{
  "abstract": "Background Protein-protein interaction (PPI) is one of the many mechanisms where individual cells communicate with nearby cells or extracellular matrix to modulate the environment. The network of interactions and the underlying stoichiometry with immune microenvironment is adversely impacted in many diseases, including cancer. While screening technologies to predict and map the protein interactome are advancing our understanding of intercellular/intracellular PPIs, no orthogonal spatial technologies exist to validate identified PPIs with known mRNA/protein markers. To bridge this gap, we developed a technology that uses modifications to RNAscope TM, a validation technology known for its high sensitivity and specificity, to visualize PPIs, proteins, and mRNA on a single tissue section.Methods Antibodies were conjugated to oligonucleotides designed to bind RNAscope signal amplification complexes, enabling in situ visualization of protein targets and PPIs alongside mRNA expression. The entire workflow was performed using the fully automated Leica BOND RX system, followed by whole-slide scanning and analysis. The assay, which interrogate up to 6 PPI/mRNA/protein target combinations, was deployed on samples from patients with localized, muscle-invasive bladder cancer who received neoadjuvant anti-PD-L1 checkpoint inhibitor therapy prior to planned surgical cystectomy on a clinical trial (NCT02451423). To characterize tumor-immune microenvironment of these patients, PD-1/PD-L1 interactions were assessed using a panel of targets to include cell phenotyping protein markers for immune and tumor cells (CD3, CD4, CD8, FoxP3, and PanCK) and effector molecule-encoding mRNA markers (IFNG, GZMB, GZMK).Results We visualized PD-1/PD-L1 interactions, proteins and mRNA targets within the tumor before and after immunotherapy treatment. Change in overall PD-1/PD-L1 interaction after treatment did not correlate with patients’ response to the treatment. The interaction between CD4+ or CD8+ T cells and CK+ tumor cells, however, showed significant reduction in responder samples than non-responder samples. Additionally, responder samples showed a decrease in percentage of tumor-infiltrating population out of total population for both regulatory and cytotoxic T cells. These same patients did not have quantitative differences in CD8+ infiltration into tumor with treatment based on orthogonal multiplex IHC quantitation. These results demonstrate that while anti-PD-L1 may not induce an increase in CD8+ T cell recruitment overall, this treatment modulates spatial interactions through cell-cell and protein-protein interactions including PD-1/PD-L1 interaction.Conclusions Our findings underscore the importance of spatial evaluation of PD-1/PD-L1 interaction alongside cell phenotyping protein and effector molecule encoding RNA in assessing anti-PD-L1 treatment responses. This approach will elucidate the complex interplay of spatial relationships, shedding light on the modulation of immune responses through PPIs.Trial Registration NCT02451423",
  "authors": [
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Ge-Ah Kim"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Sonali A Deshpande"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Li-Chong Wang"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Krutika Khinvasara"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Julia Yu"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Hamidreza Sepasizangabadi"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Ji Zhang"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Jason Au"
    },
    {
      "affiliations": [],
      "name": "Kathryn Allaire"
    },
    {
      "affiliations": [
        "University of California, San Francisco, San Francisco, CA, USA"
      ],
      "name": "Jun Yan He"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Henry Lamparski"
    },
    {
      "affiliations": [
        "Advanced Cell Diagnostics, a Bio-Techne brand, Newark, CA, USA"
      ],
      "name": "Maithreyan Srinivasan"
    },
    {
      "affiliations": [],
      "name": "David Y Oh"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Lawrence Fong"
    }
  ],
  "title": "87 Evaluating anti-PD-L1 treatment response in bladder cancer patients by spatially detecting PD-1/PD-L1 interaction, protein and mRNA",
  "uid": "b0b4fb66-ac4b-5dc4-908e-76e8663b093b"
}