Infiltrating Myeloid Cells Drive Osteosarcoma Progression via GRM4 Regulation of IL23.

The glutamate metabotropic receptor 4 (GRM4) locus is linked to susceptibility to human osteosarcoma, through unknown mechanisms. We show that Grm4-/- gene-targeted mice demonstrate accelerated radiation-induced tumor development to an extent comparable with Rb1+/- mice. GRM4 is expressed in myeloid cells, selectively regulating expression of IL23 and the related cytokine IL12. Osteosarcoma-conditioned media induce myeloid cell Il23 expression in a GRM4-dependent fashion, while suppressing the related cytokine Il12. Both human and mouse osteosarcomas express an increased IL23:IL12 ratio, whereas higher IL23 expression is associated with worse survival in humans. Consistent with an oncogenic role, Il23 -/- mice are strikingly resistant to osteosarcoma development. Agonists of GRM4 or a neutralizing antibody to IL23 suppressed osteosarcoma growth in mice. These findings identify a novel, druggable myeloid suppressor pathway linking GRM4 to the proinflammatory IL23/IL12 axis. SIGNIFICANCE: Few novel systemic therapies targeting osteosarcoma have emerged in the last four decades. Using insights gained from a genome-wide association study and mouse modeling, we show that GRM4 plays a role in driving osteosarcoma via a non-cell-autonomous mechanism regulating IL23, opening new avenues for therapeutic intervention.See related commentary by Jones, p. 1484.This article is highlighted in the In This Issue feature, p. 1469.

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

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.

Tumor-derived autophagosome vaccine: mechanism of cross-presentation and therapeutic efficacy.

PURPOSE: We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8(+) T cells. Here, we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), which is derived from tumor cells after inhibition of protein degradation, and to provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy. EXPERIMENTAL DESIGN: DRibbles were characterized by Western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole-tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed, and the antitumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma. RESULTS: The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiP), and damage-associated molecular pattern molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional and plasmacytoid dendritic cells (DC), and cross-presentation was partially CLEC9A dependent. Furthermore, this autophagy-assisted antigen cross-presentation pathway involved both caveolae- and clathrin-mediated endocytosis and endoplasmic reticulum-associated degradation machinery. It depends on proteasome and TAP1, but not lysosome functions of antigen-presenting cells. Importantly, DCs loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma. CONCLUSIONS: These data documented the unique characteristics and potent antitumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials.

Adjuvant therapy with agonistic antibodies to CD134 (OX40) increases local control after surgical or radiation therapy of cancer in mice.

The tumor recurrence from residual local or micrometastatic disease remains a problem in cancer therapy. In patients with soft tissue sarcoma and the patients with inoperable nonsmall cell lung cancer, local recurrence is common and significant mortality is caused by the subsequent emergence of metastatic disease. Thus, although the aim of the primary therapy is curative, the outcome may be improved by additional targeting of residual microscopic disease. We display in a murine model that surgical removal of a large primary sarcoma results in local recurrence in approximately 50% of animals. Depletion of CD8 T cells results in local recurrence in 100% of animals, indicating that these cells are involved in the control of residual disease. We further show that systemic adjuvant administration of αOX40 at surgery eliminates local recurrences. In this model, αOX40 acts to directly enhance tumor antigen-specific CD8 T-cell proliferation in the lymph node draining the surgical site, and results in increased tumor antigen-specific cytotoxicity in vivo. These results are also corroborated in a murine model of hypofractionated radiation therapy of lung cancer. Administration of αOX40 in combination with radiation significantly extended the survival compared with either agent alone, and resulted in a significant proportion of long-term tumor-free survivors. We conclude that αOX40 increases tumor antigen-specific CD8 T-cell cytotoxic activity resulting in improved endogenous immune control of residual microscopic disease, and we propose that adjuvant αOX40 administration may be a valuable addition to surgical and radiation therapy for cancer.

CD122+CD8+ Treg suppress vaccine-induced antitumor immune responses in lymphodepleted mice.

Lymphodeleption prior to adoptive transfer of tumor-specific T cells greatly improves the clinical efficacy of adoptive T-cell therapy for patients with advanced melanoma, and increases the therapeutic efficacy of cancer vaccines in animal models. Lymphodepletion reduces competition between lymphocytes, and thus creates "space" for enhanced expansion and survival of tumor-specific T cells. Within the lymphodepleted host, Ag-specific T cells still need to compete with other lymphocytes that undergo lymphopenia-driven proliferation. Herein, we describe the relative capacity of naïve T cells, Treg, and NK cells to undergo lymphopenia-driven proliferation. We found that the major population that underwent lymphopenia-driven proliferation was the CD122+ memory-like T-cell population (CD122+CD8+ Treg), and these cells competed with Ag-driven proliferation of melanoma-specific T cells. Removal of CD122+CD8+ Treg resulted in a greater expansion of tumor-specific T cells and tumor infiltration of functional effector/memory T cells. Our results demonstrate the lymphopenia-driven proliferation of CD122+CD8+ Treg in reconstituted lymphodepleted mice limited the antitumor efficacy of DC vaccination in conjunction with adoptive transfer of tumor-specific T cells.

Cross-presentation of tumor associated antigens through tumor-derived autophagosomes.

Cross-presentation of exogenous antigens by host professional antigen-presenting cells (APCs) plays a pivotal role in the initiation and development of T-cell immune responses to tumor-associated antigens, including self or mutated self-antigens derived from tumor cells, and foreign antigens derived from infectious agents. Cross-presentation requires multiple steps that involve the antigens' synthesis and compartmentalization in donor cells, packaging and delivery, and processing and presentation by MHC class I molecules on professional APCs. The intricate pathways that lead to protein degradation and the formation of MHC I-peptide complexes inside the APC are well documented for both soluble and particulate antigens. However, much less is known about how cross-presentation is regulated by the protein degradation pathways in antigen-donor cells (ADCs), including autophagy-mediated lysosomal proteolysis and proteasomal degradation. The exact nature or form of the antigens derived from donor cells at the time of delivery to the APC for cross-presentation is very controversial.