Abstract The primary objective of this proposal is to develop a non-invasive and effective tumor-targeted chemo- immunotherapy as a novel combination regimen for treating metastatic osteosarcoma tumors in the lungs. It is well known that recurrent osteosarcoma almost exclusively metastasizes to the lungs and is resistant to single- agent and combined chemotherapy. New treatment options such as immunotherapy are urgently needed for combination with chemotherapy to control and eliminate these tumors. We hypothesize that tumor-targeted delivery of a combination therapy that includes a chemo-drug (doxorubicin, routinely used in clinic to treat osteosarcoma) and an immune stimulant (IL-12 protein) will demonstrate an improved anti-tumor efficacy against metastatic osteosarcoma. To test this hypothesis, we will synthesize a novel tumor-targeted RBC- membrane-cloaked nanoparticle (ttRBC-NP) delivery system and then test the anti-tumor efficacy of the doxorubicin(DOX)-loaded ttRBC-NP, denoted ttRBC-NP(DOX), using a spontaneously formed post-surgery metastatic osteosarcoma mouse lung model. The ttRBC-NP(DOX) will next be combined with tumor-targeted IL-12 protein therapy, denoted ttIL-12, to examine potential synergistic effects on treating metastatic osteosarcoma. Furthermore, we will explore the impact of this tumor-targeted chemo-immunotherapy on tumor microenvironment in order to decipher the underlying mechanism. Overall, two specific aims will be pursued in this proposal, including: (i) to synthesize ttRBC-NP(DOX) and test its anti-tumor efficacy in combination with ttIL-12 protein therapy against metastatic osteosarcoma; and (ii) to study the underlying mechanisms by which the targeted combination therapy boosts anti-tumor efficacy against metastatic osteosarcoma. The success of this project will provide a new and effective treatment option for metastatic osteosarcoma by combining a novel nanoparticle-based chemotherapy with a powerful IL-12 protein therapy. This work will also advance the research of nanotechnology in medicine by developing a unique and robust biomimetic nanoparticle delivery platform that utilizes natural RBC membranes to coat and thus camouflage synthetic drug nanocarriers to evade the immune system. This work will also significantly improve the understanding of how combinatorial chemo-immunotherapy impacts the tumor microenvironment and thus enhances the anti-tumor efficacy.