{
  "abstract": "Background PD-1/PD-L1 blockade is being used for the treatment of several cancer types and the mechanisms underlying efficacy in many of them have been extensively studied. However, in bladder cancer these mechanisms remain ill defined, limiting the ability to improve immunotherapy outcomes. Prior work has shown that fibroblast growth factor receptor-3 (FGFR3) activation is associated with a non-T cell-inflamed tumor microenvironment and with resistance to immune checkpoint blockade. Using both human bladder cancers and murine modeling, we investigated mediators of immunotherapy efficacy, and how FGFR3 affects the immune landscape of bladder tumors and its impact on PD-1/PD-L1 blockade efficacy.Methods We performed multiplex immunofluorescence (mIF) and RNAscope on 47 localized human muscle-invasive bladder cancer (MIBC) samples to characterize immune infiltrates and FGFR3 expression. A second cohort of 21 MIBC samples obtained prior to anti-PD-1/PD-L1 treatment was also analyzed by mIF. Spatial clustering of CD8+ T cells and DC1s was quantified via a modified Ripley’s K function (K-cross), an unbiased computational analysis. In vivo, we used subcutaneous and orthotopic MB49 models engineered to express the FGFR3 G370C activating mutation or control vector, with and without anti-PD-L1 treatment. Tumor growth measurements and immune phenotypic analysis by spectral flow cytometry were performed. Analysis of spatial transcriptomics (10x Visium CytAssist) on 8 human MIBC samples evaluated FGFR3 effects on immune cells distribution and gene expression.Results Clinical response to PD-1 blockade was associated with clustering of CD8+ T cells and DC1s, but not with their absolute numbers. FGFR3 expression inversely correlated with CD8+ T cells and Batf3+DC1s in human tumors. In mice, FGFR3-activated tumors (G370C) showed reduced CD86+ DC1s in tumor-draining lymph nodes and impaired CD8+ T cell activation, increased exhaustion and decreased TCF-1+ CD8+ TILs following anti-PD-L1 treatment, compared to control tumors. Spatial transcriptomics analysis revealed an inverse correlation between the proportion of FGFR3+ regions and DC1 cell presence and the proportion of DC1+CD8+ co-localization. Within tumors, FGFR3+ areas had fewer DC1+, CD8+, and DC1+CD8+ spots, along with reduced expression of chemokines that recruit these cells. CD8+ T cells in FGFR3+ regions exhibited diminished expression of activation/effector and progenitor-exhausted gene signatures, and higher expression of exhaustion/inhibitory genes compared to those in FGFR3 negative areas.Conclusions Our results indicate that in bladder cancer DC1/CD8+ T cell clustering is a key requirement for PD-1/PD-L1 blockade efficacy, and FGFR3 activation mediates resistance by disrupting this interaction, preventing CD8+ T cells from acquiring the phenotypes needed for treatment efficacy.Ethics Approval This study was approved by the University of Chicago Institutional Review Board IRB#15-550 and Institutional Animal Care and Use Committee (IACUC) #72602.",
  "authors": [
    {
      "affiliations": [
        "The University of Chicago, Chicago, IL, USA"
      ],
      "name": "Andrea Ziblat"
    },
    {
      "affiliations": [
        "Merck Sharp and Dohme Corp., Rahway, NJ, USA"
      ],
      "name": "Ken Hatogai"
    },
    {
      "affiliations": [
        "University of Chicago Medicine, Chicago, IL, USA"
      ],
      "name": "Anthony A Fernald"
    },
    {
      "affiliations": [
        "University of Chicago Medicine, Chicago, IL, USA"
      ],
      "name": "Danny EC Kim"
    },
    {
      "affiliations": [
        "University of Chicago Medicine, Chicago, IL, USA"
      ],
      "name": "Hyunsik Lee"
    },
    {
      "affiliations": [
        "University of Chicago Medicine, Chicago, IL, USA"
      ],
      "name": "Alexander T Pearson"
    },
    {
      "affiliations": [
        "University of Chicago Medicine, Chicago, IL, USA"
      ],
      "name": "Madeleine Torcasso"
    },
    {
      "affiliations": [
        "University of Chicago Medicine, Chicago, IL, USA"
      ],
      "name": "Randy F Sweis"
    }
  ],
  "title": "482 Deciphering mediators of PD-1/PD-L1 blockade efficacy in bladder cancer and the role of FGFR3 in immunotherapy resistance",
  "uid": "d5c5039a-4c3a-52a5-939a-a758aeb9bda4"
}