Extracellular Matrix Protein Tenascin C Increases Phagocytosis Mediated by CD47 Loss of Function in Glioblastoma

Abstract

Glioblastomas (GBM) are highly infiltrated by myeloid-derived innate immune cells that contribute to the immunosuppressive nature of the brain tumor microenvironment (TME). CD47 has been shown to mediate immune evasion, as the CD47-SIRPα axis prevents phagocytosis of tumor cells by macrophages and other myeloid cells. In this study, we established CD47 homozygous deletion (CD47^-/-) in human and mouse GBM cells and investigated the impact of eliminating the "don't eat me" signal on tumor growth and tumor-TME interactions. CD47 knockout (KO) did not significantly alter tumor cell proliferation in vitro but significantly increased phagocytosis of tumor cells by macrophages in cocultures. Compared with CD47 wild-type xenografts, orthotopic xenografts derived from CD47^-/- tumor cells grew significantly slower with enhanced tumor cell phagocytosis and increased recruitment of M2-like tumor-associated microglia/macrophages (TAM). CD47 KO increased tumor-associated extracellular matrix protein tenascin C (TNC) in xenografts, which was further examined in vitro. CD47 loss of function upregulated TNC expression in tumor cells via a Notch pathway-mediated mechanism. Depletion of TNC in tumor cells enhanced the growth of CD47^-/- xenografts in vivo and decreased the number of TAM. TNC knockdown also inhibited phagocytosis of CD47^-/- tumor cells in cocultures. Furthermore, TNC stimulated release of proinflammatory factors including TNFα via a Toll-like receptor 4 and STAT3-dependent mechanism in human macrophage cells. These results reveal a vital role for TNC in immunomodulation in brain tumor biology and demonstrate the prominence of the TME extracellular matrix in affecting the antitumor function of brain innate immune cells. SIGNIFICANCE: These findings link TNC to CD47-driven phagocytosis and demonstrate that TNC affects the antitumor function of brain TAM, facilitating the development of novel innate immune system-based therapies for brain tumors.

Fig. 1.. Establish CD47 KO human GBM cells by genome editing.

A. Schematic graph of genome editing strategy with two guide RNAs to knockout CD47. B. PCR product from genomic DNA of selected clones showing heterozygous and homozygous deletion of CD47. C. Sanger sequencing of clone 21 showing deletion of part of the CD47 coding sequence. D. Flow cytometry analysis with a CD47 antibody indicated no CD47 expression on cell membrane in CD47^−/− cells. Representative data of three measurements. E. Immunocytostaining of CD47 in control and CD47^−/− cells. Bar = 20 μm. F, CD47 KO minimally affected cell proliferation in vitro. G. CD47 KO decreased cell migration of clone 7, but had no effect on clone 21 (G), n=3. H. Phagocytosis analysis of THP-1 cells co-cultured with control and CD47^−/− cells. I. Quantification of phagocytosis rate of THP-1 cells against CD47 WT and CD47^−/− tumor cells. *: P<0.05, ***: P<0.001, n=6.

Fig. 2.. Effect of CD47 KO on tumor growth.

A. Representative H &E staining of xenografts derived from control and CD47^−/− cells. Bar = 500 μm. B. Quantification of the size of WT and CD47^−/− xenografts. C. Immunofluorescent staining confirmed that CD47 expression was eliminated in CD47^−/− xenografs. Bar = 20 μm. D. E. Ki67 staining and quantification in WT and CD47^−/− xenografts. Bar = 20 μm. F. Representative microphotographs of H& E staining of well-demarcated margins in WT tumors (left) and irregular CD47^−/− tumor margins (right). Bar = 100 μm. G. Xenografts were immunostained with an antibody against human nuclear specific antigen to show tumor margins. Bar = 200 μm. H, I. Laminin staining to show blood vessels in control and CD47^−/− xenografts. Bar = 100 μm. ***: P<0.001. In vivo experiments were repeated once. N=8 in total.

Fig. 3.. Distribution of TAMs in xenografts.

A. Microglial/macrophage marker Iba-1 staining indicated higher density of TAMs in CD47^−/− xenografts. B. Quantification of Iba⁺ cells per microscopic field in control and CD47^−/− xenografts. C, D. Co-staining of the M2 marker Arg-1 (red) and Iba-1 (green) in xenografts and quantification of Arg-1+ cells per field. E. Double-staining of TAMs (Iba-1⁺, green) and tumor cells (HuNu⁺, red) showing host immune cells with human tumor nuclei (Arrows) in CD47^−/− xenografts. F. Confocal microscopic imaging of the double staining of Iba-1 and HuNu in a CD47^−/− xenograft showing the two markers were from the same cells (Arrows). G. Quantification of Iba-1⁺ cells with HuNu⁺ staining per microscopic field. *: P<0.05, n=8. Bar = 20 μm.

Fig. 4.. Tenascin C (TNC) was upregulate in CD47^−/− xenografts.

A. Immunofluorescence staining of TNC with an antibody recognizing both mouse and human TNC. B. Staining of TNC using an antibody that only recognizes human TNC. Bar = 100 μm. C. Quantification of human TNC staining intensity. D, E. RT-PCR and Western blot analysis confirmed TNC was upregulated at the mRNA and protein level (Mw 200-250 KD) in CD47^−/− clonal cells. F. Signaling pathways activated in CD47^−/− clones. The Notch but not Wnt and AKT pathways were elevated in CD47^−/− cells. G. Jagged-1 and NOTCH1 were upregulated by CD47 KO at the mRNA level. H. Notch pathway inhibitor DAPT blunted TNC upregulation in CD47^−/− cells. In vitro experiments were replicated at least three times. *: P<0.05, ***: P<0.001, n=8.

Fig 5.. CD47 KO increased TNC expression in immunocompetent mouse models.

A. Flow cytometry analysis with an anti-mouse CD47 antibody indicated no CD47 expression on cell membrane in Cd47^−/− cells. B. Phagocytosis analysis of THP-1 cells co-cultured with GL261 Cd47 WT and Cd47^−/− cells. C, D. Representative H &E staining of tumors derived from GL261 cells grown in syngeneic immunocompetent mice C56BL/6. Bar = 500 μm. E. H&E staining of well-demarcated margins in WT tumors (left) and irregular Cd47^−/− tumor margins (right) in immunocompetent mice. Bar = 100 μm. F. Immunostaining showed TNC was upregulated in Cd47^−/− tumor. Bar = 100 μm. G. Quantification of intensity of TNC in GL261 tumors. H. Western blot analysis confirmed that TNC was upregulated in CD47 KO GL261 cells. *: P<0.05; ***: P<0.001, n=4.

Fig. 6.. The effect of TNC loss-of-function on the anti-tumor function of CD47 KO.

A. Knocking down TNC expression in WT cells and clone 21 CD47^−/− cells using TNC specific shRNA (TNCKD). Non-silencing shRNA (NS) was used as a control. B. Growth curve of TNCKD cells in comparison to their counterparts. C. H&E staining of xenografts derived from CD47 WT and CD47^−/− cells harboring TNCKD. Bar = 500 μm. D. Quantification of tumor size. E. Staining of human specific TNC confirmed TNC downregulation in xenografts derived from cells receiving TNC shRNA. Bar = 100 μm. F. Staining of the microglial/macrophage marker Iba-1 in TNCKD xenografts. Bar = 20 μm. G. Quantification of the number of Iba-1⁺ cells in control and CD47^−/− xenografts with and without TNCKD. TNCKD decreased Iba1⁺ cells in CD47^−/− xenografts. *: P<0.05, n=5.

Fig. 7.. Effect of TNC on phagocytosis and macrophage cells.

A. TNCKD in tumor cells decreased phagocytosis of WT and CD47^−/− cells by macrophages. B. Quantification of phagocytosis, n=3. C. RT-PCR indicated that exogenous TNC induced expression of pro-inflammatory genes including IL-1β, IL-6 and TNF-α in THP-1 cells in a dose dependent manner. LPS was used as a positive control, n=6. D. ELISA showed TNC increased TNF-α secretion in THP-1 cells in a dose dependent manner, n=3. E. Immunohistochemistry staining of TNF-α in CD47 WT +/− NS and CD47^−/− +/− TNCKD xenografts. Methyl green (green) was used to counterstain nuclei. Bar = 20 μm. F. Quantification of % of cells with TNF-α staining in the xenografts. n=5. G. Immunoblot analysis indicated that TNC treatment activated STAT3 in THP-1 cells, which was blocked by the STAT3 inhibitor stattic. H. Stattic prevented TNF-α production in THP-1 cells treated with TNC, n=3. I. TLR4 inhibitor TAK242 blocked STAT3 activation in THP-1 cells in response to TNC. J. TAK242 decreased TNC-induced TNF-α production in THP-1 cells, n=3. *: P<0.05.