Clinical Utility of Patient-Derived Cell-Based In Vitro Drug Sensitivity Testing for Optimizing Adjuvant Therapy in Dogs with Solid Tumors: A Retrospective Study (2019–2023)

Comments : The study does not mention blinding, which could impact outcomes. If researchers or clinicians are aware of the treatment type (empirical or guided), it may introduce bias in treatment decisions and data interpretation, potentially skewing results.

Response: We thank the reviewer for comments. Due to the retrospective nature of our study, group allocation was determined in the past. Therefore, blinding was not feasible, as the researcher cannot control the assignment of participants to treatment groups at the time of treatment initiation. However, in retrospective studies, there are methods to implement blinding in order to reduce bias or ensure that the interpretation of results is not influenced by the researcher. The outcome assessor can be influenced by knowing which treatment was given, so it is essential to keep them blinded. In our study, the researcher measured time to tumor progression (TTP) without knowing which treatment group the dogs belonged to, ensuring that personal preferences or biases did not influence the results. We have added the sentence as follows.

Line 130-131: “To ensure that bias did not influence the results, TTP was measured by a researcher blinded to the treatment group assignment.”

Comments : The follow-up duration should be sufficient to capture any late-onset therapeutic side effects or tumor recurrence. Although the study reports time to tumor progression (TTP), a longer follow-up period is essential to evaluate long-term survival, recurrence, and the treatment's safety and effectiveness over time. This would provide a more thorough and comprehensive assessment of the treatment's overall impact.

Response: We thank the reviewer for comments. As the reviewer rightly pointed out, the follow-up time in our study was relatively short (median, 386 days) and may have insufficient to identify recurrence or long-term survival in this population, despite using TTP as the primary endpoint. Nevertheless, this study provides preliminary evidence for the feasibility of in vitro drug sensitivity test in veterinary oncology. Therefore, we have added this explanation in limitation to justify the short follow-up period as a background for a more extensive study.

Line 379-380: “Second, the short follow-up duration may have been insufficient to identify tumor recurrence, despite using TTP as the primary endpoint.”

Line 392-394: “Nevertheless, our study provides preliminary evidence for the feasibility of patient-derived cell-based in vitro drug sensitivity testing in veterinary oncology.”

Comments : The empirical group’s treatment is based on histopathological examination, but the study does not specify whether a standard protocol was followed for evaluating tumor characteristics, such as mitotic count, surgical margin status, and histological grade. Clarifying this would ensure consistency and reliability in the treatment decision-making process.

Response: We thank the reviewer for comments. The treatment in the empirical group is indeed based on histopathological examination, including diagnosis and characteristics. To improve clarity, we have revised the sentence as follows.

Line 129-130: “In contrast, dogs in the empirical group received anticancer drugs based solely on the histopathologic diagnosis and features (Figure 1).”

Comments : Standardization of the in vitro drug-sensitivity testing process, including the drug concentrations, incubation times, and assessment of drug response, are essential to ensure consistency across cases.

Response: We appreciate the reviewer’s insightful comment. We have revised the methods (section 2.3) to clarify the in vitro drug-sensitivity testing process. We evaluated the dose-dependent effects of candidate drugs by testing at least six concentrations under the condition of 80% confluence of cells. To compare the efficacy of candidate drugs, we generated dose-response curves using GraphPad Prism software, in which the logarithmic drug concentrations were plotted against the relative cell viability (%) normalized to vehicle control-treated cells as shown in supplementary materials (Table S1). These modifications have been added to the Materials and Methods section as follows.

 Line 129-130: “After allowing 16–24 hours for attachment and settling, the medium was replaced with a medium containing serially dilute candidate drugs once cells reach approximately 80% confluence per well. At least six concentrations ranging from 1 to 100 µM (e.g., 1, 5, 10, 20, 50, 100 µM) were tested for each drug, and vehicle controls of DMSO were included. Each condition was assessed in triplicate, and results were expressed as mean ± standard error. After 24 hours of drug exposure, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA). The efficacy of can-didate drugs was compared using dose-response curves generated in GraphPad Prism (GraphPad Software, San Diego, CA, USA). Dose-dependent effects were demonstrated by plotting the logarithmic drug concentrations against the relative cell viability (%), normalized to the vehicle control group (Table S1).”

Comments : The study does not explore why certain medications are more effective than others in specific cases, which is a critical factor for understanding the mechanisms behind treatment responses. Investigating these variations would offer valuable insights into how treatments work, helping to identify the factors that contribute to treatment success or failure in individual patients. This understanding could ultimately guide more personalized and effective therapeutic strategies.

Response: We thank the reviewer for comments. Cytotoxic chemotherapeutic agents and tyrosine kinase inhibitors (TKIs), which were used in our study, exert their effects through distinct mechanisms and are applied to specific tumor types accordingly. However, as described in the ‘Introduction’ section, variations in treatment response are frequently observed due to tumor heterogeneity, making it difficult to attribute efficacy solely to known mechanisms. Given this complexity, our study aims to indirectly assess which anticancer agents may be effective in vitro, rather than identifying a specific mechanistic basis for their activity. Nonetheless, this approach could serve as a foundation for understanding the inherent variability in tumor biology.

Comments : The methodology has a solid foundation, with a clear experimental design and relevant outcome measures. However, there are areas for improvement, including:

- Larger sample sizes for each group.

- Clearer description of the treatment selection process and possible confounding factors.

- More detailed statistical analysis and consideration of additional outcome measures.

- Providing more transparency regarding diagnostic test accuracy.

Response: We thank the reviewer for comments. Most of the suggestions made by the reviewer correspond to the limitation of our study.

1) Although large-scale randomized controlled trials are required, our study provide preliminary evidence for the feasibility of in vitro drug sensitivity in veterinary oncology and serve as foundation for further research.

2) Furthermore, due to the retrospective nature of our study, group allocation was determined in the past. As a result, blinding was not feasible, as the researcher cannot control the assignment of participants to treatment groups at the time of treatment initiation. Instead, TTP was measured by a researcher blinded to the treatment group assignment to ensure that bias, possible confounding factors, did not influence the results.

3) Additionally, our study only compares TTP between the groups due to the short follow-up time. However, follow-up studies will allow for consideration of additional outcome measures (e.g., overall survival).

4) In our study, only diagnostic test was histopathological examination, which was confirmed by veterinary diagnostic laboratories. If we have not fully understood the reviewer’s comment, we would appreciate further clarification, and we will address it accordingly.

Comments : The conclusions are consistent with the evidence presented in the study and directly address the main research question. The observed positive correlation between in vitro drug-sensitivity testing and improved clinical outcomes supports the argument that this approach can enhance adjuvant therapy for dogs with solid tumors. However, the authors rightly emphasize the need for further research, including larger-scale, randomized controlled trials, to fully validate these findings and explore the broader applicability of this approach across different tumor types.

Response: We thank the reviewer for comments. We have already described the need for large-scale randomized controlled trials to strength our results in the ‘Conclusions’section. However, as the reviewer suggested, we have revised the following sentence to acknowledge the need for further research, while emphasizing the significance and value of our study.

Line 403-404: “Although large-scale randomized controlled trials and further validation for specific tumor types are needed, our findings serve as a valuable foundation for future research.”

Line 65 : delete ‘well’

Response: We thank the reviewer for comments. We revised the sentence as follows.

Line 64-65: “Furthermore, the relationship between target inhibition and clinical response is not established in canine tumors”

Line 201-204 : Not clear and confusing.

Response: We thank the reviewer for comments. To improve clarity, we revised the sentence as follows.

Line 207-211: “Of 22 dogs, including the five dogs for whom cell culture was not possible and 17 dogs whose owners did not request testing, five dogs were excluded due to absence of adjuvant therapy after surgery (n = 2), loss to follow-up after surgery (n = 2), and duration of adjuvant therapy of ≤ 1 month (n = 1). Consequently, 17 dogs were included in the empirical group (Figure 2)”

Comments: I thank the authors for revising the manuscript and responding point-by-point to my comments on the previous version. I have confirmed that the manuscript has been sufficiently improved.

Response: We thank the reviewer for their comments. We appreciate the constructive feedback provided in the first round, which has helped improve the manuscript.