Summary of review of Manuscript # 637217 Submitted to Advances in Cancer: Research & Treatment Reviewer #1 Major Comments The manuscript “Confluentic acid inhibits the growth of rat glioblastoma cells in vitro” represents another attempt to test a biological product against GBM. The manuscript contains several flaws: a) The statement “Reinfection (sic) of tissue with cancer cells…” (P1,p2,L1-2) needs to be modified. b) Although the authors state that “we must turn to the secondary metabolites of the lichens…” (P1,p2,L7-8) at no time they explain why the lichens might be antineoplastic; or why they studied them; or what antecedents were considered; or why they think that lichens could be used for cancer research. The absence of this information decreases the scientific value of the findings. c) The whole paragraph 3 on page 1 is inexact in oncological terms. d) What is the “social arrangement” of lichens? (P1,p3,L1-2). e) Legend for fig. 2 says “Dose-dependent effects of confluentic acid on the growth of the rat glioblastoma cell …” what is obvious in the figure is that it is NOT a dose-dependent effect but a constant effect not related with the dose. f) In contrast, the graphic on fig. 3 shows a dose-dependent effect which is not explained. g) The fact that the substance tested (confluentic acid) was diluted in ethanol makes it very toxic for cultured cells. It is possible that the cytolytic effect observed was due to this. Reviewer #2 Major Comments Several aspects of the manuscript should be improved, especially the quality of analysis and evidence. For ex. in order to improve the paper, the authors should evaluate the cell viability by a specific method such as MTT assay; the toxic effect of the extract against a normal cell or tissue (for example, control astrocytes or organotypic culture) should be also evaluated; Some points are given below: - Methods: 1) How the “pure extracts” were prepared? How pure is the extract? How is the concentration of Confluentic acid in the extract? Which chemical class this chemical substance belongs? 2) What is the final concentration of ethanol used? This should be indicated in the legends. 3) Which cell line of glioma was used? 4) Why the authors used 5 days of treatment. Probably if they treat the cells for 48h, they could see if the effect was dose-dependent. 5) Although the authors present the statistical method used, they did not put the significance in the figures. - Results: 1) The presentation of the figures should be improved. The numbers presented in the axis should be bigger. 2) The title of Figure 2 should be correct because the presented results do not demonstrate a “dose-dependent” effect. 3) In the Figure 1, what is the concentration of ethanol in the control? - Discussion The discussion should be improved. The points discussed are purely speculative. Ex: - The sentence “We propose the biological activity of confluentic acid is related to lichen ecology. It is known that lichens are adapted for the manipulation of radiation, and also adapted for defense against the foragers . Therefore, it is not surprising that the secondary metabolites of the lichen can enhance the effect of radiation and inhibit foreign cells” does not appear to be consistent, since if the lichens produce some protective substances, such as Confluentic acid, how this substance can increase the cytotoxic effect of radiation against glioma cells?