ABSTRACT The interplay between glioma stem cells (GSCs) and the tumor microenvironment plays crucial roles in promoting malignant growth of glioblastoma (GBM), the most lethal brain tumor.

However, the molecular mechanisms underlying this crosstalk are incompletely understood. Here, we show that GSCs secrete the Wnt‐induced signaling protein 1 (WISP1) to facilitate a pro-tumor

microenvironment by promoting the survival of both GSCs and tumor-associated macrophages (TAMs). WISP1 is preferentially expressed and secreted by GSCs. Silencing WISP1 markedly disrupts

GSC maintenance, reduces tumor-supportive TAMs (M2), and potently inhibits GBM growth. WISP1 signals through Integrin α6β1-Akt to maintain GSCs by an autocrine mechanism and M2 TAMs through

CONTENT BEING VIEWED BY OTHERS ARS2/MAGL SIGNALING IN GLIOBLASTOMA STEM CELLS PROMOTES SELF-RENEWAL AND M2-LIKE POLARIZATION OF TUMOR-ASSOCIATED MACROPHAGES Article Open access 12 June 2020

IFI35 REGULATES NON-CANONICAL NF-ΚB SIGNALING TO MAINTAIN GLIOBLASTOMA STEM CELLS AND RECRUIT TUMOR-ASSOCIATED MACROPHAGES Article 09 April 2024 CCL2 MEDIATED IKZF1 EXPRESSION PROMOTES M2

POLARIZATION OF GLIOMA-ASSOCIATED MACROPHAGES THROUGH CD84-SHP2 PATHWAY Article 07 August 2024 INTRODUCTION Glioblastoma (GBM), the WHO grade IV glioma, is the most common and lethal type of

primary brain tumor. Despite aggressive treatments, including surgical resection, radiotherapy, and chemotherapy, the median survival of GBM patients remains less than 16 months1,2. GBM

displays striking cellular heterogeneity and hierarchy, with heterogeneous cancer cells, including glioma stem cells (GSCs) and non-stem tumor cells (NSTCs), in the tumor microenvironment,

which also includes endothelial cells, vascular pericytes, abundant tumor-associated macrophages (TAMs), and other immune cells3,4. GSCs, comprising a small fraction of cancer cells at the

apex of the differentiation hierarchy, play crucial roles in tumor initiation, cancer invasion, tumor angiogenesis, immune evasion, and therapeutic resistance3,5,6. GSCs actively interact

with other cells in the tumor microenvironment to promote malignant progression in GBMs4,7,8. Thus, targeting GSCs and their interactions with other components of the tumor microenvironment

has the potential for improving GBM treatment. TAMs are abundant in the GBM microenvironment, and are important in supporting malignant growth and progression. The density of TAMs correlates

positively with glioma grade and negatively with prognosis9,10. TAMs are the main source of cytokines that promote tumor cell growth in GBMs, including IL-611. In addition, TAMs closely

interact with GSCs12,13, as both cell types are enriched in perivascular regions and hypoxia niches in GBMs14,15,16,17,18. Interestingly, both GSC and TAM populations are increased in

recurrent tumors after irradiation19,20. Recent studies have shown that TAMs secrete cytokines, such as Pleiotrophin (PTN) and TGF-β1, to promote GSC maintenance and invasion21,22.

Furthermore, GSCs recruit monocyte-derived TAMs from peripheral blood to promote GBM growth through paracrine Periostin (POSTN) and Osteopontin signaling18,23. It is well recognized that

TAMs include two major populations: tumor-supportive M2 macrophages and tumor-suppressive M1 macrophages24, although each population may contain subpopulations. M2 TAMs play immune

suppressive roles in the tumor microenvironment to promote tumor growth25. The majority of TAMs in GBMs display M2-like properties26. These M2 TAMs have been shown to support malignant

growth in GBM tumors18,21,27. Despite the significant effect of M2 TAMs on GBM progression, the molecular mechanisms underlying the maintenance of M2 TAMs in the tumor microenvironment

remain unclear. We have previously shown that GSCs secrete Periostin to recruit monocyte-derived TAMs into GBMs18, but how TAMs are educated and maintained as M2 tumor-supportive macrophages

in the tumor microenvironment in GBM has not been defined. The Wnt/β-catenin signaling regulates cell proliferation, migration, and death and plays key roles in development, tissue

homeostasis, and cancer progression28. The activation of the Wnt/β-catenin pathway leads to the stabilization of β-catenin, which is subsequently translocated into the nucleus, where it

activates the transcription of Wnt target genes28. A previous report showed that tumor-intrinsic Wnt/β-catenin signaling could regulate the tumor microenvironment to promote malignant

progression29. In lymphoma, Wnt/β-catenin signaling is activated and promotes lymphoma cell chemotaxis towards endothelial cells and adhesion to the endothelial cell layers30. Activation of

Wnt/β-catenin signaling in melanoma inhibits T cell infiltration to promote tumor growth and therapeutic resistance by regulating CCL4 secretion31. In addition, activation of Wnt/β-catenin

signaling in osteoblasts promotes metastasis in prostate cancer through paracrine production of WISP1, and activates its receptor on prostate cancer cells in bone metastases32. In GBMs,

Wnt/β-catenin signaling is highly activated in GSCs, promoting malignant transformation and tumor progression33,34. However, how Wnt/β-catenin signaling promotes tumor growth in GBMs is not

fully understood. In addition, whether Wnt/β-catenin signaling can regulate the GBM microenvironment to promote malignant progression remains elusive. To interrogate the potential

relationship between Wnt/β-catenin activation and regulation of the tumor microenvironment in GBMs, we analyzed the expression of Wnt/β-catenin-induced secretory proteins, finding that

_WISP1_ is the only highly expressed gene in GBMs relative to normal brains. WISP1, first discovered as a target gene of the Wnt/β-catenin pathway35, is a secreted cysteine-rich protein that

belongs to the CCN family of matri-cellular proteins. It is involved in cell adhesion, survival, proliferation, differentiation, and migration36. Increased WISP1 expression is associated

with tumor progression in certain tumor types and predicts poor prognosis37. A recent study demonstrated that WISP1 is highly expressed in colon cancer and promotes proliferation and

invasion38. WISP1 is also upregulated in breast cancer to promote cell proliferation, invasion, and epithelial-mesenchymal-transition (EMT)39. Here, we investigate the role of WISP1 in

regulating GBM growth, finding that WISP1 plays a dual role in promoting GBM growth through both autocrine and paracrine effects. WISP1 promotes GSC maintenance in an autocrine loop.

Importantly, it also promotes the survival of tumor-supportive TAMs (M2) to support tumor growth in a paracrine fashion. Inhibition of Wnt/β-catenin-WISP1 signaling by carnosic acid (CA)

disrupts the GSC maintenance, inhibits survival of tumor-supportive TAMs, and suppresses GBM growth, suggesting that targeting this signaling axis may effectively improve GBM treatment.

RESULTS WISP1 IS PREFERENTIALLY SECRETED BY GLIOMA STEM CELLS To investigate the potential molecular link between Wnt/β-catenin signaling and regulation of the tumor microenvironment in

GBMs, we analyzed the expression of Wnt/β-catenin target genes, especially secretory proteins, including _CXCL12_, _DKK1_, _WISP1_, _FGF20_, and _EDN1_40,41, in GBMs, using the TCGA42 and

Gravendeel43 databases. These analyses revealed that _WISP1_ is the only Wnt/β-catenin target gene preferentially expressed in human GBMs relative to normal brain tissues (Fig. 1a, b and

Supplementary Fig. 1a, b). Bioinformatic analyses of these databases indicated that high expression of _WISP1_ correlates with poor survival (Fig. 1c, d). To assess whether WISP1 is

expressed in GBMs, we initially examined WISP1 expression in 5 pairs of matched GSCs and non-stem tumor cells (NSTCs). Matched GSCs and NSTCs were isolated from human GBM surgical specimens

or patient-derived GBM xenografts through cell sorting (CD15+/CD133+ for GSCs and CD15−/CD133− for NSTCs). Isolated GSCs were characterized by the expression of the GSC markers (SOX2, OLIG2,

CD133, L1CAM) and functional assays including serial neurosphere formation assay, in vitro cell differentiation assay and in vivo limiting dilution tumor formation assay. Immunoblot

analyses showed that WISP1, active β-catenin, total β-catenin and the GSC markers including SOX2 and OLIG2 were preferentially expressed in GSCs relative to matched NSTCs (Fig. 1e).

Consistently, immunofluorescent staining of WISP1 and the GSC marker SOX2 in matched GSCs and NSTCs validated the preferential expression of WISP1 in GSCs (Fig. 1f). As WISP1 is a secreted

protein, we determined the levels of WISP1 in the conditioned media from paired GSCs and NSTCs, confirming that conditioned medium from GSCs contains much more WISP1 than that from matched

NSTCs (Fig. 1g). To further verify the preferential expression of WISP1 by GSCs in vivo, we examined the expression patterns of WISP1 in several human GBM specimens and GSC-derived GBM

xenografts. Immunofluorescent staining confirmed that WISP1 was preferentially expressed in glioma cells expressing the GSC markers SOX2 and OLIG2, and was enriched in the proximity of GSCs

(Fig. 1h, i and Supplementary Fig. 1c,d). Taken together, these data demonstrate that WISP1 is preferentially expressed and secreted by GSCs in human GBMs. WISP1 SUPPORTS THE MAINTENANCE OF

GLIOMA STEM CELLS To determine the functional significance of the preferential expression of WISP1 in GSCs, we examined the effect of WISP1 disruption by shRNA on the GSC maintenance.

Silencing WISP1 by two independent shRNAs significantly reduced WISP1 expression in GSCs (Fig. 2a), resulting in decreased GSC proliferation in T4121 and T387 GSCs (Fig. 2b). Furthermore,

disruption of WISP1 impaired the self-renewal of GSCs, as assessed by tumorsphere formation (Fig. 2c, d) and in vitro limiting dilution assays (Fig. 2e). These data indicate that WISP1 is

required for the GSC maintenance. To further validate the function of WISP1 in GSC maintenance, we also examined whether the exogenous recombinant human WISP1 (rWISP1) protein could replace

autocrine WISP1 to rescue GSC proliferation and tumorsphere formation that had been impaired by silencing endogenous WISP1. Consistently, addition of exogenous rWISP1 partially rescued the

decreased GSC proliferation and tumorsphere formation caused by WISP1 disruption in a dose-dependent manner (Fig. 2f and Supplementary Fig. 2a). In addition, treatment of GSCs with exogenous

rWISP1 increased GSC proliferation in a dose-dependent manner (Supplementary Fig. 2b). Moreover, forced expression of WISP1 in GSCs further augmented cell proliferation and tumorsphere

formation (Fig. 2g-i and Supplementary Fig. 2c–e). Collectively, these data indicate that WISP1, secreted by GSCs, promotes cell proliferation and self-renewal of GSCs through an autocrine

loop. SILENCING WISP1 INHIBITS GSC-DRIVEN TUMOR GROWTH As WISP1 plays a critical role in maintaining GSCs, which potentially promotes malignant growth, we examined the impact of disrupting

WISP1 on GSC-driven tumor growth in vivo. GSCs (T4121 or T387) expressing firefly luciferase along with WISP1 shRNAs or non-targeting control shRNA (shNT) were transplanted into the brains

of immunocompromised mice. Bioluminescent imaging showed that WISP1 disruption markedly impaired GSC-driven tumor growth (Fig. 3a, b and Supplementary Fig. 3a, b). As a consequence, mice

bearing xenografts derived from shWISP1-expressing GSCs survived significantly longer than control mice (Fig. 3c and Supplementary Fig. 3c). Immunofluorescent staining indicated that the

xenografts from shWISP1-expressing GSCs contained fewer Ki67-postive proliferative cells (Fig. 3d, e and Supplementary Fig. 3d,e) and more apoptotic cells, marked by cleaved-caspase-3 (Fig. 

3f, g and Supplementary Fig. 3f, g). In addition, WISP1 disruption by shRNA significantly decreased the GSC population as measured by SOX2 immunofluorescence in tumor xenografts (Fig. 3h, i

and Supplementary Fig. 3h,i). Collectively, these results demonstrate that WISP1 plays an essential role in promoting GSC-driven tumor growth in GBMs. WISP1 ACTIVATES THE AKT PATHWAY TO

PROMOTE GSC PROLIFERATION To understand how WISP1 promotes GSC maintenance and tumor growth, we used a protein phospho-kinase array to identify downstream mediators of WISP1 function. The

result showed that the activating phosphorylation of Akt (pAkt-Ser473) was dramatically reduced by disrupting WISP1 (Fig. 4a), indicating that WISP1 may regulate Akt activity in GSCs.

Immunoblot analysis confirmed that knockdown of WISP1 reduced Akt-activating phosphorylation (pAkt-Ser473) in GSCs (Fig. 4b), whereas overexpression of WISP1 enhanced this phosphorylation

(Fig. 4c). Decreased Akt phosphorylation was also detected in GSC-derived xenografts expressing shWISP1 relative to the shNT control (Supplementary Fig. 4a). As WISP knockdown reduced Akt

phosphorylation (pAKT-Ser473), we further examined whether ectopic expression of WISP1 rescues the effect induced by WISP1 disruption. As the shWISP1-2 targets the 3’-end non-coding region

of endogenous _WISP1_ mRNA, and the WISP1 overexpression construct does not contain the 3’-end non-coding sequence, we were able to simultaneously silence endogenous WISP1 and overexpress

exogenous WISP1 in GSCs. Immunoblot analyses showed that ectopic expression of WISP1 in GSCs rescued the decreased Akt phosphorylation (pAkt-Ser473) caused by knockdown of endogenous WISP1

(Supplementary Fig. 4b). To further address whether WISP1 is an autocrine agonist of Akt signaling in GSCs, we examined the effect of rWISP1 protein on Akt activation. Consistently,

stimulation with rWISP1 induced significant phosphorylation of Akt in GSCs (Fig. 4d). rWISP1 treatment also rescued the decreased Akt phosphorylation (pAkt-Ser473) caused by WISP1 disruption

in a dose-dependent manner (Supplementary Fig. 4c). These results demonstrate that WISP1 secreted by GSCs regulates Akt signaling in an autocrine manner. We next explored whether Akt

signaling is required for WISP1-mediated GSC maintenance and tumorigenic potential. As Akt1 is the predominant isoform expressed in GSCs, a constitutively active form of Akt1 (Myr-Akt1) was

introduced into GSCs expressing shWISP1, or a shNT control (Fig. 4e and Supplementary Fig. 4d). Ectopic expression of Myr-Akt1 restored the proliferation and tumorsphere formation of GSCs

impaired by WISP1 disruption (Fig. 4f, g and Supplementary Fig. 4e, f). Similarly, ectopic expression of Myr-Akt1 in GSCs expressing shWISP1 partially restored tumor growth and reduced the

survival of mice bearing GSC-derived GBMs (Fig. 4h–j). Immunofluorescent staining showed that overexpression of Myr-Akt1 partially rescued the proliferation of GSCs expressing shWISP1 in

vivo, as indicated by elevated Ki67-positive staining in xenografts (Supplementary Fig. 4g, h). Collectively, these data suggest that WISP1 activates Akt signaling in GSCs to promote cell

proliferation and survival, which may partially augment tumor growth in vivo. INTEGRIN Α6Β1 IS A RECEPTOR FOR AUTOCRINE WISP1 IN GSCS To understand the molecular mechanisms underlying

WISP1-mediated Akt activation in GSCs, we sought to identify the receptor for the autocrine function of WISP1. Emerging evidence suggests that WISP1 may trigger its downstream signaling by

binding to versatile cell surface receptors, the Integrins44,45. As a superfamily of cell adhesion receptors, Integrins regulate a variety of cellular responses through various combinations

of α and β subunits in a cell-specific manner46,47. However, the specific Integrin mediating the function of WISP1 in GSCs was unclear, although Integrins α3, α6, and α7 have been reported

to be preferentially expressed in GSCs and promote GSC maintenance and tumor growth48,49,50. To determine specific Integrins that participate in the function of WISP1 in GSCs, we utilized

blocking antibodies. Immunoblot analysis showed that anti-Integrin α6 antibody dramatically attenuated Akt activity induced by WISP1 overexpression in GSCs, while the other two blocking

antibodies against Integrin α3 or α7 had little effect (Fig. 5a, b). Treatment with the Integrin α6-blocking antibody also reversed the enhanced GSC proliferation and tumorsphere formation

induced by ectopic expression of WISP1 (Fig. 5c, d and Supplementary Fig. 5a, b). Because Integrin α6 forms a functional dimer with Integrin β1 in GSCs49, we used Integrin β1-blocking

antibody to probe the role of Integrin β1. As expected, anti-Integrin β1 inhibited the GSC proliferation and tumorsphere formation induced by WISP1 overexpression (Fig. 5e, f and

Supplementary Fig. 5c, d). Moreover, treatment of GSCs with Integrin α6- or β1-blocking antibody significantly decreased GSC proliferation and tumorsphere formation (Fig. 5g, h and

Supplementary Fig. 5e, f). However, blocking Integrin β4, the other binding partner of Integrin α6, had no effect on GSC proliferation and tumorsphere formation (Fig. 5g, h and Supplementary

Fig. 5e,f). Immunoblot analysis confirmed that inhibiting Integrin α6 or β1 by blocking antibody reduced Akt phosphorylation (pAkt-Ser473) in GSCs, while inhibiting Integrin β4 had no

effect on the Akt phosphorylation (Supplementary Fig. 5g). We next examined the effects of Integrin α6 disruption by shRNA on GSC proliferation and Akt phosphorylation. shRNAs targeting α6

significantly decreased Integrin α6 expression and Akt phosphorylation (pAkt-Ser473) in GSCs (Fig. 5i and Supplementary Fig. 5h). Disruption of Integrin α6 also significantly inhibited GSC

proliferation and tumorsphere formation (Fig. 5j, k and Supplementary Fig. 5i, j). Taken together, these data demonstrate that WISP1 enhances Akt activating phosphorylation and GSC

proliferation through Integrin α6β1. To validate that Integrin α6β1 is a receptor for WISP1, we performed co-immunoprecipitation (CoIP) assay to confirm their binding. To increase the

potential binding for detection, we overexpressed WISP1 in GSCs and then performed CoIP with anti-Integrin α6 or β1 antibody. Anti-Integrin α6 antibody pulled down the Integrin α6 along with

WISP1 and Integrin β1 (Fig. 6a, b). In addition, the anti-Integrin β1 antibody pulled down the Integrin β1 along with WISP1 and Integrin α6 (Fig. 6c, d). To test the specificity of the

interaction between WISP1 and the receptor Integrin α6β1, we examined Akt phosphorylation in GSCs treated with rWISP1 along with Integrin blocking antibody at different ratios. Immunoblot

analysis showed that 5 μg/ml of Integrin α6 or β1 blocking antibody dramatically prevented the Akt phosphorylation (pAkt-Ser473) induced by 0.2 μg/ml of rWISP1, while this dose of antibody

had a relatively little effect on the Akt phosphorylation (pAkt-Ser473) induced by 0.8 μg/ml of rWISP1 (Fig. 6e). However, 10 μg/ml of Integrin α6 or β1 blocking antibody dramatically

prevented the increased Akt phosphorylation (pAkt-Ser473) induced by both doses of rWISP1 (Fig. 6e). These results indicate that Integrin α6β1 is relatively specific to WISP1. Moreover,

immunofluorescent analysis showed the co-expression of WISP1 and Integrin α6 proteins in primary human GBM samples (Fig. 6f). A recent study showed that malignant cells in human GBM exist in

four main cellular states that recapitulate neural-progenitor-like (NPC-like), oligodendrocyte-progenitor-like (OPC-like), astrocyte-like (AC-like), and mesenchymal-like (MES-like)

states51. Thus, we also assessed the expression of WISP1 and Integrin α6β1 across the four GBM cellular states. The results showed that WISP1 is enriched in some AC-like and MES-like cells,

while Integrin α6 and β1 are widely expressed in all four states. These data suggest that WISP1 and Integrin α6β1 are co-expressed by some AC-like and MES-like cells in GBM (Supplementary

Fig. 5k). Collectively, these data indicate that Integrin α6β1 is the receptor for autocrine signaling in response to WISP1 in GSCs. WISP1 PROMOTES THE SURVIVAL OF TUMOR-SUPPORTIVE TAMS IN

VIVO Because overexpression of Myr-Akt1 in GSCs expressing shWISP partially rescued the impaired tumor growth caused by WISP1 disruption, we speculated that other mechanisms may be involved

in the growth of GBMs promoted by WISP1, in addition to its autocrine signaling in GSCs. Therefore, we explored whether secreted WISP1 could also affect other cell types in GBMs, in a

paracrine manner. First, we examined the impact of rWISP1 on the viability of NSTCs in vitro. In a cell titer assay, exogenous rWISP1 treatment had no obvious effect on the growth or

survival of NSTCs (Supplementary Fig. 2f). We also analyzed whether WISP1 disruption could impact tumor angiogenesis. Immunofluorescent staining using the endothelial marker Glut1 showed

that WISP1 knockdown had little effect on vascular density in GSC-derived tumors (Supplementary Fig. 6a–d). Because GBMs usually contain abundant TAMs that mainly promote malignant

progression12,52, we examined whether WISP1 disruption could affect TAM density and subtype distribution in GBMs. Immunofluorescent staining using the total TAM markers Iba1 and CD11b

demonstrated that knockdown of WISP1 markedly decreased TAM density in GSC-derived xenografts (Fig. 7a–f and Supplementary Fig. 6c, e), and ectopic expression of Myr-Akt1 did not rescue the

decreased TAM density caused by WISP1 disruption in the GSC-derived xenografts (Supplementary Fig. 6f, g). The expression of WISP1 were indeed significantly decreased in xenografts

expressing WISP1 shRNA, demonstrated that these tumors were not derived from the GSCs that escaped form shRNA knockdown (Fig. 7a–f and Supplementary Fig. 6f, g). As both GSCs and TAMs are

enriched in perivascular niches in GBMs, we next examined the potential correlation between WISP1 expression and TAM density in primary GBM specimens. Immunofluorescent analysis showed that

TAMs are enriched in the WISP1-abundant regions (Supplementary Fig. 7a), supporting the idea that WISP1 may play a role in the TAM maintenance. As TAMs include both tumor-supportive

macrophages (M2 TAMs) and tumor-suppressive macrophages (M1 TAMs)53,54, we investigated which subtype of TAMs is affected by WISP1 disruption in GSC-derived xenografts. We used several

specific M2 markers (CD206, CD163, Arg1, and Fizz1) and M1 markers (CD11c, CD16/32, iNOS, and MHCII) for the study, as those markers have been used to distinguish M2/M1 TAMs in GBM xenograft

models from our group18 and others55,56,57. We found that WISP1 disruption markedly reduced M2 TAMs in GSC-derived tumors (Fig. 7g–l and Supplementary Fig. 7b–g). Interestingly, disrupting

WISP1 had little effect on M1 TAMs (Supplementary Fig. 8a–l). Immunofluorescent analyses further demonstrated that disrupting WISP1 increased apoptosis of M2 TAMs (Cleaved Caspase-3+/CD206+)

(Supplementary Fig. 9a, b) and showed no effect on M1 TAMs (Cleaved Caspase-3+/CD16/32+) (Supplementary Fig. 9c, d). Consistently, WISP1 disruption resulted in a significant increase in

total apoptotic TAMs (Cleaved Caspase-3+/Iba1+) in the xenografts (Supplementary Fig. 9e, f). Taken together, these data demonstrate that WISP1 secreted by GSCs potently promotes the

survival of M2 TAMs in GBMs. To further confirm the WISP1 function in M2 TAM maintenance and GBM tumor growth, we applied a Tet-On inducible expression system to examine whether inducible

overexpression of WISP1 in response to doxycycline (Dox) affects the TAM population and GBM tumor growth. Immunoblot analysis confirmed that Dox treatment enhanced WISP1 expression in GSCs

(Supplementary Fig. 10a). Bioluminescent imaging demonstrated that induced overexpression of WISP1 by Dox treatment significantly promoted GSC-driven tumor growth in mouse brains

(Supplementary Fig. 10b–d). Importantly, induced expression of WISP1 by Dox also increased the density of M2 and total TAMs (Supplementary Fig. 10e–h). These results validate that WISP1

plays a critical role in maintaining M2 TAMs to support GBM tumor growth. WISP1 SIGNALS VIA Α6Β1-AKT TO PROMOTE M2 TAM SURVIVAL As our data suggest that Integrin α6β1 is the key receptor for

WISP1-mediated signaling in GSCs, we next examined whether Integrin α6β1 is also expressed on cell surface of TAMs. Immunofluorescent staining demonstrated that Integrin α6 or β1 was also

expressed by tumor-supportive M2 TAMs (Supplementary Fig. 11a, b) but rarely colocalized with the M1 TAM markers in human GBMs (Supplementary Fig. 11c, d). To confirm the preferential

expression of α6β1 in M2 TAMs, we further examined α6β1 expression in primed M1 and M2 macrophages in vitro. Since U937 monocyte-like cells can be primed to differentiate into

macrophages18,21, we polarized U937 cells into M1 or M2 macrophages to mimic TAMs for our in vitro study. Immunoblot analyses of the M2 markers (CD163, CD206, and Arg-1) or M1 markers (MHC

II and iNOS) validated that U937 cells were successfully polarized into M1 or M2 macrophages (Supplementary Fig. 12a). Consistently, both Integrins α6 and β1 were preferentially expressed in

M2 macrophages relative to M1 macrophages (Supplementary Fig. 12a). To verify the function of WISP1 in macrophage survival, we examined whether exogenous rWISP1 protein could rescue the

macrophages from serum starvation-induced cell death. Indeed, rWISP1 treatment significantly prevented the death of M2 macrophages in a dose-dependent manner, while having no effect on M1

macrophages (Supplementary Fig. 12b). In addition, rWISP1 treatment enhanced Akt-activating phosphorylation (pAkt-Ser473) in M2 macrophages, but not in M1 macrophages (Supplementary Fig. 

12c). To assess whether WISP1 promotes the survival of M2 TAMs through Integrin α6β1 signaling, we applied Integrin α6 shRNAs to knockdown its expression (Supplementary Fig. 12d). Disruption

of Integrin α6 by shRNAs inhibited the rWISP1-enhanced survival of M2 macrophages cultured under serum starvation condition (Supplementary Fig. 12e). To further validate this result, we

then applied Integrin α6- or β1-neutralizing antibodies to block this function. Anti-Integrin α6 or anti-β1 also substantially inhibited the rWISP1-enhanced survival of M2 macrophages

cultured under serum starvation condition (Supplementary Fig. 12f). Consistently, Integrin α6 or β1 blocking antibody attenuated rWISP1-induced Akt-activating phosphorylation in M2

macrophages (Supplementary Fig. 12g). Collectively, these results indicate that WISP1 promotes the survival of tumor-supportive M2 macrophages by activating Integrin α6β1-Akt signaling.

DISRUPTING THE WNT/Β-CATENIN-WISP1 AXIS INHIBITS GBM GROWTH To evaluate the therapeutic potential of targeting Wnt/β-catenin-WISP1 signaling in GBM, we examined whether pharmacologic

inhibition of this pathway by carnosic acid (CA), a small molecule inhibitor of β‐catenin activity58, could impact GSCs and M2 TAMs to inhibit GBM tumor growth. We selected carnosic acid in

our preclinical study, because it can penetrate the blood-brain barrier59,60, and it has been reported to improve the treatment of medulloblastoma in a mouse model60. When GSCs were treated

with different doses of CA, the expression levels of active β-catenin and WISP1 were significantly reduced in a dose-dependent manner (Supplementary Fig. 13a, b). Consistently, CA treatment

markedly reduced GSC viability (Supplementary Fig. 13c, d) and suppressed GSC tumorsphere formation (Supplementary Fig. 13e, f) in a dose-dependent manner. Next, we examined the effect of CA

on the growth of orthotopic GBM xenografts, based on its in vitro efficacy and known ability to penetrate the blood-brain barrier59,60. In vivo bioluminescent imaging indicated that CA

significantly inhibited the growth of GSC-derived xenografts (Fig. 8a–c). Consequently, mice treated with CA had a significantly extended survival relative to the control group (Fig. 8d).

Immunofluorescent staining showed that CA administration reduced Ki67-postive proliferative cells (Fig. 8e, f) and increased the number of apoptotic cells, marked by cleaved-caspase-3, in

GSC-derived xenografts (Fig. 8g, h). In addition, CA treatment significantly reduced the GSC population in GBM xenografts, as demonstrated by SOX2 immunofluorescence (Fig. 8i, j). Moreover,

CA treatment resulted in a significant decrease in WISP1 expression, in the number of M2 TAMs (CD206+ or CD163+) and total TAMs (Iba1+) in GSC-derived xenografts (Fig. 8k, l and

Supplementary Fig. 13g–i). Collectively, these data demonstrate that inhibition of Wnt/β-catenin-WISP1 signaling by CA disrupts GSC maintenance, impairs M2 TAM survival, and potently

suppresses GBM tumor growth, indicating that targeting this pathway may effectively improve GBM treatment. DISCUSSION Wnt/β-catenin signaling has been implicated in the regulation of

malignant growth in several cancer types, but less is known regarding its role in mediating crosstalk between GSCs and other cells in the tumor microenvironment. In this study, we identified

WISP1 as a key mediator of the GSC-GSC and GSC-TAM crosstalk in GBMs (Fig. 9). We demonstrate that WISP1 plays crucial roles in promoting the maintenance of GSCs and survival of

tumor-supportive M2 TAMs by activating Akt (Fig. 9). Moreover, Inhibition of Wnt/β-catenin-WISP1 signaling markedly suppresses GBM tumor growth, suggesting that targeting this signaling axis

represents an attractive therapeutic strategy. Our findings indicate that WISP1 promotes GSC proliferation and self-renewal in an autocrine loop. Several studies reported that autocrine

WISP1 signaling enhanced cell growth in various cancers such as breast cancer and oral squamous cell carcinoma61,62,63, but its autocrine role in GBM has not been defined. A recent study

showed that WISP1 is an oncogene in GBM and inhibition of WISP1 suppressed the proliferation of GBM cells64. However, the origin of WISP1 in GBM and the role of WISP1 in regulating of GSC

properties remain unclear. Our study reveals that WISP1 is preferentially expressed by GSCs and activates Akt signaling to promote GSC proliferation. As re-activating Akt signaling in

shWISP-expressing GSCs only partially rescues tumor growth, we further investigated additional mechanisms that might be involved in WISP1-promoted GBM tumor growth and surprisingly found

that the WISP1-mediated activation of Akt is crucial for maintaining tumor-supportive M2 TAMs. TAMs are critical immune cells in the GBM microenvironment and play important roles in

facilitating GBM growth65. Our study reveals a paracrine mechanism that drives the survival of tumor-supportive M2 TAMs in which the WISP1 produced by GSCs supports GBM malignant

progression. Our results demonstrate that disruption of WISP1 dramatically reduces density of M2 TAMs. We fully recognized that the M1/M2 dichotomy is an oversimplification of TAMs in

tumors. In this study, we used the term “M2 TAMs” to indicate the tumor-supportive macrophages that may contain several subpopulations, and used “M1 TAMs” to represent the tumor-suppressive

macrophages that may also contain subpopulations. The M1/M2 dichotomy used here does not mean that there are only two simple types of TAMs in GBM tumors. We believe that there is a

heterogeneity of TAMs in GBM tumors. However, our studies confirmed that silencing WISP1 indeed reduced tumor-supportive macrophages (M2 TAMs) in our xenograft models. According to our

previous studies18,21 and current data, it is reasonable to conclude that M2/M1 TAMs indeed represent two major but functionally different macrophage populations (tumor-supportive and

tumor-suppressive) in our tumor models, although we can’t rule out that each major population (M2 or M1) may contain several subpopulations. It will be interesting to further analyze

subpopulations in M2 TAMs and M1 TAMs in GBMs in the future. A preclinical study reported that blocking CSF-1R (macrophage colony-stimulating factor 1 receptor) did not impact total TAM

density in GBMs, indicating that other survival factors from the tumor microenvironment may provide compensatory growth and survival signals27. However, we found that silencing WISP1 in GSCs

markedly decreased TAM density in GSC-derived GBM tumors. It is possible that silencing WISP1 may result in an altered tumor microenvironment, which may contribute to decreased TAMs.

However, our in vitro data suggest that WISP1 has a direct effect on the survival of macrophages. It would be interesting to further investigate whether WISP1 can regulate the tumor

microenvironment in GBMs in the future. Our previous study demonstrated that GSCs secrete Periostin to recruit monocyte-derived TAMs into GBMs18, but how these TAMs are maintained as M2 TAMs

in GBM was not clear. In this study, we discover that WISP1, secreted by GSCs, promotes the survival of M2 TAMs and thus maintains tumor-supportive macrophages in GBM tumors, indicating

that the recruitment of TAMs and the maintenance of M2 TAMs are regulated by different molecules secreted by GSCs in the tumor microenvironment. Therefore, GSCs play vital roles not only in

TAM recruitment but also in the maintenance of M2 TAMs, indicating that GSCs could manipulate their niches through multiple paracrine functions. Similarly, tumor-supportive M2 TAMs may

secrete several factors to impact several aspects of GSCs. Our previous study demonstrated that M2 TAMs secrete PTN to promote self-renewal and survival of GSCs in GBMs21. These studies

confirm that the molecular and cellular interactions between GSCs and TAMs are bi-directional. Although CSF-1R inhibition has been shown to inhibits GBM tumor growth in a preclinical

study27, clinical trials using a CSF-1R inhibitor for cancer treatment failed due to its toxicity66,67,68, because CSF-1R is expressed by many types of immune cells including monocytes69,70.

Our study indicates that disruption of M2 TAM survival by targeting WISP1-related signaling may effectively suppress GBM tumor growth. As WISP1 is preferentially secreted by GSCs and

maintains M2 TAM survival, and silencing WISP1 promotes apoptosis of M2 TAMs, targeting this GSC-specific paracrine signaling pathway to disrupt M2 TAMs may offer a therapeutic strategy to

improve GBM treatment. Because there is no available WISP1 inhibitor so far and the Wnt/β-catenin signaling is activated in GSCs, we targeted the WISP1 upstream signaling with the β-catenin

inhibitor carnosic acid for GBM treatment. As Wnt/β-catenin signaling induces multiple downstream targets to promote tumor growth, the inhibition of GBM growth by carnosic acid may be a

comprehensive result. Nevertheless, carnosic acid treatment reduces WISP1 expression in vitro and in vivo, suggesting that WISP1 inhibition at least partially contributes to the therapeutic

effect of carnosic acid. Because WISP1 is also expressed in other tumors38,39 and may play a similar role in maintaining tumor-supportive M2 TAMs, targeting WISP1-associated signaling may

improve treatment for other malignant tumors as well. In summary, our study defined WISP1 as a key regulator in mediating the molecular crosstalk between GSCs and tumor-supportive M2 TAMs in

the tumor microenvironment in GBMs. We demonstrated that WISP1 plays both autocrine and paracrine roles in the maintenance of GSCs and in the survival of M2 TAMs, to promote malignant

growth in GBMs. Disrupting WISP1 signaling or targeting its upstream regulators could potently suppress GBM growth through inhibition on both GSCs and tumor-supportive M2 TAMs, which may

provide an effective therapeutic approach to improve treatment for GBMs and potentially other malignant tumors. In addition, as WISP1 is a secretory protein highly expressed by GBM tumors,

WISP1 in serum or cerebrospinal fluid may serve as a promising diagnostic biomarker for GBMs or other cancers. METHODS HUMAN GBM SPECIMENS AND GLIOMA STEM CELLS (GSCS) Human primary GBM

specimens in this study were collected from the Brain Tumor and Neuro-Oncology Center at Cleveland Clinic and University Hospitals of Case Western Reserve University in accordance with the

Institutional Review Board-approved protocol. All procedures performed using human tissues were approved by the ethics committee of Cleveland Clinic and University Hospitals. Informed

consent was obtained from individuals. GSCs and matched NSTCs were isolated from primary GBM specimens or patient-derived GBM xenografts and functionally characterized. Briefly, tumor cells

were isolated from GBM tumors using Papain Dissociation System (Worthing Biochemical) according to the manufacturer’s instructions and then were recovered in Neurobasal-A medium (Gibco) with

B27 supplement (Gibco), 10 ng/ml EGF (Gold Biotech), 10 ng/ml bFGF (R&D), 1 mM sodium pyruvate (Gibco), and 2 mM L-glutamine (Gibco) at least 6 h. Isolated cells were labeled with a

PE-conjugated anti-CD133 antibody (Miltenyi Biotec, 130-098-826) and a FITC-conjugated anti-CD15 antibody (BD, 347423) followed by FACs to sort the GSCs (CD15+/CD133+) or NSTCs

(CD15-/CD133−). The cancer stem cell characteristics of isolated GSCs were validated by the expressions of GSC markers (SOX2, OLIG2, CD133, L1CAM) and a seried of functional assays including

serial neurosphere formation assay (in vitro limiting dilution assay), serum-induced cell differentiation assay and in vivo tumor formation limiting dilution assay. All experiments conform

to relevant regulatory standards. Specifically, T387 GSCs and NSTCs were derived from a GBM from a 76-year old female patient. D456 GSCs and NSTCs were derived from a GBM from an 8-year old

female patient. T4121 GSCs and NSTCs were derived from a GBM from a 53-year old male patient. T3094 GSCs and NSTCs were derived from a GBM from a 63-year old male patient. T3565 GSCs and

NSTCs were derived from a GBM from a 32-year old male patient. T3359 GSCs and NSTCs were derived from a GBM from a 31-year old male patient. CW1797 GBM specimens were collected from a

57-year old male patient. CW1798 GBM specimens were collected from a 47-year old male patient. CW2360 GBM specimens were collected from a 26-year old male patient. DI-74 GBM specimens were

collected from a 52-year old male patient. CCF2445 GBM specimens were collected from a 50-year old male patient. CELL DIFFERENTIATION AND IN VIVO LIMITING DILUTION ASSAYS For cell

differentiation assay, GSCs were cultured on the Matrigel-coated dishes and induced for differentiation through withdrawal of all growth factors and by addition of serum (10% FBS in DMEM).

At day 0, 2, 4, 6, 8, cells were harvested for immunoblot analysis or fixed for immunofluorescent staining of the GSC (SOX2, OLIG2) and differentiation markers (GFAP, MAP2). For in vivo

limiting dilution assay, GSCs were counted and certain number cells (100, 500, 1000, 5000 or 10000) were implanted into the right frontal lobes of NSG mice. Mice were maintained up to 25

weeks or until the development of neurological signs. Brains of mice were collected, fixed in 4% paraformaldehyde, and paraffin embedded for hematoxylin-eosin staining. INTRACRANIAL

TUMORIGENESIS AND TREATMENT All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Cleveland Clinic and were conducted in accordance with IACUC

guidelines. Mice used in these studies were 6–7 weeks old female or male mice. NSG mice (The Jackson Laboratory) were housed under a 12-h light/12-h dark cycle in a temperature (20–26 °C)

and humidity (30–-70%) controlled environment and were fed ad libitum. 5000 GSCs were transplanted into the right cerebral cortex of NSG mice at a depth of 3.5 mm. Mice were monitored by the

bioluminescent imaging or maintained until neurological signs were observed. For inducible overexpression, 5000 GSCs were transplanted intracranially into NSG mice for 10 days. The mice

were then supplied with drinking water containing 2 mg/ml doxycycline or control water for 6 days. For the carnosic acid (Enzo Life Tech) treatment, 50 μL of 10 mg/kg carnosic acid was

dissolved in DMSO and was administrated daily via intraperitoneal injection. CELL VIABILITY AND TUMORSPHERE FORMATION ASSAYS For cell viability assay, 1000 cells were plated into each well

of the 96-well plate, cell viability were determined at the indicated days after cell seeding using the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) according to the

manufacturer’s protocol. For tumorsphere formation assay, 1000 GSCs were plated into each well of the 96-well plate, tumorsphere number was calculated at the sixth day after cell seeding. IN

VITRO LIMITING DILUTION ASSAYS GSCs were plated into one well of 96-well plates at an indicated density (0, 4, 8, 12, 16 cells) with 30 replicates for each concentration. Six days later,

the presence and number of tumorspheres in each well were recorded and analyzed using the software at http://bioinf.wehi.edu.au/software/elda/. IMMUNOBLOT ANALYSIS AND PHOSPHO-KINASE ARRAY

For immunoblot analysis, we directly lysed cells or homogenised tissues in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS and supplemented with protease

inhibitors). Lystes were centrifuged for 10 min at 16,900 × _g_ and 4 °C. The resulting supernatant fraction was separated by SDS-PAGE and transferred onto PVDF membranes. The membranes

were blocked with 5% non-fat milk for 1 h and then immunoblotted with relative antibodies overnight at 4 °C followed by the HRP-conjugated antibody at room temperature for 1 h. Blots were

imaged using BioRad Image Lab software. Phospho-kinase array was determined using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems). Briefly, cells were lysed in RIPA

lysis buffer. Lystes were centrifuged for 10 min at 16,900 × _g_ and 4 °C. Further analysis was performed according to the manufacturer’s protocol. A complete list of antibodies including

dilutions is shown in Supplementary Table 1. Uncropped images are shown in Supplementary Fig. 14. CO-IMMUNOPRECIPITATION (COIP) Cells were collected in IP lysis buffer (50 mM Tris-HCl pH

7.8, 137 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol and supplemented with protease inhibitors) for 30 min and pre-cleared by centrifugation at 16,900 × _g_ for 10 min. Protein lysates

were incubated with primary antibody or isotype IgG overnight at 4 °C and then captured by protein A/G Plus agarose beads (Santa cruz, sc-2003) for 2 h at 4 °C. The precipitants were washed

with wash buffer (20 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 100 mM KCl, 2 mM MgCl2, 0.1% Tween 20, 10% glycerol) for 4 times, boiled with SDS sample buffer (50 mM Tris-HCL pH = 6.8, 2% SDS, 10%

glycerol, 1% β-mercapitalethanol, 0.1% bromophenol blue) at 95 °C for ten minutes and subjected to immunoblot analysis. A complete list of antibodies including dilutions is shown in

Supplementary Table 1. Uncropped images are shown in Supplementary Fig. 14. CONDITIONED MEDIUM PREPARATION GSCs and matched NSTCs were cultured in neurobasal media without supplements and

growth factors for 40 h. Conditioned medium was collected from cultures at a density of 2 × 106 cells/mL. The cells were removed by centrifugation (300 × _g_, 5 min), and the conditioned

medium was sterile filtered through a 0.2 μm filter. Samples were then concentrated to dryness by vacuum centrifugation using Eppendorf Concentrator plus/Vacufuge plus system (Eppendorf).

Resulting residues were then dissolved in SDS sample buffer, denatured at 95 °C for ten minutes and then subjected to immunoblot analysis. DNA CONSTRUCTS AND LENTIVIRAL TRANSDUCTION

Lentiviral clones expressing two non-overlapping shRNAs against human WISP1 (TRCN0000373969, TRCN0000373970), human Integrin α6 (TRCN0000296162, TRCN000057775) and non-targeting shRNA

(SHC002) were obtained from Sigma-Aldrich. A lentiviral construct expressing WISP1 was generated by cloning the human WISP1 open reading frame into the PCDH-MCs-T2A-Puro-MSCV vector (System

Biosciences, CD522A-1) or PCW57.1 (Addgene, 50661). A lentiviral construct expressing myr-Akt1 was generated by cloning Akt1 with an N-terminal src myristoyation sequence into the

PCDH-MCs-T2A-Puro-MSCV vector. Viral particles were produced in 293FT cells with pPACK set of helper plasmids (System Biosciences) in Neurobasal-A medium. The viruses were then concentrated

by precipitation with PEG8000 (Fisher Scientific) according to the manufacturer’s instructions. For lentiviral transduction, GSCs were transducted with lentivirus expressing the shRNA, WISP1

or Akt for 48 h, and then processed for next analysis. IN VIVO BIOLUMINESCENCE ANALYSIS To monitor tumor growth in living mice, GSCs were transduced with firefly luciferase through

lentiviral infection. 48 h after shRNA infection, 5000 GSCs were intracranially transplanted into NSG mice. Then, mice were intraperitoneal injected with 120 mg/kg D-luciferin (Gold Biotech)

and anesthetized with isoflurane at the indicated days. The size of the tumor was monitored by bioluminescence channel of IVIS Spectrum imaging system. IMMUNOFLUORESCENT STAINING

Immunofluorescent staining were performed in tissues and cultured cells. Mouse GBM xenografts were collected from mice when neurological signs occur after GSC transplantation. Human GBM

specimens were obtained from GBM patients through surgical resection. Briefly, clutured cells or tumor sectons were fixed in 4% PFA for 15 min and washed with PBS twice after that. Samples

were blocked with a PBS solution containing 1% BSA plus 0.3% Triton X-100 for 30 minutes at room temperature, and then incubated with indicated primary antibody onvernight at 4 °C followed

by the fluorescent second antibody (1:200) at room temperature for 2 h. Nuclei were counterstained with DAPI for 5 min, and then sections were mounted on glass and subjected to microscopy.

Image J 1.47v (NIH) was used to quantify the positive cells. A complete list of antibodies including dilutions is shown in Supplementary Table 1. U937 CELLS AND U937-DERIVED M1 OR M2

MACROPHAGES U937 cells were maintained in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. U937-derived M1 or M2 macrophages were

generated as a macrophage model. Briefly, U937 cells were primed with PMA (Sigma, 5 nM) for 48 h to become unpolarized macrophages. To establish the M1 macrophages, the unpolarized

macrophages were stimulated with 20 ng/ml of IFN-γ (Peprotech) and 100 ng/ml of LPS (Sigma) for an additional 48 h. To establish the M2 macrophages, the unpolarized macrophages were

stimulated with IL4, IL10, and TGF- β (20 ng/ml, Peprotech) for additional 72 h. Cells were then harvested for immunoblot analysis or fixed for immunofluorescent staining of the indicated

markers. STATISTICS AND REPRODUCIBILITY Statistical differences were determined by two-tailed unpaired Student’s _t_-test for two groups, or by two-way ANOVA for multiple groups. The data

used in this study are presented as the mean ± s.d. or mean ± s.e.m. For Kaplan–Meier survival curves, statistical differences were determined by log-rank test. All analysis were carried out

using Microsoft excel 2010 or GraphPad Prism 7 software. _p_ < 0.05 was considered statistically significant. Detailed information is described in each figure legends. Except for the

results from the public database, similar results were obtained from three independent experiments for all other results. REPORTING SUMMARY Further information on research design is

available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY The TCGA database (Agilent-4502A platform) and Gravendeel database can be downloaded from GlioVis

data portal (http://gliovis.bioinfo.cnio.es/). The gene expression in cluster of two-dimensional representation of cellular states can be downloaded from Single Cell Portal

(http://singlecell.broadinstitute.org/single_cell/study/SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma/). All other data supporting the findings of this study are available

within the article and its Supplementary information files. All remaining data are available from the corresponding author upon reasonable request. The source data underlying Fig.1a–d, i,

2b, d–f, h, i, 3b, c, e, g, i, 4f, g, i, j, 5c–h, j, k, 7b, c, e, f, h, i, k, l, 8c, d, f, h, j, l, and Supplementary Figs. 1a, b, 2a, b, d–f, 3b, c, e, g, i, 4e, f, h, 5a–f, i, j, 6b, d, e,

g, 7c, d, f, g, 8b, c, e, f, h, i, k, l, 9b, d, f, 10d, f, h, 12b, e, f, 13c–f, h, i, are provided as a Source data file. Source data are provided with this paper. REFERENCES * Stupp, R. et

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3824–3824 (2015). Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the Brain Tumor and Neuro-Oncology Centers at Cleveland Clinic and University Hospitals of Case

Western Reserve University for providing GBM specimens for this study. We also thank the Flow Cytometry Core, Imaging Core, and Central Cell Services Core at Cleveland Clinic Lerner Research

Institute for their assistance. This work was supported by Cleveland Clinic Foundation and the NIH R01 grants (NS091080 and NS099175) to S.B. and the NIH Shared Instrument Grant

(S10OD018205) to the Cleveland Clinic Lerner Research Institute. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic,

Cleveland, OH, 44195, USA Weiwei Tao, Chengwei Chu, Wenchao Zhou, Zhi Huang, Kui Zhai, Xiaoguang Fang, Qian Huang, Aili Zhang, Xingjiang Yu, Haidong Huang, Jennifer S. Yu, George R. Stark 

& Shideng Bao * Division of Regenerative Medicine, Department of Medicine, University of California, San Diego, San Diego, CA, 92037, USA Xiuxing Wang, Qiulian Wu & Jeremy N. Rich *

Brain Tumor and Neuro-Oncology Center, Seidman Cancer Center, University Hospitals, Case Western Reserve University, Cleveland, OH, 44106, USA Andrew E. Sloan * Case Comprehensive Cancer

Center, Case Western Reserve University School of Medicine, Cleveland, OH, 44106, USA Andrew E. Sloan, Jennifer S. Yu, Xiaoxia Li, George R. Stark & Shideng Bao * Department of Radiation

Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, 44195, USA Jennifer S. Yu * Center for Cancer Stem Cell Research, Lerner Research Institute, Cleveland Clinic,

Cleveland, OH, 44195, USA Jennifer S. Yu & Shideng Bao * Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 44195, USA Xiaoxia Li

Authors * Weiwei Tao View author publications You can also search for this author inPubMed Google Scholar * Chengwei Chu View author publications You can also search for this author inPubMed

 Google Scholar * Wenchao Zhou View author publications You can also search for this author inPubMed Google Scholar * Zhi Huang View author publications You can also search for this author

inPubMed Google Scholar * Kui Zhai View author publications You can also search for this author inPubMed Google Scholar * Xiaoguang Fang View author publications You can also search for this

author inPubMed Google Scholar * Qian Huang View author publications You can also search for this author inPubMed Google Scholar * Aili Zhang View author publications You can also search

for this author inPubMed Google Scholar * Xiuxing Wang View author publications You can also search for this author inPubMed Google Scholar * Xingjiang Yu View author publications You can

also search for this author inPubMed Google Scholar * Haidong Huang View author publications You can also search for this author inPubMed Google Scholar * Qiulian Wu View author publications

You can also search for this author inPubMed Google Scholar * Andrew E. Sloan View author publications You can also search for this author inPubMed Google Scholar * Jennifer S. Yu View

author publications You can also search for this author inPubMed Google Scholar * Xiaoxia Li View author publications You can also search for this author inPubMed Google Scholar * George R.

Stark View author publications You can also search for this author inPubMed Google Scholar * Jeremy N. Rich View author publications You can also search for this author inPubMed Google

Scholar * Shideng Bao View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS W.T. and S.B. designed the experiments. W.T., C.C., W.Z., Z.H., K.Z.,

X.F., Q.H., A.Z., X.W., X.Y., H.H., and Q.W. performed the experiments. J.S.Y., X.L., G.R.S., and J.N.R. provided scientific input. A.E.S. provided some GBM surgical specimens. G.R.S.

edited the paper. W.T. and S.B. analyzed the data and wrote the paper. CORRESPONDING AUTHOR Correspondence to Shideng Bao. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_ thanks Shi-Yuan Cheng, Anjali Shiras and the other, anonymous, reviewer(s) for their contribution

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in maintaining glioma stem cells and tumor-supportive macrophages in glioblastoma. _Nat Commun_ 11, 3015 (2020). https://doi.org/10.1038/s41467-020-16827-z Download citation * Received: 07

October 2019 * Accepted: 28 May 2020 * Published: 15 June 2020 * DOI: https://doi.org/10.1038/s41467-020-16827-z SHARE THIS ARTICLE Anyone you share the following link with will be able to

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