In Vitro Model Characterizing Carcinogenic Progression of HPV-Positive Oropharyngeal Cancer
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Line 394: “p120 expression levels was strongly noted” is an unclear construction. Perhaps they mean “high p120 levels were noted”. Overall I am opposed to the description of evaluation of comparative protein levels as “expression levels”, as most readers interpret expression levels as transcription levels, but I guess this terminology is not strictly incorrect.
Line 423: “respectfully” should be “respectively”
Guidry et al., 2019 and Makielski et al., 2019 do utilize oral keratinocyte raft cultures transfected with HPV18 and HPV16 respectively, and should be appropriately cited in this work.
Fig 2 – the resolution of very poor and must be improved – it was nearly unreadable.
The title refers to “Head and Neck Cancer”, while HPV cancers are indeed head and neck cancers they only make up about 25% of all head and neck cancers, and are more specifically oropharyngeal cancers (>60%), so it is strongly recommended that this term be used in the title.
Throughout the document the authors refer to keratinocytes transfected with HPV16 genomes as “infected”. While this transfection likely mimics HPV16 infection in most ways, it bypasses the normal stages of infection of attachment, entry and uptake mediated by the capsid. Also the HPV16 DNA is transfected as naked DNA episomes, and any chromatin (and chromatin markers) coming into the cell from the previous host cell are bypassed. While I would not imagine this would have a large effect, it remains possible. Hence, the term “HPV16 infected” should be replaced with “HPV16 transfected” throughout the document.
I find it interesting that genomic instability was noted in early passages of HPV16 transfected oral cell line differentiation, but subsided following integration (line 489). Why would this be given that the HPV16 oncoproteins are still expressed after integration? The manuscript would benefit from addressing or at least discussing this finding.
The observations that the keratin K8 transformation-associated keratin, as well as p16 both appear to be present at substantially higher levels in the later stage raft cultures created from male cells as compared to female cells should be noted. Particularly as oropharyngeal HPV cancers show higher incidence in males as compared to females.
Author Response
Reviewer 1
Comment 1: Line 394: “p120 expression levels was strongly noted” is an unclear construction. Perhaps they mean “high p120 levels were noted”. Overall I am opposed to the description of evaluation of comparative protein levels as “expression levels”, as most readers interpret expression levels as transcription levels, but I guess this terminology is not strictly incorrect.
Response 1: Thank you for the comment and suggestion. We have revised the sentences to incorporate your recommended wording (Lines 425-429). While we understand the concern regarding the use of “expression levels” to describe comparative protein levels, we believe this terminology remains the most effective way to convey the findings of this study.
Comments 2: Line 423: “respectfully” should be “respectively”
Response 2: Thank you for the comment. We have revised the manuscript to correctly use the term “respectively” and apologize for using the incorrect word preceding “respectively”.
Comments 3: Guidry et al., 2019 and Makielski et al., 2019 do utilize oral keratinocyte raft cultures transfected with HPV18 and HPV16 respectively, and should be appropriately cited in this work.
Response 3: Thank you for the comment and suggestion. We revised the sentence and added the previous study (Guidry et al., 2019), which derived raft tissues from HPV16/18 transfected oral keratinocytes, as a reference. The Makielski et al., 2019 paper could not be located; if the reviewer can provide a DOI, we will gladly include it (Lines 490-493).
Comments 4: Fig 2 – the resolution of very poor and must be improved – it was nearly unreadable.
Response 4: Thank you for the comment. When we submitted the manuscript, the figures were high quality and high resolution on our end; however, the resolution appears to have decreased during processing by the editorial office. We have contacted the editorial office to resolve this issue and ensure that both the editors and reviewers receive the high resolution versions. With the revised submission, we also provided individual files for each figure, and the editorial office confirmed that if the issue occurs again, they will supply the high resolution files to the reviewers and replace any low resolution images for publication. We apologize for this issue and understand that it made the review process difficult.
Comment 5: The title refers to “Head and Neck Cancer”, while HPV cancers are indeed head and neck cancers they only make up about 25% of all head and neck cancers, and are more specifically oropharyngeal cancers (>60%), so it is strongly recommended that this term be used in the title.
Response 5: Thank you for the comment and suggestion. We agree with your recommendation and have revised the manuscript title, replacing “Head and Neck” with “Oropharyngeal.”
Comment 6: Throughout the document the authors refer to keratinocytes transfected with HPV16 genomes as “infected”. While this transfection likely mimics HPV16 infection in most ways, it bypasses the normal stages of infection of attachment, entry and uptake mediated by the capsid. Also the HPV16 DNA is transfected as naked DNA episomes, and any chromatin (and chromatin markers) coming into the cell from the previous host cell are bypassed. While I would not imagine this would have a large effect, it remains possible. Hence, the term “HPV16 infected” should be replaced with “HPV16 transfected” throughout the document.
Response 6: Thank you for the comment and suggestion. We have revised the manuscript by replacing “HPV16 infected” with “HPV16 transfected” throughout the document. We also note that natural infection was used to produce HPV16 tonsillar keratinocyte lines, as indicated on Line 277; however, transfection was more efficient.
Comment 7: I find it interesting that genomic instability was noted in early passages of HPV16 transfected oral cell line differentiation, but subsided following integration (line 489). Why would this be given that the HPV16 oncoproteins are still expressed after integration? The manuscript would benefit from addressing or at least discussing this finding.
Response 7: Thank you for the comment and suggestion. We have addressed the subsiding of genomic instability following integration in the manuscript and added the appropriate references (Lines 537-545). This observation was first identified and discussed in our collaborative work (Chan et al., 2024), which is cited in the text. For context, genomic instability diminished after HPV16 integration because the primary source of ongoing DNA damage, episomal viral replication, was eliminated. In the study, all tonsillar keratinocyte lines accumulated structural variants rapidly during early passages while HPV16 remained episomal, demonstrating that instability preceded integration. After integration, four of the five lines showed a marked reduction in new structural variant accumulation, indicating that the genome stabilized post integration. These findings support the conclusion that although E6/E7 remain expressed, the loss of episomal replication stress following integration results in a more genetically stable cellular state.
Comment 8: The observations that the keratin K8 transformation-associated keratin, as well as p16 both appear to be present at substantially higher levels in the later stage raft cultures created from male cells as compared to female cells should be noted. Particularly as oropharyngeal HPV cancers show higher incidence in males as compared to females.
Response 8: Thank you for the comment and suggestion. We agree with your recommendation and have noted the increased presence of K8 and p16 in the later stage male raft cultures compared to the female cultures in Lines 621–624.
Reviewer 2 Report
Comments and Suggestions for Authors
This manuscript aims to describe a novel in vitro biological system to study progression of human papillomavirus-positive oropharyngeal cancers. This is a very important endeavour since no precancers have been identified in vivo to allow molecular studies. The authors establish HPV genome-transfected tonsillar keratinocytes of increasing passages in 3D raft cultures. This approach aims to mimic cancer progression, with higher passages potentially inducing cellular transformation.
The main issue with the manuscript is the poor quality of the figures, which makes it hard to assess the data. In the various immunofluorescence images, it is difficult to identify the features that the authors wish to point out. Higher magnifications of each image, separation of the antibody stains from the DAPI staining, scale bars and annotation of the basal layer and the top of the epithelium is required in each case. Where various features are discussed, these should be indicated with arrows. At present, the data do not support the conclusions of the manuscript.
In some cases, the raft tissues look very disrupted. For example, in Figure 3B, tissues grown from passage 32 and 37 keratinocytes look necrotic, while in Figure 4A P76 and Figure 4B P30, 48 and 76, and Figure 5 F and H, there are “blobs” of DAPI staining which obscure the antibody staining and suggest nuclear leakage has occurred. It would be important to stain tissues with antibodies against cell death markers to resolve this.
The authors used p16, p120 and COX5B staining as markers of cancer progression. However, the most important factor in HPV-associated cervical cancer progression is increased expression of the viral oncoproteins E6 and E7. Staining raft tissues green from cells of increasing passage to detect surrogate biomarkers of these, p53 and Rb, would give a far better analysis of the relevance of progression of the tissues to HPV-associated oropharyngeal cancer.
Author Response
Comments 1: The main issue with the manuscript is the poor quality of the figures, which makes it hard to assess the data. In the various immunofluorescence images, it is difficult to identify the features that the authors wish to point out. Higher magnifications of each image, separation of the antibody stains from the DAPI staining, scale bars and annotation of the basal layer and the top of the epithelium is required in each case. Where various features are discussed, these should be indicated with arrows. At present, the data do not support the conclusions of the manuscript.
Response 1: Thank you for the comment. When we submitted the manuscript, the figures were high quality and high resolution on our end; however, the resolution appears to have decreased during processing by the editorial office. We have contacted the editorial office to address this issue and ensure that both the editors and reviewers receive the correct high resolution versions. In the revised submission, we also provided individual files for each figure, and the editorial office confirmed that, should the issue recur, they will supply the high resolution files to the reviewers and replace any low resolution images for publication. We apologize for this problem and understand that it made the review process difficult. Additionally, we have revised all figures to include separate staining panels (antibody stain, DAPI, and merged images), annotations of the basal layer, and arrows in the images highlighting features discussed in the text. We hope these improvements meet your expectations, and we appreciate your feedback.
Comments 2: In some cases, the raft tissues look very disrupted. For example, in Figure 3B, tissues grown from passage 32 and 37 keratinocytes look necrotic, while in Figure 4A P76 and Figure 4B P30, 48 and 76, and Figure 5 F and H, there are “blobs” of DAPI staining which obscure the antibody staining and suggest nuclear leakage has occurred. It would be important to stain tissues with antibodies against cell death markers to resolve this.
Response 2: Thank you for the comment. Regarding the H&E stainings in Figure 3B (passages 32 and 37), although the tissue may appear disrupted, we disagree with the interpretation that it is necrotic. The progressive loss of epithelial organization observed in these sections reflects the dysplastic and carcinogenic changes inherent to our raft culture system. As the tissues advance toward malignant transformation, architectural integrity becomes increasingly disrupted; however, this pattern is distinct from true necrosis. Necrotic oral epithelium typically exhibits ghost cell morphology, karyolysis, and complete loss of cellular detail, none of which are present in our samples. Thus, the observed disruption is consistent with malignant progression rather than necrotic tissue.
Regarding the “blobs” of DAPI staining in Figure 4A (P76) and Figure 4B (P30, P48, and P76), these signals are not indicative of nuclear leakage. Instead, they represent the formation of keratin pearls, a hallmark feature of squamous differentiation during progression toward carcinoma. Keratin pearl formation is a well documented phenomenon in oral squamous cell carcinoma, arising from concentric layers of keratinizing malignant squamous cells. As described in SCC histopathology, keratin pearls may contain retained nuclei due to incomplete keratinization, dyskeratosis, or entrapment of malignant squamous cells within the keratinizing layers—processes characteristic of aberrant squamous maturation rather than tissue necrosis. The DAPI positive structures observed in our raft cultures are therefore consistent with keratin pearl formation and not nuclear leakage.
Refs:
- Narayan B, Rajoria S, Urs AB, Kumar P, Augustine J. Lifting the lid over the pearl: A histological insight. J Oral Maxillofac Pathol. 2023 Apr-Jun;27(2):399-401. doi: 10.4103/jomfp.jomfp_389_22. Epub 2023 Jul 13. PMID: 37854906; PMCID: PMC10581307.
- Keerthika R, Chandra A, Agrawal R. Exotic Keratin Pearl Degradation Mechanism by Giant Cells in Oral Squamous Cell Carcinoma and its Plausible Hypothesis. J Maxillofac Oral Surg. 2024 Oct;23(5):1093-1095. doi: 10.1007/s12663-024-02319-w. Epub 2024 Aug 31. PMID: 39376769; PMCID: PMC11455730.
- Jaimes N, Zalaudek I, Braun RP, Tan BH, Busam KJ, Marghoob AA. Pearls of Keratinizing Tumors. Arch Dermatol. 2012;148(8):976. doi:10.1001/archdermatol.2011.3475
Comments 3: The authors used p16, p120 and COX5B staining as markers of cancer progression. However, the most important factor in HPV-associated cervical cancer progression is increased expression of the viral oncoproteins E6 and E7. Staining raft tissues green from cells of increasing passage to detect surrogate biomarkers of these, p53 and Rb, would give a far better analysis of the relevance of progression of the tissues to HPV-associated oropharyngeal cancer.
Response 3: Thank you for the comment and suggestion. We agree with your recommendation and have added two additional figures containing p53 and Rb immunofluorescence stainings as surrogate markers for E6 and E7 activity. In these stainings, there is a clear overall decrease in p53 and Rb signal in the later passages, supporting that increased E6 and E7 activity is present in our in vitro model.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
The authors are to be thanked for their efforts to address reviewers’ comments and for including new, important data. However, there are still some issues to be addressed.
Figure 3: the scale bars would be better shown in white, and should be shown on all images. The sizes represented by the scale bars should be indicated in the figure legend.
Figure 4 and line 330: keratin 5 expression is clearly present in the basal layer of the tissues but is also present throughout the other epithelial layers at low passage (P7) as well as high passage. In fact, in the tissues derived from male keratinocytes, there is more keratin 5 expression in the upper layers of P7 compared to P37. Can the authors explain this?
Figure 5: why is there no keratin 10 expression in early passage female tissues but it is present in early passage male tissues?
In reference to Figs 4-8 the authors compared the results of HPV16-transfected tissues to primary tonsil rafts but there are no images of untransfected primary tonsil rafts.
Can the authors explain why they observe keratin pearls only in female-derived rafts, but not in the male-derived rafts, in the images in Figures 4-7. Why are there no keratin pearls in the rafts shown in the remaining figures?
Line 402: “Lower passages revealed no expression of p16 across the male and female raft tissues “. There is clearly expression of p16 at lower passages, shown by the green colour in the immunofluorescence images.
Minor points
Line 294: urogenital=anogenital
In Figure 2b and c there are inconsistencies in font sizes that should be corrected.
Is it appropriate to define the basal layer with a dotted line in tissues of high passage given that in a cancer-progressed tissue, the epithelium will become filled with basal cells.
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
