Supplementary Figure 16 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 16 shows that CP-d/n-ATF5 inhibits growth of BE(2)-C tumors and increases apoptosis</p

Supplementary Figure 5 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 5 shows that overexpression of ATF5 promotes anoikis resistance of CHLA-255 in vitro</p

Supplementary Figure 15 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 15 shows that CP-d/n-ATF5 treatment does not induce BMF under adherent conditions</p

Supplementary Figure 9 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 9 shows that BMF knockdown rescues ATF5 loss-induced reduction of anchorage-independent cell viability</p

Combining immunotherapy with high-dose radiation therapy (HDRT) significantly inhibits tumor growth in a syngeneic mouse model of high-risk neuroblastoma

Purpose: The mortality in patients with MYCN-amplified high-risk neuroblastoma remains greater than 50% despite advances in multimodal therapy. Novel therapies are urgently needed that requires preclinical evaluation in appropriate mice models. Combinatorial treatment with high-dose radiotherapy (HDRT) and immunotherapy has emerged as an effective treatment option in a variety of cancers. Current models of neuroblastoma do not recapitulate the anatomic and immune environment in which multimodal therapies can be effectively tested, and there is a need for an appropriate syngeneic neuroblastoma mice model to study interaction of immunotherapy with host immune cells. Here, we develop a novel syngeneic mouse model of MYCN-amplified neuroblastoma and report the relevance and opportunities of this model to study radiotherapy and immunotherapy. Materials and methods: A syngeneic allograft tumor model was developed using the murine neuroblastoma cell line 9464D derived a tumor from TH-MYCN transgenic mouse. Tumors were generated by transplanting 1 mm3 portions of 9464D flank tumors into the left kidney of C57Bl/6 mice. We investigated the effect of combining HDRT with anti-PD1 antibody on tumor growth and tumor microenvironment. HDRT (8 Gy x 3) was delivered by the small animal radiation research platform (SARRP). Tumor growth was monitored by ultrasound. To assess the effect on immune cells tumors sections were co-imuunostained for six biomarkers using the Vectra multispectral imaging platform. Results: Tumor growth was uniform and confined to the kidney in 100% of transplanted tumors. HDRT was largely restricted to the tumor region with minimal scattered out-of-field dose. Combinatorial treatment with HDRT and PD-1 blockade significantly inhibited tumor growth and prolonged mice survival. We observed augmented T-lymphocyte infiltration, especially CD3+CD8+ lymphocytes, in tumors of mice which received combination treatment. Conclusion: We have developed a novel syngeneic mouse model of MYCN amplified high-risk neuroblastoma. We have utilized this model to show that combining immunotherapy with HDRT inhibits tumor growth and prolongs mice survival

Supplementary Figure 17 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 17 shows that CP-dn-ATF5 reduces tumor growth and metastasis of SK-N-DZ in vivo</p

FIGURE 3 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Depletion of ATF5 induces anoikis and decreases metastasis of neuroblastoma cells. A and B, Viability of BE(2)-C and SK-N-DZ suspension cells in poly-HEMA–coated plates, expressing Dox-inducible shATF5-1 or shATF5-2 at different timepoints after Dox treatment. Mean ± SD. C and D, Quantification of anoikis of BE(2)-C and SK-N-DZ suspension cells cultured as in A and B at different timepoints after Dox addition. Mean ± SD. E, Quantification of whole-body bioluminescence flux (photons/second) in mice 24 hours after intracardiac injection of BE(2)-C-shATF5-2 cells, + Dox (n = 9); −Dox, (n = 9). Mice were maintained on drinking water containing Dox (2 mg/mL) from 3 days before injection to the time of euthanasia. F, Bioluminescent images at 24 hours after intracardiac injection under conditions described in E. G, Quantification of bioluminescence of blood from mice collected 12 and 24 hours after intracardiac injection under conditions described in E. H, Quantification of apoptosis of BE(2)-C-shATF5-2 CTCs isolated from mice 12 hours after intracardiac injection and treatment ± Dox (see E and Materials and Methods), + Dox (n = 5), −Dox, (n = 5). I, RT-PCR analyses of ATF5 and ACTB in circulating BE(2)-C-shATF5-2 cells isolated after 12 hours. J, Time course of whole-body bioluminescence flux in mice treated as in E. + Dox (n = 8); −Dox, (n = 9). The mice were monitored for metastatic spread by bioluminescence and euthanized at day 31. K, Quantification of total flux (photons/second) by ex vivo liver bioluminescence at the time of euthanasia (day 31). L, Quantification of bioluminescence in bone marrow homogenates at the time of euthanasia (day 31). *, P P P P < 0.0001.</p

Supplementary Figure 12 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 12 shows that CP-d/n-ATF5 inhibits the growth of MYCN-amplified and MYCN-non-amplified cell lines in a dose-dependent manner</p

Supplementary Figure 7 from Activating Transcription Factor 5 Promotes Neuroblastoma Metastasis by Inducing Anoikis Resistance

Supplementary Figure 7 shows that ATF5 knockdown decreases SK-N-DZ CTC survival</p