PREreview estructurada del Brief report on the development of patient-derived lung cancer organoids with keratinizing squamous cell carcinoma morphology

Does the introduction explain the objective of the research presented in the preprint?

Yes

1. Establishes Clinical Unmet Need: It highlights that lung squamous cell carcinoma (LUSC) has high mortality, lacks driver-mutation targeted therapies, and possesses a poor prognosis when displaying keratinization, a histological hallmark associated with impaired immunotherapy efficacy. 2. Identifies Current Model Limitations: It notes that traditional preclinical models fail to replicate complex 3D human tumor architecture, intratumor heterogeneity, and the tumor microenvironment. 3. States the Direct Research Goal: It introduces patient-derived organoids (PDOs) as a faithful 3D alternative and explicitly states the goal in the closing sentence.

Are the methods well-suited for this research?

Somewhat appropriate

Strengths: The protocols for tissue digestion, PDO generation in Cultrex basement membrane matrix, confocal imaging, and immunofluorescent staining for key squamous/differentiation markers (p63, Ki-67, involucrin, pan-cytokeratin) follow established standard procedures in the 3D organoid field. The techniques successfully demonstrate phenotypic fidelity and morphological retention of keratin pearls in 3D culture. Limitations: The methodology relies on a very small sample size (n=2 patient samples). Furthermore, critical experimental details such as the exact composition/formulation of the growth media are omitted from the main text and relegated to supplementary material, and it would be even better if they had provided genomic matching, such as NGS, if it was performed, to methodologically confirm genomic conservation alongside the immunofluorescent markers.

Are the conclusions supported by the data?

Somewhat supported

What is supported: The primary data (bright-field imaging, confocal microscopy, and immunofluorescent staining for p63, Ki-67, Involucrin, and Pan-cytokeratin) clearly support the claim that these organoid lines preserve squamous marker expression and spontaneously recapitulate 3D keratin pearl structures in culture. Where conclusions overreach slightly: The authors claim PDOs serve as a drug testing platform, but provide no experimental drug testing data. Claims of "invasion" are based on 2D cell outgrowth onto plastic rather than functional 3D matrix invasion assays.

Are the data presentations, including visualizations, well-suited to represent the data?

Somewhat appropriate and clear

How clearly do the authors discuss, explain, and interpret their findings and potential next steps for the research?

Somewhat clearly

The authors clearly explained the biological significance of p63, Ki-67, Involucrin, and cytokeratins in confirming squamous cell lineage, proliferation capacity, and terminal keratinization. They also provided a thoughtful comparison between their findings and existing literature, noting key similarities and spatial differences in protein localization. Aside from not addressing the sample size, such as why only n=2, and providing somewhat vague future directions.

Is the preprint likely to advance academic knowledge?

Somewhat likely

This study provides a valuable proof of concept by demonstrating that patient-derived organoids (PDOs) from keratinizing lung squamous cell carcinoma can spontaneously form 3D keratin pearls in vitro without induction. Still, it has some limitations, such as the small sample size (n=2), a lack of genomic sequencing to confirm fidelity to parent tumors, and reliance on 2D plastic outgrowth rather than 3D matrix assays to assess invasion.

Would it benefit from language editing?

No

The manuscript is written in clear, professional scientific English that is easy to understand throughout. While there are minor typographical oversights in figure labels and brief phrasing choices that could be refined for conciseness, these minor issues do not hinder comprehension or obscure the scientific findings. Full copyediting or extensive language revision is unnecessary before publication.

Would you recommend this preprint to others?

Yes, it’s of high quality

This study demonstrates that patient-derived organoids from keratinizing lung squamous cell carcinoma spontaneously recapitulate 3D keratin pearls in vitro. However, if they address the claims I mentioned earlier, it will greatly enhance the strength of the manuscript.

Is it ready for attention from an editor, publisher or broader audience?

Yes, after minor changes

Yes, addressing the claims I mentioned earlier could significantly strengthen the manuscript. For instance, providing a brief explanation for the small sample size and offering more supporting conclusions would be beneficial, including why they used a small sample size and more supporting conclusions.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.