Poster T5 - Comparison of spatial and temporal dynamics of immunogenic cell death induced by radiofrequency ablation in in vitro biomimetic thermochromic phantoms to in vivo murine flank tumors

Introduction: Ablative therapies generate an in situ source of tumor-associated antigens and unique immunogenic environment that is poised to be synergistically combined with immunotherapeutics to modulate the tumor-immune landscape and facilitate a systemic antitumor response. Incomplete ablations in which viable tumor remains may enhances antigen uptake due to the concurrent display of immunogenic cell death and thermal stress markers as well as increased immune cell infiltration in the peri-ablation zone. Biomimetic thermochromic phantoms, in which cells are incorporated into an agarose-based hydrogel containing an ink that undergoes a color change from white to magenta upon heating, are an appealing platform to investigate the temporal and spatial evolution of ablation-induced immunogenic cell death and reduce the need for in vivo tumor models. The size of ablation zones predicted by thermochromic phantoms have been validated to correspond to ex vivo and in vivo tissue for a broad range of modalities and ablation parameters. In this study, spatial transcriptomics and immunohistochemistry will be used to visualize classical markers of thermal stress, damage associated molecular patterns (DAMPs), and pro-inflammatory cytokines in the peri-ablation zone of biomimetic thermochromic phantoms and in vivo murine flank tumors.

Materials and

Methods: Biomimetic thermochromic phantoms are composed of 3% (w/w) low-melting point agarose, 1% (w/w) PVA (as a cryoprotectant to aid in sectioning), 8% thermochromic ink, and CT26 cells at a concentration of 1-2x106 cells/mL. Initial formulations of thermochromic phantoms were polyacrylamide-based which is cytotoxic and was substituted with agarose. The impact of this alteration on the ablation zone was visualized by the ink color change following radiofrequency ablation experiments (90˚ C for 5 min with a 4 mm probe). Incorporated cell viability in agarose-based phantoms was assessed with NADH-diaphorase. The peri-ablation zone was characterized with HMGB1, TNF-alpha, calreticulin, and CCL2 as spatial transcriptomics (RNAScope) markers and calreticulin, HSP70, and DAPI as immunohistochemistry markers. These markers were validated by submerging flasks of cells in a water bath heated to 37˚ C, 43˚ C, or 50˚ C. Cells were then incubated at 37⁰C for 30 minutes, 3 hours, or 24 hours before being harvested, pelleted, and fixed in formalin. The same markers and incubation times were used to evaluate ablated phantoms that were frozen post-ablation. The spatial and temporal evolution of these markers in phantoms was compared to those of murine CT26 flank tumors that underwent the same ablation procedure and were excised at either 3- or 24-hours post-ablation.

Results, Conclusions, and Discussions: The schematic in Fig. 1A depicts the agarose formulation that is a modification from an original design that included polyacrylamide and has undergone extensive validation with ex vivo and in vivo tissue for a broad range of ablation modalities. The size of the ablation zone as defined by the white-to-magenta color was similar for both formulations. Representative cross-sectional images illustrate the value of thermochromic phantoms for ablation planning (Fig. 1B). Using agarose heated to slightly above its gelling point (~40⁰C) ensured that incorporated cells remained viable as visualized with NADH-diaphorase staining which displays viable cells as blue while dead cells remain unstained (Fig. 1C). The distribution of dead cells corresponds with the ablation zone indicated by the ink color change. Analysis of RNA quality with cyclophilin B (PPIB) demonstrates that the RNA in incorporated cells remains intact after freezing indicating that spatial transcriptomics analysis is possible. The response of biomimetic thermochromic phantoms to radiofrequency ablation will be compared to in vivo-ablated murine flank tumors. Phantoms are incubated at 37⁰C for either 30 minutes, 3 hours, or 24 hours post-ablation and compared to flank tumors that were excised 3- or 24-hours post-ablation (Fig. 1D).
Biomimetic thermochromic phantoms are poised to inform the rational combination of immunotherapeutics with local ablations with the goal of potentiating a systemic anti-tumor response. Profiling the spatial and temporal evolution of immunogenic cell death and thermal stress following ablation could guide the selection and scheduling of drugs for follow-up in vivo studies, as well as the selection of tumor types that may be particularly appealing for combination therapy. Future studies could extend the value of biomimetic thermochromic phantoms by exposing cells to immunomodulators prior to incorporation into the phantom or delivering them via direct injection post-ablation. Further, analysis of innate immune components (e.g., dendritic cells, macrophages) in excised tumors with immunohistochemistry can buttress the characterization of immunogenic cell death and thermal stress.

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