Immune response to RB1-regulated senescence limits radiation-induced osteosarcoma formation
Maya Kansara, … , Mark J. Smyth, David M. Thomas
Maya Kansara, … , Mark J. Smyth, David M. Thomas
Published November 15, 2013
Citation Information: J Clin Invest. 2013;123(12):5351-5360. https://doi.org/10.1172/JCI70559.
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Research Article Oncology

Immune response to RB1-regulated senescence limits radiation-induced osteosarcoma formation

Abstract

Ionizing radiation (IR) and germline mutations in the retinoblastoma tumor suppressor gene (RB1) are the strongest risk factors for developing osteosarcoma. Recapitulating the human predisposition, we found that Rb1+/– mice exhibited accelerated development of IR-induced osteosarcoma, with a latency of 39 weeks. Initial exposure of osteoblasts to carcinogenic doses of IR in vitro and in vivo induced RB1-dependent senescence and the expression of a panel of proteins known as senescence-associated secretory phenotype (SASP), dominated by IL-6. RB1 expression closely correlated with that of the SASP cassette in human osteosarcomas, and low expression of both RB1 and the SASP genes was associated with poor prognosis. In vivo, IL-6 was required for IR-induced senescence, which elicited NKT cell infiltration and a host inflammatory response. Mice lacking IL-6 or NKT cells had accelerated development of IR-induced osteosarcomas. These data elucidate an important link between senescence, which is a cell-autonomous tumor suppressor response, and the activation of host-dependent cancer immunosurveillance. Our findings indicate that overcoming the immune response to senescence is a rate-limiting step in the formation of IR-induced osteosarcoma.

Authors

Maya Kansara, Huei San Leong, Dan Mei Lin, Sophie Popkiss, Puiyi Pang, Dale W. Garsed, Carl R. Walkley, Carleen Cullinane, Jason Ellul, Nicole M. Haynes, Rod Hicks, Marieke L. Kuijjer, Anne-Marie Cleton-Jansen, Philip W. Hinds, Mark J. Smyth, David M. Thomas

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Figure 3

In vivo and ex vivo studies show differential regulation of cytokines and senescence response in bone following IR in wild-type and Rb1fl/fl and Rb1+/+ mice.

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In vivo and ex vivo studies show differential regulation of cytokines an...
(A) C57/Bl6 mice injected with saline or 4 μCi 45Ca were sacrificed at day 14 after injection and stained for SA-β-Gal expression. Representative image shows vertebrae with SA-β-Gal–positive cells (blue) (original magnification, ×100). (B) SA-β-Gal staining was quantified using MetaMorph. Box-and-whisker plot shows the percentage of blue pixels in the whole image and standard error (saline vs. 45Ca, 0.68 ± 0.05 vs. 4.12 ± 1.12, respectively; mean ± SEM). ***P ≤ 0.0001, 2-tailed Student’s t test. (C and D) C57/Bl6 calvarial cells were plated and exposed to IR at 4 Gy. At day 10, condition media was collected and assayed for the expression IL-6 and MCP-1 using CB bead arrays. Values shown are representative of at least 3 experiments and SEM. (E) SA-β-Gal staining is attenuated in Rb1fl/fl mice compared with that in Rb1+/+ control mice. Mice were injected with 4 μCi 45Ca and sacrificed at day 14 after injection. Sections of spine were stained for SA-β-Gal. Box-and-whisker plots show mean percentage blue pixels in the whole image ± SEM (IR Rb1+/+ vs. IR Rb1fl/fl, mean 4.5 ± 0.3 vs. 0.69 ± 0.10, respectively; ***P < 0.0001). (F) Transcript level analysis by qRT-PCR of irradiated bone (tibiae and femurs) from Rb1+/+ and Rb1fl/fl mice. Values expressed relative to wild-type bone ± SEM and are representative of 3 independent experiments. (G and H) Expression of cytokines by CB bead arrays as described for C and D. (C, D, F, and H) *P < 0.05, (B and E) ***P < 0.0001, 2-tailed Student’s t test.