Investigating the Role of SNAI1 and ZEB1 Expression in Prostate Cancer Progression and Immune Modulation of the Tumor Microenvironment

Round 1

Reviewer 1 Report

The manuscript "Epithelial-to-mesenchymal transition markers SNAI1 and ZEB1 are associated with immune modulation of the tumor microenvironment during prostate cancer progression." attempts at identification of Differentially Expressed Genes (DEGs) and pathways associated with biochemical recurrence (BCR) and clinical risk. However, the study lacks robust experimental support for the proposed hypothesis. The manuscript primarily relies on available data from various databases, and there is a deficiency in incorporating distinct experiments to assess signaling pathways for COL1A1, COL1A2, and COL3A1,etc protein in colon cancer cells/tumors. Depending solely on database screening appears inadequate to substantiate the hypothesis presented in the manuscript. A notable gap exists in the form of more in-depth experimentation, which is crucial for a comprehensive evaluation of the hypothesis including risk analysis. There are too many unnecessary tables and database figures in the manuscript that are irrelevant. These extraneous elements should be removed to enhance the clarity and relevance of the manuscript.  Therefore, the manuscript requires significant attention, particularly in improving punctuation, grammar, and overall readability. Additionally, incorporating substantive experimental evidence will strengthen the validity and impact of the study.

Please see comments to author.

Author Response

The text was revised in many places to improve reading clarity.

Reviewer 2 Report

1.       Lines 142-144- RNA profiling was performed using both the NanoString PanCancer and the Imune Profiling Panels (NanoString Technologies Inc., Seattle, WA, USA) [32] according to the manufacturer’s instructions and previously described [33].

I would briefly explain the method/machine as it is not that well-known method.

2.       Lines 162-164 It is a bit unclear about how the quartiles were assigned, especially the syntagma “below Q3, the upper quartile“. I understood that you asigned as low expression everything bellow Q3, and the rest (q4) was high. Is that correct? If yea, I would erase „the upper quartile“ as it is confusing.

3.       Line 268- the same as above

4.       I am a bit puzzled why you chose high expression ZEB1 to identify genes (Figure 2A-D) when reduced ZEB1 expression is associated with aggressive PC?

5.       I would include Table S5 into the manuscript as I find it valuable.

6.       Several other DEGs have been previously associated with higher Gleason scores, including COL1A2, INHBA (subunit of Activin A) [44,45].

I believe this sentence is missing and.

7.       Line 407 extra comma

8.       Line 412 increased

9.       I think the last segment of the discussion could benefit from adding a sentence about how prostate cancer recruits more mast cells than normal prostate epithelial cells, leading prostate cancer more resistant to chemotherapy and radiotherapy (Xie H., Li C., Dang Q., Chang L. S., Li L. Infiltrating mast cells increase prostate cancer chemotherapy and radiotherapy resistances via modulation of p38/p53/p21 and ATM signals. Oncotarget. 2016; 7: 1341-1353. Retrieved from https://www.oncotarget.com/article/6372/text/).

Author Response

1. Lines 142-144- RNA profiling was performed using both the NanoString PanCancer and the Imune Profiling Panels (NanoString Technologies Inc., Seattle, WA, USA) [32] according to the manufacturer’s instructions and previously described [33]. I would briefly explain the method/machine as it is not that well-known method.

At lines 144-148 we have added the following text to briefly explain the principle of the NanoString methodology:

“Briefly, RNA profiling using the NanoString methodology relies on digital molecular barcoding and direct hybridization to quantify gene expression levels across multiple genes simultaneously. This methodology has been shown to offer high sensitivity, specificity, and the ability to analyze of gene expression patterns from small amounts of RNA, as described previously [33].”

2. Lines 166-168 It is a bit unclear about how the quartiles were assigned, especially the syntagma “below Q3, the upper quartile“. I understood that you assigned as low expression everything below Q3, and the rest (q4) was high. Is that correct? If yes, I would erase, the upper quartile“ as it is confusing.
3. Line 252 - the same as above

Yes, low expression everything was assigned below Q3. We thank the reviewer for pointing out this confusing wording, which has been corrected in both the suggested places in comments 2 and 3.

4. I am a bit puzzled why you chose high expression ZEB1 to identify genes (Figure 2A-D) when reduced ZEB1 expression is associated with aggressive PC?

Part of our analysis was investigating the role of ZEB1 downstream genes, so we expected increased expression of this transcription factor to identify regulated target genes.  Consistent with this rationale, many of our significantly expressed DEGs were found to be known ZEB1 target genes in the Harmonizome database. The prognostic role of reduced expression of ZEB1 is discussed at Lines 355-361.

5. I would include Table S5 into the manuscript as I find it valuable. This table is now Table 3 in the main manuscript at Line 300
6. Several other DEGs have been previously associated with higher Gleason scores, including COL1A2, INHBA (subunit of Activin A) [44,45]. I believe this sentence is missing and.

Corrected

7. Line 407 extra comma

Corrected

8. Line 412 increased

Corrected

9. I think the last segment of the discussion could benefit from adding a sentence about how prostate cancer recruits more mast cells than normal prostate epithelial cells, leading prostate cancer more resistant to chemotherapy and radiotherapy (Xie H., Li C., Dang Q., Chang L. S., Li L. Infiltrating mast cells increase prostate cancer chemotherapy and radiotherapy resistances viamodulation of p38/p53/p21 and ATM signals. Oncotarget. 2016; 7: 1341-1353. Retrieved from https://www.oncotarget.com/article/6372/text/).

The following text and the suggested new citation have been added at the end of the discussion (lines 436-437):

“Interestingly, mast cell infiltration in PCa has been associated with chemotherapy resistance through activation of p38/p53/p21 signaling [82].”

Author Response File: Author Response.pdf

Reviewer 3 Report

The paper deals with biomarkers discovery regarding TME and the progression of PCa. The paper is well written and uses technologically sound methodology including NanoString gene expression analysis. The reported results demonstrate that the increased expression of both ZEB1 and SNAI1 (TME related genes) inSNAI1 and ZEB1 was associated with immune checkpoint expression, thus suggesting the future manipulations of these genes as relevant tools to fight PCa progression. Other biomarkers found are less novel but also of interest. The study includes confirmatory analysis using TCGA-PRAD cohort.

Points of potential improvement:

1.        The series of 51 prostate cases is limited since PCa is quite frequent as is the procedure of radical prostatectomy used to access the tissue. Probably a larger number of cases might be necessary for better results.  Regarding translation to clinically valuable research for instance. I suggest the authors to add a paragraph to comment on the power of sample size used in the current study.

2.        CAPRA score was used to split cases in risk groups of tumors progression, but this has limitations, and the authors should add a comment of this method to predict PCa progression.

3.        One problem with PCa research is the heterogeneity of this tumor which include different tumor nodules in each prostatectomy, each of the nodule with different Gleason score,  therefore, it is necessary to include in the manuscript how the authors handled this issue…How was selected the nodule? What was the Gleason score; was it related to the index lesion (larger nodule with highest GS). This has been recognized as important prognostic player in PCa.

4.        Was used paraffin embedded material or fresh frozen tissue?

5.        Was taken into account the presence of +surgical margins?

6.        Were the cases split in organ-confined or locally advanced for analysis…This type of studies need to avoid confounding factor as T stage category. Also, it could make the paper more understandible for the urologists. 

Author Response

Reviewer 3

The paper deals with biomarkers discovery regarding TME and the progression of PCa. The paper is well written and uses technologically sound methodology including NanoString gene expression analysis. The reported results demonstrate that the increased expression of both ZEB1 and SNAI1 (TME related genes) inSNAI1 and ZEB1 was associated with immune checkpoint expression, thus suggesting the future manipulations of these genes as relevant tools to fight PCa progression. Other biomarkers found are less novel but also of interest. The study includes confirmatory analysis using TCGA-PRAD cohort.

Points of potential improvement:

1. The series of 51 prostate cases is limited since PCa is quite frequent as is the procedure of radical prostatectomy used to access the tissue. Probably a larger number of cases might be necessary for better results.  Regarding translation to clinically valuable research for instance. I suggest the authors to add a paragraph to comment on the power of sample size used in the current study.

This study limitation is addressed in response to point 2 below.

2. CAPRA score was used to split cases in risk groups of tumors progression, but this has limitations, and the authors should add a comment of this method to predict PCa progression.

We thank the reviewer for pointing out the limitations of the CAPRA-S score. The following limitations of this study have been added to the discussion at lines 450-456:

“This study has some limitations. The CAPRA-S score, which was used to classify the groups according to tumor progression relies on pathological factors like Gleason score and tumor stage. While these are important, they may not fully capture the complexity of prostate cancer biology and its interaction with the host environment. Furthermore, our sample size of 51 cases, while limited, was aimed at providing pilot data to establish a connection between EMT and the immune TME in prostate cancer, thereby providing a basis for future clinical investigations with larger cohorts.” 

3. One problem with PCa research is the heterogeneity of this tumor which include different tumor nodules in each prostatectomy, each of the nodule with different Gleason score, therefore, it is necessary to include in the manuscript how the authors handled this issue…How was selected the nodule? What was the Gleason score; was it related to the index lesion (larger nodule with highest GS). This has been recognized as important prognostic player in PCa.

At lines 111-121:

“Smaller prostates were submitted for pathological assessment in its entirety according to the guidelines of the American College of Pathology. In cases where larger glands were partially sampled, we followed the protocol by submitting the entire tumor if grossly visible, along with the tumor, surrounding periprostatic tissue, and margins, including the entire apical and bladder neck margins. Additionally, we included the junction of each seminal vesicle with the prostate proper. If there was no grossly visible tumor, a systematic sampling strategy was used.  This involved taking slices from the posterior aspect of each transverse section, along with a mid-anterior block from each side. Additionally, we submitted samples including the entire apical and bladder neck margins, as well as the junction of each seminal vesicle with the prostate.”

At lines 136-138 :

“RNA extraction was performed on tissues containing tumor-rich areas, which were previously identified and marked by a pathologist (FPS) to represent the highest Gleason pattern.”

4. Was used paraffin-embedded material or fresh frozen tissue?

Paraffin material was used. Please see line 138.

5. Was taken into account the presence of +surgical margins?

The cohort under study comprises intermediate-risk disease, which is usually diagnosed at an earlier clinical stage compared to high-risk disease. However, the reviewer is correct it may still involve locally advanced disease with possible extension beyond the prostate gland.  In Supplementary Table S1 we detail the clinical findings for all patients in our cohort including TNM, ISUP, and identity cases with locally advanced disease.

6. Were the cases split in organ-confined or locally advanced for analysis…This type of studies need to avoid confounding factor as T stage category. Also, it could make the paper more understandible for the urologists. 

See above comment in response to question 5.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

In the updated manuscript “Investigating the Role of SNAI1 and ZEB1 Expression in Prostate Cancer Progression and Immune Modulation of the Tumor Microenvironment”, the authors did address all the previous concerns and now the manuscript is convincing and would help advance the understanding of role of SNAI1 and ZEB1 in prostate cancer. The manuscript is now updated, and I recommend this article for publication.

Reviewer 3 Report

The authors have addressed properly the requests done by this reviewer.