Peer review of: FGF2 alters macrophage polarization, tumour immunity and growth and can be targeted (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The manuscript by Buzzelli at al. focuses on understanding the contributions of host-derived 
FGF2LMW to tumor growth and macrophage polarization using various tumor models. Initial 
studies demonstrate a significant decrease in tumor growth following subcutaneous injection into 
FGF2LMW-/- mice using various models (MC38, KPC and LLC). Further studies demonstrate 
alterations in immune profiles favoring an increased anti-tumor response in the FGF2-/-LMW mice 
driven by T cells. In addition, macrophages isolated from control and FGF2-/-LMW mice show 
altered expression of various cytokines and chemokines. To further define the contributions of 
macrophages to this phenotype, tumor cells were co-injected with control or FGF2-/-LMW bone 
marrow derived macrophages, which demonstrates reduced tumor growth although in a T cell-
independent manner. Finally, irradiation was found to promote increased levels of FGF2, and 
treatment with anti-FGF2 led to better response to irradiation, which correlated with an increased 
iNOS:CD206 ratio in macrophages. Based on these findings, the authors conclude that FGF2 
regulates tumor immunity and growth through altering macrophage polarization. While the link 
between FGFs and immune modulation within the tumor microenvironment is not necessarily 
novel, the findings that macrophage-derived FGF2 impacts tumor growth and that anti-FGF2 
enhances efficacy of irradiation are of interest. While these studies demonstrate a role for host-
derived FGF2LMW in promoting tumor growth, and effectively demonstrate efficacy of anti-FGF2 in 
the context of irradiation, some concerns are noted as described below. 
 
The authors should provide information in the Materials and Methods regarding the specific 
substrains of C57BL/6 mice that were used for these studies. As written, it appears that the 
FGF2LMW-/- mice were purchased from Jackson Laboratories on the C57BL/6J background and 
maintained in-house, and the control mice were purchased from Charles River, which provides the 
C57BL/6N substrain. If this is correct, this should be noted in the methods section. Furthermore, in 
light of recent observations that differences in substrain can impact immune phenotypes (for 
example, PMID 27210752), if different substrains were indeed used in these studies, this should 
be discussed as a potential caveat particularly of the immune phenotype analysis. Ideally, key 
experiments involving immune profiling, including the macrophage polarization studies, should be 
repeated using mice of the same substrain. 
 
Figure 3: While basal levels of a number of the factors shown are significantly different than 
control, the magnitude of induction seems to be similar. Does restoration of FGF2 to the 
FGF2LMW-/- cells impact basal expression levels of these factors? 
 
Figure 5C: The conclusion that the majority of FGF2-producing cells appear to be TAMs is not 
supported by the images provided as there are substantial areas of green FGF2 staining that do 
not appear to be associated with F4/80 staining. Quantification of dual-staining or flow cytometry-
based quantification should be provided to support these conclusions. 
 
The authors conclude several times (in the title, lines 215-216 and 352) that “macrophage 
polarization by FGF2LMW mediates tumor growth”. However, this conclusion seems premature 
given the studies provided. There are several other possible explanations for the phenotypes that 
have not been ruled out, such as reduced tumor growth due to loss of macrophage-derived FGF2-
mediated effects on tumor cells, or other non-immune host cells, which could ultimately impact 
both tumor growth rates and the immune environment. Thus, additional potential explanations for 
the phenotypes observed should be thoroughly considered. 
 
The authors demonstrate that expression levels of FGFR1 and FGFR2 are higher on TAMs 
compared with other immune cell populations. Does FGF2 directly activate FGFR signaling in TAMs? 
 
In Figure 1, the authors demonstrate that the reduction in tumor growth observed in the 

FGF2LMW-/- mice is T cell-dependent. However, in Figure 4, T cells are found to be indispensible 
for the reduction in tumor growth induced by the FGF2LMW-/- macrophages. Potential reasons for 
these differences (such as the possibility that non-macrophage FGF2LMW-expressing host cells are 
impacting the T cell population in the FGF2LMW-/- mice) should be discussed. 
 
Because the flow plots are presented in the context of FSC or SSC, the authors should provide the 
gating schemes used to identify the different immune cell populations (as Supplementary data). 
 
Please provide the number of mice used for each in vivo experiment. 
 
Figure 1: Figures J and K are switched when referred to in the text. 
 
Figure 2: Please clarify whether this antibody recognizes only the secreted form of FGF2, or 
whether it also recognizes the intracellular form of FGF2, and whether this impacts interpretation 
of the data. 
 
Figure 3A: The shifts are difficult to see in the panels provided and may be more easily interpreted 
as histograms. Also, please provide the iNOS:CD206 ratios along with statistical analysis. 
 
Figure 4E: The authors conclude that there is a reduction in MDSCs in tumors with FGF2LMW-/- 
macrophages, although this does not appear to be supported by the data presented in the graph. 
 
Figure 6: For clarity, please provide the cell number count for each plot. 
 
Although a general statement regarding statistical analysis is included in the materials and 
methods section, it is not clear which test was used for each graph, which could be provided in the 
Figure Legends. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Buzzelli et al present experimental evidence that suggests a role of FGF2 as a key modulator of 
macrophage polarization in tumors, as part of the "wound response" associated with localized 
radio-therapy. They identify FGF2 as a potential target of translational interest, since antibodies 
against FGF2 could remove one of radio-therapy immune-suppressive effects, while exploiting 
other pro-immunogenic ones. 
 
Several concerns: 
 
1. The mechanism of radio-therapy induction of FGF2 is not clear. In vitro experiments are needed 
to specifically define the nature of signaling after irradiation, whether it is different in different cells 
types and whether it is irradiation-does dependent. 
2. Is FGF2 induction part of DDR? A heat map for FGF2 null versus parental lines could help 
understanding the pathway. It could also provide important hints regarding the component of 
cross talk with innate immunity 
3. The previous work of the authors in reference 25 shows an effect of FGF2 on angiogenesis, 
independent of irradiation. Understanding the mechanism of FGF2 induction by irradiation is key, 
since it may also have clinical impact in cancer metastasis. 
 
4. A main concern is the use of only one radiotherapy regimen (9GyX2), without clear justification 
for this selection. Particularly in the in vivo experiments a different dose could have overcome the 
progression of tumors and/or the type of TAMs infiltration. Since no dose response curves were 
provided in the model used, one can not exclude that re-population because of inadequate dose 
may have also recruited M2 and/or induced polarization of TAMs. The choice of this radio-therapy 

dose and number of fractions needs a much more explicit justification, since others have shown 
very different results for instance when low doses are used. (Klug et al Cancer Cell. 2013 Nov 
11;24(5):589-602) 
5. Figure 5 Shows modest difference in HT29 model and the delay in MC38 is only transient. From 
the images it is hard to conclude a sustained superior effect by the combination 
6. References on irradiation and macrophages are incomplete (missing key papers of Deng L et al, 
Xu J et al etc.). 
 
 
 
Reviewer #4: 
Remarks to the Author: 
In this manuscript, Buzzelli et al. show slower growth of primary tumors and decreased liver 
metastasis in FGF2lmw deleted mice. Mechanistic studies showed that the deletion induces T cell 
recruitment into the tumors and that this is critical for the decreased tumor growth. Although the 
authors were not able to detect FGFR1 or 2 in the T cells, they found that the majority of tumor-
associated macrophages expressed FGFR1 and 2. Both FGF2lmw deletion and anti-FGF2 antibody 
induced TAM polarization towards a more inflammatory phenotype. Decreased tumor xenograft 
growth was seen in nude mice when anti-FGF2 blocking antibody was used in combination with 
irradiation. The manuscript reports many interesting, but somewhat dissociated results that raise 
several questions. 
 
In the previous paper by the same authors, metastasis-associated neutrophils expressed 
substantially more fibroblast growth factor 2 (FGF2) in comparison to naïve neutrophils, indicating 
neutrophil polarization by the tumor microenvironment. Administration of FGF2 neutralizing 
antibody to mice bearing experimental liver metastases phenocopied neutrophil depletion by 
reducing liver metastatic colony growth, vascular density, and branching (Hepatology 
2017;65:1920-1935). 
 
It has also been shown that FGFR1 inhibition by gene silencing in tumor cells or administration of a 
compound (SSR128129E at nanomolar concentrations) inhibiting FGF-induced signaling in tumor 
xenografts increases the efficiency of radiotherapy (pubmed/26896280). 
 
Thus, an alternative possibility to explain the authors’ results is that FGF2lmw depletion directly 
causes initial TC death in the tumors in vivo, and that the resulting danger signals invite 
inflammatory/immune cells to the tumors. This possibility is consistent with the “T-cell 
independent alteration” that “resulted in less rapid tumor growth in FGF2lmw-/- mice than in WT 
mice”. Although the authors show that anti-FGF2 antibody treatment after irradiation did not alter 
clonogenic survival of SW1222 and HT29 cells, it would be essential to also analyze the effect of 
FGF2 silencing. 
 
In the manuscript, a limited analysis is reported on the role of TC produced FGF, and the 
expression of FGF receptors. A comparison of the amounts of FGF2 produced by TCs and 
macrophages should be shown. A more careful mapping of the autocrine pathway in the TCs is 
also needed. It would be essential to compare the survival of the control, FGF2 and FGFR1 silenced 
as well as FGF2 antibody treated tumor cell cultures after irradiation. 
 
On pages 5 and 6 the authors show that T-cells are required for tumor regression in the FGF2lmw-
/- mice. However, later, in Figure 5 F-H and Supplementary Figure 4, they show that T-cells are 
not required for the effects of the anti-FGF2 antibody on human SW1222 and HT29 tumor 
xenografts in immunodeficient mice. The authors should analyze the mouse tumor cell lines in 
using irradiation with and without the anti-FGF2 antibody in an immunocompromised background. 
Alternatively, they could use another inhibitor of the FGF2-FGFR pathway, such as SSR128129E, a 
FGFR1 blocking antibody, soluble receptor or FGFR1 gene silencing in the tumor cells. In addition, 
the authors should discuss why anti-FGF2 as a monotherapy does not decrease xenograft growth 

(Fig 5 F-H), although FGF2lmw deletion decreases tumor growth in the isogenic mice (Fig. 1). 
 
As the authors indicate in the introduction, FGF2lmw is a highly potent angiogenic molecule. 
However, here the authors don`t provide any tumor vessel analysis. This should be analyzed to 
exclude the possible role of decreased vascular density to the decreased tumor growth. If anti-
FGF2 antibody treatment decreases vascular density as shown previously in the case of liver 
metastasis, it could explain why anti-FGF2 antibody treatment decreases SW1222 and HT29 tumor 
growth in immunodeficient mice. 
 
Specific comments: 
 
Another question concerns the specificity of the effects reported here. The authors should perhaps 
mention that the other 21 members of FGF family are not expressed or of concern here. Similarly, 
they should describe which of the four receptors are present in the tumor cells they use. 
 
When reporting immune infiltration to the metastases/tumors, the authors should also report if 
this concerned normal tissues or circulating immune cells. 
 
The authors should re-isolate the escape clones that resumed growth and test if they regrow in 
corresponding experiments in FGF2lmw-/- mice. 
 
The LLC growth curves should be added to Supplementary Figure 1. 
 
Did TAMs express FGFR3 or FGFR4? Was phosphorylation of FGFR1 correlated with FGF2lmw 
expression or irradiation in TCs or TAMs? 
 
The anti-FGF2 antibody was used for the MC38 clonogenic assay in Supplementary Figure 5. 
However, only human tumor cells were used in the in vivo experiments. Why were the mouse 
tumor cells not used in the in vivo tumor experiments? 
 
As the authors explain, FGF2 expression has been reported to be induced in irradiated human 
tumors. Thus, Figs 5A and B are of little value. In Fig. 5E, much of the macrophage and FGF2 
staining appears to be eradicated by the irradiation. The authors should show the specificity of 
anti-FGF2 by staining macrophages of the corresponding tumors grown in FGF2lmw-/- mice. 
 
Minor comments 
 
• Fig. 1 J and K are mixed on page 6? 
• How was liver tumor burden measured? 
• How was tumor volume calculated? The authors should study M1/M2 ratio in tumors beyond the 
(very small) volume of 500mm3. 
• Fig. 1 B: Please show the invasion front in high magnification, with the identification of the tumor 
cells (by IHC). 
• In all experiments, the number of mice should be mentioned in each group. The individual tumor 
growth curves are difficult to interpret, for example in Fig. 5F.