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. 2012 Aug 15;72(16):4119-29.
doi: 10.1158/0008-5472.CAN-12-0546. Epub 2012 Jun 12.

Resistance of glioblastoma-initiating cells to radiation mediated by the tumor microenvironment can be abolished by inhibiting transforming growth factor-β

Matthew E Hardee  1 , Ariel E Marciscano, Christina M Medina-Ramirez, David Zagzag, Ashwatha Narayana, Scott M Lonning, Mary Helen Barcellos-Hoff
Affiliations

Resistance of glioblastoma-initiating cells to radiation mediated by the tumor microenvironment can be abolished by inhibiting transforming growth factor-β

Matthew E Hardee et al. Cancer Res. .

Abstract

The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma-initiating cells (GIC). Other studies indicate that although GIC are sensitive, the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-β (TGF-β), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased by approximately 25% when treated with LY364947, a small-molecule inhibitor of TGF-β type I receptor kinase, before irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGF-β neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on the primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 treatment before IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGF-β per cell compared with conventional culture, suggesting that TGF-β production by GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGF-β inhibitors with radiation to improve therapeutic response in patients with GBM.

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Figures

Figure 1
Figure 1. TGFβ inhibition radiosensitizes glioma cells independent of effects on proliferation
(A) Human (U251 and U87MG) and mouse (GL261) glioma cell lines respond to exogenous TGFβ1 (2 ng/mL), as measured by Smad phosphorylation, an effect that was blocked with the addition of the TGFβ type 1 receptor kinase inhibitor, LY364947. Representative immunoblots of three replicates and bar plots of densitometry quantitation of mean + S.E. are shown. (B) Clonogenic assay of GL261, U251, and U87MG glioma cells with (open symbols) and without (closed symbols) pre-treatment with LY364947 24 hours before radiation exposure shows that TGFβ inhibition significantly radiosensitizes all three glioma cell lines. The DER at 10% survival is between 1.25-1.30. Mean ± SD values of triplicate determinations are shown. GL261, P = 0.04; U251, P = 0.03; U87MG, P = 0.03, ANOVA with Tukey post-test.
Figure 2
Figure 2. Radiosensitization of glioma cells by TGFβ inhibition correlates with abrogation of the DNA damage response
(A) Aspects of the DNA damage response of the murine GL261 and human U251 and U87 MG glioma cell lines assessed by immunoblotting cell lysates obtained 30 minutes after 2 Gy of ionizing radiation. Pre-treatment with TGFβ inhibitor LY364947 decreased IR-induced ATM phosphorylation at serine 1981 and p53 phosphorylation at serine 15 in all 3 cell lines. Representative immunoblots of 2-3 replicates and bar plots of densitometry quantitation of mean + S.E. are shown. (B) TGFβ inhibition with LY364947 significantly decreased γH2AX foci (green) in murine GL261 and human U251 and U87 MG glioma cells. Nuclei are counterstained with DAPI (blue). Quantification of γH2AX foci shown below each panel revealed a significant reduction in the number of radiation-induced γH2AX foci with LY354947 in GL261 cells (19.2+0.5 vs. 8.6+0.4) U251 cells (11.1+0.4 vs 6.4+0.2) and U87MG cells (10.8+0.7 vs 4.7+0.6). *** p<0.0001, ANOVA.
Figure 3
Figure 3. Treatment with 1D11 pan-specific TGFβ monoclonal antibody radiosensitizes glioma cells in vitro and in vivo
(A) Inhibition of TGFβ using a pan-specific monoclonal antibody, 1D11, resulted in similar level of radiosensitization (1.22 at 10% cell survival) as seen with the use of the small molecule inhibitor LY364947. P = 0.04, ANOVA with Tukey post-test. (B) Treatment with 1D11 neutralizing antibody resulted in decreased γH2AX immunofluorescence (green) 1 hr after 2 Gy compared to irradiated mice receiving control antibody 13C4. Nuclei are counterstained with DAPI (blue). (C) A single intraperitoneal injection of 1D11 antibody 24 hours prior to irradiation (6 Gy) of GL261 flank tumors resulted in greater tumor growth delay compared to mice receiving 13C4 control antibody. The y-axis represents tumor volume normalized to pre-treatment volume. RT/13C4 vs RT/1D11, p<0.05; RT/1D11 vs Sham/1D11, p<0.01; ANOVA with Newman-Keuls multiple comparison post-test (D) The time-to-reach 3 times pre-treatment volume was significantly increased by 1D11 treatment compared to antibody control 13C4. * p<0.05 and ** p<0.01, ANOVA with Newman-Keuls multiple comparison post-test.
Figure 4
Figure 4. TGFβ inhibition in conjunction with radiation decreases GL261 neurosphere-forming capacity and radiosensitizes neurosphere-derived cells
GL261 murine glioma cells were cultured under neurosphere conditions. (A) Treatment with LY364947 (24hrs, 400nM) alone had no effect on primary neurosphere formation. Irradiation (2Gy) decreased primary neurosphere-forming capacity by 28%, and LY364947 treatment for 24 hours prior to irradiation decreased neurosphere formation by an additional 47%, resulting in 75% fewer neurospheres. (B) Treatment with LY364947 (24hrs, 400nM) alone had no effect on secondary neurosphere formation. Irradiation of primary neurospheres had no effect on secondary neurosphere-forming capacity, yet LY364947 treatment of primary neurospheres before irradiation decreased secondary neurosphere formation by 68%. (C) Pre-treatment with LY364947 for 24 hr decreased neurosphere-derived clonogenic cell survival after irradiation (2Gy). After 2 Gy irradiation the survival fraction of untreated neurospheres was reduced by 43% while the survival fraction of neurospheres treated with 2Gy and LY354947 was further reduced by an additional 20%. (D) Representative images of neurospheres from sham, control-treated, sham, LY364947 -treated, 2Gy, control-treated, and 2Gy, LY364947 -treated. Data are means+S.D. of triplicate determinations and representative three experiments. NS, not significant, * p<0.05 and *** p<0.0001, ANOVA.
Figure 5
Figure 5. TGFβ inhibition affects DNA damage response and self-renewal pathways up-regulated by ionizing radiation
(A) LY364947 pre-treatment for 24 hours prior to 2 Gy decreased radiation-induced γH2AX foci in GL261 cells derived from neurosphere. (B) Quantification revealed an 81% decrease in the number of radiation-induced γH2AX foci with LY364947 treatment (6.2 +0.5 vs 1.2+0.3). (C) Comet assay was used to determine that LY364947 inhibited DNA repair following fractionated (3×2 Gy) daily radiation exposure of neuropsphere cultures. Data shown are representative of three experiments. Comets measured for control, N= 317; LY364947 treated, N=74; fractionated 3×2 Gy, N=52; LY364947 treated and fractionated 3×2, N=197. (D) TGFβ production by GL261 cells was measured in conditioned media obtained following 48 hours in monolayer or neurosphere growth conditions. GL261 cells grown under neurosphere conditions produced 3.7 fold more total and 1.9 fold more active TGFβ per cell than cells grown in monolayer culture. *** indicates p<0.0001, single-tailed unpaired t-test. (E) Inhibition of TGFβ with LY364947 also blocked radiation-induced Notch1 immunofluorescence (red), and (F) CXCR4 immunofluorescence (red), in GL261 cells dissociated from neurospheres three hours after irradiation with 2 Gy. Nuclei are counterstained with DAPI (blue).
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