance of our work, future studies should evaluate the impact of DSTYK on resistance to additional chemotherapeutic agents beyond doxorubicin and docetaxel.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eStatistical analysis of the patient dataset indicates that a high DSTYK level is correlated with an increased risk of mortality in breast cancer patients, TNBC patients, and TNBC patients treated only with chemotherapy\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanisms by which DSTYK mediates chemoresistance remain poorly understood\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Notably, we identified a strong positive correlation between DSTYK and Trop2 expression. Trop2 is a well-established therapeutic target in TNBC, as it is highly expressed on the surface of most TNBC cells while exhibiting minimal expression in normal tissues. Trop2 is a transmembrane glycoprotein that functions as an intracellular calcium signal transducer and plays a critical role in tumor progression by regulating oncogenic signaling pathways, including AKT, ERK, and JAK/STAT. Trop2 overexpression is consistently associated with poor prognosis and aggressive TNBC phenotypes\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Trop2 antibody drug conjugates sacituzumab govitecan (Trodelvy) and datopotomab deruxtecan (Datroway) have been approved by FDA not long ago for the clinical treatment of TNBC patients, which is a groundbreaking achievement to significantly increase both progression-free survival (PFS) and overall survival (OS) probability of metastatic TNBC patients according to a statistical analysis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our study, TNBC specimens with high DSTYK expression consistently exhibited elevated Trop2 levels, whereas tumors with low DSTYK expression showed correspondingly low Trop2 expression. This statistically significant correlation suggests a previously unrecognized link between DSTYK and Trop2 and highlights a compelling direction for future mechanistic studies aimed at elucidating how DSTYK promotes chemoresistance. Given that DSTYK is a protein kinase, it represents a highly druggable target. The development of cell-permeable DSTYK inhibitors may therefore offer a novel therapeutic strategy to overcome chemoresistance and enhance the efficacy of existing treatments, including Trop2-targeted therapies, ultimately improving clinical outcomes for patients with TNBC.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our findings provide important advances in therapeutic strategies for the ongoing challenge of chemotherapy resistance in TNBC. By further validating, we identify DSTYK as a promising druggable target for targeting and eliminating chemoresistant TNBC in clinical patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with institutional regulations after protocol review and approval by the University of Toledo\u0026apos;s Institutional Animal Care and Use Committee. The authors complied with the ARRIVE guidelines\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Z., S.R., and Y.J. wrote the manuscript; Y.J. revised the manuscript according to the comments and suggestions of G.S. and Y.Z. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by National Institutes of Health grants R01CA272760 and startup funding from the University of Toledo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLai, R. C.\u003cem\u003e et al.\u003c/em\u003e Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. \u003cem\u003eStem Cell Res\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 214-222 (2010). https://doi.org/10.1016/j.scr.2009.12.003\u003c/li\u003e\n\u003cli\u003eAbdolahi, S.\u003cem\u003e et al.\u003c/em\u003e Patient-derived xenograft (PDX) models, applications and challenges in cancer research. \u003cem\u003eJ Transl Med\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 206 (2022). https://doi.org/10.1186/s12967-022-03405-8\u003c/li\u003e\n\u003cli\u003eAgelidis, A., Ter-Zakarian, A. \u0026amp; Jaloudi, M. Triple-Negative Breast Cancer on the Rise: Breakthroughs and Beyond. \u003cem\u003eBreast Cancer (Dove Med Press)\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 523-529 (2025). https://doi.org/10.2147/BCTT.S516125\u003c/li\u003e\n\u003cli\u003eKim, C.\u003cem\u003e et al.\u003c/em\u003e Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e173\u003c/strong\u003e, 879-893 e813 (2018). https://doi.org/10.1016/j.cell.2018.03.041\u003c/li\u003e\n\u003cli\u003eLusby, R., Zhang, Z., Mahesh, A. \u0026amp; Tiwari, V. K. Decoding gene regulatory circuitry underlying TNBC chemoresistance reveals biomarkers for therapy response and therapeutic targets. \u003cem\u003eNPJ Precis Oncol\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 64 (2024). https://doi.org/10.1038/s41698-024-00529-6\u003c/li\u003e\n\u003cli\u003eDhara, A.\u003cem\u003e et al.\u003c/em\u003e Enhanced ETS1 stability by DNAPKcs orchestrates transcriptional changes during chemoresistance in triple negative breast cancer. \u003cem\u003eNPJ Breast Cancer\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 114 (2025). https://doi.org/10.1038/s41523-025-00782-3\u003c/li\u003e\n\u003cli\u003eTrnkova, L.\u003cem\u003e et al.\u003c/em\u003e Molecular profiling of chemotherapy-resistant breast cancer reveals DNA methylation remodeling associated with the acquisition of paclitaxel resistance. \u003cem\u003eDrug Resist Updat\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 101350 (2026). https://doi.org/10.1016/j.drup.2026.101350\u003c/li\u003e\n\u003cli\u003eFleisher, B., Clarke, C. \u0026amp; Ait-Oudhia, S. Current advances in biomarkers for targeted therapy in triple-negative breast cancer. \u003cem\u003eBreast Cancer (Dove Med Press)\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 183-197 (2016). https://doi.org/10.2147/BCTT.S114659\u003c/li\u003e\n\u003cli\u003eBaltali, E., Ozisik, Y., Guler, N., Firat, D. \u0026amp; Altundag, K. Combination of docetaxel and doxorubicin as first-line chemotherapy in metastatic breast cancer. \u003cem\u003eTumori\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 18-19 (2001). https://doi.org/10.1177/030089160108700104\u003c/li\u003e\n\u003cli\u003eRicci, M. S. \u0026amp; Zong, W. X. Chemotherapeutic approaches for targeting cell death pathways. \u003cem\u003eOncologist\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 342-357 (2006). https://doi.org/10.1634/theoncologist.11-4-342\u003c/li\u003e\n\u003cli\u003eZafar, A., Khatoon, S., Khan, M. J., Abu, J. \u0026amp; Naeem, A. Advancements and limitations in traditional anti-cancer therapies: a comprehensive review of surgery, chemotherapy, radiation therapy, and hormonal therapy. \u003cem\u003eDiscov Oncol\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 607 (2025). https://doi.org/10.1007/s12672-025-02198-8\u003c/li\u003e\n\u003cli\u003eLiu, Y.\u003cem\u003e et al.\u003c/em\u003e Programmed cell death in triple-negative breast cancer. \u003cem\u003eCell Mol Biol Lett\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 111 (2025). https://doi.org/10.1186/s11658-025-00789-5\u003c/li\u003e\n\u003cli\u003eNedeljkovic, M. \u0026amp; Damjanovic, A. Mechanisms of Chemotherapy Resistance in Triple-Negative Breast Cancer-How We Can Rise to the Challenge. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2019). https://doi.org/10.3390/cells8090957\u003c/li\u003e\n\u003cli\u003eLoftus, L. V., Amend, S. R. \u0026amp; Pienta, K. J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e (2022). https://doi.org/10.3390/ijms23094723\u003c/li\u003e\n\u003cli\u003eWei, Y., Hankey, W., Xu, D. \u0026amp; Yuan, F. Programmed Cell Death in Cancer. \u003cem\u003eMedComm (2020)\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e70357 (2025). https://doi.org/10.1002/mco2.70357\u003c/li\u003e\n\u003cli\u003eGielecinska, A.\u003cem\u003e et al.\u003c/em\u003e Apoptosis, necroptosis, and pyroptosis as alternative cell death pathways induced by chemotherapeutic agents? \u003cem\u003eBiochim Biophys Acta Rev Cancer\u003c/em\u003e \u003cstrong\u003e1878\u003c/strong\u003e, 189024 (2023). https://doi.org/10.1016/j.bbcan.2023.189024\u003c/li\u003e\n\u003cli\u003eMantri, S. \u0026amp; Doshi, G. M. Reactivating apoptotic pathways in cancer: A review of novel therapeutic approaches. \u003cem\u003eEur J Pharmacol\u003c/em\u003e \u003cstrong\u003e1003\u003c/strong\u003e, 177965 (2025). https://doi.org/10.1016/j.ejphar.2025.177965\u003c/li\u003e\n\u003cli\u003eKrajewski, S.\u003cem\u003e et al.\u003c/em\u003e Reduced expression of proapoptotic gene BAX is associated with poor response rates to combination chemotherapy and shorter survival in women with metastatic breast adenocarcinoma. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 4471-4478 (1995).\u003c/li\u003e\n\u003cli\u003eMcCurrach, M. E., Connor, T. M., Knudson, C. M., Korsmeyer, S. J. \u0026amp; Lowe, S. W. bax-deficiency promotes drug resistance and oncogenic transformation by attenuating p53-dependent apoptosis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 2345-2349 (1997). https://doi.org/10.1073/pnas.94.6.2345\u003c/li\u003e\n\u003cli\u003eJeon, Y., Jo, U., Hong, J., Gong, G. \u0026amp; Lee, H. J. Trophoblast cell-surface antigen 2 (TROP2) expression in triple-negative breast cancer. \u003cem\u003eBMC Cancer\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1014 (2022). https://doi.org/10.1186/s12885-022-10076-7\u003c/li\u003e\n\u003cli\u003eLiao, Q.\u003cem\u003e et al.\u003c/em\u003e TROP2 is highly expressed in triple-negative breast cancer CTCs and is a potential marker for epithelial mesenchymal CTCs. \u003cem\u003eMol Ther Oncol\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 200762 (2024). https://doi.org/10.1016/j.omton.2024.200762\u003c/li\u003e\n\u003cli\u003eAlhamed, A. S.\u003cem\u003e et al.\u003c/em\u003e Modulation of Chemotherapy Sensitivity of Breast Cancer Cells through Transforming Growth Factor-beta Pathway-mediated Alterations in DNA Damage Response. \u003cem\u003eInt J Med Sci\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 2031-2039 (2025). https://doi.org/10.7150/ijms.111217\u003c/li\u003e\n\u003cli\u003eSakach, E., Sacks, R. \u0026amp; Kalinsky, K. Trop-2 as a Therapeutic Target in Breast Cancer. \u003cem\u003eCancers (Basel)\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e (2022). https://doi.org/10.3390/cancers14235936\u003c/li\u003e\n\u003cli\u003eAlhushki, S., Deshmukh, S., Levy, B. \u0026amp; Desai, A. A tale of two TROP-2 antibody-drug conjugates: a comparative saga of datopotamab deruxtecan and sacituzumab govitecan. \u003cem\u003eOncologist\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e (2025). https://doi.org/10.1093/oncolo/oyaf251\u003c/li\u003e\n\u003cli\u003eLiu, Z.\u003cem\u003e et al.\u003c/em\u003e A fast, simple, and cost-effective method of expanding patient-derived xenograft mouse models of pancreatic ductal adenocarcinoma. \u003cem\u003eJ Transl Med\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 255 (2020). https://doi.org/10.1186/s12967-020-02414-9\u003c/li\u003e\n\u003cli\u003eGu, A., Li, J., Li, M. Y. \u0026amp; Liu, Y. Patient-derived xenograft model in cancer: establishment and applications. \u003cem\u003eMedComm (2020)\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e70059 (2025). https://doi.org/10.1002/mco2.70059\u003c/li\u003e\n\u003cli\u003eZhang, J.\u003cem\u003e et al.\u003c/em\u003e DSTYK Promotes Metastasis and Chemoresistance via EMT in Colorectal Cancer. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 1250 (2020). https://doi.org/10.3389/fphar.2020.01250\u003c/li\u003e\n\u003cli\u003eOgbu, S. C.\u003cem\u003e et al.\u003c/em\u003e DSTYK Enhances Chemoresistance in Triple-Negative Breast Cancer Cells. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e (2021). https://doi.org/10.3390/cells11010097\u003c/li\u003e\n\u003cli\u003eEbrahim, N. A. A., Hussein, M. A., Sobeih, M. E. \u0026amp; Amin, N. H. From biomarker to targeted therapy: investigating trophoblast cell-surface antigen 2 expression in triple-negative breast cancer- insights from the national cancer institute. \u003cem\u003eBMC Cancer\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1008 (2025). https://doi.org/10.1186/s12885-025-14402-7\u003c/li\u003e\n\u003cli\u003eLopez-Gonzalez, L.\u003cem\u003e et al.\u003c/em\u003e Exploring Biomarkers in Breast Cancer: Hallmarks of Diagnosis, Treatment, and Follow-Up in Clinical Practice. \u003cem\u003eMedicina (Kaunas)\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e (2024). https://doi.org/10.3390/medicina60010168\u003c/li\u003e\n\u003cli\u003eBlanchard, Z., Brown, E. A., Ghazaryan, A. \u0026amp; Welm, A. L. PDX models for functional precision oncology and discovery science. \u003cem\u003eNat Rev Cancer\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 153-166 (2025). https://doi.org/10.1038/s41568-024-00779-3\u003c/li\u003e\n\u003cli\u003eEl-Sahli, S.\u003cem\u003e et al.\u003c/em\u003e Nanoparticle-Mediated mRNA Delivery to Triple-Negative Breast Cancer (TNBC) Patient-Derived Xenograft (PDX) Tumors. \u003cem\u003eACS Pharmacol Transl Sci\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 460-469 (2025). https://doi.org/10.1021/acsptsci.4c00597\u003c/li\u003e\n\u003cli\u003eEchepare, M.\u003cem\u003e et al.\u003c/em\u003e DSTYK Inhibition Sensitizes NSCLC to Taxane-Based Chemotherapy. \u003cem\u003eJ Thorac Oncol\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 345-365 (2025). https://doi.org/10.1016/j.jtho.2024.11.003\u003c/li\u003e\n\u003cli\u003eGoldenberg, D. M., Stein, R. \u0026amp; Sharkey, R. M. The emergence of trophoblast cell-surface antigen 2 (TROP-2) as a novel cancer target. \u003cem\u003eOncotarget\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 28989-29006 (2018). https://doi.org/10.18632/oncotarget.25615\u003c/li\u003e\n\u003cli\u003eAslan, M.\u003cem\u003e et al.\u003c/em\u003e Oncogene-mediated metabolic gene signature predicts breast cancer outcome. \u003cem\u003eNPJ Breast Cancer\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 141 (2021). https://doi.org/10.1038/s41523-021-00341-6\u003c/li\u003e\n\u003cli\u003eHuang, J., Huang, T., Guo, J., Hu, K. \u0026amp; Zhou, H. The efficacy and safety of datopotamab deruxtecan (Dato-DXd) in advanced solid tumors: a systematic review and meta-analysis. \u003cem\u003eEur J Med Res\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1265 (2025). https://doi.org/10.1186/s40001-025-03538-8\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brcr","sideBox":"Learn more about [Breast Cancer Research](http://breast-cancer-research.biomedcentral.com)","snPcode":"13058","submissionUrl":"https://submission.nature.com/new-submission/13058/3","title":"Breast Cancer Research","twitterHandle":"@BCRJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Orthotopic PDX mouse model, PDX expansion, Subcutaneous PDX mouse model, TNBC","lastPublishedDoi":"10.21203/rs.3.rs-9321136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9321136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePatient-derived xenograft (PDX) models are widely regarded as robust preclinical platforms because they preserve the histopathological and molecular features of primary tumors. In this study, we established twenty triple-negative breast cancer (TNBC) PDX models using freshly resected patient tumors, including ten with high DSTYK expression and ten with low DSTYK expression. Tumor fragments were orthotopically implanted into the fourth mammary fat pads of NSG mice. DSTYK expression was validated by immunohistochemistry and western blotting. Histological evaluation across three serial passages demonstrated that PDX tumors retained the cellular morphology, stromal architecture, and lineage characteristics of their corresponding primary tumors. Using these models, we assessed \u003cem\u003ein vivo\u003c/em\u003e responses to combination chemotherapy with doxorubicin and docetaxel. DSTYK-low PDX tumors exhibited significantly greater chemosensitivity, whereas DSTYK-high tumors showed marked resistance, indicating a critical role for DSTYK in mediating chemotherapy resistance. Consistent with these findings, analyses from The Human Protein Atlas and Kaplan-Meier Plotter databases revealed that high DSTYK expression is associated with poor survival outcomes in TNBC patients. Collectively, our results from clinical specimens, PDX models, and public datasets identify DSTYK as a promising prognostic biomarker and predictor of chemotherapeutic response in TNBC, with potential implications for patient stratification and treatment optimization.\u003c/p\u003e","manuscriptTitle":"DSTYK predicts Chemoresistance in Triple-Negative Breast Cancer Patient-Derived Xenograft Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 20:01:45","doi":"10.21203/rs.3.rs-9321136/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"34890728195843824720204286627063404855","date":"2026-04-27T04:07:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119787660307275569513842623933987857383","date":"2026-04-23T09:14:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T08:41:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-14T14:14:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-14T11:21:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer Research","date":"2026-04-04T14:19:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brcr","sideBox":"Learn more about [Breast Cancer Research](http://breast-cancer-research.biomedcentral.com)","snPcode":"13058","submissionUrl":"https://submission.nature.com/new-submission/13058/3","title":"Breast Cancer Research","twitterHandle":"@BCRJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d550dfa-30b0-46fd-b68b-f491bc7daa2d","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T20:01:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 20:01:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9321136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9321136","identity":"rs-9321136","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}