Gal-3 Protein Expression and Localization in Prostate Tumours

Round 1

Reviewer 1 Report (Previous Reviewer 1)

I consider that the manuscript has improved considerably and has an adequate approach.

The only observation I have is the format. The discussion section is long, but it is only divided into 3 paragraphs, of which 2 paragraphs are too long, which makes the reading a bit more complex. I suggest dividing the content of the "discussion" section into more paragraphs (More final points in the writing). Also the last paragraph is about limitations and perspectives, which is fine, but I suggest that the last paragraph be a short conclusion.

Author Response

Comments and Suggestions for Authors (Reviewer 1)

I consider that the manuscript has improved considerably and has an adequate approach.

The only observation I have is the format. The discussion section is long, but it is only divided into 3 paragraphs, of which 2 paragraphs are too long, which makes the reading a bit more complex. I suggest dividing the content of the "discussion" section into more paragraphs (More final points in the writing). Also the last paragraph is about limitations and perspectives, which is fine, but I suggest that the last paragraph be a short conclusion.

In light of this comment, the discussion section has been changed, having been split into more paragraphs for readability. Additionally, the following short conclusion was added as the last paragraph: “Given these results, Gal-3 deserves to be further explored in the context of PCa, especially its diagnostic role.”

Reviewer 2 Report (Previous Reviewer 2)

The manuscript has been significantly improved and now warrants publication in Current Oncology

Author Response

Please see the attachment.

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript reports a series of data that are relevant from the point of view of the molecular biology of prostate cancer. However, I have several observations:

Title: Preclinical validation of urinary Gal-3 protein as a target for PCa in prostate tissue

1.- The title does not reflect the content or the usefulness of what was reported: the word "Preclinical" generally denotes in vitro models or animal models, which does not agree with the study of patient samples used.

2.- It is not observed that they are validating its clinical utility, but rather reporting molecular characteristics of PCa, but its use is not validated in any way.

The cut-off point for low or high expression is not clear. They mention that it is by location, but one would expect them to use percentage of cell positivity or intensity of positivity. With values on a numerical scale (ordinal or numerical) they could obtain a cut-off point with a ROC CURVE and analyze whether it is a predictive factor for cancerous tissue. Additionally, they do not make comparisons with serum or urinary levels, which could have a clinical utility. The latter is made clear in discussions, however, it is felt that the article should include it because of the idea generated by the title of the manuscript.

I also consider that due to the low expression of GAL-3 in tumor tissue, it might not be useful as a target. It would serve more as a protein that would have to be re-activated, while the word target is more used for molecules that have to be blocked or inactivated.

I consider that it is a good report that describes the expression of GAL3 in cancerous and normal tissue. This has its value, since it would be consistent with previous studies, but I consider that the title and the expectation of this generation are not consistent. For this reason, I suggest restructuring it by adding correlations with values of serum or urinary levels to assess its possible clinical utility, or else restructuring it as a manuscript whose objective is only to assess the variations in expression of GAL 3 in prostate tissue.

Author Response

Comments and Suggestions for Authors (Reviewer 1)

The manuscript reports a series of data that are relevant from the point of view of the molecular biology of prostate cancer. However, I have several observations:

Title: Preclinical validation of urinary Gal-3 protein as a target for PCa in prostate tissue

1.- The title does not reflect the content or the usefulness of what was reported: the word "Preclinical" generally denotes in vitro models or animal models, which does not agree with the study of patient samples used.

2.- It is not observed that they are validating its clinical utility, but rather reporting molecular characteristics of PCa, but its use is not validated in any way.

In light of these comments, the title of the article has been changed to “Gal-3 protein expression and localization in prostate tumours”.

The cut-off point for low or high expression is not clear. They mention that it is by location, but one would expect them to use percentage of cell positivity or intensity of positivity. With values on a numerical scale (ordinal or numerical) they could obtain a cut-off point with a ROC CURVE and analyze whether it is a predictive factor for cancerous tissue. Additionally, they do not make comparisons with serum or urinary levels, which could have a clinical utility. The latter is made clear in discussions, however, it is felt that the article should include it because of the idea generated by the title of the manuscript.

We appreciate this comment. To clarify the cut-off point for low or high expression the following sentence was added to the manuscript: “Based on frequency distribution of data, tumour samples were categorized as low or high Gal-3 expression. For that, an IHC score of 2 corresponding to the percentile 75, was defined as a cut-off for high and low expression.” The cut-off point was only used for performing the survival curves. Gal-3 expression graphs were constructed using data from the IHC score (the sum of the intensity and extension scores). Regarding Gal-3 localization graphs, a numerical scale (1 or 2) was adopted to translate its cytoplasmic (1) or nuclear + cytoplasmic localization (2), respectively. We apologize if this information is not clear in the manuscript, and we hope this explanation clarifies.

A ROC curve using Gal-3 protein expression in tumour and normal prostate tissue was performed. It was observed that Gal-3 protein expression levels are useful in discriminating between tumour and normal prostate tissues and are a predictive factor for cancerous tissue (AUC= 0.9583, p-value<0.001). The following section was added to the results section of the manuscript: “To assess the utility of Gal-3 in discriminating between tumour and normal prostate tissues, a receiver operating characteristic (ROC) curve using Gal-3 protein expression levels in theses samples was performed. This ROC curve yielded an area under the curve (AUC) value of 0.9583 (95% CI: 0.9042 – 1.012) and a p-value<0.001, demonstrating that Gal-3 protein expression levels are useful in discriminating between tumour and normal prostate tissues (Figure 3)”.

I also consider that due to the low expression of GAL-3 in tumor tissue, it might not be useful as a target. It would serve more as a protein that would have to be re-activated, while the word target is more used for molecules that have to be blocked or inactivated.

Considering this comment, the target word referring to Galectin-3 is no longer used.

I consider that it is a good report that describes the expression of GAL3 in cancerous and normal tissue. This has its value, since it would be consistent with previous studies, but I consider that the title and the expectation of this generation are not consistent. For this reason, I suggest restructuring it by adding correlations with values of serum or urinary levels to assess its possible clinical utility, or else restructuring it as a manuscript whose objective is only to assess the variations in expression of GAL 3 in prostate tissue.

According to this comment, the manuscript was restructured with the aim of assessing only variations in Gal-3 expression in prostate tissue.

 

Reviewer 2 Report

In this manuscript, the authors demonstrated that Gal-3 protein expression significantly decreased in tumor prostate tissue compared with normal adjacent normal prostate, which is consistent with their previous study showing the urinary levels of Gal-3 from PCa patients. Furthermore, hypermethylation of the Gal-3 promoter could be one of the MOAs underlying its downregulation in tumor tissue compared to normal tissue However, Gal-3 protein levels did not significantly impact the survival or relapse of PCa patients. Therefore, these findings highlight the diagnostic potential of Gal-3 in PCa, but it seems not to be a prognostic biomarker on patient survival. Although the results are potentially interesting, there are two significant issues to be addressed before considering its acceptance.

 Major comments:

1. Could the authors supplement some experiment to elucidate what’s the MOA (mechanism of action) causing hypermethylation of the Gal-3 promoter in PCa tissues if possible?

If not, the authors should provide some reasonable explanations in the discussion section.

2. A recent publication (doi: 10.1007/s11033-022-08207-1) indicated that the degree of Gal-3 methylation varied by disease stage, with early stages (I and II) showing strong methylation and advanced stages (III and IV) showing mild methylation. This methylation profile seems to be more significant for the early diagnosis of PCa. Did the authors observe the similar results in this study?

Author Response

Comments and Suggestions for Authors (Reviewer 2)

In this manuscript, the authors demonstrated that Gal-3 protein expression significantly decreased in tumor prostate tissue compared with normal adjacent normal prostate, which is consistent with their previous study showing the urinary levels of Gal-3 from PCa patients. Furthermore, hypermethylation of the Gal-3 promoter could be one of the MOAs underlying its downregulation in tumor tissue compared to normal tissue. However, Gal-3 protein levels did not significantly impact the survival or relapse of PCa patients. Therefore, these findings highlight the diagnostic potential of Gal-3 in PCa, but it seems not to be a prognostic biomarker on patient survival. Although the results are potentially interesting, there are two significant issues to be addressed before considering its acceptance.

 Major comments:

1. Could the authors supplement some experiment to elucidate what’s the MOA (mechanism of action) causing hypermethylation of the Gal-3 promoter in PCa tissues if possible?

If not, the authors should provide some reasonable explanations in the discussion section.

In response to this comment, the discussion section has been changed and the following section has been added:” The mode of operation of Gal-3 promoter hypermethylation in relation to the survival or relapse of PCa patients is not fully understood. However, it is believed that the methylation of the Gal-3 promoter could potentially impair the expression of Gal-3 protein in PCa cells, leading to increased tumorigenic activity. Furthermore, the inhibition of Gal-3 protein expression could reduce the tumour suppressor activity of Gal-3, resulting in a more aggressive cancer phenotype. Additionally, the reduced expression of Gal-3 protein in PCa cells could lead to a decrease in the antitumor immune response, which could contribute to the poor prognosis of PCa patients.”.

2. A recent publication (doi: 10.1007/s11033-022-08207-1) indicated that the degree of Gal-3 methylation varied by disease stage, with early stages (I and II) showing strong methylation and advanced stages (III and IV) showing mild methylation. This methylation profile seems to be more significant for the early diagnosis of PCa. Did the authors observe the similar results in this study?

In line with this comment, the discussion section has been changed and the following section has been added: “The scope of this study was to evaluate variations in Gal-3 expression in tumour tissue not the mechanism of action behind it. However, it would be very interesting to evaluate Gal-3 promoter methylation in the tissue samples analysed in this study. As in PCa, a different degree of Gal-3 methylation has been reported according to the stage of the disease, with stages I and II showing strong methylation and stages III and IV a mild methylation [37], which is in accordance with different degrees of Gal-3 protein expression in stages II and III reported elsewhere [44]. In fact, in our study, the decrease in Gal-3 expression was more pronounced in stage II than in stage III tumours.”

Reviewer 3 Report

The authors have extended their previous work where they show gene and protein expression levels and their involvement in PCa-related biological processes. In this study  the authors the expression of Gal-3  in tumours and normal prostate tissue using  immunohistochemistry. The study utilized  48 tumour prostate tissues, 8 normal prostate tissues and 14 adjacent normal prostate tissues. Gal-3 staining was found to be decreased in tumour tissues with GS 5-8 and pT2 and pT3 stages compared with normal prostate, no correlation was found between Gal-3 expression and PCa progression. The expression of  Gal-3 did not significantly affect the survival and relapse of these PCa patients. In conclusion, the authors propose Gal-3 as a promising urinary marker for PCa diagnosis.

The study is a continuation of the authors' study, the finding is matching their previous findings. The experiments were done meticulously.

Some points

1.     The legends of the table should be described in detail so easy for readers.

2.     The scale of the   Figure is not visible  

 

3.     The p-value of Figure 4 should be mentioned ( although it is not significant)

Author Response

Comments and Suggestions for Authors (Reviewer 3)

The authors have extended their previous work where they show gene and protein expression levels and their involvement in PCa-related biological processes. In this study the authors the expression of Gal-3 in tumours and normal prostate tissue using  immunohistochemistry. The study utilized 48 tumour prostate tissues, 8 normal prostate tissues and 14 adjacent normal prostate tissues. Gal-3 staining was found to be decreased in tumour tissues with GS 5-8 and pT2 and pT3 stages compared with normal prostate, no correlation was found between Gal-3 expression and PCa progression. The expression of Gal-3 did not significantly affect the survival and relapse of these PCa patients. In conclusion, the authors propose Gal-3 as a promising urinary marker for PCa diagnosis.

The study is a continuation of the authors' study, the finding is matching their previous findings. The experiments were done meticulously.

Some points

 

1. The legends of the table should be described in detail so easy for readers.

The following caption has been added to Table 1: “Galectin-3 protein expression was evaluated in tissue slides comprising normal prostate tissues removed from patients without PCa (tumour-free prostates) (n=8), adjacent normal prostate (only normal tissue removed from patients diagnosed with PCa) (n=14) and tumour prostate tissues removed from PCa patients (n=48). This table presents the clinical data of the subjects enrolled in this study, including survival and relapse data. pT2a stage refers to a tumour in one half or less of only one side of the prostate, in pT2b stage the tumour is localized in more than half  of  one  side  of  the  prostate and in pT2c the tumour involves both lobes of the prostate gland.  The pT3a and pT3b stages implies that tumour extends beyond the prostate capsule or invades seminal vesicle(s), respectively and in pT4 the tumour has spread into other body organs. Abbreviations: GS: Gleason Score; PCa, prostate cancer.”

2. The scale of the   Figure is not visible  

Figure 1 has been enhanced to make the scale of the figure visible.

3. The p-value of Figure 4 should be mentioned (although it is not significant)

Following this request, the p-value of Figure 4 was added.