Differentiated glioma cell-derived fibromodulin activates integrin-dependent Notch signaling in endothelial cells to promote tumor angiogenesis and growth
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Evaluation Summary:
The authors shed light on the role that non-cancer stem cell exerts in promoting cancer progression, revealing that non-cancer stem cell-secreted fibromodulin is crucial in mediating angiogenesis in glioma via integrin-dependent Notch signaling. The logic is smooth and clear and the results are solid, and the findings should be interesting for those who are expertized in the field of cancer biology and cancer stem cell.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Cancer stem cells (CSCs) alone can initiate and maintain tumors, but the function of non-cancer stem cells (non-CSCs) that form the tumor bulk remains poorly understood. Proteomic analysis showed a higher abundance of the extracellular matrix small leucine-rich proteoglycan fibromodulin (FMOD) in the conditioned medium of differentiated glioma cells (DGCs), the equivalent of glioma non-CSCs, compared to that of glioma stem-like cells (GSCs). DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth. FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. DGC-secreted FMOD promotes angiogenesis by activating integrin-dependent Notch signaling in endothelial cells. Furthermore, conditional silencing of FMOD in newly generated DGCs in vivo inhibits the growth of GSC-initiated tumors due to poorly developed vasculature and increases mouse survival. Collectively, these findings demonstrate that DGC-secreted FMOD promotes glioma tumor angiogenesis and growth through paracrine signaling in endothelial cells and identifies a DGC-produced protein as a potential therapeutic target in glioma.
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Author Response
Reviewer #2 (Public Review):
Tumors such as glioblastoma contain several types of cells: cancerous and reactive non-cancerous cells, and among cancerous cells, cancer cells with tumorigenic properties so-called "stem" and pseudo-differentiated cancer cells.
Strengths: a multidisciplinary international cooperation gathering complementary expertises. An impressive quantity of experiments and presented data (28 supplementary figures with multiple panels!). First description of Fibromodulin as a secreted factor acting in a paracrine manner to activate an Integrin-dependent Notch signaling in endothelial cells. A detailed analysis of the molecular signaling triggered by integrin activation. Most of the results support this claim.
Weaknesses: Several formulations in the introduction are controversial. Several results …
Author Response
Reviewer #2 (Public Review):
Tumors such as glioblastoma contain several types of cells: cancerous and reactive non-cancerous cells, and among cancerous cells, cancer cells with tumorigenic properties so-called "stem" and pseudo-differentiated cancer cells.
Strengths: a multidisciplinary international cooperation gathering complementary expertises. An impressive quantity of experiments and presented data (28 supplementary figures with multiple panels!). First description of Fibromodulin as a secreted factor acting in a paracrine manner to activate an Integrin-dependent Notch signaling in endothelial cells. A detailed analysis of the molecular signaling triggered by integrin activation. Most of the results support this claim.
Weaknesses: Several formulations in the introduction are controversial. Several results should be more clearly explained and the precise methods used are difficult to find since they are dispersed between the text, the "methods section" and often lacking in the legend of the figure.
These points are addressed appropriately; in particular, the figure legends are extensively modified. Changes are also added in the methods section.
More precisely the following points should be addressed:
1- The formulation "non-cancer stem cells" is confusing since these are cancer cells but without the functional characteristics of cancer stem cells and within the tumor exist non-cancer cells co-opted to the tumor, such cells being called "microenvironment" even if they are bona fide part of the tumor.
We understand the possibility that the use of "non-cancer-stem cells (non-CSCs)" may create confusion as it may sound like referring to stromal cells of cancer that are actually non-cancer cells of the tumor. This was rightly pointed out by reviewer # 2. After obtaining advice from Dr. Caigang Liu, Reviewing Editor, we decided to use "glioma stem-like cells" (GSCs) and "differentiated glioma cells" (DGCs) in place of CSCs and non-CSCs respectively.
2- Lines 91 to 95 are particularly controversial and even erroneous since CD133- GSC have been reported by several authors and nestin is not a selective marker of CSC. This is most-likely due to referencing reviews of a single group that promoted this dichotomy that do not correspond to most of the reported results. Furthermore it is well known that GSC proliferation or DGC reprogrammation to GSC are favored by hypoxia, illustrated in vivo by the failure of anti-VEGF treatment to increase life expectancy.
3- As soon as 2012-2013 (thus before the referenced Suva et al 2014 paper), the group of Thierry Virolle demonstrated that stem cell-like properties of GSC fuel glioblastoma development by providing the different cell types that comprise the tumor. Reference to their work is surprisingly missing. Of note, after describing that the miR-302-367 cluster is strongly induced during stemness suppression, they showed that stable miR-302-367 cluster expression is sufficient to suppress the stemness signature, self-renewal, and cell infiltration within a host brain tissue, through inhibition of the CXCR4 pathway involving the SHH-GLI-NANOG network. Micro-RNA profiling studies to search for regulators of stem cell plasticity, allowed them to identified miR-18a* as a potential candidate and its expression correlated with the stemness state. MiR-18a* expression promotes clonal proliferation in vitro and tumorigenicity in vivo.
Turchi L, Debruyne DN, Almairac F, Virolle V, Fareh M, Neirijnck Y, BurelVandenbos F, Paquis P, Junier MP, Van Obberghen-Schilling E, Chneiweiss H, Virolle T. Tumorigenic potential of miR-18A* in glioma initiating cells requires NOTCH-1 signaling. Stem Cells. 2013 Jul;31(7):1252-65. doi: 10.1002/stem.1373. PMID: 23533157
Fareh M, Turchi L, Virolle V, Debruyne D, Almairac F, de-la-Forest Divonne S, Paquis P, Preynat-Seauve O, Krause KH, Chneiweiss H, Virolle T. The miR 302367 cluster drastically affects self-renewal and infiltration properties of gliomainitiating cells through CXCR4 repression and consequent disruption of the SHHGLI-NANOG network. Cell Death Differ. 2012 Feb;19(2):232-44. doi: 10.1038/cdd.2011.89.
We agree with reviewer # 2 (points 2 and 3) that there exists heterogeneity among GSCs of glioma and that multiple markers can characterize the GSCs. Hence, the statement regarding biomarker of GSC is addressed appropriately, taking the recommendations. Accordingly, references that reported the stemness properties of CD133- glioma cells (Beier et al., 2007; Chen et al., 2010; Joo et al., 2008; Ogden et al., 2008; Wang et al., 2008), involvement of miR302-367 cluster and miR18A* in the regulation of stemness (Fareh et al., 2012, Turchi et al., 2013), and the existence of stem cell-associated heterogeneity in GBM (Dirkse et al., 2019) are referred in the revised manuscript.
4- A main question arises from the use of multiple cellular models, some highly valuables such as MGG4, MGG6 and MGG8, that correspond to patient-derived cell line maintained in culture conditions known to preserve the phenotype and genotype encountered in real patient tumors, and other cell lines (LN229, U251, U87) known to be highly unrepresentative since grown for a long time in serum conditions. However, after the first set of experiments, only MGG8 is used in the rest of the paper, with no validation on MGG4 and MGG6 and one should wonder why.
We agree with the reviewer that more human patient-derived GSC lines could have been used in animal models. However, we would present our views on the usage of GSC lines as below:
We have used three patient-derived GSC lines (MGG4, MGG6, and MGG8) for the initial discovery and in vitro validation of the DGC-specific expression of FMOD.
We have used one human patient-derived GSC line (MGG8) and two murine GSC lines (AGR53 and DBT-Luc) in an intracranial orthotopic mouse model experiment to prove the importance of FMOD induced angiogenesis in tumor growth. While the MGG8 line silenced for FMOD using short hairpin RNA (shRNA) was used to prove the importance of FMOD in tumor angiogenesis and growth, AGR53 and DBT-Luc lines carrying doxycycline-inducible shRNAs proved the importance of FMOD secreted by de novo generated DGCs from a GSC initiated tumor in angiogenesis and growth.
The only mouse model experiment carried out using the U251 cell line is now moved to the supplementary section as these cell lines are highly unrepresentative since they are grown for a long time in serum conditions, as recommended by reviewer # 2
The other places where the established glioma cell lines, LN229, U251, and U87 used are in main figure 4 and the associated supplementary figures. In these experiments, we used these cells only as a source of secreted FMOD to study the angiogenesis-inducing property. However, we would like to point out that conditioned media from MGG4, MGG6, and MGG8 also showed the angiogenesis-inducing property of FMOD.
5- Results presented in Fig2C and 2D are really strange and do not support the claim that "results indicate that FMOD secreted by DGCs is essential for the growth of tumors initiated by GSCs. FMOD induces angiogenesis of host-derived and tumor-derived endothelial cells" First, one should wonder about the subcutaneous model used since such xenograft do not raise a glioblastoma-like tumor but a mesenchymal-like highly undifferentiated tumor. Second, considering the development of the graft at day 5 and the growth curve of GSC alone, one should wonder why inhibiting expression of FMOD in DGC triggers a necrosis of the initial tumor and not a slower growth parallel to the one of GSC alone.
We agree with the reviewer in principle. However, we would like to explain the possible reason for the acute reduction in tumor growth after FMOD silencing in DGCs as below. The FMOD provided by the coinjected DGCs may induce extensive angiogenesis and may result in faster tumor growth. In such conditions, silencing FMOD through doxycycline injection may lead to phenomena like necrosis, thus resulting in a drastic reduction in the tumor size.
The subcutaneous model was used only for the co-implantation experiment. The intracranial orthotopic mouse model is used in all our animal experiments.
6- Line 445: "Cellular hierarchy is well established in GBM." This is an old view mimicking normal differentiation. Since GSC can pseudo-differentiate into DGC and DGC can be reprogrammed into GSC, no hierarchy exists, only cells with different properties and functions in tumor growth. GSC is not the origin of glioblastoma but the ultimate state of aggressiveness.
Keeping the views of the reviewer, we have now removed the sentence "Cellular hierarchy is well established in GBM" which appeared in the beginning of the Discussion. We have indeed discussed the cellular plasticity and the formation of GSCs from DGCs during chemotherapy and hypoxia in 4th paragraph in the revised manuscript.
7- Lines 452-53 "GSCs are known to promote the establishment of a highly vascularized microenvironment by being in close physical contact with endothelial cells (Calabrese et al., 2007)." This in only partially true since many soluble factors have been described to support the dialog between endothelial cells and GSC: secreted proteins such as VEGF, HDGF, GDF15, and multiple types of microRNA.
We have now added the references that describe the soluble factors from GSCs that induce angiogenesis in the first paragraph of the Discussion of the revised manuscript.
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Evaluation Summary:
The authors shed light on the role that non-cancer stem cell exerts in promoting cancer progression, revealing that non-cancer stem cell-secreted fibromodulin is crucial in mediating angiogenesis in glioma via integrin-dependent Notch signaling. The logic is smooth and clear and the results are solid, and the findings should be interesting for those who are expertized in the field of cancer biology and cancer stem cell.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors shed light on the role that non-CSC exerts in promoting cancer progression, revealing that non-CSC secreted fibromodulin is crucial in mediating angiogenesis in glioma via integrin-dependent Notch signaling. The data volume is sufficient and the argumentation is rigorous enough to support the conclusions. The results are important for gaining insight into the less concerned non-CSC component in promoting cancer, and would potentially enrich the treatment strategy for glioma.
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Reviewer #2 (Public Review):
Tumors such as glioblastoma contain several types of cells: cancerous and reactive non-cancerous cells, and among cancerous cells, cancer cells with tumorigenic properties so-called "stem" and pseudo-differentiated cancer cells.
Strengths: a multidisciplinary international cooperation gathering complementary expertises. An impressive quantity of experiments and presented data (28 supplementary figures with multiple panels!). First description of Fibromodulin as a secreted factor acting in a paracrine manner to activate an Integrin-dependent Notch signaling in endothelial cells. A detailed analysis of the molecular signaling triggered by integrin activation. Most of the results support this claim.
Weaknesses: Several formulations in the introduction are controversial. Several results should be more clearly …
Reviewer #2 (Public Review):
Tumors such as glioblastoma contain several types of cells: cancerous and reactive non-cancerous cells, and among cancerous cells, cancer cells with tumorigenic properties so-called "stem" and pseudo-differentiated cancer cells.
Strengths: a multidisciplinary international cooperation gathering complementary expertises. An impressive quantity of experiments and presented data (28 supplementary figures with multiple panels!). First description of Fibromodulin as a secreted factor acting in a paracrine manner to activate an Integrin-dependent Notch signaling in endothelial cells. A detailed analysis of the molecular signaling triggered by integrin activation. Most of the results support this claim.
Weaknesses: Several formulations in the introduction are controversial. Several results should be more clearly explained and the precise methods used are difficult to find since they are dispersed between the text, the "methods section" and often lacking in the legend of the figure. More precisely the following points should be addressed:
1- The formulation "non-cancer stem cells" is confusing since these are cancer cells but without the functional characteristics of cancer stem cells and within the tumor exist non-cancer cells co-opted to the tumor, such cells being called "microenvironment" even if they are bona fide part of the tumor.
2- Lines 91 to 95 are particularly controversial and even erroneous since CD133- GSC have been reported by several authors and nestin is not a selective marker of CSC. This is most-likely due to referencing reviews of a single group that promoted this dichotomy that do not correspond to most of the reported results. Furthermore it is well known that GSC proliferation or DGC reprogrammation to GSC are favored by hypoxia, illustrated in vivo by the failure of anti-VEGF treatment to increase life expectancy.
3- As soon as 2012-2013 (thus before the referenced Suva et al 2014 paper), the group of Thierry Virolle demonstrated that stem cell-like properties of GSC fuel glioblastoma development by providing the different cell types that comprise the tumor. Reference to their work is surprisingly missing. Of note, after describing that the miR-302-367 cluster is strongly induced during stemness suppression, they showed that stable miR-302-367 cluster expression is sufficient to suppress the stemness signature, self-renewal, and cell infiltration within a host brain tissue, through inhibition of the CXCR4 pathway involving the SHH-GLI-NANOG network. Micro-RNA profiling studies to search for regulators of stem cell plasticity, allowed them to identified miR-18a* as a potential candidate and its expression correlated with the stemness state. MiR-18a* expression promotes clonal proliferation in vitro and tumorigenicity in vivo. Turchi L, Debruyne DN, Almairac F, Virolle V, Fareh M, Neirijnck Y, Burel-Vandenbos F, Paquis P, Junier MP, Van Obberghen-Schilling E, Chneiweiss H, Virolle T. Tumorigenic potential of miR-18A* in glioma initiating cells requires NOTCH-1 signaling. Stem Cells. 2013 Jul;31(7):1252-65. doi: 10.1002/stem.1373. PMID: 23533157 Fareh M, Turchi L, Virolle V, Debruyne D, Almairac F, de-la-Forest Divonne S, Paquis P, Preynat-Seauve O, Krause KH, Chneiweiss H, Virolle T. The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network. Cell Death Differ. 2012 Feb;19(2):232-44. doi: 10.1038/cdd.2011.89.
4- A main question arises from the use of multiple cellular models, some highly valuables such as MGG4, MGG6 and MGG8, that correspond to patient-derived cell line maintained in culture conditions known to preserve the phenotype and genotype encountered in real patient tumors, and other cell lines (LN229, U251, U87) known to be highly unrepresentative since grown for a long time in serum conditions. However, after the first set of experiments, only MGG8 is used in the rest of the paper, with no validation on MGG4 and MGG6 and one should wonder why.
5- Results presented in Fig2C and 2D are really strange and do not support the claim that "results indicate that FMOD secreted by DGCs is essential for the growth of tumors initiated by GSCs. FMOD induces angiogenesis of host-derived and tumor-derived endothelial cells" First, one should wonder about the subcutaneous model used since such xenograft do not raise a glioblastoma-like tumor but a mesenchymal-like highly undifferentiated tumor. Second, considering the development of the graft at day 5 and the growth curve of GSC alone, one should wonder why inhibiting expression of FMOD in DGC triggers a necrosis of the initial tumor and not a slower growth parallel to the one of GSC alone.
6- Line 445: "Cellular hierarchy is well established in GBM." This is an old view mimicking normal differentiation. Since GSC can pseudo-differentiate into DGC and DGC can be reprogrammed into GSC, no hierarchy exists, only cells with different properties and functions in tumor growth. GSC is not the origin of glioblastoma but the ultimate state of aggressiveness.
7- Lines 452-53 "GSCs are known to promote the establishment of a highly vascularized microenvironment by being in close physical contact with endothelial cells (Calabrese et al., 2007)." This in only partially true since many soluble factors have been described to support the dialog between endothelial cells and GSC: secreted proteins such as VEGF, HDGF, GDF15, and multiple types of microRNA. -
Reviewer #3 (Public Review):
This article proposed a hypothesis that non-cancer stem cells secreted factor-FMOD could impressively promote angiogenesis to induce tumor growth in vivo. The finding uncovered a potential interesting and important protein therapeutic target from non-cancer stem cells but not from the glioma stem-like cells. Authors utilized diverse in vitro and in vivo methods to elucidate their hypothesis. The logic is smooth and clear and the results are solid. This article showed us that it might be worth also looking at non-cancer stem cells more in tumor growth.
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