Abstract 4142: Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models
Authors: Azari Feredun , Kennedy Gregory , Chang Ashley , Nadeem Bilal , Sullivan Neil , Encarnado Emanuel , Albelda Steven , Eruslanov Evgeniy , Singhal Sunil 
Published: June 2022

Abstract
Objective: Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and affect neoplastic progression by stromal remodeling, paracrine signaling, potentiating cancer cell proliferation, and immune system evasion. As such, they are a potential target for optimizing therapeutic and diagnostic strategies. The purpose of our study was to evaluate if Fibroblast Activation Protein (FAP) can be a suitable target for molecular imaging guided cancer localization during surgery because FAP is overwhelmingly expressed by CAFs.
Methods: Patient derived lung adenocarcinoma FAP+/alpha SMA+ myofibroblasts (CAFs) were isolated by flow cytometry and cultured according to institutional best practices. Normal fibroblasts from 3T3 cell line were used as negative control whereas normal lung fibroblasts transfected with human FAP cDNA were used as positive control. Cell expression of FAP was compared using SDS Page analysis and flow cytometry. FAP ligand coupled to NIR S0456 fluorochrome (FAP-S0456) was then evaluated with in-vitro dose escalation, cell-toxicity study, fluorescence parameters, and immunofluorescence microscopy.
Results: Patient derived lung cancer (LC) CAFs had lower FAP-S0456 dye binding expression compared to transfected fibroblasts (TF), while normal fibroblasts (FAP-, aSMA-) had no FAP-S0456 dye binding detected on FACS direct antibody analysis (MFI 1033 vs 244, p&lt;0.05). SDS page immunofluorescence protein analysis with FAP-S0456 showed 5.33x relative expression of FAP in LC CAFs and transfected fibroblasts compared to normal controls on densitometric analysis (p&lt;0.05). Both the LC CAFs and FAP transfected fibroblasts demonstrated targeted fluorochrome endosomal internalization at 798 nm wavelength with mean MFI of 418 (SD 41) compared to minimal uptake by normal fibroblasts (MFI 49 (SD 5.12)p&lt;0.05). FAP-S0456 was internalized by FAP-expressing fibroblasts in a dose- and time-dependent manner, as compared to normal fibroblasts, which did not internalize the dye. Matrix co-culturing FAP+/GFP+ fibroblasts with A549 cells showed FAP-S0456 uptake in the GFP+ fibroblasts with no fluorescence observed in neoplastic cells. Evaluation of FAP-S0456 specificity for FAP transmembrane protein by competitive inhibition via 1000x free FAP ligand showed no uptake in the FAP expressing fibroblasts (LC CAF, FAP+ TF) with MFI similar to negative controls (normal fibroblasts, (39 vs 48, p=0.77).
Conclusion: FAP transmembrane receptor is highly expressed in cancer associated fibroblasts and can serve as a potential target for FAP ligand conjugated near-infrared tracers. This study demonstrates proof of principle of utilizing FAP-S0456 in CAF in-vitro models and paves a path for pre-clinical diagnostic and therapeutic validation for surgical applications.
Citation Format: Feredun Azari, Gregory Kennedy, Ashley Chang, Bilal Nadeem, Neil Sullivan, Emanuel Encarnado, Steven Albelda, Evgeniy Eruslanov, Sunil Singhal. Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4142.