Transcriptomic Profiling of Carboplatin- and Paclitaxel-Resistant Lung Adenocarcinoma Cells Reveals CSF3 as a Potential Biomarker for the Carboplatin Plus Paclitaxel Doublet Regimens
, Suchanan Tanyapattrapong 1, Thipphanet Masjon 1, Saowanee Maungchanburi 1,†and Paramee Thongsuksai 2
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript is important for Car and Pac resistant lung cancer treatment.
Comments:
1. Please explain why chose H1792 cells.
2. Does H1792 cells have WT or mutatedp53?
3. Does Car or Pac change p53 level?
4. What dosage was used in Figure 1A and 1B?
5. Needs to show CSF3 protein level in Figure 8. 12-16 fold increase does not mean CSF3 protein also increases 12-16 fold?
Author Response
Comment 1: Please explain why chose H1792 cells.
Response 1: The author used H1792 cells as a model for the development of Car and Pac resistance, as these cells were derived from the pleural effusion of a White male patient with stage 4 adenocarcinoma. This can illustrate an actual condition in which doublet chemotherapy is necessary for advanced lung cancer.
Comment 2: Does H1792 cells have WT or mutatedp53?
Response 2: H1792 cells exhibit a P53 mutation. Given that p53 mutations are frequently observed in patients with advanced lung cancer, using cells with p53 mutations may serve as an effective model for investigating genes associated with drug resistance or the mechanisms of drug resistance.
Comment 3: Does Car or Pac change p53 level?
Response 3: Our present study did not measure p53 level following Car and Pac therapy in parental cells, H1792/Car cells, and H1792/Pac cells. Numerous prior studies across various cancer types indicate that Car and Pac are implicated in DNA damage and apoptosis through the activation of the p53 signaling pathway. Pac enhanced the expression of P53 in prostate and lung cancer cells. Increased expression of p53 was also seen in breast cancer and head and neck squamous cell carcinoma cells after carboplatin treatment. Consequently, we hypothesize that P53 is implicated in the sensitivity to Car and Pac responses; however, to elucidate this matter, an investigation of P53 expression levels following single and combined Car and Pac treatments in both H1792 parental and resistant cells must be conducted in our future research.
Comment 4: What dosage was used in Figure 1A and 1B?
Response 4: (A) Schematic representation of the experimental workflow used to generate H1792/Car and H1792/Pac cell lines using a stepwise method. (B) Morphological characteristics of parental H1792 cells, H1792 cells in the recovery phase post-treatment with Pac or Car at concentration of 10 nM and 5 µM, respectively, and drug-resistant cells following recovery, were observed under a microscope at 20× magnification.
Comment 5: Needs to show CSF3 protein level in Figure 8. 12-16 fold increase does not mean CSF3 protein also increases 12-16 fold?
Response 5:
1) The expression level of CSF3 protein will be assessed in our forthcoming study. We will assess the expression level in cell lysate via western blot analysis and in tissue cells via immunohistochemistry. Furthermore, given that the CSF3 gene encodes granulocyte colony-stimulating factor (G-CSF), which is secreted into the bloodstream, we will conduct additional experiments to ascertain the CSF3 levels in the secretion media of parental H1792 cells compared to resistant cells after drug treatment, as well as in the serum of patients following a combination of Car and Pac regimens.
2) The increase in the dosage of Car and Pac treatment, or the fold change in drug resistance, did not correspond with the fold change of expression in the CSF3 gene or protein. This may be owing to the complexity of the mechanisms involved in drug resistance. Some targeted proteins are downstream molecules that require the induction of numerous upstream molecules before their expression changes.
Reviewer 2 Report
Comments and Suggestions for Authorsinitially, it has a significant acceptable text match estimated via iThenticate of 22%. The paper is interesting and well-written.
The aim of this study was to determine the establishment of two chemoresistant cell lines derived from the 61 human H1792 adenocarcinoma cell line: carboplatin-resistant cells (H1792/Car) and paclitaxel-62-resistant cells (H1792/Pac). We assessed the cross-resistance of these cell lines to other 63 chemotherapy drugs, including Cys, Car, Gem, and Pac, as well as to doublet regimens 64 such as Car plus Pac, Car plus Gem, and Cys plus Gem. Transcriptome sequencing was 65 used to identify differentially expressed genes (DEGs) between the resistant cell lines 66 (H1792/Car and H1792/Pac) and their parental H1792 cells.
The methodology was adequate and aligned with the objectives. Institutional ethical approval is lacking that the experiments were performed according to good clinical practice guidelines.
The paper may be acceptable for publication because it will be of scientific interest to the readers.
Author Response
Comment 1: Initially, it has a significant acceptable text match estimated via iThenticate of 22%. The paper is interesting and well-written. The aim of this study was to determine the establishment of two chemoresistant cell lines derived from the 61 human H1792 adenocarcinoma cell line: carboplatin-resistant cells (H1792/Car) and paclitaxel-62-resistant cells (H1792/Pac). We assessed the cross-resistance of these cell lines to other 63 chemotherapy drugs, including Cys, Car, Gem, and Pac, as well as to doublet regimens 64 such as Car plus Pac, Car plus Gem, and Cys plus Gem. Transcriptome sequencing was 65 used to identify differentially expressed genes (DEGs) between the resistant cell lines 66 (H1792/Car and H1792/Pac) and their parental H1792 cells. The methodology was adequate and aligned with the objectives. Institutional ethical approval is lacking that the experiments were performed according to good clinical practice guidelines. The paper may be acceptable for publication because it will be of scientific interest to the readers.
Response 1: This research received approval from the Ethics Committee on Human Research at the Faculty of Medicine, Prince of Songkla University (REC 66243381) as mentioned in Institutional Review Board Statement. Moreover, all investigators have undertaken GCP training to verify their ethical standards for conducting research.
Reviewer 3 Report
Comments and Suggestions for Authors1. I suggest that the authors provide a more detailed explanation of the methods used for generating drug-resistant cell lines.
2. I think the inclusion of a comparison of transcriptomic profiles with other cancer cell lines resistant to different chemotherapy drugs would enhance the generalizability of the findings.
3. The authors need to address the potential mechanisms underlying cross-resistance observed in H1792/Car and H1792/Pac cells.
4. I suggest that the discussion expand on the clinical implications of CSF3 as a biomarker.
5. I think the authors should validate additional hub genes identified through transcriptomic analysis using RT-qPCR.
6. The authors need to explore the role of apoptosis resistance mechanisms in greater detail.
7. I suggest that the authors include additional visual representations of the pathways identified in KEGG enrichment analyses.
8. I think the study would benefit from further analysis of the tumor microenvironment's impact on the observed resistance.
9. The authors need to ensure that the limitations of the study are adequately discussed.
Author Response
Comment 1: I suggest that the authors provide a more detailed explanation of the methods used for generating drug-resistant cell lines.
Response 1: Information regarding the dosage used, particularly the initial dose that generates resistance to Car and Pac cells, has been included. Other details have been fully described in the article.
Comment 2: I think the inclusion of a comparison of transcriptomic profiles with other cancer cell lines resistant to different chemotherapy drugs would enhance the generalizability of the findings.
Response 2: The suggestion has been included in the conclusion section.
“We successfully established drug-resistant cell lines, H1792/Car and H1792/Pac, specific to the Car plus Pac regimen. This finding may validate earlier research in other cancers that employed the creation of drug-resistant cell lines as a model for identifying potential biomarkers for predicting response. Our findings indicate that resistance to a single drug also results in resistance to combinations treatment. In addition, our transcriptomic sequencing, bioinformatics analysis, and technical validation of gene expression revealed a marked increase in CSF3 expression in both cell lines. These findings suggest that CSF3 may serve as a novel biomarker for resistance to the Car plus Pac regimen and a potential molecular target for addressing chemoresistance in advanced NSCLC patients.”
Comment 3: The authors need to address the potential mechanisms underlying cross-resistance observed in H1792/Car and H1792/Pac cells.
Response 3: In this investigation, we proved the effects of Car and Pac on the cell cycle and apoptosis by flow cytometry, as well as the associated apoptotic proteins via western blotting, to confirm the resistant properties in our drug-resistant cells. This study was not intended to investigate the potential mechanism of cross-resistance in our drug-resistant cells. However, we intend to explore additional resistance mechanisms linked to cross-resistance in both single-agent and combination treatment in future studies.
Comment 4: I suggest that the discussion expand on the clinical implications of CSF3 as a biomarker.
Response 4: The suggestion has been included in the discussion section.
“Consequently, these findings suggest that G-CSF may serve as a possible biomarker for cancer prognosis and could act as a predictive biomarker for treatment regimen selection in advanced NSCLC.”
Comment 5: I think the authors should validate additional hub genes identified through transcriptomic analysis using RT-qPCR.
Response 5: Due to the limited budget, the gene selection for this validation was based on the use of bioinformatics tools and the Kaplan-Meier plotter. The selected genes, IL1A and CSF3, exhibited differential expression levels in cancerous tissues relative to normal tissues and were correlated with PPS in patients receiving chemotherapy.
Comment 6: The authors need to explore the role of apoptosis resistance mechanisms in greater detail.
Response 6: I fully agree with this suggestion. Our subsequent investigation will examine associated proteins in apoptotic pathways, including p53, Bax, and BCl2, as well as proteins implicated in drug resistance, such as p-glycoprotein, beta-tubulin, PI3K/AKT, and Glutathione S-transferase 1.
Comment 7: I suggest that the authors include additional visual representations of the pathways identified in KEGG enrichment analyses.
Response 7: The suggestion has been included in the supplementary.
Comment 8: I think the study would benefit from further analysis of the tumor microenvironment's impact on the observed resistance.
Response 8: I fully agree with this suggestion. G-CSF exerts a direct effect on tumor cells and can enhance pro-tumorigenic immune cell phenotypes within the tumor microenvironment. Therefore, the effect of G-CSF on chemotherapy resistance, both in direct tumor cells and through modulation of the tumor microenvironment, is possible.
Comment 9: The authors need to ensure that the limitations of the study are adequately discussed.
Response 9: The suggestion has been included in the discussion section.
“Nevertheless, our current investigation is limited by the lack of tumor tissue samples from patients who received this specific doublet regimen. Furthermore, the functions of CSF3 regarding chemotherapeutic response remain unexplored. Consequently, addi-tional functional study in both parental H1792 and resistant cells, H1792/Car and H1792/Pac, as well as clinical studies in tissue samples are necessary to validate the role of CSF3 as a biomarker in this context.”
Reviewer 4 Report
Comments and Suggestions for AuthorsPritsana et al. submitted the manuscript entitled: Transcriptomic Profiling of Carboplatin- and Paclitaxel-Resistant Lung Adenocarcinoma Cells Reveals CSF3 as a Potential Biomarker for the Carboplatin plus Paclitaxel Doublet Regimens, in which the authors investigated on unrevealed potential targets in drug resistant lung adenocarcinoma cells. The authors first induced and validated H1792 cells with Car and Pac resistance. The drug-resistant cell lines showed different cell cycle profiles when treated with drugs. Related apoptosis comparison together with cell cycle pattern implied potential targets exist in related pathway. Then, the authors performed transcriptomic analysis combining with bioinformatic analysis and identified multiple DEGs. Further, the authors performed survival analysis and identified IL1A and CSF3 as potential biomarkers, and downregulations of mRNA expression were validated by RT-qPCR. I believe this work will be of interest to potential readers of CIMB provided the authors can address some minor issues properly.
My comments are as follows:
1. The authors claimed CSF3 as a biomarker of Lung Adenocarcinoma but the experiments are based on H1792. So this conclusion is applicable to H1792 only or common to all (or most) of clinical patients? The authors are suggested to check and analysis other dataset for further validation.
2. The authors did cell cycle analysis and found cell cycle arrest in the parental cell line. But the authors seemed not to address well on the relations between identified targets and cell cycle. Is CSF3 (or other identified targets) related to cell cycle change? If not, why did the authors want to check the cell cycles and apoptosis?
3. The authors focused on mRNA expression changes of IL1A and CSF3 between parental and drug-resistant cells. 3a) did the authors check expression in normal cells as well? Will targeted therapy on CSF3 lead to toxicity? 3b) It is not appropriate to validate the expression change with RT-PCR, because the two targets identified by transcriptomic analysis, which is also based on mRNA expression. In this case, the authors should evaluate the expression from another aspect, eg. protein expression.
Author Response
Pritsana et al. submitted the manuscript entitled: Transcriptomic Profiling of Carboplatin- and Paclitaxel-Resistant Lung Adenocarcinoma Cells Reveals CSF3 as a Potential Biomarker for the Carboplatin plus Paclitaxel Doublet Regimens, in which the authors investigated on unrevealed potential targets in drug resistant lung adenocarcinoma cells. The authors first induced and validated H1792 cells with Car and Pac resistance. The drug-resistant cell lines showed different cell cycle profiles when treated with drugs. Related apoptosis comparison together with cell cycle pattern implied potential targets exist in related pathway. Then, the authors performed transcriptomic analysis combining with bioinformatic analysis and identified multiple DEGs. Further, the authors performed survival analysis and identified IL1A and CSF3 as potential biomarkers, and downregulations of mRNA expression were validated by RT-qPCR. I believe this work will be of interest to potential readers of CIMB provided the authors can address some minor issues properly.
Comment 1: The authors claimed CSF3 as a biomarker of Lung Adenocarcinoma but the experiments are based on H1792. So this conclusion is applicable to H1792 only or common to all (or most) of clinical patients? The authors are suggested to check and analysis other dataset for further validation.
Response 1: Certainly, H1792 cannot fully explain the drug resistance mechanism in all patients, as the emergence of drug resistance may result from multiple genes or different risk factors. The author chose H1792 due to its comparability to patients undergoing doublet chemotherapy, as it was derived from a patient with advanced stage. For other profiling datasets in other ADC cell lines which are model of combined drug usage, there are currently no constructed. To increase confidence in the application of CSF in clinical practice, the author will conduct a further evaluation of CSF protein levels in Formalin-Fixed Paraffin-Embedded (FFPE) tissue of patients receiving doublet chemotherapy with the CAR plus Pac and Cis plus Gem combination regimens. This assessment aims to determine the discriminatory power of CSF as a predictor for chemotherapy response or regimen selection.
Comment 2: The authors did cell cycle analysis and found cell cycle arrest in the parental cell line. But the authors seemed not to address well on the relations between identified targets and cell cycle. Is CSF3 (or other identified targets) related to cell cycle change? If not, why did the authors want to check the cell cycles and apoptosis?
Response 2: The author examined the cell cycle and apoptosis to verify that the generated drug-resistant cells were exactly drug-resistant. The results showed that the drug-resistant cells did not undergo cell cycle arrest and had less death cells compared to parental cells at the same dose. The author will further investigate the relationship of CSF3 with the cell cycle and apoptosis through functional analysis, focusing on both loss and gain of expression of this gene, as well as examining the associated pathways involved in drug resistance mechanisms.
Comment 3: The authors focused on mRNA expression changes of IL1A and CSF3 between parental and drug-resistant cells.
Comment 3a: did the authors check expression in normal cells as well? Will targeted therapy on CSF3 lead to toxicity?
Response 3a: In this study, the objective was to establish a resistant model for doublet chemotherapy and to find the genes involved in drug resistance. For the gene expression level, the author used GEPIA analysis and found that CSF3 showed significantly reduced expression in lung adenocarcinoma tissues compared to normal tissues. However, for the expression level in clinical samples, the author will further study in FFPE tissue in adjacent normal tissue of patient compared to tumor tissues.
Comment 3b: It is not appropriate to validate the expression change with RT-PCR, because the two targets identified by transcriptomic analysis, which is also based on mRNA expression. In this case, the authors should evaluate the expression from another aspect, eg. protein expression.
Response 3b: It is crucial to conduct technical validation using a technique that measures gene expression level, as this study employed transcriptomic sequencing techniques to identify differentially expressed genes between parental H1792 cells and resistant cells, H1792/Car or H1792/Pac. Nevertheless, the author will investigate protein expression in both cell lines using western blotting and FFPE tissue using immunohistochemistry, as protein expression is the endpoint of central dogma.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe WB data is important for the conclusion of the current study.
Reviewer 3 Report
Comments and Suggestions for AuthorsNo more comments
