This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research article
Akihiro Inoue
Ehime University Graduate School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7438-2369
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive and highly migratory glioma stem-like cells (GSCs) in tumors. Upon GBM recurrence or progression, the highly invasive phenotype of GSCs changes to a less-motile, proliferative phenotype, thus generating tumor mass. Elucidating the molecular mechanism underlying this phenotypic transition could lead to the identification of effective molecular targets for treating GBM.
Methods
We examined mRNA expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, CD44, and osteopontin in GBM tissues and investigated the effect of hypoxia (1% O2: severe or 5% O2: moderate) on expression of these molecules using two lines of cultured GSCs. We also analyzed the effect of osteopontin on the invasiveness, migration, and proliferation of GSCs under hypoxic conditions. In addition, the effect of CD44 knockdown on tumor growth and survival were investigated in vitro and in vivo using a mouse xenograft model.
Results
Severe hypoxia upregulates CD44 expression via activation of HIF-1α, inducing GSCs to assume a highly invasive phenotype. In contrast, moderate hypoxia upregulates osteopontin expression via activation of HIF-2α. Osteopontin in turn binds to CD44, simultaneously inhibiting CD44-promoted GSC migration and invasion and stimulating GSC proliferation, resulting in GSCs assuming a less-invasive, highly proliferative phenotype. CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo. However, although CD44 knockdown did not affect tumor growth in vitro, mouse brain tumors generated from GSCs with CD44 knockdown exhibited diminished invasiveness, and the mice survived significantly longer than control mice. In contrast, siRNA-mediated silencing of the osteopontin gene led to decreased GSC proliferation, but the osteopontin-mediated inhibition of high GSC migratory behavior and invasiveness was diminished.
Conclusion
The highly invasive phenotype of GSCs can be reversed by switching from severe to moderate hypoxia, leading to less-invasive and proliferative tumors. CD44 and osteopontin, which are expressed in a mutually exclusive manner under severe or moderate hypoxia, play a central role in regulating GSC invasion and proliferation by inducing a phenotypic transition, suggesting that these molecules could be effective targets for treating both primary and recurrent GBM.
Keywords
glioma stem cell, glioblastoma, CD44, osteopontin, hypoxia, invasion
Figure 1
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Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- ARRIVEchecklist.docx
- FigureS1.tif
Supplementary Figure 1. Evaluation of the CD44 silencing efficacy of five clones of CD44 shRNA.
- FigureS2.TIF
Supplementary Figure 2. MRI findings and expression of CD44 and VEGF of HI and LI tumor in GBMs. (A) MR images showing two phenotypic types of GBM. (left) HI tumor presents inhomogeneously gadrinium (Gd)-enhanced tumor with irregular margin wall and diffuse pritumoral edema, suggesting GBM with high invasive nature. (right) LI tumor discloses intensely Gd-enhanced tumor with relatively clear margin and focal edema, implying GBM with low invasive tumor. (B) Expression of CD44 (amounts of mRNA in the tumor periphery) (far left), VEGF (amounts of mRNA in the tumor periphery) (middle), and CD44 (periphery/core ratio of CD44 expression). HI tumor expressed significantly much more CD44 in the tumor periphery than LI tumor, while LI tumor expressed significantly much higher VEGF in the tumor periphery than CD44. *p<0.05 **p<0.001. HI, high invasive; LI, low invasive/ high proliferative.
- FigureS3.tif
Supplementary Figure 3. Features of glioblastoma stem-like cell, GDC40. (A) Ability of GDC40 to induce multilineage differentiation into neurons (TUJ1; green) and astrocytes (GFAP; red). The nuclei are labeled with Hoechst (blue). (bar 100um) Sphere formation of GDC40 is presented in Figure S4. (B) Expression of CD44 and OPN mRNA in GDC40 cells. The expression of CD44 was markedly increased under severe (1% O2) hypoxia, but the level of CD44 expression in GDC40 cells was markedly lower than that in the GSL1 and GSL2 lines (The data are presented in Fig. 1c. Note that the scale for the relative mRNA expression for the GSC lines is 10 times higher than that for GDC40 cells). There was no difference in the expression of OPN between the GDC40 and GSC lines. (C) Effect of inhibition of OPN expression on the expression of CD44 and VEGF in GDC40 cells. Silencing of OPN expression increased the expression of CD44 but decreased the expression of VEGF. *p<0.05 **p<0.01 *** p<0.001.
- FigureS4.tif
Supplementary Figure 4. Silencing OPN in GDC40 cells. (A) Inhibitory effect of OPN knockdown on the proliferation of GDC40 cells in sphere culture. (B) Inhibition of OPN induced diffuse infiltration of tumor cells around the sphere of GDC40 cells. Scale bar, 100 µm.
- additionalfile.pdf
We provide all original blot images with figure legends as additional files. Figure legends Original blot images-1. Western blot showing expressions of HIF-1α and CD44 under normoxia and 1% hypoxia with HIF-1α siRNA inhibition in two GSC lines, GSL-1 and GSL-2. Lanes: M; molecular weight size markers, 1; Control (normoxia), 2; Control (1% hypoxia), 3; treated with control siRNA (1% hypoxia), 4; treated with HIF-1α siRNA (1% hypoxia). Original blot images-2. Western blot showing expressions of HIF-2α and osteopontin (OPN) under normoxia and 1% hypoxia with HIF-2α siRNA inhibition in two GSC lines, GSL-1 and GSL-2. Lanes: M; molecular weight size markers, 1; Control (normoxia), 2; Control (5% hypoxia), 3; treated with control siRNA (5% hypoxia), 4; treated with HIF-2α siRNA (5% hypoxia).
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research article
Akihiro Inoue
Ehime University Graduate School of Medicine
Corresponding Author
ORCiD: https://orcid.org/0000-0002-7438-2369
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 22 Sep, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
The poor prognosis of glioblastoma multiforme (GBM) is primarily due to highly invasive and highly migratory glioma stem-like cells (GSCs) in tumors. Upon GBM recurrence or progression, the highly invasive phenotype of GSCs changes to a less-motile, proliferative phenotype, thus generating tumor mass. Elucidating the molecular mechanism underlying this phenotypic transition could lead to the identification of effective molecular targets for treating GBM.
Methods
We examined mRNA expression of hypoxia-inducible factor (HIF)-1α, HIF-2α, CD44, and osteopontin in GBM tissues and investigated the effect of hypoxia (1% O2: severe or 5% O2: moderate) on expression of these molecules using two lines of cultured GSCs. We also analyzed the effect of osteopontin on the invasiveness, migration, and proliferation of GSCs under hypoxic conditions. In addition, the effect of CD44 knockdown on tumor growth and survival were investigated in vitro and in vivo using a mouse xenograft model.
Results
Severe hypoxia upregulates CD44 expression via activation of HIF-1α, inducing GSCs to assume a highly invasive phenotype. In contrast, moderate hypoxia upregulates osteopontin expression via activation of HIF-2α. Osteopontin in turn binds to CD44, simultaneously inhibiting CD44-promoted GSC migration and invasion and stimulating GSC proliferation, resulting in GSCs assuming a less-invasive, highly proliferative phenotype. CD44 knockdown significantly inhibited GSC migration and invasion both in vitro and in vivo. However, although CD44 knockdown did not affect tumor growth in vitro, mouse brain tumors generated from GSCs with CD44 knockdown exhibited diminished invasiveness, and the mice survived significantly longer than control mice. In contrast, siRNA-mediated silencing of the osteopontin gene led to decreased GSC proliferation, but the osteopontin-mediated inhibition of high GSC migratory behavior and invasiveness was diminished.
Conclusion
The highly invasive phenotype of GSCs can be reversed by switching from severe to moderate hypoxia, leading to less-invasive and proliferative tumors. CD44 and osteopontin, which are expressed in a mutually exclusive manner under severe or moderate hypoxia, play a central role in regulating GSC invasion and proliferation by inducing a phenotypic transition, suggesting that these molecules could be effective targets for treating both primary and recurrent GBM.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- ARRIVEchecklist.docx
- FigureS1.tif
Supplementary Figure 1. Evaluation of the CD44 silencing efficacy of five clones of CD44 shRNA.
- FigureS2.TIF
Supplementary Figure 2. MRI findings and expression of CD44 and VEGF of HI and LI tumor in GBMs. (A) MR images showing two phenotypic types of GBM. (left) HI tumor presents inhomogeneously gadrinium (Gd)-enhanced tumor with irregular margin wall and diffuse pritumoral edema, suggesting GBM with high invasive nature. (right) LI tumor discloses intensely Gd-enhanced tumor with relatively clear margin and focal edema, implying GBM with low invasive tumor. (B) Expression of CD44 (amounts of mRNA in the tumor periphery) (far left), VEGF (amounts of mRNA in the tumor periphery) (middle), and CD44 (periphery/core ratio of CD44 expression). HI tumor expressed significantly much more CD44 in the tumor periphery than LI tumor, while LI tumor expressed significantly much higher VEGF in the tumor periphery than CD44. *p<0.05 **p<0.001. HI, high invasive; LI, low invasive/ high proliferative.
- FigureS3.tif
Supplementary Figure 3. Features of glioblastoma stem-like cell, GDC40. (A) Ability of GDC40 to induce multilineage differentiation into neurons (TUJ1; green) and astrocytes (GFAP; red). The nuclei are labeled with Hoechst (blue). (bar 100um) Sphere formation of GDC40 is presented in Figure S4. (B) Expression of CD44 and OPN mRNA in GDC40 cells. The expression of CD44 was markedly increased under severe (1% O2) hypoxia, but the level of CD44 expression in GDC40 cells was markedly lower than that in the GSL1 and GSL2 lines (The data are presented in Fig. 1c. Note that the scale for the relative mRNA expression for the GSC lines is 10 times higher than that for GDC40 cells). There was no difference in the expression of OPN between the GDC40 and GSC lines. (C) Effect of inhibition of OPN expression on the expression of CD44 and VEGF in GDC40 cells. Silencing of OPN expression increased the expression of CD44 but decreased the expression of VEGF. *p<0.05 **p<0.01 *** p<0.001.
- FigureS4.tif
Supplementary Figure 4. Silencing OPN in GDC40 cells. (A) Inhibitory effect of OPN knockdown on the proliferation of GDC40 cells in sphere culture. (B) Inhibition of OPN induced diffuse infiltration of tumor cells around the sphere of GDC40 cells. Scale bar, 100 µm.
- additionalfile.pdf
We provide all original blot images with figure legends as additional files. Figure legends Original blot images-1. Western blot showing expressions of HIF-1α and CD44 under normoxia and 1% hypoxia with HIF-1α siRNA inhibition in two GSC lines, GSL-1 and GSL-2. Lanes: M; molecular weight size markers, 1; Control (normoxia), 2; Control (1% hypoxia), 3; treated with control siRNA (1% hypoxia), 4; treated with HIF-1α siRNA (1% hypoxia). Original blot images-2. Western blot showing expressions of HIF-2α and osteopontin (OPN) under normoxia and 1% hypoxia with HIF-2α siRNA inhibition in two GSC lines, GSL-1 and GSL-2. Lanes: M; molecular weight size markers, 1; Control (normoxia), 2; Control (5% hypoxia), 3; treated with control siRNA (5% hypoxia), 4; treated with HIF-2α siRNA (5% hypoxia).
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