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. 2007 Jul;9(3):259-70.
doi: 10.1215/15228517-2007-010. Epub 2007 May 23.

Inhibiting TGF-beta signaling restores immune surveillance in the SMA-560 glioma model

Thomas-Toan Tran  1 , Martin Uhl, Jing Ying Ma, Lisa Janssen, Venkataraman Sriram, Steffen Aulwurm, Irene Kerr, Andrew Lam, Heather K Webb, Ann M Kapoun, Darin E Kizer, Glenn McEnroe, Barry Hart, Jonathan Axon, Alison Murphy, Sarvajit Chakravarty, Sundeep Dugar, Andrew A Protter, Linda S Higgins, Wolfgang Wick, Michael Weller, Darren H Wong
Affiliations

Inhibiting TGF-beta signaling restores immune surveillance in the SMA-560 glioma model

Thomas-Toan Tran et al. Neuro Oncol. 2007 Jul.

Erratum in

  • Neuro Oncol. 2007 Oct;9(4):465

Abstract

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.

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Figures

Fig. 1
Fig. 1
SX-007 inhibits TGF-β signaling in cells. (A) TGF-β–sensitive CCL64 cells were treated with recombinant TGF-β 1 or TGF-β 2 (10 ng/ml) for 72 h in the absence or presence of SX-007. Cell number in the absence of TGF-β and SX-007 was defined as 100% (mean ± SD, n =3). (B) SMA-560 cells express the target, TGF-βRI. SMA-560 cells were stained with a polyclonal antiserum against TGF-βRI. Cells were analyzed by flow cytometry. Control = secondary antibody only. (C) SX-007 does not affect proliferation of SMA-560 cells. SMA-560 cells were treated with indicated concentrations of SX-007 for three days. Proliferation was assessed by measuring MTS formazan production. (D) SMA-560 cells were starved for 2 h in serum-free medium. Smad phosphorylation was induced with 10% serum in the presence or absence of SX-007. Smad phosphorylation was measured by enzyme-linked immunosorbent assay. (E) Protein lysates from untreated cells or cells preexposed to SX-007 (0.3 μM) for 24 h and then exposed (or not exposed) to TGF-β 2 (5 ng/ml) for 1 h in the further absence or presence of SX-007 were assessed for the levels of p-Smad 2 or total Smad 2/3. Note that the antibodies are specific for p-Smad 2 but not for total Smad 2 and 3.
Fig. 2
Fig. 2
SX-007 prolongs survival in SMA-560 model of glioma. (A) Summary graph of efficacy studies. SMA-560 cells were implanted into the right striatum of VM/Dk mice. On day 3, animals were dosed with vehicle or 20 mg/kg SX-007. Although dosing ended on day 35, animals survived past day 200. The results of 12 studies were pooled together and evaluated by Kaplan-Meier analysis. Median survival after pooling data in the vehicle (n = 114) and dosed (n = 112) groups, was 19 and 23 days, respectively. The increase in median survival between vehicle and treated animals ranged from −0.5 to 4.5 days. The percentage of long-term survivors of treated animals ranged from 0 to 60%. For the vehicle-treated animals, the percentage of long-term survivors ranged from 0 to 25%. (B) SX-007 is efficacious in late-stage disease. Animals (n = 12 for each arm) were dosed with vehicle or 20 mg/kg SX-007 p.o. once daily (q.d.) beginning day 11. Median survival of vehicle- and SX-007–treated animals was 18 and 22 days, respectively (p < 0.05, log-rank posttest). (C) Dose response with SX-007. Animals were dosed as above with vehicle or 5, 20, or 40 mg/kg p.o. q.d. Median survival in vehicle, 5 mg/kg, 20 mg/kg (*p = 0.008), and 40 mg/kg (**p = 0.009, log-rank posttest) groups was 19.5, 19.5, 23, and 24.5 days, respectively (n = 12 for each group). (D) Pharmacokinetics of SX-007 in VM/Dk mice dosed with 20 mg/kg SX-007 p.o. (E) Prolonged coverage with SX-007 does not improve efficacy. Animals were dosed as above with either vehicle (p.o. twice daily [b.i.d.]) or 20 mg/kg SX-007 (p.o. q.d. and p.o. b.i.d.; n = 12 for each group). There was no difference between the q.d. and b.i.d. groups.
Fig. 3
Fig. 3
SX-007 inhibits TGF-β signaling in the brain. Animals were treated with 20 mg/kg SX-007 p.o. once daily as described in Fig. 2. On day 15, the animals were sacrificed 2 h postdose, and the brain was divided into contralateral and ipsilateral hemispheres. The bulk of the tumor was also separated from the ipsilateral hemisphere. Five brains were analyzed. (A) Smad phosphorylation analysis. *p < 0.05, **p < 0.01 (two-tailed unpaired t-test). (B) TaqMan analysis of PAI-1 transcription. *p < 0.05 (two-tailed unpaired t-test).
Fig. 4
Fig. 4
SX-007 inhibits SMA-560 motility. (A) Invasiveness was analyzed with Matrigel-coated membranes in a Boyden chemotaxis chamber assay using 104 SMA-560 cells, untreated or treated with SX-007 (0.3 μM), TGF-β 2 (5 ng/ml), or both for 24 h prior to and during the experiment, in the upper chamber. Invasive cells were counted after 24 h (mean ± SD, n = 3). (B) The number of satellite tumors (tumor foci not connected to main body of tumor) was counted in sections from animals sacrificed at day 15 of the study (n = 17 for vehicle, n = 18 for SX-007; p < 0.05, two-tailed unpaired t-test). (C) A representative micrograph showing reduced satellite tumors in SX-007–treated animals. Scale bar = 1 mm.
Fig. 5
Fig. 5
SX-007 increases infiltrating CD3+ cells. The number of CD3+ cells was counted in sections from animals sacrificed at day 15 of the study (n = 17 for vehicle; n = 18 for SX-007) (p < 0.05, two-tailed unpaired t-test).
Fig. 6
Fig. 6
Long-term survivors reject tumor rechallenge. (A) Kaplan-Meier analysis of long-term survivors rechallenged with SMA-560 cells (n = 22 per group). (B) Reinjected tumor is rejected in long-term survivors. Naive animals or long-term survivors were rechallenged with SMA-560 cells in the contralateral hemisphere. Animals were sacrificed 15 days postimplantation, and the brains were harvested for histological analysis. Representative images are shown. Scale bar = 1 mm. (C) High concentration of CD3+ cells in the vicinity of the rechallenge site. Representative images are shown. Scale bar = 20 μm.
Fig. 7
Fig. 7
SX-007 increases SMA-560–specific CD8+ T-cell degranulation. (A) Representative histograms of surface CD107a staining on CD8+ T-cells (CD8α+CD3ɛ+) from lymph nodes. (B) Scatter plots of percentage of tumor-specific CD8+ T-cells that mobilize CD107a to the surface. The percentage of cells that spontaneously mobilize CD107a (in the absence of SMA-560 cells) was subtracted to calculate tumor-specific activity.
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