Secretion of Extracellular Microvesicles Induced by a Fraction of Escherichia coli: Possible Role in Ovarian Cancer with Bacterial Coinfections

Response 1: We sincerely appreciate you excellent feedback and very positive evaluation of the manuscript. We are truly honored by your acknowledgement that the work is well-done and for applauding our efforts. We have meticulously reviewed and addressed de minor points you suggested, ensuring the integrity of the analysis is maintained.

Comments 2: Please revise the abstract scientific, logical and to the point. Please arrange keywords in ascending alphabetic order. The introduction should be shortened as it contains some irrelevant information. I suggest only three to four paragraphs are enough with last paragraph including hypothesis, aims and objectives. Please add some statistical analysis to the figure 3B, C. The discussion should focus on the interplay between bacteria and ovarian cancer. Also add one graphical abstract or schematic illustration to the study.

Response 2: Agree. We have, accordingly, modified the abstract, keywords in ascending alphabetic order, the introduction and discussion to emphasize this point.

The statistical analysis was added to figure 3B, C. n=3 (B) and n=6 (C) were used to one-way one-way Anova with Bonferroni’s post hoc test, * P<0.05, ** P<0.01

The graphical abstract is uploaded to the journal's system, so we can add the image to this document.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1:    (in red)

Comments 1:

The authors investigate the role of extracellular vesicles polydispersity secreted by ovarian cancer cells in response to bacterial components, aiming to elucidate a potential communication pathway between ovarian cancer and the bacterial microenvironment. For that, authors incubate a fraction of Gram-negative E. coli bacteria with SKOV-3 cell line, and profile the proteomic composition of polydisperse extracellular vesicles (PVEs). Then, they study the morphology of SKOV-3 cells and compare it with healthy and ovarian cancer histological sections. Subsequently, they evaluated the location and characteristics of large PVEs in cells. Finally, they evaluated the enzymatic activity of the PEV extract and intact PEV vesicles, finding increased catalytic activity in intact PEV.

While experiments show that E. coli membrane is an inducer of PEV in SKOV-3 cells, there is a lack of sufficient experimental results that support this mechanism as a potential communication pathway between the ovarian tumor cells and the tumor microenvironment.

I suggest the authors to expand the experimental setup to other ovarian cancer cell lines for robustness of the observed effects, and see whether it is a cell-specific phenomenon or it is universal to ovarian cancer cell lines.

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have modified the abstract (line 29 and 41) and analyses that our results suggest that in cases where an interaction between ovarian cancer cells, with characteristics like those of the SKOV-3 cell line, occurs in contact with a set of bacterial components similar to the one we used, these cells could well increase the overall secretion of polydisperse extracellular vesicles. The stimulation of cells or alterations in them that favor tumor progression have already been observed and reported previously in other types of cancer by several research groups, as described in the introduction (paragraphs 2, 3, and 4) and later in the discussion.

We currently do not have other ovarian cancer cell lines at our disposal, abd therefore cannot promptly conduct those experiments in the coming months, but, we have considered this for future studies. The Escherichia coli fraction that we have used in the present work stimulates other cell lines to secrete polydisperse extracellular vesicles, among them we have been able to test it to stimulate the J774A.1 line (originally started from histiocytic sarcoma and is widely used as a model for studying macrophages) which resulted in the publication Sierra-López et al 2025, likewise, so we believe that there may be multiple cell lines and different types of cancers that could be stimulated to secrete PEVs.

Comments 2:

I also suggest they include additional experiments to prove cell-cell communication and the potential role of PVE in ovarian cancer progression, such as viability assay with PBMCs or K562, invasion and migration assay in Matrigel.

Response 2:

Agreed. Consequently, we have tested the interaction between SKOV-3 EVPs and EAhy926 cells, which we have added to the supplementary material. Interestingly, we have observed EAhy926 cells actively moving, others secreting large EVPs, and some cells detaching from the cell culture surface. These are aspects that we believe should be studied in depth in future work.

4. Response to Comments on the Quality of English Language:

Besides, I strongly recommend they revise the text for typos, clarification, and English. For example:

Point 1:

·         Lines 65-66, this sentence lacks meaning: Detect bacteria such as Mycoplasma hominis and Chlamydia trachomatis has in ovarian cancer, suggesting a possible role in chronic inflammation or co-carcinogenesis

Response 1: We have changed the sentence:

Detection of bacteria such as Mycoplasma hominis and Chlamydia trachomatis has been reported in ovarian cancer, suggesting a possible role in chronic inflammation or co-carcinogenesis.

Point 2:

·         Lines 105-107, "in abundance in terms of size" is incorrect: We have previously reported that a fraction of E. coli bacteria is capable of inducing macrophages to release PEVs in abundance in terms of size.

·          

Response 2: We have changed the sentence:

We have previously reported that a fraction of E. coli bacteria is able to induce macrophages to release abundant PEVs,

Point 3:

·         Lines 114-117, there is dot at the end at the end with a repetitive sentence: Our findings could offer a new perspective on the pathogenesis of this disease and the role of bacterial infections as stimulants to cancer cells with phenotypes related to greater aggressiveness.to cancer cells with phenotypes related to greater aggressiveness.

·          

·         Response 3:    (in red)

We have looked for places in the text where statements have been repeated, and corrected them.

·         Point 4:

·         Figure 1, Results stated in the text do not match with Figure 1A since the most abundant is at ~60 KDa : The most intense band of SBMF corresponds to the 39.3 kD 125 OmpF protein.

Response 4: Thank you for reviewing the description of the SDS-PAGE gels. We've included the image and annotated description below:

The most intense band of SBMF corresponds to the 39.3 kD OmpF protein. Other proteins were also identified, such as OmpA (37.2 kD), OmpC (53.7 kD), ATP synthase subunits (atpD of 50.3 kD, atpA of 55.2 kD), maltoporin LamB (49.9 kD), and several lipoproteins and other outer membrane proteins (OmpX, Lpp, RcsF) (Sierra-López et al., 2025).

Column 4 showed the profile of polydisperse extracellular vesicles (PEVs) obtained from unstimulated cells, with a main band near 45 kDa, and other bands above 45 kDa and even above 200 kDa, as well as between 14 and 21.5 kDa. Column 5, with vesicles from stimulated cells, revealed a similar profile, but with more intense bands.