Tumor Associated Macrophages Promote Glioma Stem Cells Invasion Via TGF-β1Signaling Pathway
|School||Third Military Medical University|
|Course||Pathology and Pathophysiology|
|Keywords||Malignant glioma Cancer stem cells Tumor associated macrophages Invasion Smad3 p38 Transforming growth factor-β1(TGF-β1) Transforming growthfactor-β type Ⅱ receptor (TGFBR2) Matrix metalloproteinase-9(MMP-9)|
Malignant gliomas are the most common primary brain tumors. One of the most lethalproperties of malignant gliomas is their capability of invading the surrounding normal braintissues. The infiltrating glioma cells often escape from surgical resection and subsequentlyresist radio-/chemo-therapy, which inevitably leads to tumor recurrence. Therefore, theinvasion of malignant glioma cells into the surrounding normal brain tissues is a crucial factorcausing the poor outcome and high mortality in patients.‘Cancer stem cells (CSCs)’ hypothesis opens up a new orientation in understandingmalignant gliomas and their biological characteristics. The results of our group demonstratethe existence of glioma stem cells (GSCs) in malignant glioma. GSCs are usually present inextremely small amounts and possess the properties of self-renewal and differentiation thatare similar to neural stem cells. These cells play a key role in the process of tumordevelopment and progression. Study on the biological characteristics of GSCs is useful forunderstanding the invasive property of malignant gliomas.The emerging evidence suggests that inflammatory cells are involved in the developmentand progression of tumors. The immune-microenvironment around the tumor and variousinfiltrating immune cells are known to be involved in many aspects of tumor progression,such as angiogenesis, immune-suppression, invasion and metastasis. Tumor associatedmacrophages (TAMs), which play an important role during tumor progression, are the majorproportion among these infiltrating immune cells. However, the impacts of tumorimmune-microenvironment and the infiltrating immune cells, such as TAMs, on thebiological characteristics of CSCs remains elusive..To further explore the influence of TAMs on the invasive property of GSCs and thepossible mechanisms underlying this process, this study first identified and characterizedCD133~+GSCs from the transplantation of murine malignant glioma cell line GL261andprimary glioblastoma specimens. Further, we successfully separated TAMs from the graft ofmurine malignant glioma in animal models, and the phenotype of TAMs was identified. We observed the spatial distribution of TAMs and GSCs in the marginal area of primary gliomaspecimens and orthotropic transplanted glioma, and then examined the impact of TAMs onthe invasiveness of GSCs. Finally, we studied the role of transforming growth factor-β1(TGF-β1) signaling pathway and its receptor, transforming growth factor-β type II receptor(TGFBR2) in promoting the invasiveness of GSCs mediated by TAMs. Our data will pave theway to identify new therapeutic targets for developing novel regimens for the treatment ofgliomas.The experiment data and main conclusions are listed as follows:1. Isolation and characterization of GSCs from malignant gliomas.①A heterogeneous contents of CD133~+glioma cells, ranging from0.1%to14.6%, wereexhibited in5cases of human primary glioma specimens.②A significantly higher proportionof CD133~+cells was detected in the transplanted tumor, which showed a steady state in vivo.CD133~+cells significantly increased in GL261derived tumors comparing with GL261cellline (1.83%±0.5%vs.0.68%±0.46%, p<0.01). However, there is no difference between thepercentages of CD133~+cells in transplanted tumor derived from GL261cell line and sortedCD133~+cells (1.83%±0.5%vs.2.05%±0.52%, p>0.05).③The purities of sorted CD133~+cells could reach above80%and these CD133~+cells could re-form tumor-spheres upon serialpassage in neural stem cells conditioned medium without serum.④CD133~+glioma cellsdisplayed a higher capability for tumor colony formation than CD133－counterparts.⑤Thesorted CD133~+cells expressed abundant nestin and Sox2, and lower levels of astrocyticmarker GFAP and oligodendrocytic marker MBP. In contrast, CD133－cells contained verylittle nestin and Sox2, and high levels of GFAP and MBP.⑥CD133~+cells with multipledifferentiation capacity could be induced to differentiate into cells expressing GFAP, MBP orβ-tubulin Ⅲ. ⅢAs few as1×103CD133~+cells were able to initiate tumor formation. Incontrast,10-fold higher number of CD133－cells were needed to form tumor in C57BL/6mice and500-fold higher number of CD133－cells were required in NOD-SCID mice,compared with CD133~+cells2. Maligant glioma infiltrating TAMs with a pro-tumor M2phenotype.①TAMs were the major group of malignant glioma infiltrating immune cells. Usingflow cytometry, we found that the percentage of malignant glioma infiltrating immune cells(i.e. CD45+cells) was about9.3%±2.7%, while that of infiltrating TAMs (i.e. CD11b+cells) could reach6.8%±1.1%. In addition, the distribution of a large number of CD68+cells couldalso be found in clinical specimens of human glioblastoma, especially in the invasive front oftumor.②CD11b+F4/80+TAMs could be purified by flow cytometry sorting fromGL261-derived tumors to the purity at94.6%. And these cells were round or oval, the typicalmacrophage morphology under the light microscope. CD11b antigen could be observed to beexpressed on the cell surface under the confocal microscope.③The same phenotype ofCD11b+F4/80+macrophages (Mφ) could be isolated from the spleen of normal mice.④TAMs exhibited a M2phenotype and released high levels of IL-10and TGF-β1, whileexpressed little cytokines associated with the M1phenotype, such as IL-1β, IL-6, IL-12p40and TNF-α under unstimulated condition, as well as LPS (100ng/ml) plus IFN-γ (20ng/ml)or IL-4(20ng/ml) induced condition. These results suggest that glioma infiltrating TAMswith M2phenotype.3. Presence of GSCs in the marginal area of glioma correlates with the location ofTAMs in the marginal area of glioma. Invasive promotion effect of isolated TAMs onGSCs could be blocked by TGF-β1neutralizing antibody.①No matter clinical specimens of human glioblastoma or orthotropic transplanted gliomarevealed that the presence of GSCs in the marginal area of glioma showed a positiverelationship with the location of TAMs in the marginal area of glioma. And the distribution ofTGF-β1in this region was consistent with that of TAMs.②Comparing with CD133－cells,CD133~+cells displayed notably increased invasive ability after co-culture with TAMs (p<0.01).③After the use of TGF-β1neutralizing antibody in co-culture, the invasiveness of CD133~+cells was significantly reduced (p<0.01). In contrast, the invasive ability of CD133－cells onlyslightly decreased (p>0.05).④Invasive capabilities of CD133~+cells of primary gliomas fromtwo different patients were found remarkably increased after pre-incubation with TGF-β1(p<0.01), while only sight influence on the invasiveness of CD133－cells (p<0.05).4. GSCs preferentially expressed high level of TGFBR2, which induce the release ofMMP-9from GSCs and play a crucial role on the invasion of GSCs mediated by TAMs.①TGFBR2mRNA and protein expression levels were found much higher in CD133~+cells than that in CD133－cells.②Matrix metalloproteinase-9(MMP-9) mRNA expressionlevel was found much higher in CD133~+cells than CD133－ones.③In the presence ofTAMs, additional increase of MMP-9mRNA was detected in the CD133~+GSCs. Neutralization of TGF-β1by TGF-β1neutralizing antibody resulted in a reduction of MMP-9mRNA in CD133~+tumor cells.④Theexogenous TGF-β1significantly promoted MMP-9expression in CD133~+cells, but had less effect on CD133－tumor cells.5. Disruption of TGF-β1signaling pathway by knocking down TGFBR2decreasedthe expression of MMP-9and reduced the invasion capability of GSCs.①The inducible effects of TGF-β1on MMP-9expression (p<0.01) and invasioncapability (p<0.01) of CD133~+tumor cells were blocked by SIS3(a specific inhibitor ofSmad3) or SB203580(a specific inhibitor of p38).②TGFBR2protein was remarkablydecreased by61%in the glioma cells that were transduced with shRNA2. TGFBR2knock-down by shRNA2significant decreased the expression of MMP-9in CD133~+tumorcells (p<0.01).③AlthoughTGFBR2knock-down in CD133－cells had no effect on theirinvasion capability (p>0.05), CD133~+tumor cells of which TGFBR2was knocked down aftertransduction of shRNA2significantly loss their invasion capability when they wereco-cultured with TAMs (p<0.01).④The invasion of orthotropic transplanted glioma derivedfrom CD133~+tumor cells transduced with shRNA2was significant decreased in vivo, andCD133~+tumor cells were remarkably reduced in the marginal area of transplanting tumorscompared to the transplanting tumor derived from the mock cells (p<0.01).Conclusion: The invasion of GSCs had a positive correlation with the distribution ofTAMs in the marginal area of malignant glioma. M2polarized TAMs could promote theinvasion of GSCs via paracrine TGF-β1. GSCs preferentially expressed high level ofTGFBR2, which was the key molecule during the invasive process of GSCs mediated byTAMs. Once activation of TGFBR2, the release of MMP-9from GSCs increased, whichpromoted the invasiveness of GSCs. Disruption of TGF-β1signaling pathway by usinginhibitors and knocking down TGFBR2both could decrease the expression of MMP-9andreduce the invasion capability of GSCs in vivo and in vitro. The results above suggest thatTGF-β1signaling pathway and its receptors TGFBR2are potential therapeutic targets forlimiting the invasion of GSCs.