Breast cancer is the most prevalent cancer among females with different molecular subtypes. Corosolic acid is a pentacyclic triterpenoid with anti-cancer properties. The MTT assay was used to assess the cytotoxic activity of corosolic acid on MDA-MB-231 and MCF7 cell lines. To determine the apoptotic cells, the flow cytometry technique was utilized. The expression levels of apoptosis-related genes and proteins were quantified using quantitative real time-PCR (qRT-PCR) and Western blotting methods. The activity of caspase enzymes was measured by spectrophotometry. Corosolic acid significantly inhibited the proliferation of both cell lines compared with controls. This agent markedly induced apoptosis in MDA-MB-231 cells but did not affect MCF7 cells compared with controls. Treating the MADA-MB-231 and MCF7 cell lines with corosolic acid showed an inducing effect on apoptosis-associated caspases, including Caspase-8, 9, and -3, in MADA-MB-231 cells with no effect on apoptotic markers in MCF7 cells. Further experiments uncovered corosolic acid-induced apoptosis in MADA-MB-231 cells by decreasing the expression of the phosphorylated form of JAK2 and STAT3 proteins.The present data suggested that corosolic acid is an apoptosis-inducing phytochemical in triple-negative breast cancer MADA-MB-231 cells. Also, corosolic acid triggered apoptosis in these cells by stimulating both pathways of apoptosis and inhibiting the JAK/STAT signaling. Furthermore, corosolic acid was found to inhibit MCF7 cell proliferation by a non-apoptotic mechanism. Breast cancer is the most prevalent type of cancer among females and ranks as the second cause of cancer-dependent death in women worldwide . Breast Lagerstroemiaspeciosa with antiinflammatory, antiobesity, antidiabetic, antihyperlipidemic, anti-cancer, and antiviral activities . Both MCF7 and MDA-MB-231 cell lines were seeded at the density of 2×104 cells/well into 96-well cell culture plates. Subsequently, the seeded cells were treated with different concentrations of corosolic acid  for 24, 48, and 72 hr.Both MCF7 and MDA-MB-231 cell lines were purchased from National Center for Cell Science . The cells were then cultured and incubated in a humidified incubator at 37 Cell viability After incubation of the cell lines with increasing concentrations of corosolic acid, the viability of the cells was quantified using Vybrant 3--2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit  according to the manufacturer’s instructions. Briefly, the cell lines were incubated with 5 mg/ml MTT solution for 4 hr to evaluate the influence of corosolic acid on their viability. Subsequently, the MTT solution was removed and 200 µl of dimethyl sulfoxide (DMSO) solution was added to each well. After 15 min, the quantity of formazan product was detected by measuring the absorbance at 570 and 630 nm using a plate reader spectrophotometer .Real-time PCR-ΔΔCt method and the GAPDH transcript was used as the housekeeping gene. The primer sequences used in the present study are shown in The primary primer sequences were extracted from the NCBI database and the specific primers were designed by using Primer Express software v1.5 (Applied Biosystems). The total RNA content of both cell lines was extracted using an RNeasy mini kit . To synthesize the complementary DNA (cDNA) from 1 µg/ml of single-stranded RNA, the oligo-d (T) 15 primer  and M-MLV reverse transcriptase were used. Real-time PCR analyses were carried out on cDNA samples by using SYBR-green master mix  on ABI PRISM 7900HT  under thermocycling conditions. Furthermore, melt curve analysis was carried out for each gene to prove the specificity of the primers and the lack of non-specific products. The expression levels of all genes were analyzed using the 2Flow cytometry°C) for 5 min, washed once with 5 ml of a phosphate-buffered saline (PBS), and resuspended in the binding buffer. Afterward, 5 µl of PI and 5 µl of FITC-annexin V were added to the cell suspension and incubated in the dark for 15 min at room temperature. Analysis of FITC-annexin V binding was carried out on a FAC scan flow cytometer (BD Biosciences) with an excitation wavelength of 488 nm and an emission wavelength of 350 nm. PI-negative/Annexin V-positive cells were presented as early apoptotic cells and the cells with positive PI and Annexin V were reported as late apoptotic cells. The effect of corosolic acid on apoptosis was studied using a FITC/annexin-V-propidium iodide (PI) kit  according to the manufacturer’s protocol. Briefly, MCF7 and MDA-MB-231 cells were treated with corosolic acid at concentrations equal to IC50 and IC25 for 48 hr. Then, the treated cells were centrifuged (1000 g) at room temperature  to prepare the whole-cell lysates. The lysates were then centrifuged and the supernatants were utilized for Western blot analysis.To measure the expression of proteins by Western blotting technique, both MCF7 and MDA-MB-231 cell lines were seeded in 6-well culture plates at the density of 2×10°C. All membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody in the dark for 1 hr at room temperature. All antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Finally, the immunoblots were developed by using an enhanced chemiluminescent kit  followed by exposure to autoradiography film.A bicinchoninic acid (BCA) assay kit  was used to estimate the total protein content of the samples. 40 µg of the total protein was loaded onto 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane  through electroblotting. The membranes were blocked with 5% non-fat milk for 1 hr at room temperature. Subsequently, the membranes were exposed to Caspase-8, Caspase-9, Caspase-3, Bax, Bcl-2, STAT3, JAK2, Phosphorylated STAT3, Phosphorylated JAK2, and GAPDH primary antibodies overnight at 4 Measurement of the activity of caspase enzymes°C. Subsequently, 200 μg of the total protein content of the cell lysate was mixed with the assay buffer  to a final volume of 100 μl. Then, 20 μl of each substrate , ,  was added to the mixture. The reaction mixtures were incubated for 30 min at 37 °C and the absorbance of the enzyme products was measured at 405 nm using an Epoch microplate spectrophotometer.To measure the activity of Caspase-8, -3, and -9, both MCF7 and MDA-MB-231 cell lines were lysed in the lysis buffer  with vortexing for 30 min at 4 Statistical analysispost hoc test. These statistical analyses were done using SPSS software version 19 . The statistical difference between the data was considered significant at P<0.05.All experiments were repeated at least three times and the obtained data were expressed as mean±standard deviation (SD). The results were analyzed using the one-way analysis of variance (ANOVA) test followed by Duncan’s multiple range tests as the Effect of corosolic acid on the viability of MDA-MB-231 and MCF7 cell linesThe results of the MTT assay revealed that treating both MDA-MB-231 and MCF7 cell lines with increasing concentrations of corosolic acid for 48 and 72 hr could mitigate the proliferation of these breast cancer cell lines in a dose-dependent manner compared with the untreated control cells. Corosolic acid treatment for 24 hr had no significant effects on either cancer cell line  and 2A. Effects ofcorosolic acid on apoptosis induction in MDA-MB-231 and MCF7 cell linesTo evaluate the apoptosis-inducing activity of corosolic acid on MDA-MB-231 and MCF7 cell lines, the cells were treated with IC25 and IC50 concentrations of this natural compound. In the MDA-MB-231 cell line, a marked increase in the late and early stages of apoptosis was observed after treatment of the cell line with 10 μM (IC25) and 20 μM (IC50) of corosolic acid for 48 hr . CorosolEffects of corosolic acid on caspase enzyme activity in MDA-MB-231 and MCF7 cell linesAfter corosolic acid treatment, the activity of three key caspase enzymes, Caspase-8, -3, and -9 was measured in MDA-MB-231 and MCF7 cell lines to evaluate the activation and function of these enzymes in the presence of this compound. As depicted in Effects of corosolic acid on expression of apoptosis-related factorsin MDA-MB-231 cell lineTo assess the possible effect of corosolic acid on key factors involved in the apoptosis of MDA-MB-231 cells, the expression levels of some apoptosis-related genes and proteins were measured. The results of quantitative real time-PCR analyses provided evidence to show that corosolic acid, at concentrations equal to IC25 and IC50 for 48 hr treatment, considerably elevated the transcript levels of CASP-8, -9, and -3 in the MDA-MB-231 cell line in comparison with untreated control cells. However, it had no significant effect on the expression levels of JAK2 and STAT3 genes compared with the controls . The resEffects of corosolic acid on expression of apoptosis-related factorsin MCF7 cell lineTo unravel the potential effect of corosolic acid on apoptosis markers in MCF7 cells, the gene and protein expression of these markers were quantified using real time-PCR and western blotting. The results of gene expression analysis uncovered that corosolic acid treatment of MCF7 cells with IC25 (14 µM) and IC50 (28 µM) concentrations of this agent for 48 hr did not alter the transcript expression of CASP-8, -9, and -3 as well as BAX and BCL-2 as compared with the untreated control cell line . The resAs described before, TNBC is the most aggressive type of breast cancer and develops resistance to treatment methods due to the absence of ER, PR, and HER-2 receptors . AlthougIn the first step, MDA-MB-231 and MCF7 cell lines were treated with increasing concentrations of corosolic acid for 24, 48, and 72 hr. Corosolic acid treatment of both MDA-MB-231 and MCF7 cell lines led to a significant decrease in the viability of the cells in a dose- and time-dependent manner. Of note, this triterpenoid hindered the MDA-MB-231 cell proliferation rate at lower concentrations  than those in the MCF7 cell line . This suggests that the TNBC cell line, MDA-MB-231, is more sensitive to corosolic acid than the PR- and ER-positive MCF7 cell line. Additionally, it can be implied that the anti-breast cancer effect of corosolic acid is not necessarily receptor-dependent. In the second step, we hypothesized that the antiproliferative activity of corosolic acid may arise from its effect on the induction of apoptosis in both cell lines. Hence, the cell lines were treated with IC50 and IC25 concentrations of corosolic acid. The results clarified that corosolic acid considerably augmented both early and late apoptotic cell populations of the MDA-MB-231 cell line, however, it showed no remarkable pro-apoptotic activity against MCF7 cells. To affirm the role of the key caspase enzymes in this effect, the cell lines were exposed to IC50 and IC25 concentrations of corosolic acid, and then the activity of Caspase-8, -9, and -3 was measured. The obtained findings confirmed the elevated activity of all three caspases in the MDA-MB-231 cell line due to corosolic acid treatment. However, the compound did not affect the activity of the enzymes in the MCF7 cell line. The process of apoptosis functions through two main pathways that include mitochondria-dependent (intrinsic) and death receptor-mediated (extrinsic) pathways. The former pathway initiates apoptosis via activating Caspase-9 and the latter induces apoptosis by stimulating Caspase-8. These activated caspases trigger the activation of Caspase-3, as the executioner in apoptosis . AccordiIn the third step, the effect of corosolic acid treatment on the expression levels of some apoptosis-related genes and proteins was quantified in MDA-MB-231 and MCF7 cell lines. The results of real-time PCR and western blotting methods evidenced that corosolic acid had no marked effect on the expression levels of Caspase-8, -9, -3, Bax, and Bcl-2 at either mRNA or protein levels. However, treating the MDA-MB-231 cell line with IC50 and IC25 concentrations of corosolic acid significantly amplified the levels of Caspase-8, -9, and -3 in comparison with untreated control cells. et al. identified that corosolic acid decreased the proliferation and increased apoptosis of MDA-MB-231 cells in a dose-dependent manner. They revealed that the apoptosis-inducing effect of corosolic acid on MDA-MB-231 cells was mediated by a mechanism that involves reactive oxygen species (ROS)  activity. However, they did not study the apoptosis-inducing activity of corosolic acid in this cell line . Son et es (ROS) . However pathway . They me pathway . Sung an pathway . However pathway -29. It set al. demonstrated that corosolic acid suppressed the proliferation of glioblastoma cells and tumor-associated macrophages by blocking STAT3 and NF-κB in these cells (An increasing body of evidence in the literature shows that targeting the JAK/STAT signaling pathway in TNBC cell lines is a novel and efficient approach to stimulating apoptotic cell death in these cancer cells -32. Consse cells . It has se cells . CorosolNatural products that target both pathways of apoptosis are the most competent anti-cancer drugs because they prevent drug resistance that arises from the cross-talks between the signaling pathways in the cancer cells . Therefoin vitro investigation of the effects of corosolic acid on breast cancer cell lines. Thus, future works should focus on the development of in vivo models of breast cancer using both cancer cell lines. Then, the present factors can be evaluated in those animal models to establish the present results.Although the present study has valuable data and offers beneficial contributions to current literature, we note some limitations of our research and suggest some extra evaluations for future studies. As indicated in the title of the manuscript, the present study is an KH, ATJ, and SZ designed the experiments and methodology. SAJ, OZK, RSZ, QAQ, and YFM performed the experiments and collected the data. SHA, CMW, YABJ, and MSM were involved in data analysis and manuscript writing. KH edited the manuscript. The authors have no conflicts of interest to declare.


The optimal radiobiological model, which assesses the biological effects of novel radiotherapy techniques that concurrently modify multiple physical factors, has not yet been defined. This study aimed to investigate the impact of intensity‐modulated radiotherapy (IMRT) and volumetric‐modulated arc therapy (VMAT) on cellular response in head and neck cancer and melanoma models.Clonogenic analysis, DNA double‐strand break analysis, apoptosis, and cell cycle analysis were performed on cancer stem cell models, cancer models, and normal tissue cell models to assess radiation sensitivity.The segmented radiation approach used in IMRT applications enhanced radiosensitivity and cytotoxicity in the cancer models, while changes in dose rate had varying effects on cytotoxicity depending on the tumor cell type. VMAT increased cellular resistance, favoring treatment outcomes.The biological processes were influenced differently by dose rate, IMRT, and VMAT depending on the tumor cell type. The selection of the most appropriate technique is crucial in representing new radiotherapy approaches. The obtained data can serve as a model to address clinical questions in daily practice. The integration of non‐standard outcomes with standard applications should be considered in clinical settings. Among the primary treatment modalities for cancer patients, radiotherapy plays a vital role.The main objective of this study was to develop a radiobiology model that accurately reproduces the biological effects of advanced radiation procedures. Previous literature has identified the lack of a suitable model for these state‐of‐the‐art techniques. Therefore, our investigation focused on assessing the cellular response to complex dose distributions generated using the IMRT approach, considering various physical factors. Our ultimate goal was to establish a model that closely reflects the daily applications of these techniques.In this study, we conducted a comprehensive examination of the impact of parameters commonly used in everyday clinical practice, utilizing novel technological devices, on in vitro cell survival. To simulate real‐life scenarios, we employed three distinct cell models: two tumor cell lines known for their robust radioresistance properties, tumor stem cell lines recognized as the primary contributors to radiotherapy resistance, and normal cell lines susceptible to the effects of radiotherapy. After establishing appropriate dose ranges, we meticulously investigated the effects of dose, dose rate, and non‐uniform dose distribution on cellular survival using various assays, including clonogenic assay, DNA double‐strand break assay, apoptosis assay, cell cycle assay, and autophagy assay. Additionally, we explored the in vitro effects of volumetric‐modulated arc therapy (VMAT) application using a phantom that closely mimics daily clinical applications. Through this comprehensive study, our aim was to address the existing gap in the literature by developing an appropriate radiobiology model for advanced radiation techniques. The findings from our research contribute to a better understanding of the cellular response to complex dose distributions and have significant implications for improving treatment planning and enhancing patient outcomes in clinical practice.22.13), it provided a complete scattering medium with varying density levels for irradiating the flasks  was used to verify the calculated monitor unit (MU) values, ensuring that the dose values fell within ±3% of the planned values. Additionally, Gafchromic EBT3 films were placed at the bottom of each cell flask during irradiation to confirm the targeted doses using a secondary dosimetry method. The dose values obtained from the Gafchromic films also confirmed that the doses delivered to the flasks were within ±5%. All analyses were performed in triplicate.A cylindrical water phantom was specifically designed for the VMAT irradiation technique to accommodate T25 cell culture flasks, allowing for the simultaneous irradiation of two flasks. The phantom was designed to maintain physical conditions similar to the VMAT technique, ensuring reproducible irradiation setups. To mimic different density levels of the human body and create a scenario similar to daily applications, materials with four different densities were placed inside the phantom. When the phantom was filled with water  and human head and neck CSC line  were utilized as CSC models. Additionally, a melanoma cell line (NM2C5/RRID:CVCL_B064) and head and neck cancer cell lines (A253−/RRID:CVCL_1060 and FADU/RRID:CVCL_1218) were established as cancer cell models. Fibroblast cell line (WI‐38/RRID:CVCL_0579) and epithelial cell line (WSS‐1/RRID:CVCL_2767) were employed as control models representing normal tissue cells. All cell lines were cultured in a standard cell culture incubator at 37°C, under a 5% CO2.2.2All cancer cell models and control cells were exposed to irradiation at doses of 2, 4, 6, 8, and 10 Gy for clonogenic assays. The untreated group consisted of 100 cells, while the 2 Gy treatment group had 200 cells, the 4 Gy treatment group had 600 cells, the 6 Gy treatment group had 1000 cells, the 8 Gy treatment group had 1200 cells, and the 10 Gy treatment group had 1400 cells. After 3 weeks of irradiation, colonies containing at least 50 cells (>1 mm) were evaluated in the clonogenic assay. All analyses were conducted in triplicate. Statistical analyses were performed using the SPSS program with the following procedures. For the quadratic term, “D2” was defined as the product of the dose and dose (D2 = dose × dose). For survival analysis, the variable “S” was calculated as the natural logarithm of the ratio of colonies to cells (ln(colonies/cells)) minus the natural logarithm of the plating efficiency (ln(PE)). The weight of observed colonies was calculated using the formula “W” = colony × cells/(cells−colony). Linear regression analysis was conducted using the SPSS software . The dependent variable “S” was used, while the independent variables “dose,” “D2,” and “W” (under WLS in SPSS) were included. The analysis included confidence intervals and R‐squared values (Statistics in SPSS). Since the regression line passes through the origin, the option to include a constant in the equation (Options in SPSS) was not selected.2.2.34 cells/100 μL. The cells were then exposed to various doses of ionizing radiation and incubated for 24, 48, and 72 h. Cell proliferation was assessed using the WST‐1 assay method, which involved adding the WST‐1 Cell Proliferation Reagent to each well, followed by measuring the absorbance at 450‐nm and 620‐nm wavelengths using a microplate reader. The effects of irradiation on cell proliferation were quantified by analyzing the dose–response curve using Sigmoidal dose–response curve analysis in the Calcusyn 2.0 software.Cells were seeded in 96‐well plates at an initial density of 1 × 102.2.45 cells/mL and exposed to radiation doses ranging from 0 to 10 Gy. Annexin V (Annexin V‐EGFP Apoptosis Detection Kit‐Biovision) and Mitoprobe JC‐1 (MitoScreen JC‐1 kit‐BD Biosciences) methods were employed to study apoptosis in three replicates, following the manufacturer's instructions. The untreated group served as the control group. Flow cytometry analysis was conducted using the BD Acuri 6 system to evaluate the results.Cell lines were seeded in six microplates at a concentration of 5 × 102.2.55 cells/mL in six microplates. The analysis was performed at the 48th hour, a time point at which significant DNA double‐strand breaks could be detected. Flow cytometry was employed for evaluation.The impact of varying radiation doses on DNA double‐strand breaks in cancer models was examined using γ‐H2AX analysis (Alexa Fluor 488 Mouse anti‐H2AX kit). Cell lines exposed to radiation doses ranging from 0 to 10 Gy were seeded at a concentration of 5 × 102.2.6Cell lines were seeded at a concentration of 5 × 105 cells/mL in six microplates and exposed to radiation doses ranging from 0 to 10 Gy. The cell cycle distribution in cancer cells was investigated using cell cycle analysis (BD Cycletest Plus kit) at 24, 48, and 72 h after radiation exposure. Propidium iodide (PI) bound to DNA was quantified using flow cytometry with 488‐nm excitation and 586/42 bandpass emission filters. The FACSuite software was used to determine the number of cells in the sub G0/G1, G0/G1, S, G2/M, and >4n stages, and the percentages were calculated accordingly.2.2.7p < 0.05.Descriptive statistics were employed for data analysis. Nonlinear regression analysis based on the linear‐quadratic model was used to investigate the relationship between radiation doses and the survival fraction of cells. The same analysis was conducted to assess the impact on cell survival response using the phantom. A one‐way analysis of variance (ANOVA) was performed to compare the effects of different dose rates after 4 Gy irradiation. Apart from the aforementioned statistical analyses, all other statistical analyses were carried out using GraphPad Prism 9.5. The significance level was set at 33.13.1.1R2 = 0.978, p = 0.011, Figure In the head and neck cancer model, the coating efficiency was 17.20% in the A253 cell line and 40.33% in the FADU cell line. The melanoma cancer model (NM2C5 cell line) exhibited a coating efficiency of 36.33%. A significant correlation was observed between the dose and survival  head and neck CSC line was 99.67%. In contrast, the coating efficiency in the human  melanoma CSC line was 66.33%. A significant correlation was observed between the dose and survival . The head and neck cancer model demonstrated higher radiosensitivity compared to the melanoma model. The WSS‐1 epithelial cell line and WI‐38 fibroblast cell line were chosen to represent early and late responding normal tissues in radiobiological modeling due to their distinct behavioral traits, accurately reflecting the characteristics of normal tissues. Notably, CSC models displayed different radiosensitivity properties in comparison with tumor cell models . Interestingly, apoptosis was not observed in the NM2C5 cell line when exposed to radiation doses of 2–10 Gy from 24th to 72nd hours. In the human (parent) head and neck CSC line, apoptosis was triggered at 2–10 Gy after 48th hours, and in the human  melanoma CSC line, apoptosis occurred at 6–10 Gy after 72nd hours, both compared to the control group. Additionally, in the WI‐38 cell line, apoptosis was induced at 2–10 Gy after 24th hours, while in the WSS‐1 cell line, apoptosis was induced at 8 and 10 Gy from 24th to 72nd hours compared to the control group  head and neck CSC line, and at 6–8 Gy at 72nd hours in the human  melanoma CSC line compared to the control group. Consistent with the annexin V results, compared to the control group, apoptosis was induced at 2–10 Gy at 24th hours in the WI‐38 fibroblast cell line and induced at 6–10 Gy at 24th and 72nd hours in the WSS‐1 cell line  head and neck CSC line at doses of 2–10 Gy and in the human  melanoma CSC line at doses of 4–10 Gy. Interestingly, DNA double‐strand breaks were not detected in the WI‐38 and WSS‐1 cell lines  melanoma CSC line and the human (parent) head and neck CSC line exhibited cell cycle arrest at the G2/M stage at doses of 2–10 Gy after 24 and 72 h. No significant difference was found in the cell cycle of the WI‐38 cell line compared to the control group at doses of 2–10 Gy after 24 and 72 h. The WSS‐1 cell line showed differences in the G0/G1, S, and G2/M phases of the cell cycle compared to the control group at doses of 2–10 Gy after 24 and 72 h.3.22 flasks. A comparison of cell survival responses between non‐uniform and uniform areas was conducted using the clonogenic assay method.The effective dose of 4 Gy was determined for the cancer cell models, and the cancer cells were exposed to this dosage in partially and fully open 25 cm3.2.1t = −0.378, p = 0.742). Similarly, in the FADU cell line, the coating effectiveness and survival rates were 2.89% and 57.78% following fractionated 4 Gy treatment, and 2.11% and 42.22% after non‐fractionated 4 Gy exposure. Again, the difference was not significant . In the NM2C5 cell line, the coating effectiveness and survival rates after fractionated 4 Gy exposure were 0.87% and 7.74%, respectively, while they were 3.4% and 30.36% after non‐fractionated 4 Gy exposure. The difference was significant . This indicated a 22.62% decrease in survival following fractional irradiation in the melanoma cell line NM2C5. In summary, it was observed that the fractional irradiation technique improved cytotoxicity in the melanoma cell model, but it did not produce a significant difference in the head and neck tumor models . In the human  melanoma CSC line, no colonies were detected after fractionated 4 Gy treatment, resulting in a 0% survival rate. After non‐fractionated 4 Gy treatment, the coating efficiency and survival rates were 35.00% and 38.18%, respectively. The difference was highly significant . There was a 38.18% reduction in survival due to fractional irradiation in the melanoma stem cell line. Overall, it was observed that fractional irradiation increased cytotoxicity in the melanoma CSC model, while the survival effect of gradual irradiation was not observed in the CSC models, similar to the tumor cell models  head and neck CSC line, the coating efficiency and survival rates were 70.42% and 83.17% after fractionated 4 Gy, and 68.08% and 80.41% after non‐fractionated 4 Gy, respectively. However, the difference was not significant . In the WSS‐1 cell line, the coating efficacy and survival rates were 2.67% and 11.11%, respectively, after fractionated 4 Gy exposure, and 3.67% and 15.28% after non‐fractionated 4 Gy exposure. However, the difference was not significant . Overall analysis of the data showed that fractional irradiation in the WI‐38 cell line resulted in a 27.35% reduction in survival. It was observed that the more radioresistant normal tissue cells (WI‐38) exhibited a higher level of cytotoxicity, similar to what was seen in the melanoma tumor and CSC model , 300 MU/min (standard irradiation), or 600 MU/min (high‐dose rate) in 25 cm3.3.1t = 0.281, p = 0.805). Similarly, in the FADU cell line, the survival rates at 4 Gy were 45.2% with a dose rate of 50 MU/min, 42.23% with a dose rate of 300 MU/min, and 34.32% with a dose rate of 600 MU/min. Again, the difference was not significant . In the melanoma cancer model, the survival rates after treatment at 4 Gy were 13.15% with a dose rate of 50 MU/min, 22.02% with a dose rate of 300 MU/min, and 51.02% with a dose rate of 600 MU/min. The difference was significant . It was observed that cytotoxicity decreased as the dose rate increased. When considering all the data from the tumor cell models, it was found that the cytotoxicity of head and neck tumor models increased with increasing dose rate, while the cytotoxicity of melanoma cell models decreased with increasing dose rate . Overall, the findings indicate that compared to standard dose‐rate treatments, cell survival decreased at high‐dose rates in head and neck stem cells, as expected. It was observed that low‐dose rates had a more negative effect on survival in melanoma stem cells compared to standard dose‐rate irradiation  head and neck CSC line was 28.28% at a dose rate of 50 MU/min, 42.91% at a dose rate of 300 MU/min, and 33.24% at a dose rate of 600 MU/min. However, the difference was not significant. In the human  melanoma CSC line, the survival rate after treatment at 4 Gy was 29.24% with a dose rate of 50 MU/min, 65.30% with a dose rate of 300 MU/min, and 43.33% with a dose rate of 600 MU/min. The difference was significant . In the FADU cell line, there was a 38.67% increase in survival after 2 Gy irradiation. However, this difference was no longer present at 4 Gy, and no significant difference in viability was detected using the VMAT technique (p = 0.06). In the melanoma cancer model, there was an 8.21% decrease in survival after 2 Gy irradiation compared to the control group. However, there was a 32.17% improvement in survival following high‐dose 6 Gy irradiation compared to the control group. In the NM2C5 cell line, significant differences in vitality were observed compared to the control group at 2 Gy, 6 Gy, and 8 Gy (p < 0.0001). Radiosensitivity in epithelial cell lines increased with the use of VMAT at 2 Gy, while radiosensitivity in the melanoma cell model increased at low doses and decreased considerably at doses above 4 Gy . In the melanoma CSC line, there was an 81.87% decrease in survival at 2 Gy compared to normal cells. Radiosensitivity was observed to increase at higher dosages (p < 0.001), and it was also observed to increase at 2 Gy with the use of VMAT technique. Furthermore, the VMAT technique in the phantom was observed to enhance radiosensitivity and viability in the head and neck stem cell model, similar to the effects observed in tumor cell models. In melanoma stem cell lines, the VMAT technique exerted different effects at low and high doses  head and neck CSC line, with a 40.76% decrease in survival observed after 2 Gy irradiation. This decrease in survival was found to be significant compared to the control group . On the contrary, the WI‐38 cell line exhibited a 62.02% increase in survival compared to the control group after 2 Gy irradiation, and this difference was found to be significant (p < 0.0001). When the results of normal tissue cell models were analyzed collectively, it was observed that the VMAT technique reduced radiosensitivity in normal tissue cells, unlike in tumor and CSCs , normal tissues (lung epithelium and fibroblast cells), and CSC models (head and neck and melanoma CSCs). Upon reviewing the collective results, it was observed that the radiosensitivity differed among the various tumor cells, normal tissue cells, and CSCs. Notably, the radiosensitivity of CSC models was found to be lower compared to tumor cell models. CSCs have been shown to exhibit increased resistance to radiation compared to non‐CSC cells, and their presence is believed to contribute to treatment failure and tumor recurrence.4.2The multi‐leaf collimator (MLC) system is employed in daily radiotherapy applications to deliver radiation to the target at different intensities and at different times, enabling the modification of dose intensity in both tumors and normal tissues and achieving desired dose distributions. This technique, known as IMRT, involves delivering the radiation beam as a cumulative sum of segmented irradiation to the tumor and surrounding normal tissues throughout the treatment course. Interplay between intercellular signaling systems and this segmented irradiation could influence the cellular response, potentially leading to either an increase or decrease in radiation sensitivity.The application of IMRT has provided valuable insights into its physical characteristics, such as its ability to deliver high doses to the target while minimizing radiation exposure to nearby critical organs. This capability has enabled the successful treatment of patients with complex‐shaped tumors, surpassing the limitations of traditional radiation therapy. Nevertheless, it should be noted that most IMRT techniques involve the delivery of treatment fractions over extended time periods and at lower dose rates. The radiobiological implications of reduced dose rates and fractionated irradiation techniques are not yet fully elucidated, and further research is needed to comprehensively understand their impact on treatment outcomes.In our study, we investigated the effects of two‐dimensional fractionated irradiation techniques, representing both uniform and non‐uniform irradiation patterns, on the survival curve's 4 Gy shoulder region as the test dose. Following the completion of cell clonogenic survival studies, the findings revealed that the fractionated irradiation approach resulted in increased radio‐cytotoxicity and radiosensitivity in both melanoma and melanoma stem cell models. To the best of our knowledge, this is the first study to demonstrate that segmental irradiation enhances the radio‐cytotoxicity of melanoma cells. While long‐term therapy involving segmental irradiation has been reported to decrease radiosensitivity in various tumor types, it is noteworthy that our study observed an increase in radiosensitivity.4.3The biological response to radiation is influenced by factors such as radiation energy, dose, and dose rate.In this study, the efficacy of dose rate was evaluated in CSC models, marking the first instance in the literature. Interestingly, it was observed that lower dose rates had a more detrimental impact on survival compared to standard dose‐rate irradiations. It is advisable to consider these variances for therapeutic advantages in clinical practice and verify them through appropriate clinical protocols.4.4In this paper, we employed a cylindrical water phantom that is compatible with a reproducible irradiation system to investigate the impact of complex dose distributions obtained through IMRT techniques on cellular response. This system created physical conditions that are in line with the VMAT technique. However, determining the optimal experimental design that closely resembles daily clinical practice is not straightforward. Various researchers have proposed solid phantom models in the literature for this purpose.In volumetric irradiation techniques such as the VMAT technique, high doses are physically delivered to the tumor tissue, resulting in sharp dose gradients in normal tissues. While a high dose is maintained within the tumor, reduced dose regions are applied to normal tissues. One of the crucial radiobiological considerations associated with these new physical parameters is that irradiation can cause side effects in normal tissues beyond the expected levels. Dose constraints for normal tissues have been established based on clinical observations obtained from three‐dimensional radiotherapy applications.In conclusion, the findings of this study need to be validated through in vivo testing before incorporating them into the current treatment regimens of clinical practice. It is essential to consider the physical and radiobiological aspects of applying stereotactic radiotherapy, especially for melanoma cells, which involves modifying the dose rate and segmental irradiation at high rates. By doing so, we can better understand and manage the potential side effects in normal tissues and optimize treatment outcomes for patients.Serra Kamer: Funding acquisition (lead); investigation ; methodology ; resources ; supervision ; validation ; writing – review and editing (supporting). Sunde Yilmaz Susluer: Methodology ; validation ; writing – original draft (lead); writing – review and editing (lead). Tugce Balci Okcanoglu: Methodology ; validation . Cagla Kayabasi: Methodology ; validation . Besra Ozmen Yelken: Methodology ; validation . Sinan Hoca: Investigation ; methodology ; visualization . Emin Tavlayan: Investigation ; methodology ; visualization . Nezahat Olacak: Conceptualization ; investigation . Yavuz Anacak: Conceptualization ; supervision . Murat Olukman: Conceptualization ; supervision . Cumhur Gunduz: Investigation ; methodology ; supervision ; validation .The authors have no conflicts of interest to declare.No ethical approval statement required.


Being the standard-of-care for four decades, cisplatin-based chemotherapy is highly efficient in treating germ cell tumors (GCT). However, often refractory patients present with a remaining (resistant) yolk-sac tumor (YST(-R)) component, resulting in poor prognosis due to lack of novel treatment options besides chemotherapy and surgery. The aim of this study was to identify novel targets for the treatment of YST by deciphering the molecular mechanisms of therapy resistance. Additionally, we screened the cytotoxic efficacy of a novel antibody-drug-conjugate targeting CLDN6 (CLDN6-ADC), as well as pharmacological inhibitors to target specifically YST.Protein and mRNA levels of putative targets were measured by flow cytometry, immunohistochemical stainings, mass spectrometry of formalin-fixed paraffin-embedded tissues, phospho-kinase arrays, or qRT-PCR. Cell viability, apoptosis and cell cycle assays of GCT and non-cancerous cells were performed using XTT cell viability assays or Annexin V / propidium iodide flow cytometry, respectively. Druggable genomic alterations of YST(-R) tissues were identified by the TrueSight Oncology 500 assay.+ GCT cells in comparison with non-cancerous controls. In a cell line-dependent manner, either an accumulation in the G2 / M cell cycle phase or a mitotic catastrophe was observed. Based on mutational and proteome profiling, this study identified drugs targeting the FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling pathways as promising approaches to target YST. Further, we identified factors relevant for MAPK signaling, translational initiation and RNA binding, extracellular matrix-related processes as well as oxidative stress and immune response to be involved in therapy resistance.We demonstrated that treatment with a CLDN6-ADC enhanced apoptosis induction specifically in CLDN6In summary, this study offers a novel CLDN6-ADC to target GCT. Additionally, this study presents novel pharmacological inhibitors blocking FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling for the treatment of (refractory) YST patients. Finally, this study shed light on the mechanisms of therapy resistance in YST.The online version contains supplementary material available at 10.1186/s10020-023-00636-3. Germ cell tumors (GCT) are the most common solid tumors among young men aged between 17 and 45 years  expression profiles , high MDM2 levels, and phosphorylation-dependent translocation of p21 from the nucleus to the cytoplasm  YST to further increase therapy efficacy.The tetraspanin membrane protein CLDN6 has been identified as a cancer-associated cell surface biomarker, which is rarely expressed in healthy adult tissues   against CLDN6 to examine the cytotoxicity and specificity to target GCT.Beyond that, since specifically YST cells represent the remaining non-responsive component in refractory GCT, we further aimed at deciphering the molecular mechanisms resulting in the formation of cisplatin-resistant YST. This approach was chosen for the identification of putative multikinase inhibitor-based therapeutic options, which are already in clinical trials and / or approved for the treatment of other tumor entities.FOXA2, SOX17, APOA1 / A2 / B, ALB, FGA / B / G, and GATA3 / 4 / 6 . Additionally, we found similarities in signaling pathway activities when comparing YST and HCC   regarding expression of endodermal factors like https://imagej.nih.gov/ij/)  cell lines were cultured as described previously and summarized in Additional file 2B antibody  has been conjugated to MMAE via the drug-linker OSu-Glu-vc-PAB (CLDN6-Glu-vc-PAB-MMAE) by Levena Biopharma , resulting in an antibody-drug-ratio of at least 1 : 3.Anti-human CLDN6 monoclonal mouse IgGDNA and RNA were extracted from tumor enriched 2 × 5 µm formalin-fixed, paraffin-embedded (FFPE) slices using the ˋInnuPREP FFPE DNA Kit´ on the ˋInnuPure C16 System´  or the ˋMaxwell RNA extraction kit´  according to manufacturer’s recommendations, respectively.DNA libraries were prepared using the hybrid capture-based ˋTruSight Oncology 500 Library Preparation Kit´  following ˋIllumina’s TruSight Oncology 500 Reference Guide´  and sequenced on an ˋIllumina NextSeq 500´ instrument. FastQ files were analyzed using ‘CLC Genomics Workbench’ . The reads were mapped on hg19 followed by an initial variant calling.For sample preparation, a modified FFPE tissue lysis protocol of Ikeda et al. was applied   for principal component analyses (PCA) (Gerst and Hölzer https://david.ncifcrf.gov)    was used to generate dot plots  has been used to screen for the absorption, distribution, metabolism, excretion and toxicity features of tested drugs  GCT cohort was analyzed using cBioportal  derived from SEM (TCam-2), EC , CC , and an EC-YST-intermediate (1411H) , which were comparable to those of non-cancerous control cells   and induced apoptosis in most CLDN6+ GCT(-R) cells  in comparison to the monoclonal antibody alone  , 2102EP, NCCIT(-R), JAR, JEG-3(-R), 1411H), but not in CLDN6− cells , BRCA2 (c.7397T > C), IL7R , and SPTA1 (c.5077A > C) were observed in all YST-R samples. Furthermore, CHEK1, FGF6, FGF23, and KRAS were amplified, but with a low fold change (max 2.2), and SNVs were detected in FGFR4 (c.1162G > A), KMT2A (c.10841T > C), NTRK1 , and TSC2  in at least 50 % of the evaluated samples  tissues n = 6) were performed to identify druggable genomic alterations. We detected a mean of 3.2 mut / Mb (0.8 - 5.6 mut / Mb) in the YST-R samples, though, the tumor mutational burden (TMB) did not correlate to the microsatellite instability score ) , 1411H) has been performed to identify signaling molecules and putative YST-specific targets, which were not present in non-cancerous control cells (MPAF)  were noted in ABL1, FGFR1 / 4, FLT4, MTOR, PARP, PDK1, RET, while CSK, EGFR, ERBB2, and RAF1 harbored missense mutations , Nintedanib FGFR1 - 3), AZD7762, MK-8776 (both CHEK1), and Rapamycin (mTOR) as potential inhibitory drugs to target YST(-R) , Nintedanib , Sorafenib , Talazoparib (PARP1 / 2), OSU-03012 (PDK1), AZD4547 (FGFR1 - 3), AZD7762 (CHEK1), or Rapamycin (mTOR) resulted in LDOf the four tested YST-like cell lines, the GCT72 and 1411H showed the highest apoptosis induction under most conditions, while the female NOY-1(-R) were the least sensitive YST-like cells Fig. E. Induct2 = 0.95)  and p53 S15, S46) were seen in GCT72-R cells, while activity of ERK1 / 2 , FGR (Y412), GSK3α / β (S21 / S9), p38α (T180 / Y182), p53 (S392), PDGFRβ (Y751), SRC (Y419), STAT5a / b (Y694 / Y699), WNK1 (T60), and YES (Y426) was reduced in the resistant subline  cells  inhibitors or an ADC targeting CLDN6.Additional file 1: Figure S1. A) Mutational status and mRNA expression profile of CLDN6 in the GCT TCGA (The Cancer Genome Atlas) cohort as analyzed through https://www.cbioportal.org/. B) XTT cell viability assays of CLDN6 antibody, CLDN6-ADC, or MMAE treated GCT cell lines and non-cancerous fibroblasts (MPAF) (24  - 96 h). C) Raw flow cytometry data indicating cell cycle distributions of GCT cell lines and MPAF treated with either CLDN6 antibody alone (red), CLDN6-ADC (green), or MMAE (blue) in comparison with untreated controls (grey). D) Immunohistochemical evaluation of CLDN6 in YST-R tissues (n = 10).Additional file 2: Figure S2. A) Raw human phospho-kinase array of various cell lysates . Corresponding membrane layout with the most prominent dots (marked in red) was used for quantification using ImageJ as shown in Fig. ACTB and GAPDH were used as housekeeping genes. C) Raw data and D) densitometric evaluation of GCT72(-R) cells treated with AZD7762, Danusertib, Nintedanib, OSU-03012, or SNS-314  in comparison to the solvent control (DMSO).Additional file 3: Figure S3. A, B) Mutational status (A) and mRNA expression profiles (B) of potential targets to treat YST as found in the TCGA GCT cohort.Additional file 4: Figure S4. A) XTT cell viability assays of GCT72 cells treated with indicated drugs for 24 - 96 h. B) XTT cell viability assays of GCT cell lines GCT72(-R), NOY-1(-R), 1411H, and non-cancerous fibroblasts (MPAF) treated with indicated drugs for 24 - 96 h. C) Raw flow cytometry data of cell cycle distributions of GCT cell lines and MPAF treated with indicated drugs in comparison with solvent controls.Additional file 5: Table S1. List of used A) cell lines, B) inhibitors, C), oligonucleotides, and D) antibodies.Additional file 6: Data S1. Raw and processed mass spectrometry data of YST(-R) tissues and TSO assay data.Additional file 7: Data S2. Structures and ADMET  prediction of AZD4647, AZD7762, Danusertib, Nintedanib, OSU-03012, SNS-314, Sorafenib and Talazoparib as analyzed by the `ADMETlab 2.0’ web platform (https://admet.scbdd.com/).


Following the publication of this article , concernLanes 1–4 of the Fig 3B HMGB1 panel appear similar to the Fig 4A HMGB1 panel (left) when rotated 180°The Fig 4A HMGB1 panel (right) appears similar to the Fig 4A Tubulin panel (right) when rotated 180°The Fig 3B and Fig 4A western blot results report Tubulin controls, but the article’s methodology section reports β-actin primers and antibodies instead.○ M6 , M6/mock , and M6/Mock ○ M6/S2  and M3 The following panels appear to partially overlap despite being used to represent different experimental conditions:In addition to these concerns, it has come to the journal’s attention that several cell lines reported in this study have been reported to be misclassified or contaminated. Specifically,The HepG2 cell line reported in this study as a human hepatocellular carcinoma cell line has been misclassified, and should be reported as a hepatoblastoma cell line instead [The BEL-7404 cell line reported in this study as a human hepatocellular carcinoma cell line is a contaminated cell line, and has since the publication of this study been shown to be a HeLa derivative cell line instead .The L02 cell line reported in this study as a normal (non-cancerous) hepatocyte line is a contaminated cell line, and has since the publication of this study been shown to be a HeLa derivative cell line instead .The first author commented that the image overlap was the result of errors during figure preparation and provided underlying data and updated figures. Furthermore, the first author stated that they omitted to include the use of tubulin in the methodology section, but this did not clarify why the study’s methodology reports on the use of β-actin instead, when no β-actin results have been presented in this study. In response to the contaminated cell line concerns, the first author states that the BEL-7404 and L02 cell lines were sent to a company for identification, but they did not provide further specification regarding the company or the identification procedure, and they did not provide evidence to confirm the cell lines used were not contaminated.The first author provided underlying image data for the results presented in Figs 3, 4, 6, and 7, but the individual level data underlying the results in these figures were not provided for editorial review.PLOS ONE Editors retract this article.In light of the concerns affecting multiple figure panels that question the reliability of these image data, as well as the concerns pertaining to the contamination of cell lines including the control cell line that questions the reliability of the overall results, the JX did not agree with the retraction. YD, JH, QL, YL, WN, YZhang, YZhu, LC, and BC either did not respond directly or could not be reached.


Canine pulmonary adenocarcinoma (PAC) resembles human lung tumors in never-smokers, but it is rarer than human pulmonary adenocarcinoma. Therefore, research on canine PAC is challenging. In the present study, we successfully established various novel canine PAC cell lines from a single lesion in a dog, including two parent cell lines and fourteen cloned cell lines, and characterized their cellular properties in vitro. Several of these cell lines showed epithelial–mesenchymal transition (EMT)-like and/or cancer stem cell (CSCs)-like phenotypes. We additionally assessed the sensitivity of the cells to vinorelbine in vitro. Three clonal lines, two of which showed EMT- and CSC-like phenotypes, were resistant to vinorelbine. Furthermore, we evaluated the expression and activation status of EGFR, HER2, and Ras signaling factors. The findings indicated that the cell lines we established preserved the expression and activation of these factors to varying extents. These novel canine PAC cell lines can be utilized in future research for understanding the pathogenesis and development of treatments for canine PAC. Canine pulmonary adenocarcinoma (PAC) is the most frequently encountered tumor (87.1%), followed by sarcoma (7.6%), adenoma (3.2%), and pulmonary neuroendocrine tumor (1.5%) in dogs with primary pulmonary mass4. Locoregional tumors spread to other lung areas or lymph nodes, and distant metastasis via hematogenous and lymphatic routes are common in canine lung tumors. Metastasis is a crucial prognostic factor. In one study, the rates of local vascular or lymphatic invasion and distant metastases were 71% and 23%, respectively5. Surgical resection is the first choice of treatment, and patients without metastasis appear to have a favorable prognosis. However, the prognosis for advanced disease is very poor, and the median survival time has been reported to be less than 1 year6. In humans, cisplatin-based chemotherapy combined with surgery is available and effective in patients with non-small-cell lung cancer7. However, the efficacy of chemotherapy in dogs with lung tumors remains unclear. In a previous study, two of seven canine PAC cases partially responded to vinorelbine, a vinca alkaloid with high transferability to the lung tissue8. Toceranib, a multi-target tyrosine kinase inhibitor, has been reported to be partially effective in some canine PAC cases10. However, reliable clinical trials with larger sample sizes are lacking because cases of naturally developed PAC are rare. Moreover, only a few types of canine PAC cell lines are currently available13, and their cellular characteristics have not been clearly described. Therefore, a novel experimental model is required for further research on canine PAC.Canine primary lung tumors share clinicopathologic features with human lung tumors in never-smokers but is rare compared with that in humans15. Once EMT occurs, tumor cells migrate from the primary lesion to release circulating tumor cells into the bloodstream. Subsequently, mesenchymal–epithelial transition occurs, followed by metastasis. In human breast and pancreatic cancer models, EMT has been reported to be indispensable for lung metastasis, and it contributes to chemoresistance17. Other reports have also described the contribution of EMT to chemoresistance in human oncology18. Although the role of EMT in metastasis is still under discussion, the importance of EMT in cancer therapy in dogs has been described in a previous review19.Epithelial–mesenchymal transition (EMT) is a key event in tumor progression and metastasis. During EMT, tumor cells acquire mesenchymal phenotypes, including fibroblast-like morphology, high motility, and vimentin expression, resulting from the downregulation of epithelial hallmarks, including adhesion properties and E-cadherin expression20. CSCs are small subsets with extensive self-renewing properties that drive tumorigenesis in tumor tissues, and they are crucial factors in tumor metastasis, recurrence, and chemoresistance22. CSCs have mesenchymal properties and invasive features, the hallmarks of EMT. CSCs can be detected using various techniques, including surface antigen analysis such as CD133 analysis, colony- or sphere-formation assays, aldehyde assays, and side population (SP) assays with Hoechst 3334223 or Vybrant DyeCycle Violet (DCV)24. CSCs have been detected in various types of human tumors24. In canine tumors, CSCs have been detected in mammary carcinoma26, osteosarcoma27, and PAC11.The relationship between EMT and cancer stem cells (CSCs), which are tumor-initiating cells, has been investigated28. The epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), the preferred heterodimer partner of EGFR, are cell surface receptor tyrosine kinases (RTK), and their activation promotes tumor cell proliferation. Their activating mutation and overexpression have been observed in various types of tumors, including human lung cancer30. In canine PAC, the activation of EGFR and HER2 has been reported in tumor tissue33. The small G protein, Ras, is an oncogenic factor. Its activating mutation has been detected in various types of tumors, including human and canine PAC35. Prenylated Ras translocates from the cytoplasm to the membrane, and it is activated by RTK, such as EGFR. Subsequently, activated Ras stimulates the downstream mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K) pathways, which promote cell proliferation. These factors are recognized as potent therapeutic targets in malignant tumors, including PAC36; however, they have not been sufficiently investigated in canine PAC.In humans, personalized treatment for PAC is based on the molecular subtypes of oncogene mutations and activation of tumor-associated proteinsIn the present study, we established two types of canine PAC cell lines under different culture conditions from a dog with naturally occurring PAC. Fourteen cloned cell lines with various morphological characteristics were established by single-cell cloning. We assessed their proliferative capacities in vitro and their characteristics, focusing on the EMT and CSC phenotypes. Additionally, sensitivity to vinorelbine in vitro was assessed. Finally, we evaluated the expression of several tumor-associated proteins.We initiated two primary cultures from canine PAC tissue under different culture conditions, and named them CPACd-p and CPACr-p cell lines. They were cultured in complete media based on Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium, respectively, and were successfully purified and propagated for more than 50 passages.Next, we cloned CPACd-p and CPACr-p using a limiting-dilution method. Fifty-three and thirty-four clones were successfully obtained from the CPACd-p and CPACr-p cells, respectively. Seven clones were selected from each of these groups according to their morphology and subculture intervals. The other clones which were not selected had similar characters to the selected clones. Only the 14 clones that were realistic in terms of number of analyses were used in subsequent experiments. The selected cloned cells were propagated for more than 30 passages .Images obtained under a light microscope are shown in Fig. Additionally, we measured the doubling time of each cell line Table . No signP = 0.00074; Fig. P = 0.0022; Fig. Since some cell lines showed a mesenchymal-like morphology, we hypothesized that EMT occurred in these cell lines. First, we assessed the expression of the epithelial and mesenchymal markers, E-cadherin and vimentin, respectively, in the tumor tissue and in each cell line Fig. A. E-cadhP = 0.0071; Fig. To assess the CSC phenotype, we first evaluated the sphere-forming abilities of the cell lines Fig. A,B. CPACP = 0.0099). CPACd-AB4, CPACd-AC12, CG11, and all cloned cell lines from CPACr-p, except for CPACr-DC3, were less sensitive to vinorelbine than their parent cell lines. In contrast, CPACd-AE3 and CPACd-BB4 were more sensitive than the parent cell line CPACd-p. The IC50 values of vinorelbine in CPACd-AB4, CPACd-AC12, and CPACd-CG11 were higher than 10 µM, suggesting that these cell lines were resistant to vinorelbine treatment. In contrast, CPACd-BB4, CPACr-p, and CPACr-DC3 were relatively sensitive , characterized by cells displaying both mesenchymal and epithelial characteristics, is recognized as a part of the EMT process38. Therefore, it is plausible that partial, rather than complete, EMT has occurred in CPACd-AB4 and CPACd-CG11. Partial EMT is believed to be regulated by tumor microenvironmental factors39 and can enhance invasive properties, generate circulating tumor cells and cancer stem cells, and confer chemoresistance40. In human PAC cells, the GRHL2 gene has been implicated in the maintenance of stable partial EMT41, although the detailed mechanisms remain incompletely understood. The cell lines we have established exhibit stable partial EMT, suggesting the potential for elucidating the mechanisms responsible for stabilizing partial EMT and developing therapeutics targeting partial EMT.EMT is associated with malignancy and is considered a crucial therapeutic target22. In the present study, we assessed CSC properties using two different methods: sphere-formation and SP assays with DCV. CPACd-AB4, CPACd-CG11, and CPACr-AD4 showed relatively high sphere-forming ability and SP population; however, in CPACd-AE3, CPACd-DC10, CPACr-AH4, and CPACr-BH12, the results of the two assays were not concordant. Assessing CSC-associated antigens is widely used for detecting CSCs. In human oncology, several antigens are used as CSC markers, including CD44, CD133, CD24, and EpCAM; however, the expression patterns appear to vary with respect to tumor origin22. Although CD44 and CD133 expression have been previously assessed in a xenograft model of canine PAC, their utility as CSC markers has not been indicated42. In human PAC, these two antigens have been reported to have poor diagnostic value in CSC detection43. Reliable markers for the detection of CSCs may be determined by further assessment using developed canine PAC cell lines.CSCs are a subset of tumor cells characterized by self-renewal, differentiation capacity, and chemoresistance. These properties make CSCs crucial targets for tumor therapy20. In our study, CPACd-AB4 and CPACd-CG11 showed both EMT and CSC phenotypes. Several transcription factors are associated with the development of EMT and maintenance of CSCs. Transforming growth factor-β (TGF-β) has pleiotropic functions in tumor progression44. In epithelial cells, TGF-β acts as a tumor suppressor by inhibiting cell growth and inducing apoptosis. In contrast, in the advanced stages of tumorigenesis, TGF-β induces EMT and stimulates cell proliferation and survival in tumor cells45. TGF-β overexpression has been reported to be associated with malignancy in human tumors46. In human liver tumor cells, autocrine stimulation of TGF-β was reported to induce EMT47. Importantly, EMT stimulated by TGF-β induces the selection and expansion of CSCs49. Moreover, previous reports have indicated that MAPK and PI3K signaling play important roles in EMT or the maintenance of CSCs by EMT53. In our study, relatively high phosphorylation of Erk1/2 and expression of vimentin were observed in CPACr-AH4 and CPACr-DC3. However, Erk1/2 phosphorylation did not appear to correlate with cellular motility. CPACd-AB4 induced phosphorylation of Akt at a relatively high level; however, the other cell lines with CSC phenotypes did not show high phosphorylation of Akt. Further assessment of other signals, including the TGF-β signal, is needed to reveal the detailed mechanism of EMT and CSCs in the canine PAC. On the other hand, Andriani et al. demonstrated that the differences in susceptibility to CSCs induction by TGF-β were dependent on the balance between epithelial and mesenchymal features and cells developing partial EMT exhibit the highest sensitivity in human PAC cells54. The cell lines we have established, exhibiting varying degrees of EMT, hold significant potential possibilities as models for elucidating the association between CSCs and EMT.EMT and CSCs are closely linked55. Chemotherapy-resistant cells contribute to relapse. Although few reports are available for canine cases, chemotherapies, including vindesine, mitoxantrone, vinorelbine, and toceranib, are not fully effective57. Additionally, the mechanisms of resistance have not been elucidated. In the present study, the sensitivity to vinorelbine differed among the cell lines. CPACr-p and the clones, appeared to be relatively sensitive to vinorelbine, and Ras prenylation levels were lower than that in CPACd-p and cloned cell lines. This finding indicates that Ras signaling may be related to the mechanism that determines susceptibility to vinorelbine. However, Erk1/2 and Akt signaling may not be involved in the mechanisms common to these cell lines. Importantly, cell lines with EMT- and CSC-like phenotypes, such as CPACd-AB4 and CPACd-CG11, were resistant to vinorelbine. This finding indicates that EMT and stemness may contribute to chemoresistance in canine PAC. In contrast, although CPACd-AC12 showed resistance to vinorelbine, it showed poor expression of EMT and CSCs phenotypes. Additional studies using the cell lines we have established may reveal the mechanisms of resistance to vinorelbine, not only in canine but also in human PAC.Canine PAC resembles human lung tumors in never-smokers. Adjuvant chemotherapy, including vinorelbine, is indicated for patients with lung cancer. However, in some lung tumors that are intrinsically resistant to chemotherapy, acquired resistance has been shown to develop rapidly even in initial responders29. Also, in canine PAC, previous reports showed overexpression or activation of RTKs33, but the utility as therapeutic targets has not been established. In our findings, the established cell lines expressed EGFR and HER2 at various levels. Therefore, some of these cell lines might retain the expression of RTKs, which could be beneficial for the development of targeted therapies focusing on RTKs. On the other hand, in human lung cancer, drug resistance to EGFR inhibitors has also become a significant concern58. Due to the high similarity between PAC in humans and dogs, further evaluation of cellular characteristics associated with RTKs of these established cell lines could potentially be applied to the research aimed at overcoming resistance to RTK inhibitors in human lung cancer.The molecular characterization of canine PAC remains limited in comparison to human lung cancer. In human NSCLC, EGFR and HER2 have been shown to be overexpressed and recognized as significant therapeutic targets37. These experiments can possibly further clarify the EMT and CSC phenotypes and the detailed mechanisms underlying these phenotypes. Third, cell lines with various properties may not reflect the heterogeneity in tumor tissue but an in vitro selective pressure during the culture process. However, this possibility is difficult to be excluded. Finally, we have not been able to verify whether the established cell lines represent a general profile of canine pulmonary adenocarcinoma. However, given the absence of a defined general profile for canine PAC, it is difficult to say that. Further comparative investigation using whole-genome analysis and epigenetics methods between the established cell lines and tumor tissues from other dogs may substantiate the representativeness of these cell lines in canine PAC.The present study had several limitations. First, we did not perform transplantation in mice. Second, we did not assess the transcription factors associated with EMT, including Zeb, SNAIL, and SLUGIn conclusion, we successfully generated novel canine PAC cell lines with various characteristics. Moreover, we assessed the cellular properties in vitro, focusing on the EMT and CSC phenotypes. Our results indicate that EMT and stemness may play a role in resistance to vinorelbine. We also elucidated that the established cell lines exhibit varying levels of expression and activity of RTKs and Ras signaling factors. Therefore, these cell lines will facilitate the development of novel therapeutic strategies targeting EMT, CSCs, and intracellular signaling pathways and elucidation of the contribution of tumor heterogeneity to tumor biology, including chemoresistance, in human and canine PAC.A 14-year-old neutered male Maltese dog weighting 4.1 kg presented with a mass in the right posterior lung lobe. The mass was 2 cm in diameter, and metastatic lesions were absent according to diagnostic imaging, including computed tomography. The neoplasm was surgically resected and histopathologically diagnosed as a pulmonary adenocarcinoma . Further clinical information is described in Supplementary information file. Fresh tissue samples were collected, in accordance with the informed written consent of the owner and the approval of the Ethical Committee of Okayama University of Science , and the methods were performed in accordance with the relevant guidelines and regulations of Okayama University of Science and ARRIVE guidelines.2 incubator. Once the cell culture reached 80–90% confluence, they were repeatedly sub-cultured with 0.25% trypsin solution at a 1:4 dilution. The cell lines were named CPACd-p and CPACr-p and were cultured in DMEM- or RPMI-1640-based media, respectively. The cell lines were maintained for more than 50 passages.Tumor tissues were washed with saline and minced (1 mm). The tumor fragments were disaggregated with dispase  at 37 °C for 5 h with continuous agitation. After washing with phosphate buffered saline (PBS), the digested tissue was resuspended in DMEM  or RPMI-1640 , which are usually used in culture of various tumor cells as basic media, containing 10% deactivated fetal bovine serum , 100 U/mL penicillin , and 100 µg/mL streptomycin . Cell suspensions were maintained at 37 °C in a humidified 5% CO59. Cells were seeded at 500 cells/well in a 24-well plate and maintained until 90% confluence under the conditions described above. The cells were then seeded at a density of 1 cell/well in a 96-well plate (384 wells). The plates were examined under a microscope immediately after dispensing the cells and culturing. Wells suspected of containing more than one cell or multiple colonies were not cultured. After 30 passages, based on cellular morphology and subculture intervals, seven lines, which were realistic in terms of number of analyses, were selected from the cloned cell lines derived from CPACd-p and CPACr-p. These cell lines, including the parent cell lines, tested negative for mycoplasma contamination .The cell lines were cloned from CPACd-p and CPACr-p cells after 20 passages using the limiting dilution method5 cells/well on microcover glasses. At 80–90% confluence, the cells were fixed with methanol and stained with Wright–Giemsa stain . The glasses were captured under a light microscope at 40 × and 400 × magnifications. Each experiment was repeated at least thrice.Microcover glasses  were placed in the wells of a 12-well plate. The glasses were coated with type I collagen . The cells were then seeded and cultured in a plate at a density of 1 × 104 cells/well in a 24-well plate. After 24, 48, 72, and 96 h, the viable cells were counted using a hemocytometer with 0.5% trypan blue solution. Doubling time was calculated during the logarithmic growth phase (24–96 h) according to the following formula.The cells were seeded at 1.0 × 10Each experiment was performed in triplicate and repeated at least thrice.60, and a canine hemangiosarcoma cell line, Re1261, were also utilized as epithelial and mesenchymal controls, respectively. CHMp-13a and Re12 were kindly provided by Dr. T. Nakagawa  and Dr. H. Sakai , respectively. The results of CHMp-13a and Re12 were exclusively presented in the Supplementary Information file. Cells were lysed with NP40 lysis buffer  supplemented with an EDTA-free protease inhibitor cocktail  and a phosphatase inhibitor cocktail . Twenty micrograms of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes . The membranes were blocked for 1 h with 5% low-fat milk in Tris-buffered saline  containing 0.01% Tween 20 and then incubated with each primary antibody overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibody for 1 h at 24 °C. The primary and secondary antibodies are listed in Supplementary Table In addition to the samples from the CPAC cell lines, protein samples from a canine mammary gland tumor cell line, CHMp-13a5 cells/well in a 24-well plate and cultured. After reaching 100% confluence, the cell monolayers were manually scratched with a sterile 200 µL pipette tip and washed with PBS to remove suspended cells. The adherent cells were cultured for 12 h. Cell migration was observed and captured under a light microscope at 40 × magnification. The migration ability was calculated relative to a gap area at 0 h. Each experiment was performed in triplicate and repeated at least thrice.To evaluate the migratory ability of each cell line, a wound-healing scratch assay was performed. The cells were seeded at 3.0 × 105 cells in 500 µL of serum-free media was added to the upper chamber. Seven hundred and fifty microliters of medium supplemented with 10% FBS were added to the lower chamber. After 20 h of incubation, non-migrating cells on the upper surface of the membrane were removed using a cotton swab. The inserts were fixed and stained with methanol and Wright–Giemsa stain . The number of migrating and invading cells was counted under a light microscope at 400 × magnification. Each experiment was performed in triplicate and repeated at least thrice.To evaluate the migration and invasion abilities of each cell line, a Boyden chamber assay was performed. Culture inserts with 8 µm pore size  were set on a 24-well companion plate (Corning). Matrigel  was added to the upper chamber and incubated for 2 h at 37 °C. Subsequently, the wells were washed with PBS. Wells without the Matrigel layer were used to count the migrating cells. A cell suspension containing 2.5 × 1062. Cells were seeded at 2000 cells/mL in an ultra-low attachment 24-well plate (Corning). These cells were cultured in serum-free DMEM/F12  supplemented with bovine serum albumin , 5 mM HEPES , 100 U/mL penicillin , 100 µg/mL streptomycin , 20 ng/mL human recombinant EGF , 10 ng/mL human recombinant bFGF (Peprotech), and 1% B27 supplement . EGF, bFGF, and B27 supplement were used to stabilise the formation and maintenance of sphere. DMEM/F12 medium supplemented with 2 ng/mL EGF and 1 ng/mL bFGF was added to the culture every other day for 10 days. Spheres were counted under a light microscope at 40 × magnification. Each experiment was performed in triplicate and repeated at least thrice.To evaluate the self-renewal activity of each cell line, a sphere-formation assay was performed using the protocol described in a previous report with a slight modification5) were labeled with 5 µM DCV alone or in combination with 50 µM verapamil . After 90 min of incubation at 37 °C, the labeled cells were washed with ice-cold PBS three times and analyzed by flow cytometer  and FlowJo software version v10.8.1 . Each experiment was repeated at least thrice.To detect the side population of each cell line, the cells were stained with DCV (Thermo Fisher Scientific) and analyzed by flow cytometer. Cells  for 48 h. Subsequently, cell counting kit-8  was added. After 2 h of incubation, the absorbance was determined at 450 nm using an SH-1300 microplate reader . The percentage of absorbance was calculated against the absorbance for untreated cells (control). Each experiment was performed in triplicate and repeated at least thrice.To evaluate the antiproliferative effect of vinorelbine on each cell line, a WST-8 assay was performed. Cells were seeded at 0.5 × 10P < 0.05.All data are presented as the mean ± standard deviation (SD). The normality and homogeneity of variances were verified using the Shapiro–Wilk and Bartlett's tests, respectively. Statistical comparisons between the CPACd-p and CPACr-p cell lines were performed using the unpaired Student’s t-test. Statistical comparisons between the parent and cloned cell lines were performed by one-way ANOVA with Dunnett’s multiple comparison post hoc test using Bell Curve for Excel . Differences were considered statistically significant at Supplementary Information.


Treatment and relevant targets for breast cancer (BC) remain limited, especially for triple-negative BC (TNBC). We identified 6091 proteins of 76 human BC cell lines using data-independent acquisition (DIA). Integrating our proteomic findings with prior multi-omics datasets, we found that including proteomics data improved drug sensitivity predictions and provided insights into the mechanisms of action. We subsequently profiled the proteomic changes in nine cell lines (five TNBC and four non-TNBC) treated with EGFR/AKT/mTOR inhibitors. In TNBC, metabolism pathways were dysregulated after EGFR/mTOR inhibitor treatment, while RNA modification and cell cycle pathways were affected by AKT inhibitor. This systematic multi-omics and in-depth analysis of the proteome of BC cells can help prioritize potential therapeutic targets and provide insights into adaptive resistance in TNBC.  •Proteomic analysis of 76 breast cancer cell lines and 9 lines with perturbation.•Machine learning-based multi-omics predicts breast cancer cells’ sensitivity to 90 drugs.•Longitudinal proteomic profiling provides insights into drug resistance. Here we present a deep proteome resource of 76 human breast cancer cell lines. Each cell line included four replicates. Then we built machine learning models using these multi-omics data to predict BC cells’ sensitivity to 90 drugs. We further performed longitudinal proteomic profiling of 9 cell lines perturbed by three drugs. Together, this study presents the so far most comprehensive proteomic investigation of BC cell lines and uncovered novel molecular insights for TNBC cells. Breast cancer (BC) is the second most common cancer in the world and the most common in women, with a mortality rate ranking fourth among all malignancies . In the HER2 (ERBB2) gene amplification and high levels of HER2 expression. Basal lines lack ER, PR, and HER2 expression.The search for novel drug targets in BC has benefited from multiple well-characterized BC cell lines . These cRecent genomic profiling studies have revealed potential novel targets for BC , 6. HoweRecent advances in data-independent acquisition (DIA)-based quantitative proteomic techniques  have enaIn this study, we applied our optimized PCT-DIA method to analyze the proteomes of 76 BC cell lines, including 39 TNBC samples. By integrative analysis of our proteomic results with genomic and transcriptomic data, we generated a model to predict the drug response of these BC cells to 90 compounds, including known BC drugs. We then further profiled over 400 BC proteomes from cells perturbed by three drugs that had been prioritized based on our multi-omics modeling of drug response. This unique proteomic dataset allowed us to identify proteins whose expression patterns change in response to these drugs, as well as potential biomarkers for drug resistance in TNBC.t test or one-way ANOVA for differentially expressed analysis and Pearson correlation test for correlation analysis, which are described in figure legends and methods where relevant.The first part of this study is the generation of the BR76 proteomic dataset where we collected 302 proteomic raw files for 76 BC cell lines. Each cell line was analyzed with three biological replicates and the third biological replcates was further subjected to one technical replicate. This BR76 proteomic dataset was then combined with the published genomic and transcriptomic datasets  to predim/z) and 30 ms for each window, respectively. The DIA window schemes were optimized to 66 variable windows. The instrument was operated in a high-sensitivity mode.PCT-DIA analysis was performed as previously described . Fresh cThe DIA MS raw data were processed using the DIA-NN (1.7.15) against an established spectral library that contains 194,899 proteotypic peptide precursors and 10,323 proteotypic SwissProt proteins  . DetaileIn total, 8952 proteins were identified with a 54.6% missing rate of the whole proteome. We then removed two outlier samples with extremely low protein identification numbers as revealed by PCA plots. After further removing the proteins with over 90% missing rate, 6091 proteins remained, with a 34.7% missing rate of the whole data set. In the meantime, the batch effect across all samples and the reproducibility of the measurement were evaluated by PCA plots and the coefficient of variation of all biological and technical replicates .We used a flexible network algorithm to develop a multivariate linear model to predict the effect of each compound in 76 BC cell lines using genomics, transcriptomics, and proteomics datasets, as previously described .The “glmnet” R package was utilized for elastic network analysis, incorporating nested cross-validation. Our analysis included the following aspects: point mutations (123 mutations), RNAseq , proteomic data sets (6091 proteins), and RPPA , which were used as input. The “impute.knn” function in the Bioconductor imputes the missing values.The detailed processes were outlined below. First, an “internal” cross-validation was performed. In this step, a set of multi-omic data from 59 cell lines was employed to determine the optimal hyperparameters (α and λ) for the elastic network. The elastic network parameters α and λ were selected by minimizing cross-validation errors within the “internal” pipeline . The selected α and λ parameters were then applied to the run of the 200 elastic network algorithms, with each time using a random subset of data obtained from 90% of the available cell lines. Then, the obtained 200 coefficient vectors were averaged, and the predicted values were sorted according to the magnitude of the average coefficient weight. To select predictors suitable for application to new data of finite size, standard linear regression and 10-fold cross-validation are used to evaluate the set of pre-k-ary predictors (by averaging coefficient weights). The cross-validation error within one standard deviation of the minimum cross-validation error, k, was set to a minimum. As part of the “external” pipeline, leave-one-way cross-validation is applied throughout the process to obtain a reliable estimate of the performance of the new data. Specifically, the elastic network model obtained using the remaining data (and the above steps) predicted the drug response of each cell line. Finally, the predicted response value was correlated with the actual response value, and for any drug/data combination, the prediction accuracy was evaluated using the Pearson correlation between the observed and the predicted drug responses .2.BT549, Hs578T, ZR75, and MDA-MB-231 cell lines were cultured in DMEM/F-12 medium ; MDA-MB-468 was maintained in L15 medium ; T47D was adapted in DMEM medium ; MCF7, MX-1, and SK-BR-3 were kept in 1640 medium . These media were all supplemented with 10% fetal bovine serum  and penicillin-streptomycin (HyClone) and incubated at 37 °C with 5% CO50) was determined using the MTT cell proliferation assay following the manufacturer’s recommendations  were obtained using Multiscan Spectrum (BioTek).Half-maximal inhibitory concentration  with every drug and (ii) showed a fold-change greater than two between the last and first time points. We then searched for pathways including these proteins using KEGG pathways in STRING database. Prioritization was given to pathways that were consistently dysregulated in at least three out of five TNBC cell lines and at least two out of four non-TNBC cell lines.The proteins were clustered in TNBC and non-TNBC cell lines using Mfuzz . We onlyA). This panel in our study shares 29 lines with the CCLE proteomics study (31 BC cell lines) (A) were obtained after quality filtering. Using stringent quantitative criteria as described in the supplemental Table S1B) across all samples with a 34.7% missing rate of 6091 proteins quantified in all samples. For each sample, we quantified an average count of around 4000 proteins , and the log10 transformed peptide intensities showed a normal distribution .We collected the 76 BC cell lines  characteFig. 1l lines) , 44 linel lines) . Only twl lines)  are incll lines) , 28. Whil lines) , 17, 18,l lines) A were obA). All replicates consistently yielded comparable protein and peptide identifications. The numbers of peptides identified per protein were highly consistent across the replicates with a CV <10% . Regarding protein quantification, the correlation among technical replicates (median r = 0.93) was slightly higher than that among biological replicates (median r = 0.91), and much higher than that among non-replicate samples (r = 0.74). The median CVs across different abundance level were all lower than 0.2 . Next, we subjected these proteins for the downstream analysis. The protein intensity distribution identified from these DIA files was highly consistent . Principal component analysis (PCA) revealed a mild batch effect in signal intensities in group 4 only, probably because these samples were analyzed by a different mass spectrometer . PCA plots of the cell line proteomic data exhibited minimal batch effect after batch correction using “limma R” package . Within the dataset, we identified 16 proteins overlapping with the PAM50 gene panel, which is a well-known breast cancer classifier (D). We then computed the average protein intensities of the four groups for subsequent analyses. Comparing the gene-wise correlation of RNA-seq (D) (C) (A), which agrees with previous observations , re, reA). A-seq (D) C (5), an (D) (C) A, which rvations . The mRNrvations , 31, 32 rvations . Should ata sets .Fig. 2MuB. Then we analyzed the top ten protein complex pairs with the highest correlation at both protein and transcription levels. They exhibited a higher correlation at the protein level than that at the transcription level (D), while that at the transcript level were associated with the processes such as endoplasmic reticulum to Golgi vesicle-mediated transport, actin nucleation, protein localization and fatty acid beta-oxidation (E). Notably, fatty acid beta-oxidation was the common pathway detected at both protein and transcription levels. Two exemplar protein complexes (C) include the HADHA/B which are potential prognostic biomarkers for BC . Of note, this analysis was based on all molecules measured in each dataset. Although the discriminative power of the transcriptome is not optimal because of the inclusion of the entire transcriptome containing 29,140 transcripts, the selection of differentially expressed transcripts  could well resolve the two groups (A). We identified 771 differentially expressed proteins (DEP)s in TNBC versus non-TNBC cell lines (B). Those DEPs were significantly enriched in 12 differentially activated pathways using Ingenuity Pathway Analysis (IPA) (C), including extracellular matrix, metabolism, and immunity network relationship. We also compared with the proteomic data of BC patients (B and C). The data indicated that proteome data obtained from cell lines are also potentially informative to the clinic.In analyzing molecular patterns unique to TNBC cells, we found that the DIA data outperformed RNA-seq and RPPA datasets in distinguishing TNBC samples from the 76 cell line samples based on 29,140 transcripts, 218 proteins identified by RPPA and 6091 proteins identified by DIA, respectively A. Of noto groups A. We idell lines B. Those is (IPA) C, includpatients , B and CD). These 38 DEPs participated in multiple biological processes (D) that are known to be involved in BC progression (E). Additionally, FASN (D–F).We next selected the 38 most significant DEPs to clearly distinguish TNBC cell lines D. These rocesses D that argression , 42. In gression , indicatgression , fatty agression , glutathgression , and clugression . These pgression E. Additily, FASN , ITGB4 , RNAseq , proteomic data sets (6091 proteins), and RPPA  (A). For any drug and data combination, the prediction accuracy was evaluated using the Pearson correlation between the observed and the predicted drug responses. The overall predictive power of the DIA proteomic data was slightly lower than that of the RNA-seq data, but superior to that of the gene mutations (B). The relatively low predictive power of the mutation data might be attributable to the low numbers of mutations detected, which likely reflects the relatively low levels of single-nucleotide variants found in breast cancer  A. For anutations B. The ret cancer .Fig. 4MuC); more information is provided in Fig. 4D). We then focused on the EGFR and Akt-mTOR signaling pathway which is crucial for many cancers including BC. Among the three EGFR inhibitors, Lapatinib exhibited the best performance. The molecules associated with the response to these three drugs  are listed in Fig. 4E). These proteins are mainly involved in cell cytoskeleton, signal transduction, and apoptosis, indicating that these biological processes might be associated with the response to the respective drugs. In particular, AKT inhibitors (such as GSK650394) have been suggested as a treatment for BRCA1-deficient BCs . For each drug/cell line combination, we first determined the IC50 (B). The cell line was then treated with each drug at its IC50 concentration for 4, 12, 24, 48, and 72 h. Three biological replicates were generated, and lysates of the respective samples were analyzed by DIA-MS (A). A total of 5621 SwissProt proteins were identified in 432 samples with a 33.6% missing rate of 5621 proteins identified in all samples . The high Pearson correlation value among the replicates, as shown in supplemental Fig. S5A demonstrated the high reproducibility of the experiment and MS measurement. In the PCA analysis of each drug, the luminal lines were well separated from the TNBC samples, indicating that the variance in BC subtypes introduces more significant variations than the drug perturbations (E).Based on our multi-omics modeling of drug response predictions, we next investigated the effect of drug-induced perturbations on the proteomes of BC cell lines. In particular, we aimed to identify protein modules mediating the response to specific drug treatments and explore the mechanisms to reverse drug resistance. We focused on the three targeted therapy drugs with the strongest correlation of half-maximal inhibitory concentration (IC50) values with multi-omic data: lapatinib, MK-2206, and everolimus. We examined their effects on nine BC cell lines: SKBR3, T47D, MCF7, Hs578T, ZR75, BT549, MDA-MB-231, MDA-MB-468, and MX-1 A. For eathe IC50 B. The cey DIA-MS A. A totaC and D; A. The hirbations E.Fig. 5PB–D). In Figure 5F, the proteins shown in red, including five proteins for lapatinib, 18 proteins for everolimus, and 12 proteins for MK-2206, were positively correlated with IC50 values, therefore they potentially contribute to drug resistance. In contrast, there are several proteins in blue, including 23 proteins for lapatinib, 11 proteins for everolimus, and 20 proteins for MK-2206. Their expression levels were negatively correlated with the IC50 values, therefore they might be correlated with drug sensitivity. Among the proteins that positively correlated with drug resistance, several have been reported to be associated with therapy response. For example, hexokinase 2 (HK2) is required for tumor progression . Aligning the proteomic data with biological pathways, the results showed that lapatinib and everolimus induced distinct metabolic responses in TNBC and non-TNBC cell lines, respectively . This result is in line with previous studies highlighting metabolism as a hallmark of cancer . The TCA cycle occurs in mitochondria, which were reported to be dysregulated at baseline in metastatic, compared with non-metastatic, TNBC .To compare the protein patterns that differed in drug response between TNBC and non-TNBC cell lines, we used Mfuzz and differential expression analysis. We identified 252 proteins with the opposite dynamic patterns between TNBC and non-TNBC cell lines . To identify the key protein determinants contributing to the response to lapatinib, MK-2206, and everolimus in TNBC cells, we compared the persistently dysregulated proteins (PDPs) after the drug treatment in TNBC and non-TNBC cell lines. We found 483 PDPs in over 60% of TNBC cell lines treated with MK-2206, while there were only 173 PDPs for everlolimus and 172 PDPs for lapatinib (C). Hence, MK-2206 treatment induced the most significant differences between the TNBC and non-TNBC cell lines at the proteome level. The set of affected proteins mainly included proteins involved in ribosome biogenesis, RNA polymerase, spliceosome, and the cell cycle (B). Among these, DNA (cytosine-5)-methyltransferase-1 (DNMT1) was significantly downregulated only in the TNBC cell lines (C). AKT has been reported to reduce DNA methylation via modulating the phosphorylation of DNMT1, and DNMT1 inhibition could also reduce the activity of AKT signaling . DUS2 is involved in tRNA modification and affects the stability of mtRNA  synthase [NAD(P)+]-like (DUS2) and DNA topoisomerase 2-alpha (TOP2A) C. DUS2 iof mtRNA . Separatof mtRNA . Notablyof mtRNA , 65.Collectively, the proteins and related pathways that we identified through analyses of these large datasets lay the foundation for future studies into potential TNBC treatment. Our observations are further reinforced by previous work on the implications of these proteins and pathways in cancer development.We collected high-quality proteomics data on 76 BC cell lines with four replicates using PCT-DIA and integrated these data with genomics and transcriptomics for drug sensitivity prediction. We observed that proteomic data improved the sensitivity prediction of multiple drugs in BC. Our analysis identified 38 proteins specifically expressed in TNBC cell lines, which were validated using independent data. Our DIA dataset contributes to the effective prediction of drug response in multi-omics modeling experiments. Additionally, we performed time-course analyses investigating protein dynamics to identify proteins and pathways that are specifically dysregulated in TNBC lines and might yield insight into adaptive drug resistance in TNBC. Although lapatinib and everolimus elicited few changes in TNBC cells, MK-2206 resulted in significant changes including ones involving RNA modification and cell cycle pathways. Our data showed significant changes in DUS2, DNMT1, and TOP2A following MK-2206 treatment in TNBC. These observations warrant future investigation to explore their suitability as drug targets or biomarkers.Although this multi-omics study included a relatively large number of samples, as well as biological and technical replicates, it did not include phosphorylation data. However, this limitation does not compromise the relevance and accuracy of our main findings. The primary focus of this publication was to report on this rich proteomic dataset and conduct an initial, exploratory bioinformatic analysis of the dataset. It is outside the scope of the intended work, and therefore a limitation, to conduct in-depth experimental validation of reported biomarkers; this work is left for future study.The 76 breast cancer cell DIA data are deposited in Pride (PXD004701), the protein matrix is included as This article contains T. G. and Y. Z. are shareholders of Westlake Omics Inc. R. A. holds shares in Biognosys. W. G. are employees of Westlake Omics Inc. The remaining authors declare no competing interests.