Research ArticleCancer

Constitutive and TNFα-inducible expression of chondroitin sulfate proteoglycan 4 in glioblastoma and neurospheres: Implications for CAR-T cell therapy

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Science Translational Medicine  28 Feb 2018:
Vol. 10, Issue 430, eaao2731
DOI: 10.1126/scitranslmed.aao2731
  • Fig. 1 CSPG4 is expressed in GBM specimens and GBM-NS and associated with more aggressive disease.

    (A to C) Immunohistochemistry of GBM specimens. (A and B) Representative GBM specimens with high CSPG4 expression [BT168 (A) and BT299 (B)]. CSPG4 is mainly located in the plasma membrane of the tumor cells (arrows), with lower signal within the cytoplasm. Tumor cells within the tumor mass and tumor cells infiltrating the parenchyma both express CSPG4. (C) Representative GBM specimen with moderate-low CSPG4 expression (BT308). Tumor clusters containing both tumor cells and tumor-associated pericytes expressing CSPG4 are identified as CSPG4–α-SMA double positive within the normal parenchyma (scale bars, 50 μm). (D) Representative GBM specimen (BT168) evaluated by immunofluorescence show the presence of blood vessels within the tumor mass. Tumor-associated pericytes are identified as CSPG4 (green) and α-SMA (red) double positive (merge) (scale bars, 50 μm). (E) Representative GBM specimen evaluated by immunofluorescence (BT168). Nuclei that have been visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining (blue), nestin (red), and CSPG4 (green) are highly coexpressed (merge) by tumor cells (scale bar, 50 μm). (F) Kaplan-Meier survival curves for patients with low-moderate and high CSPG4 expression in tumor cells (**P = 0.002). (G) Flow cytometry histograms showing the expression of CSPG4 in GBM-NS. Moderate-low expression (CSPG4ML) (left panel), moderate-high expression (CSPG4MH) (middle panel), and high expression (CSPG4H) (right panel) are represented. (H) Scatter plots showing the expression of CSPG4 (n = 23), HER-2 (n = 17), IL-13Rα2 (n = 17), and EphA2 (n = 17) in GBM-NS [**P < 0.001 and ***P < 0.0001, analysis of variance (ANOVA)].

  • Fig. 2 CSPG4.CAR-Ts target GBM-NS in vitro.

    GBM-NS were cocultured in vitro with either cTs or CSPG4.CAR-Ts in GBM-NS serum-free medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12] and B27 supplement. (A) Representative dot plots (upper panel) and microscopic images (lower panel) showing the cytotoxic activity of CSPG4.CAR-Ts compared to cTs. Flow cytometry plots show GBM-NS and T cells on day 3 of coculture as assessed by flow cytometry using CD3 and CSPG4 as markers for T cells and GBM-NS, respectively (scale bar, 200 μm). (B) Bar graph showing the percentage of residual tumor cells in cocultures of CSPG4.CAR-Ts or cTs against 19 GBM-NS at an E:T ratio of 1:5. GBM-NS are ordered by CSPG4 expression (highest to lowest CSPG4-expressing cell lines). Data are means ± SD of T cell preparations obtained from six different healthy donors. (C and D) IFN-γ (C) and IL-2 (D) released by either cTs or CSPG4.CAR-Ts into the supernatant of coculture with GBM-NS within 24 hours. The established cell line T98G was used as a CSPG4-negative target (ctrl). (E) Representative flow cytometry histograms showing the proliferation of CSPG4.CAR-Ts in response to GBM-NS compared to CSPG4.CAR-Ts alone, as evaluated by the CFSE dilution assay on day 7 of coculture in one representative of six donors. (F) Scatter plot showing the proliferation of CSPG4.CAR-Ts in response to 23 GBM-NS compared to cTs. (G) Cytotoxic activity of cTs and CSPG4.CAR-Ts evaluated in a 6-hour 51Cr release assay. Data are means ± SD of T cells generated from two donors against a total of four GBM-NS (BT168-NS, BT308, BT462-NS, and BT275-NS).

  • Fig. 3 CSPG4.CAR-Ts control tumor growth in GBM-NS xenograft models.

    (A) Nude mice implanted intracranially (i.c.) with GBM-NS CSPG4H (96%, BT308-NS). GBM-NS were labeled with GFP/FFluc. Mice were injected intratumorally with either CSPG4.CAR-Ts or cTs at day 15. (B) Representative in vivo imaging illustrating the growth of GFP/FFluc BT308-NS in treated mice. (C) Line graphs of tumor flux (photons per second) versus time of all BT308-NS–bearing mice treated with cTs (red lines) or CSPG4.CAR-Ts (blue lines). The dotted lines represent the mean photon flux for cTs (dotted red line) and CSPG4.CAR-Ts (dotted blue line). (D) Kaplan-Meier survival curves of BT308-NS–bearing mice injected intratumorally with either CSPG4.CAR-Ts or cTs (***P < 0.0001). (E) Nude mice implanted intracranially with GBM-NS CSPG4ML (49%, BT168-NS). GBM-NS were labeled with GFP/FFluc. Mice were injected intratumorally with either CSPG4.CAR-Ts or cTs at day 7 because BT168-NS showed faster growth in vivo as compared to BT308-NS. (F) Representative in vivo imaging illustrating the growth of GFP/FFluc BT168-NS in treated mice. (G) Line graphs of tumor flux (photons per second) versus time of all BT168-NS–bearing mice treated with cTs (red lines) or CSPG4.CAR-Ts (blue lines). The dotted lines represent the mean photon flux for cTs (dotted red line) and CSPG4.CAR-Ts (dotted blue line). (H) Kaplan-Meier survival curves of BT168-NS–bearing mice injected intratumorally with either CSPG4.CAR-Ts or cTs (***P < 0.0001).

  • Fig. 4 CSPG4.CAR-Ts do not cause tumor escape due to antigen loss.

    (A and B) Nude mice implanted intracranially with GBM-NS CSPG4H (96%, BT308-NS) were infused intratumorally with either CSPG4.CAR-Ts (A) or cTs (B) labeled with GFP/FFluc. Bioluminescence imaging shows the persistence of CSPG4.CAR-Ts (A) and cTs (B). (C) Bar graph showing human T cells detected intratumorally in xenograft gliomas removed from mice treated with CSPG4.CAR-Ts or cTs. Human CD45+CD3+ T cells were detected by flow cytometry. (D and E). Immunohistochemistry and immunofluorescence of GBM-NS CSPG4ML (49%, BT168-NS) glioma removed from mice previously treated with either CSPG4.CAR-Ts (D) or cTs (E). Hematoxylin and eosin staining of gliomas from mice treated with CSPG4.CAR-Ts shows a disruption in the architecture as compared to glioma from mice treated with cTs. Immunofluorescence of gliomas shows that CSPG4 and nestin expression is highly preserved in gliomas treated with either cTs or CSPG4.CAR-Ts (scale bars, 50 μm).

  • Fig. 5 GBM-NS up-regulate CSPG4 in vivo.

    (A) Xenograft gliomas originating from the GBM-NS CSPG4ML (49%, BT168-NS) were explanted from engrafted mice and depleted of contaminant murine cells. Representative in vitro image shows GBM-NS 12 hours after isolation and murine cell depletion (upper panel) (scale bar, 150 μm). CSPG4 expression was assessed by flow cytometry (white histogram) compared to isotype control (black histogram). (B) BT168-NS were selected to obtain CSPG4L and CSPG4H cells using immunomagnetic sorting. Black histograms indicate the expression of CSPG4 in CSPG4L and CSPG4H cells as compared to unselected BT168-NS (white histograms). Data are representative of three different experiments. (C) Expression of CSPG4 (black histograms) in BT168-NS CSPG4L and BT168-NS CSPG4H versus unselected BT168-NS (white histograms) after 3 weeks in culture. (D) Gliomas originating from BT168-NS CSPG4L and CSPG4H cells were removed from engrafted mice. Upon depletion of contaminant murine cells, the expression of CSPG4 was measured by flow cytometry. Black histograms indicate the expression of CSPG4 in BT168 gliomas from CSPG4L and CSPG4H cells, whereas white histograms represent CSPG4 expression in CSPG4L and CSPG4H cells before in vivo implantation. (E) Kinetics of in vitro growth of CSPG4L and CSPG4H as measured by WST assay. (F) Kaplan-Meier survival curves of nude mice implanted intracranially with either BT168-NS CSPG4L or BT168-NS CSPG4H.

  • Fig. 6 TNFα derived from murine microglia influences the expression of CSPG4 in GBM-NS in vitro and in vivo.

    (A) Gliomas derived from GBM-NS in vivo were removed from engrafted nude mice. The murine fraction of the removed tumors was isolated and plated in culture (upper panel) (scale bar, 200 μm). The lower panels show murine CD45lowCD11bhighLy6C+ cells removed from the glioma, which correspond to murine-derived microglia. (B) Lysates from the unfractionated glioma, selected human glioma fraction, selected murine microglia fraction, and normal brain were analyzed for the presence of murine TNFα and IL-6 by specific ELISA. (C) BT168-NS were cultured in vitro in the presence of either the murine fraction isolated from the xenograft GBM or hTNFα. CSPG4 expression was measured by flow cytometry 24 hours after incubation. Light gray, gray, and dark gray histograms indicate CSPG4 expression after hTNFα stimulation, no stimulation, and isotype control, respectively. (D) BT168-NS CSPG4L were stimulated with hTNFα in vitro. CSPG4 expression was measured by flow cytometry. Light gray and dark gray histograms indicate CSPG4 expression in the absence or presence of hTNFα, respectively. (E) Up-regulation of CSPG4 by GBM-NS CSPG4ML (BT462-NS) upon hTNFα treatment. Light gray and black histograms indicate CSPG4 expression without and with hTNFα, respectively. (F) CSPG4 expression in BT168-NS treated with or without hTNFα and with or without TNFα-blocking Ab (infliximab, 50 μg/ml). (G) Immunoblot of BT168-NS treated with or without hTNFα and with or without TNFα-blocking Ab (infliximab, 50 μg/ml). hTNFα activates NF-κB in BT168-NS after 24 hours, as measured by the increase of phospho–NF-κB p65. Vinculin is used as loading control.

  • Fig. 7 CSPG4-high GBMs show more microglia than CSPG4-low GBMs and express TNFα.

    (A) GBM expressing high amounts of CSPG4 (BT168) shows a high number of Iba1+ cells identified as microglia. (B) The only GBM negative for CSPG4 expression (BT274) in our cohort shows a total lack of Iba1+ cells. (C) Correlation of the percentage of CSPG4-positive tumor cells and Iba1-positive cells in the tumor mass (linear regression, P = 0.04). The cutoff for Iba1 expression was set at 10%. (D) Representative GBM specimen evaluated by immunofluorescence shows the presence of Iba1+ cells (green) coexpressing TNFα (merge). Two different areas are shown at two magnifications (scale bars, 25 μm). Nuclei are visualized by DAPI staining.

  • Table 1 Characteristics of patients, GBM specimens, and corresponding neurospheres.

    BT, brain tumor; MES, mesenchymal; PN, proneural; CLAS, classical; ND, not determined; Unmet, unmethylated; Met, methylated; OS, overall survival; F, female; M, male.

    GBMCSPG4 expression% CSPG4
    GBM-NS
    OSSubtypeMGMT
    methylation
    AgeGender
    SpecimensVessels
    BT168+++++49.06.0MESUnmet78F
    BT205++85.02.0PNUnmet73M
    BT423++++98.76.5CLASMet43F
    BT273++/−85.914.0CLASMet59M
    BT275+++97.415.0CLASMet67F
    BT299+++99.06.5PNUnmet50M
    BT308+++96.03.0PNUnmet76M
    BT326+++63.06.5PNUnmet28M
    BT328++/−83.26.0MESUnmet56M
    BT358++++95.622.0MESUnmet64F
    BT373++++55.43.0PNUnmet65M
    BT417+++90.011.0CLASMet53M
    BT462+++42.510.0CLASUnmet71M
    BT482+++82.68.0MESUnmet38M
    BT517++++91.98.0CLASUnmet48M
    BT137+++6.0PNUnmet37F
    BT140+++10.0MESUnmet47F
    BT150+++10.0CLASUnmet61M
    BT157++/−9.0MESUnmet54M
    BT202+++4.0MESUnmet70M
    BT206+++4.5CLASUnmet69M
    BT211+++4.5MESND61M
    BT209+++4.5PNUnmet56M
    BT219++5.0MESUnmet55M
    BT235++++24.5CLASMet70M
    BT261++++3.0CLASUnmet65M
    BT334++++9.0CLASUnmet63M
    BT513+++13.0MESUnmet58M
    BT241+++13.0NDUnmet70M
    BT248+++6.0NDMet64M
    BT347++++29.0NDMet57F
    BT302+/−++16.060.0MESUnmet19F
    BT337+/−++69.017.5PNMet65M
    BT379+/−++97.016.0CLASUnmet73M
    BT422+/−+/−59.136.0CLASUnmet70M
    BT480+/−++59.013.0MESUnmet69F
    BT483+/−++97.715.0PNMet54M
    BT487+/−++70.624.0MESUnmet72M
    BT500+/−++84.09.5CLASUnmet67F
    BT155+/−++13.0CLASUnmet46F
    BT245+/−++46.0PNMet57F
    BT274+18.0PNUnmet50M
    BT279+/−+15.0CLASUnmet60F
    BT283+/−+9.5CLASMet46F
    BT490+/−++16.0PNUnmet76F
    BT175+/−+12.5NDUnmet63M

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/430/eaao2731/DC1

    Fig. S1. Normal brain vessels do not express CSPG4.

    Fig. S2. CSPG4.CAR is expressed in T cells.

    Fig. S3. CSPG4.CAR-Ts prolonged the survival of mice injected with BT275-NS and BT462-NS.

    Fig. S4. CSPG4.CAR-Ts controlled tumor growth in the U87-MG xenograft model.

    Fig. S5. CSPG4.CAR encoding either CD28 or CD28/4-1BB showed lower antitumor activity than CSPG4.CAR encoding 4-1BB.

    Fig. S6. CSPG4.CAR encoding either CD28 or CD28/4-1BB costimulatory endodomains showed limited antitumor activity in vivo.

    Fig. S7. CSPG4.CAR-Ts encoding either CD28 or CD28 and 4-1BB persisted for less than 5 days.

    Fig. S8. GBM-NS–derived gliomas recurring in vivo retained CSPG4 expression.

    Fig. S9. GBM-NS up-regulated PD-L1 in response to IFN-γ.

    Fig. S10. HER-2 and IL-13Rα2 expression is not up-regulated in GBM-NS in xenograft models.

    Table S1. GBM characterization based on CSPG4 expression and molecular subtypes.

  • Supplementary Material for:

    Constitutive and TNFα-inducible expression of chondroitin sulfate proteoglycan 4 in glioblastoma and neurospheres: Implications for CAR-T cell therapy

    Serena Pellegatta, Barbara Savoldo, Natalia Di Ianni, Cristina Corbetta, Yuhui Chen, Monica Patané, Chuang Sun, Bianca Pollo, Soldano Ferrone, Francesco DiMeco, Gaetano Finocchiaro, Gianpietro Dotti*

    *Corresponding author. Email: gdotti{at}med.unc.edu

    Published 28 February 2018, Sci. Transl. Med. 10, eaao2731 (2018)
    DOI: 10.1126/scitranslmed.aao2731

    This PDF file includes:

    • Fig. S1. Normal brain vessels do not express CSPG4.
    • Fig. S2. CSPG4.CAR is expressed in T cells.
    • Fig. S3. CSPG4.CAR-Ts prolonged the survival of mice injected with BT275-NS and BT462-NS.
    • Fig. S4. CSPG4.CAR-Ts controlled tumor growth in the U87-MG xenograft model.
    • Fig. S5. CSPG4.CAR encoding either CD28 or CD28/4-1BB showed lower antitumor activity than CSPG4.CAR encoding 4-1BB.
    • Fig. S6. CSPG4.CAR encoding either CD28 or CD28/4-1BB costimulatory endodomains showed limited antitumor activity in vivo.
    • Fig. S7. CSPG4.CAR-Ts encoding either CD28 or CD28 and 4-1BB persisted for less than 5 days.
    • Fig. S8. GBM-NS–derived gliomas recurring in vivo retained CSPG4 expression.
    • Fig. S9. GBM-NS up-regulated PD-L1 in response to IFN-γ.
    • Fig. S10. HER-2 and IL-13Rα2 expression is not up-regulated in GBM-NS in xenograft models.
    • Table S1. GBM characterization based on CSPG4 expression and molecular subtypes.

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