Preclinical Evidence of STAT3 Inhibitor Pacritinib Overcoming Temozolomide Resistance via Downregulating miR-21-Enriched Exosomes from M2 Glioblastoma-Associated Macrophages

doi:10.3390/jcm8070959

. 2019 Jul 2;8(7):959.

doi: 10.3390/jcm8070959.

Preclinical Evidence of STAT3 Inhibitor Pacritinib Overcoming Temozolomide Resistance via Downregulating miR-21-Enriched Exosomes from M2 Glioblastoma-Associated Macrophages

Hao-Yu Chuang^{1

2

3}, Yu-Kai Su^{4

5

6

7}, Heng-Wei Liu^{4

5

6

7}, Chao-Hsuan Chen^{8

9

10

11}, Shao-Chih Chiu^{8

9

10

11}, Der-Yang Cho^{8

9

10

11}, Shinn-Zong Lin^{12

13}, Yueh-Sheng Chen¹⁴, Chien-Min Lin^{15

16

17

18}

Affiliations

¹ Graduate Institute of Clinical Medical Science, China Medical University, Taichung 404, Taiwan.
² Department of Neurosurgery, An Nan Hospital, China Medical University, Tainan 709, Taiwan.
³ Department of Neurosurgery, China Medical University Beigang Hospital, Yunlin 651, Taiwan.
⁴ Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan.
⁵ Department of Neurology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan.
⁶ Division of Neurosurgery, Department of Surgery, Taipei Medical University-Shuang Ho Hospital, New Taipei City 23561, Taiwan.
⁷ Taipei Neuroscience Institute, Taipei Medical University, Taipei 11031, Taiwan.
⁸ Center for Cell Therapy, China Medical University Hospital, Taichung 404, Taiwan.
⁹ Drug Development Center, China Medical University, Taichung 404, Taiwan.
¹⁰ Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404, Taiwan.
¹¹ Department of Neurosurgery, China Medical University Hospital, Taichung 404, Taiwan.
¹² Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan.
¹³ Department of Neurosurgery, Tzu Chi University, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan.
¹⁴ Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung 404, Taiwan. yuehsc@mail.cmu.edu.tw.
¹⁵ Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan. m513092004@tmu.edu.tw.
¹⁶ Department of Neurology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan. m513092004@tmu.edu.tw.
¹⁷ Division of Neurosurgery, Department of Surgery, Taipei Medical University-Shuang Ho Hospital, New Taipei City 23561, Taiwan. m513092004@tmu.edu.tw.
¹⁸ Taipei Neuroscience Institute, Taipei Medical University, Taipei 11031, Taiwan. m513092004@tmu.edu.tw.

PMID: 31269723
PMCID: PMC6678764
DOI: 10.3390/jcm8070959

Preclinical Evidence of STAT3 Inhibitor Pacritinib Overcoming Temozolomide Resistance via Downregulating miR-21-Enriched Exosomes from M2 Glioblastoma-Associated Macrophages

Hao-Yu Chuang et al. J Clin Med. 2019.

. 2019 Jul 2;8(7):959.

doi: 10.3390/jcm8070959.

Authors

Affiliations

¹ Graduate Institute of Clinical Medical Science, China Medical University, Taichung 404, Taiwan.
² Department of Neurosurgery, An Nan Hospital, China Medical University, Tainan 709, Taiwan.
³ Department of Neurosurgery, China Medical University Beigang Hospital, Yunlin 651, Taiwan.
⁴ Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan.
⁵ Department of Neurology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan.
⁶ Division of Neurosurgery, Department of Surgery, Taipei Medical University-Shuang Ho Hospital, New Taipei City 23561, Taiwan.
⁷ Taipei Neuroscience Institute, Taipei Medical University, Taipei 11031, Taiwan.
⁸ Center for Cell Therapy, China Medical University Hospital, Taichung 404, Taiwan.
⁹ Drug Development Center, China Medical University, Taichung 404, Taiwan.
¹⁰ Graduate Institute of Biomedical Sciences, China Medical University, Taichung 404, Taiwan.
¹¹ Department of Neurosurgery, China Medical University Hospital, Taichung 404, Taiwan.
¹² Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan.
¹³ Department of Neurosurgery, Tzu Chi University, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 97004, Taiwan.
¹⁴ Department of Biomedical Imaging and Radiological Science, China Medical University, Taichung 404, Taiwan. yuehsc@mail.cmu.edu.tw.
¹⁵ Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan. m513092004@tmu.edu.tw.
¹⁶ Department of Neurology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City 11031, Taiwan. m513092004@tmu.edu.tw.
¹⁷ Division of Neurosurgery, Department of Surgery, Taipei Medical University-Shuang Ho Hospital, New Taipei City 23561, Taiwan. m513092004@tmu.edu.tw.
¹⁸ Taipei Neuroscience Institute, Taipei Medical University, Taipei 11031, Taiwan. m513092004@tmu.edu.tw.

PMID: 31269723
PMCID: PMC6678764
DOI: 10.3390/jcm8070959

Abstract

Background: The tumor microenvironment (TME) plays a crucial role in virtually every aspect of tumorigenesis of glioblastoma multiforme (GBM). A dysfunctional TME promotes drug resistance, disease recurrence, and distant metastasis. Recent evidence indicates that exosomes released by stromal cells within the TME may promote oncogenic phenotypes via transferring signaling molecules such as cytokines, proteins, and microRNAs.

Results: In this study, clinical GBM samples were collected and analyzed. We found that GBM-associated macrophages (GAMs) secreted exosomes which were enriched with oncomiR-21. Coculture of GAMs (and GAM-derived exosomes) and GBM cell lines increased GBM cells' resistance against temozolomide (TMZ) by upregulating the prosurvival gene programmed cell death protein 4 (PDCD4) and stemness markers SRY (sex determining region y)-box 2 (Sox2), signal transducer and activator of transcription 3 (STAT3), Nestin, and miR-21-5p and increasing the M2 cytokines interleukin 6 (IL-6) and transforming growth factor beta 1(TGF-β1) secreted by GBM cells, promoting the M2 polarization of GAMs. Subsequently, pacritinib treatment suppressed GBM tumorigenesis and stemness; more importantly, pacritinib-treated GBM cells showed a markedly reduced ability to secret M2 cytokines and reduced miR-21-enriched exosomes secreted by GAMs. Pacritinib-mediated effects were accompanied by a reduction of oncomiR miR-21-5p, by which the tumor suppressor PDCD4 was targeted. We subsequently established patient-derived xenograft (PDX) models where mice bore patient GBM and GAMs. Treatment with pacritinib and the combination of pacritinib and TMZ appeared to significantly reduce the tumorigenesis of GBM/GAM PDX mice as well as overcome TMZ resistance and M2 polarization of GAMs.

Conclusion: In summation, we showed the potential of pacritinib alone or in combination with TMZ to suppress GBM tumorigenesis via modulating STAT3/miR-21/PDCD4 signaling. Further investigations are warranted for adopting pacritinib for the treatment of TMZ-resistant GBM in clinical settings.

Keywords: GBM-associated macrophages (GAMs); STAT3 inhibitor; exosomes; glioblastoma multiforme (GBM); oncomiR-21; tumor microenvironment (TME).

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Conflict of interest statement

The authors declare that they have no potential financial competing interests from which they may in any way gain or lose financially from the publication of this manuscript presently or in the future. Additionally, no nonfinancial competing interests are involved in the manuscript.

Figures

**Figure 1**
M2 glioblastoma multiforme (GBM)-associated macrophages (GAMs) promote GBM tumorigenesis. GBM cells U87MG and LN18 cocultured with M2 GAMs showed significantly increased colony forming ability (A) and tumor sphere generating ability (B) as compared to their parental controls. Comparative real-time PCR (C) and Western blots (D) showed that M2 GAM cocultured GBM cells expressed a significantly higher level of stemness markers, Sox2, Oct4, Wnt, and Nestin while GFAP was reduced. Scale lengths = 100 μm, * p < 0.05; ** p < 0.01; *** p < 0.001.

**Figure 2**
GAM-derived exosomes harbor miR-21, which promote GBM tumorigenesis. (A) Representative transmission electronic micrograph of exosomes isolated from clinical GAMs (left); Western blot validation of exosomes isolated from GAM culture medium showed the expression of CD9, CD63, and CD81. (B) Increased temozolomide (TMZ) resistance in U87MG and LN18 cells cocultured with exosomes (+exo). Enhanced colony-forming ability (C) and tumor-sphere-generating ability (D) in the presence GAM-derived exosomes. (E) MicroRNA profiling analyses showed that exosomes (two samples) isolated from M2 GAMs contained a high level of miR-21. Scale lengths = 100 μm, * p < 0.05; ** p < 0.01; *** p < 0.001.

**Figure 3**
GAM-derived exosomes promoted GBM tumorigenesis via miR-21-5p. (A) U87MG and LN18 cells incubated with GAM-derived exosomes showed a significantly increased level of miR-21-5p. GBM tumorigenesis was associated with miR-21-5p. Increased miR-21-5p level (by mimic molecules) in GBM cells showed an increased mRNA level of Sox2, Oct4, Wnt, STAT3, and Nestin or protein level of Sox2, Oct4, STAT3, Akt, Wnt, and Nestin with decreased GFAP, while a decrease in miR-21-5p (inhibitor) led to the opposite phenomenon (B,C). Incubation with GAM-derived exosomes increased colony-forming ability (D) and tumor-sphere-generating ability (E) in both U87MG and LN18 cells. (F) U87MG and LN18 cells transfected miR-21-5p mimic molecules (increased miR-21-5p level) resulted in significantly increased TMZ resistance, while there was reduced miR-21-59 and decreased TMZ resistance. Scale lengths = 100 μm, * p < 0.05; ** p < 0.01; *** p < 0.001.

**Figure 4**
MiR-21-5p targets STAT3 and PDCD4. (A) The bioinformatics tool shows miR-21-5p binding to the 3’UTR of STAT3 and PDCD4 (upper panel). (B) A negative correlation between the expression of miR-21-5p and PDCD4 in the GBM database (n = 525, TCGA). (C) An increased miR-21-5p level (by mimic molecule, lane M) led to significantly reduced PDCD4 expression in both U87MG and LN18 cells; the reverse was true with the inhibitor of miR-21-5p. (D) Flow cytometry analysis showed a significantly reduced CD206⁺/CD68⁺ population in GAMs cocultured with miR-21-5p-silenced U87MG and LN18 cells; the reverse was observed in miR-21-5p mimic transfected coculture experiments. (E) The inhibitor of miR-21-5p resulted in the reduction of VEGF, TGF- β1, and IL-6 secreted by the U87MG cells into the culture medium. *** p < 0.001.

**Figure 5**
Pacritinib treatment suppresses GBM tumorigenesis and glioma stem cell (GSC) properties. (A) Pacritinib treatment significantly suppressed both U87MG and LN18 cells (approximate IC₅₀ values 0.5 and 1.5 µM, respectively). (B) Pacritinib treatment significantly reduced GBM cells’ ability to induce M2 GAMs. CD206 mRNA in GAMs was significantly reduced, while TNF-α was increased. Pacritinib treatment significantly reduced colony formation (C) and tumor sphere generation (D) in both U87MG and LN18 cells. (E) Pacritinib treatment led to a significantly reduced mRNA level of STAT3, Akt, Sox2, PDCD4, and miR-21-5p and increased GFAP in both U87MG and LN18 cells. (F) GAMs treated with pacritinib resulted in the decreased release of exosomes. Western blot of exosomes collected from GAMs showed a significantly lower abundance of exosomes (CD63 and CD9, markers of exosomes). The exosomes collected showed a significantly lower miR-21-5p level. Scale lengths = 100 μm, * p < 0.05; ** p < 0.01; *** p < 0.001.

**Figure 6**
In vivo evaluation of pacritinib for treating GBM and reducing M2 GAMs in TMZ-resistant LN18 bearing mice. (A) Immunohistochemical staining in TMZ-resistant LN18-bearing mice showed that treatment in the pacritinib group and pacritinib/TMZ combination group suppressed tumorigenesis. (B) The tumor size showed that the significantly reduced tumor size in the pacritinib group and the combination of pacritinib and TMZ group led to the most significantly reduced tumor size. NS, statistically nonsignificant. (C) Comparative real-time PCR analyses showed the reduced mRNA level of STAT3, Sox2, PDCD4, and miR-21-5p and the increased GFAP expression in the pacritinib group and pacritinib/TMZ combination group (lane 1, control; lane 2, TMZ alone; lane 3, pacritinib alone; lane 4, pacritinib/TMZ combination). (D) M2 GAMs from tumor samples showed a significantly reduced CD206 (M2 marker) mRNA level (lane 3, pacritinib alone; lane 4, pacritinib/TMZ combination) and an increase in TNF-α (lanes 3 and 4). (E) Kaplan–Meier survival curve and (F) statistical comparisons showed increased median overall survival in TMZ, pacritinib, and pacritinib/TMZ combination groups. Scale lengths = 50 μm, * p < 0.05; ** p < 0.01; *** p < 0.001.

**Figure 7**
GAMs in the tumor microenvironment promote the survival of GBM cells via miR-21-enriched extracellular microvesicles (EVs). Mir-21 targets and suppresses the expression of tumor suppressor PDCD4 in GBM cells, leading to the elevated STAT3/Akt signaling. In turn, GBM cells secrete inflammatory cytokines TGF-β1 and IL-6 and promote M2 polarization. Pacritinib (STAT3 inhibitor) treatment suppresses GBM tumorigenesis by inhibiting STAT3 signaling and reducing M2 polarization of GAMs.

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References

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[1] Khosla D. Concurrent therapy to enhance radiotherapeutic outcomes in glioblastoma. Ann. Transl. Med. 2016;4:5. - PMC - PubMed

[2] Khosla D. Concurrent therapy to enhance radiotherapeutic outcomes in glioblastoma. Ann. Transl. Med. 2016;4:5. - PMC - PubMed

[3] Hambardzumyan D., Gutmann D.H., Kettenmann H. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 2016;19:20–27. doi: 10.1038/nn.4185. - DOI - PMC - PubMed

[4] Hambardzumyan D., Gutmann D.H., Kettenmann H. The role of microglia and macrophages in glioma maintenance and progression. Nat. Neurosci. 2016;19:20–27. doi: 10.1038/nn.4185. - DOI - PMC - PubMed

[5] Bowman R.L., A Joyce J. Therapeutic targeting of tumor-associated macrophages and microglia in glioblastoma. Immunother. 2014;6:663–666. doi: 10.2217/imt.14.48. - DOI - PubMed

[6] Bowman R.L., A Joyce J. Therapeutic targeting of tumor-associated macrophages and microglia in glioblastoma. Immunother. 2014;6:663–666. doi: 10.2217/imt.14.48. - DOI - PubMed

[7] Skog J., Würdinger T., Van Rijn S., Meijer D.H., Gainche L., Curry W.T., Carter B.S., Krichevsky A.M., Breakefield X.O., Sena-Esteves M., et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 2008;10:1470–1476. doi: 10.1038/ncb1800. - DOI - PMC - PubMed

[8] Skog J., Würdinger T., Van Rijn S., Meijer D.H., Gainche L., Curry W.T., Carter B.S., Krichevsky A.M., Breakefield X.O., Sena-Esteves M., et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 2008;10:1470–1476. doi: 10.1038/ncb1800. - DOI - PMC - PubMed

[9] Graner M.W., Cumming R.I., Bigner D.D. The Heat Shock Response and Chaperones/Heat Shock Proteins in Brain Tumors: Surface Expression, Release, and Possible Immune Consequences. J. Neurosci. 2007;27:11214–11227. doi: 10.1523/JNEUROSCI.3588-07.2007. - DOI - PMC - PubMed

[10] Graner M.W., Cumming R.I., Bigner D.D. The Heat Shock Response and Chaperones/Heat Shock Proteins in Brain Tumors: Surface Expression, Release, and Possible Immune Consequences. J. Neurosci. 2007;27:11214–11227. doi: 10.1523/JNEUROSCI.3588-07.2007. - DOI - PMC - PubMed

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Preclinical Evidence of STAT3 Inhibitor Pacritinib Overcoming Temozolomide Resistance via Downregulating miR-21-Enriched Exosomes from M2 Glioblastoma-Associated Macrophages

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Preclinical Evidence of STAT3 Inhibitor Pacritinib Overcoming Temozolomide Resistance via Downregulating miR-21-Enriched Exosomes from M2 Glioblastoma-Associated Macrophages

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