Serum-Derived Macrophage-Activating Factor Exhibits Anti-Tumor Activity via M2-to-M1 Macrophage Reprogramming
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors report about the effect of the Gc protein-derived macrophage-activating factor (GcMAF, “MAF”) on RAW macrophages and EMT6 tumor cells. They found that MAF induces the differentiation of M2 macrophages, which rather stimulate tumor cell proliferation, to the M1 typer which suppresses tumor cell growth. These results are new, interesting and of potential interest for anti-tumor therapies. However, some statements of the authors are not or not sufficiently supported by the data. This needs to be corrected. Furthermore, the description of the experimental setups could be improved. These are the detailed corresponding points:
Fig. 1A: Is the mRNA expression of RAW-MAF/EMF really larger than of RAW-MAF with p<0.01? Please provide the data.
The scaling of the ordinate could be improved.
Fig. 2 Legend: “EMT6 cells were stimulated with 0.1 μg/mL GcMAF or 1 μg/mL LPS for 24 h”
From the marks in the figure, one reads that RAW macrophages were stimulated with MAF or LPS and not the EMT6 cells. Please, clarify.
L181: “In EMT6 cells, the mRNA expression levels of COX-2 and VEGF, which are associated with tumor growth and invasion, increased after GcMAF stimulation (Figure 1E, F).”
This is true only in co-culture with RAW macrophages and this should be stated.
L202: “The inhibitory effect of RAW264.7 cells on tumor cell 202
proliferation was stronger than that of EMT6 cells.”
I do not understand this sentence.
L214: “In GcMAF-stimulated RAW264.7 cells, levels of 1735 genes, including TNF-α, iNOS, IL-1β, IL-6, C-X-C motif chemokine ligand 2, and C-C motif chemokine ligand 5, were up-regulated more than 2-fold compared to unstimulated cells. The expression of several genes related to inflammatory cytokines was upregulated. Although a few genes related to anti-inflammatory cytokines were upregulated, the increase in the expression of M1-related genes was more significant. These results suggested that GcMAF induces M1 polarization in macrophages.”
Please, show the results of all the mentioned genes.
L255: “We examined the effect of GcMAF on the M2 polarization of macrophages to verify whether GcMAF activates TAMs in the tumor microenvironment.”
The effects of MAF in the tumor environment was not investigated here.
L260: “No significant differences in cell proliferation were observed between EMT6 cells cultured alone for 24 h in GcMAF medium and EMT6 cells co-cultured with RAW264.7 cells.”
That would mean that there is a significant reduction of proliferation if EMT6 cells are co-cultured with MAF-Stimulated RAW but not at stimulation of EMT6 with MAF, i.e. there should be a significant difference between columns 3 and 5, what I do not see. Please provide the detailed data.
L273: “When co-cultured with GcMAF-stimulated RAW264.7 cells, EMT6 cells exhibited enhanced gene expression levels of COX-2 and VEGF, which are involved in cell invasion and proliferation.”
Please explain, why nevertheless the proliferation of these cells is decreased.
Further minor points:
L53: “TAMs induce immunosuppression via various factors, such as interleukin (IL)-10 and transforming growth factor-β, in tumors [15], and produce various factors, such as the vascular endothelial growth factor (VEGF), IL-8, and cyclooxygenase-2 (COX-2), under hypoxic stimulation [16,17]. They activate the factors involved in angiogenesis [18], creating a favorable tumor microenvironment.”
Here the authors should differentiate between M1 and M2 phenotype and between tumor promoting and tumor suppressing factors.
L84: “2.3. Co-culture of RAW264.7 cells and EMT6 cells”
The different culture conditions (co-culture or not, direct contact of both cell types or not) might be better described for example, by a cartoon for readers, which are not so familiar with these methods.
L182: “… COX-2 and VEGF, which are associated with tumor growth and invasion”
Please provide the related reference.
L278: “Suppression of cell proliferation by GcMAF-stimulated macrophages may be due to the action of the surrounding surviving cells, which emit survival signals.”
Please explain, which signals are emitted and provide the respective references.
Comments on the Quality of English LanguageMinor editing of English language required.
Author Response
[Question]
Fig. 1A: Is the mRNA expression of RAW-MAF/EMF really larger than of RAW-MAF with p<0.01? Please provide the data.
The scaling of the ordinate could be improved.
[Answer]
Attached is the raw data for Figure 1A. The vertical axis in Figure 1A has been corrected.
| Sample Name | TNF-α mRNA | 　 | 　 | 　 | t.test | 　 | ||
| 　 | 　 | 1 | 2 | 3 | Average | SD | vs Control | vs Co-culture |
| 　 | RAW (Control) |
1.2386 | 0.8635 | 0.8980 | 1.0000 | 0.1693 | 　 | 　 |
| 　 | RAW+MAF | 2.2649 | 2.1061 | 2.0472 | 2.1394 | 0.0920 | 0.0011 | 　 |
| Co-culture | RAW / EMT6 |
0.3838 | 0.4373 | 0.5427 | 0.4546 | 0.0660 | 0.0132 | 　 |
| 　 | RAW+MAF / EMT6 |
2.5396 | 1.8608 | 2.3739 | 2.2581 | 0.2890 | 0.0060 | 0.0010 |
| 　 | RAW+LPS / EMT6 |
2.9384 | 3.6246 | 4.0624 | 3.5418 | 0.4626 | 0.0019 | 0.0007 |
| 　 | RAW / EMT6+MAF |
0.7407 | 0.8225 | 0.3447 | 0.6360 | 0.2086 | 0.1278 | 0.3062 |
| 　 | RAW / EMT6+LPS |
0.4575 | 0.9195 | 0.1724 | 0.5165 | 0.3079 | 0.1235 | 0.7948 |
[Question]
Fig. 2 Legend: “EMT6 cells were stimulated with 0.1 μg/mL GcMAF or 1 μg/mL LPS for 24 h”
From the marks in the figure, one reads that RAW macrophages were stimulated with MAF or LPS and not the EMT6 cells. Please, clarify.
[Answer]
As you point out, the words "RAW264.7 cells" and "EMT6 cells" were reversed.
[Question]
L96: “In EMT6 cells, the mRNA expression levels of COX-2 and VEGF, which are associated with tumor growth and invasion, increased after GcMAF stimulation (Figure 1E, F).”
This is true only in co-culture with RAW macrophages and this should be stated.
[Answer]
As you point out, added wording to limit the use to "EMT6 co-cultured with RAW264.7".
[Question]
L113: “The inhibitory effect of RAW264.7 cells on tumor cell
proliferation was stronger than that of EMT6 cells.”
I do not understand this sentence.
[Answer]
As you point out, the text is difficult to understand the meaning, so I deleted it.
[Question]
L126: “In GcMAF-stimulated RAW264.7 cells, levels of 1735 genes, including TNF-α, iNOS, IL-1β, IL-6, C-X-C motif chemokine ligand 2, and C-C motif chemokine ligand 5, were up-regulated more than 2-fold compared to unstimulated cells. The expression of several genes related to inflammatory cytokines was upregulated. Although a few genes related to anti-inflammatory cytokines were upregulated, the increase in the expression of M1-related genes was more significant. These results suggested that GcMAF induces M1 polarization in macrophages.”
Please, show the results of all the mentioned genes.
[Answer]
As you point out, replaced the image with the name of each gene and its location.
[Question]
L137: “We examined the effect of GcMAF on the M2 polarization of macrophages to verify whether GcMAF activates TAMs in the tumor microenvironment.”
The effects of MAF in the tumor environment was not investigated here.
[Answer]
As you point out, since this study is in vitro only, the text has been revised.
[Question]
L171: “No significant differences in cell proliferation were observed between EMT6 cells cultured alone for 24 h in GcMAF medium and EMT6 cells co-cultured with RAW264.7 cells.”
That would mean that there is a significant reduction of proliferation if EMT6 cells are co-cultured with MAF-Stimulated RAW but not at stimulation of EMT6 with MAF, i.e. there should be a significant difference between columns 3 and 5, what I do not see. Please provide the detailed data.
[Answer]
Detailed analysis of EMT6 cells stimulated with MAF was not performed, so Figure 5 and related text have been revised.
[Question]
L181: “When co-cultured with GcMAF-stimulated RAW264.7 cells, EMT6 cells exhibited enhanced gene expression levels of COX-2 and VEGF, which are involved in cell invasion and proliferation.”
Please explain, why nevertheless the proliferation of these cells is decreased.
[Answer]
It is thought that surviving EMT6 cells produce COX-2 and VEGF for survival, but COX-2 and VEGF appear to have no effect in vitro.
Further minor points:
[Question]
L55: “TAMs induce immunosuppression via various factors, such as interleukin (IL)-10 and transforming growth factor-β, in tumors [15], and produce various factors, such as the vascular endothelial growth factor (VEGF), IL-8, and cyclooxygenase-2 (COX-2), under hypoxic stimulation [16,17]. They activate the factors involved in angiogenesis [18], creating a favorable tumor microenvironment.”
Here the authors should differentiate between M1 and M2 phenotype and between tumor promoting and tumor suppressing factors.
[Answer]
As you point out, we distinguished between M1 and M2 phenotypes, tumor promoters and tumor suppressors.
[Question]
L222: “4.3. Co-culture of RAW264.7 cells and EMT6 cells”
The different culture conditions (co-culture or not, direct contact of both cell types or not) might be better described for example, by a cartoon for readers, which are not so familiar with these methods.
[Answer]
As you point out, Figure 6 has been added to this section.
[Question]
L96: “… COX-2 and VEGF, which are associated with tumor growth and invasion”
Please provide the related reference.
[Answer]
Reference [18] added.
[Question]
L188: “Suppression of cell proliferation by GcMAF-stimulated macrophages may be due to the action of the surrounding surviving cells, which emit survival signals.”
Please explain, which signals are emitted and provide the respective references.
[Answer]
The text was deleted because of errors in content and no references.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study investigates the anti-tumor effects of GcMAFs via M2-to-M1 macrophage reprogramming. The results suggested that the anti-tumor activity of GcMAF via M2-to-M1 macrophage reprogramming could aid in developing novel cancer immunotherapies.
My recommendation is that this paper cannot be published in its current form for several reasons. Here are my comments:
1. In this study, GcMAF was extracted from human-derived Gc proteins, whereas the subject of the study was a murine-derived cell line. Is it possible that human-derived protein fragments may have some immunogenicity to murine-derived cells and thus regulate the immune function of murine-derived cells? Why not use a human cell line?
2. In part 2.11, the description of statistical analysis is too simple, the number of experiments repeated, data types and analysis software are not introduced. Please add it.
3. In EMT6 cells, the mRNA expression levels of COX-2 and VEGF increased after GcMAF stimulation. This result contradicted the antitumor effect of GcMAF. Please explain it.
4. The overall study did not go far enough and did not explain how GcMAF reversed M2-type to M1-type macrophages, which signaling pathways are regulated by GcMAF to change macrophage polarization state, and what are the key molecules involved?
5. In abstract, GcMAFs stands for group-specific component-derived macrophage-activating factors. However, in Introduction, GcMAFs stands for Gc protein-derived macrophage-activating factor. Please strive to be consistent.
Author Response
Reviewer 2
- In this study, GcMAF was extracted from human-derived Gc proteins, whereas the subject of the study was a murine-derived cell line. Is it possible that human-derived protein fragments may have some immunogenicity to murine-derived cells and thus regulate the immune function of murine-derived cells? Why not use a human cell line?
[Answer]
We have previously conducted and published studies of human-derived GcMAF using mouse macrophage cells and tumor cells, and since we did not use T cells, we did not consider immunogenicity.
- In part 2.11, the description of statistical analysis is too simple, the number of experiments repeated, data types and analysis software are not introduced. Please add it.
[Answer]
Detailed information has been added.
- In EMT6 cells, the mRNA expression levels of COX-2 and VEGF increased after GcMAF stimulation. This result contradicted the antitumor effect of GcMAF. Please explain it.
[Answer]
An explanation was added to L96. We also added that this phenomenon is limited to EMT6 cells co-cultured with RAW264.7 cells.
- The overall study did not go far enough and did not explain how GcMAF reversed M2-type to M1-type macrophages, which signaling pathways are regulated by GcMAF to change macrophage polarization state, and what are the key molecules involved?
[Answer]
At this time, the GcMAF receptor is unknown, and the signaling pathway has not been investigated, so it is unknown. This paper only clarifies the phenomenon.
- In abstract, GcMAFs stands for group-specific component-derived macrophage-activating factors. However, in Introduction, GcMAFs stands for Gc protein-derived macrophage-activating factor. Please strive to be consistent.
[Answer]
We changed the sentence in L22.
