{
  "abstract": "Introduction An adaptive immune response to cancer requires three main signals: antigen presentation and recognition (“signal 1”), costimulation (“signal 2”), and secreted immunostimulatory cytokines (“signal 3”). Expression of these signals in tumors via non-viral gene delivery represents a promising strategy to reprogram the tumor microenvironment (TME) and prime antitumor immunity.Methods We used modular polymeric poly(beta-amino ester)-based nanoparticles (NPs) to investigate codelivery of plasmids encoding signal 3s (interleukin (IL)-2, IL-12, IL-15, IL-23, IL-35, or granulocyte-macrophage colony-stimulating factor) and signal 2s (4-1BBL, CD80, CD86, or OX40L) to B16F10 melanoma tumors in vivo. Downstream immune responses and impact on the TME were assessed via flow cytometry and spatial proteomics using a 27-marker PhenoCycler panel.Results Ex vivo flow cytometry and PhenoCycler tissue analysis revealed that multiple signal 2/3 NP combinations led to decreased tumor growth, increased immune-cell infiltration, and skewing of adaptive and innate populations towards immunostimulatory phenotypes with increased CD8 + T-cell and M1 macrophage infiltration. Signal 2/3 NPs also drove antigen presentation on tumor and antigen-presenting cells (APCs) (macrophages, dendritic cells), and led to induction of major histocompatibility complex class I and class II on melanoma cells. Evaluation of putative biomarkers of treatment response to signal 2/3 NP delivery demonstrated that multiple markers of an inflamed TME correlated negatively with tumor growth, with antigen presentation induction highly correlated with tumor size reduction. Spatial analysis on top-performing NP formulations demonstrated that immune cell populations entered the TME via both the tumor–stroma interface and intratumoral vessels; CD8+ T cells and proinflammatory M1 macrophages were brought into proximity. Furthermore, top NP formulations increased the density of intratumoral CD8+-CD4+-proinflammatory APC triads, which have been identified as key structures for immunotherapy-mediated clearance of solid tumors.Conclusions These results demonstrate the utility of a modular NP design for systematic screening of signal 2/3 delivery to melanoma in vivo and highlight the benefit of in-depth profiling via spatial proteomics to evaluate local antitumor immune responses. The results provide insight into the mechanisms underpinning this therapeutic reprogramming strategy, emphasizing the relationship between signal 2/3 NPs and their ability to drive signal 1 for productive antitumor responses in vivo.",
  "authors": [
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Translational Therapeutics & Regenerative Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA"
      ],
      "name": "Kathryn M Luly"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA"
      ],
      "name": "Xin Ming Matthew Zhou"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Translational Therapeutics & Regenerative Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA"
      ],
      "name": "Sachin S Surwase"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Translational Therapeutics & Regenerative Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA"
      ],
      "name": "Erick E Rocher"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA"
      ],
      "name": "Jack Kollings"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA"
      ],
      "name": "Charles Lu"
    },
    {
      "affiliations": [
        "Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA"
      ],
      "name": "Elizabeth Will"
    },
    {
      "affiliations": [
        "Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA"
      ],
      "name": "Qingfeng Zhu"
    },
    {
      "affiliations": [
        "Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Convergence Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA"
      ],
      "name": "Robert A Anders"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Translational Therapeutics & Regenerative Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA",
        "Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, Maryland, USA",
        "Departments of Neurosurgery and Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Departments of Materials Science & Engineering and Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, USA"
      ],
      "name": "Jordan J Green"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Translational Therapeutics & Regenerative Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA"
      ],
      "name": "Stephany Y Tzeng"
    },
    {
      "affiliations": [
        "Center for Translational ImmunoEngineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Translational Therapeutics & Regenerative Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA",
        "Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA",
        "Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA"
      ],
      "name": "Joel C Sunshine"
    }
  ],
  "title": "Reprogramming the melanoma tumor immune microenvironment via combinatorial signal 2/3 gene delivery",
  "uid": "95e9c21a-c3d4-5c63-96d7-60d7e520d977"
}