{
  "abstract": "Background A major fraction of patients displays only partial responses to immune checkpoint blockade (ICB). 1 Achieving more durable responses in these patients would greatly impact overall survival. A major impediment to ICB efficacy is the spatial heterogeneity of the tumor microenvironment (TME). This heterogeneity arises from the complex network of cell-cell interactions in the TME that are remodeled by ICB. However, it is difficult to extricate the most relevant TME features that bottleneck complete responses. Fundamentally the race between tumor cell death and proliferation rates is the major determinant of ICB response, therefore precisely quantifying and mapping these rates is key to modeling the dynamics of the anti-tumor response and identifying the most crucial roadblocks to achieving complete responses.Methods We tackled these hypotheses by expressing a genetically encoded reporter of apoptosis 2 in our tumor models. This reporter offers a narrower temporal window for activation and requires no exogenous reagent to readout which is crucial for our studies. We combined this reporter with tissue clearing3 and large-volume imaging of our tumor models to not only estimate death rates on the whole-tumor scale but also determine where the most anti-tumor activity is localized and what TME features are colocalized. By timestamping immune cell arrival in the tumor, we also identify how CD8+ T cell trafficking post-ICB relates to enhanced tumor cell killing.4 Results We expressed our apoptosis reporter in the checkpoint responsive model MC-38. Through a late administration of either aCTLA-4 or aPD-1, we achieved partial vs complete responses in our model respectively. We then harvested these tumors at various timepoints post-ICB and found enhanced tumor cell killing rates in both aCTLA-4 and aPD-1 treated samples that weakly correlated with overall tumor growth kinetics. We also find that tumor cell death rates increase in specific zones we term reactive (RE) zones and are decreased in other zones termed non-reactive (NRE) zones. As the response progresses, killing advances out from RE zones centered around arrival sites of newly recruited CD8+ T cells ( figure 1).Conclusions We conclude that our use of the apoptosis reporter allows for precise quantification of tumor cell death rates in vitro and in vivo. Using this tool, we show that the anti-tumor response originates at specific focal points in the tumor composed of unique assemblages of immune cells. Spatiotemporal profiles of killing following aCTLA-4 and aPD-1 display divergent behaviors with rapid propagation from RE zones in complete responders and stalled propagation in partial responders.References Chatziioannou E, Leiter U, Thomas I, Keim U, Seeber O, Meiwes A, Boessenecker I, Gonzalez SS, Torres FM, Niessner H, Sinnberg T, Forschner A, Flatz L, Amaral T. Features and long-term outcomes of stage IV melanoma patients achieving complete response under anti-PD-1-based immunotherapy. Am J Clin Dermatol. 2023;24:453–467Zhang J, Wang X, Cui W, Wang W, Zhang H, Liu L, Zhang Z, Li Z, Ying G, Zhang N, Li B. Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage. Nat Commun. 2013;4:2157.Li W, Germain RN, Gerner MY. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc. 2019;14:1708–1733Shanahan S-L, Kunder N, Inaku C, Hagan NB, Gibbons G, Mathey-Andrews N, Anandappa G, Soares S, Pauken KE, Jacks T, Schenkel JM. longitudinal intravascular antibody labeling identified regulatory T cell recruitment as a therapeutic target in a mouse model of lung cancer. J Immunol. 2024;213:906–918Abstract 458 Figure 1Apoptosis reporter imaging in mouse tumor models reveals varying rates of tumor cell death across space",
  "authors": [
    {
      "affiliations": [
        "The University of Texas MD Anderson Cancer Center, Houston, TX, USA"
      ],
      "name": "Derya Helvaci"
    },
    {
      "affiliations": [
        "The University of Texas MD Anderson Cancer Center, Houston, TX, USA"
      ],
      "name": "Zhixuan Huang"
    },
    {
      "affiliations": [
        "The University of Texas MD Anderson Cancer Center, Houston, TX, USA",
        "James P Allison Institute, The University of Texas MD Anderson Cancer Center, Houston, TX, USA"
      ],
      "name": "Kenneth H Hu"
    }
  ],
  "title": "458 Mapping and quantifying tumor cell killing rates in 3D to determine the bottlenecks to durable responses to checkpoint blockade",
  "uid": "0278bb47-f618-539e-a3df-e4c4b47bbc4d"
}