PONE-D-20-32987

Dear Editor,

Changes in the tumor microenvironment and outcome for TME-targeting therapy in glioblastoma: A pilot study

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: English can improve further, but other than that the work is done very well. Detail of shortcomings is also included. They have used majorly surface markers to analyze the TME rather than more elaborate settings including cytokines. But this is also well explained.I recommend publication.

Reviewer #2: In the present manuscript the authors present a pilot study addressing the changes in immune cell populations of the TME of a syngeneic mouse model of GBM, in relation to the different treatments administered to recapitulate standard and novel candidate therapies for human GBM. They also measure tumor growth and survival following different treatments. In some experiments they use CSF1R KO animals to address the involvement of tumor-promoting myeloid cells. Their aim is to show associations between changes in the TME-associated cells and the use of the distinct therapeutic agents. The accumulation of bone marrow-derived cells in the GBM TME and the effect of immune-suppressive myeloid cells in causing alternative neovascularization, the revival of glioma stem cells, and recurrence is already know and well-documented. The percentages of immune cells in GBM TME have not been addressed in relation to GBM therapies before and their characterization would be beneficial for a translational application in GBM cure; yet, as it is now, the study is too preliminary for publication and requires further integrations and some corrections to enforce the solidity and informativeness of data before being accepted. The authors themselves indicate the limitations of their study and point at future experiments to be done (lines 569-585). Immunohistochemical analyses might be postponed to future characterizations, but cytofluorimetric analyses in the present study are not exhaustive and need to be revised.

No details to the antibodies used are reported in the M&M section (clone, fluorochrome, vendor); a live/dead staining should have been included, to avoid to count dead cells in the acquisition. It is not clear how many surface markers were studied within each multicolor staining panels, the latter information is not provided and the exact gating strategies are not shown per each analysis either. Without the use of multicolor staining panel it is not possible to univocally identify/distinguish the immune population of interest. It is well-known that the phenotypic distinction between M1 and M2 macrophages is not clear-cut and most large scale transcriptome analyses show that macrophages have a mixed phenotype expressing both M1 and M2 markers: therefore, the couples of markers used herein appear not sufficient; in line with this, the reciprocal percentages are almost the same. On the other hand, among TAMs, it should be important to dissect the proportions of peripherally derived infiltrating BMDMs versus resident microglia, according to already published cytofluorimetric methods that rely on differential cell-surface expression of CD45 and CD11b by these two cell types (CD45loCD11b+ defining putative microglia, CD45hiCD11b+ defining putative BMDMs). This might help explain results of treatments in Fig. 3. Multicolor flow cytometry panels to comprehensively immunoprofile TME-associated cells, should include different markers for myeloid cells (CD11b, Gr1, Ly6G, Ly6C, CD11c, Tie2, and MHC class II), for lymphoid cells (CD3, CD4, CD8 and possibly CD19) and possibly activation status and PD1 expression on T cells (as anti-PD1 therapy is evaluated as well).

Stainings with isotype control Abs should be done, resulting useful especially for myeloid cells. Plots are not indicated for each figure, for each condition tested and for each control. The cytofluorimetric plots provided do not seem to be correctly compensated and often many events are found in the diagonal, as if they were dead cells. The Ab used to detect CSFR gives a very low staining, so it is very difficult to appreciate positive cells (Suppl fig 2: all plots for all mice should be shown, including plots of wt mice, where CD45+CSFR1+ cells are present).

Compensation and gating might be checked and consequently the percentages should be recalculated. Some of them already appear overestimated (e.g., in Fig. 5, the sum of CD45+CD4+, CD45+CD8+ and CD45+CD11b+ cells in the condition TMZ is far over 100%….). Gating strategies and plots should be always shown (they are not shown for related graphs in Fig. 5 and 6) for clarity. Graphs of cumulative percentages of different CD45+ subpopulations for each condition could be less ambiguous.

The authors should better detail figure legends: every legend should include the timepoint of assay, the number of mice/group and the number of repeated measures. The number of mice (n) is not always indicated and sometimes there are discrepancies (Fig1B legend: n=8 and not 10; Fig 3: the number of dots shown in panel B do not match to the number of mice shown in panel A for the corresponding condition).

The tumor growth and survival data are not measured for the same treatments analyzed for TME-associated cells. A systematic investigation should be conducted, including CSF1R KO animals, as also said by the authors. For the tumor mass studies, too few mice are used per group (n=3), resulting in high SEM and impeding the identification of significant differences among treatments. More importantly, not all the combinations of treatments have been addressed, therefore controls are missing. Radiation plus TMZ plus/minus antiangiogenic drug is missing as well, as a reference of standard therapies for patients. Currently, surgical resection followed by radiation and TMZ therapies is the standard of care for GBM patients.

Further on, the observation that “the animals that received TME-associated cell-directed therapy showed significantly lower tumor growth 2 weeks following treatments” is correct compared to the condition “vehicle” (=no treatment), but not compared to radiation, that exerts a similar effect (Fig. 7), arguing the advantage of preferring selective TME-associated cell-directed therapy. Finally one of the conclusion “instead of concurrent therapy, a sequential strategy would be best to target TME associated cells” is not really sustained by data, to prove this the authors should have included sequential administration of different treatments in their experimental setting.

Some figures could be pooled together; the introduction and the discussion are quite long compared to results; the discussion is too speculative.

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Reviewer #1: No

Reviewer #2: No

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Changes in the tumor microenvironment and outcome for TME-targeting therapy in glioblastoma: A pilot study

PONE-D-20-32987R1

Dear Dr. Arbab,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Ilya Ulasov, Ph.D

Academic Editor

PLOS ONE