A Tumor-Imaging Method Targeting Cancer-Associated Fibroblasts

Abstract

The tumor stroma, which accounts for a large part of the tumor mass, represents an attractive target for the delivery of diagnostic and therapeutic compounds. Here, the focus is notably on a subpopulation of stromal cells, known as cancer-associated fibroblasts, which are present in more than 90% of epithelial carcinomas, including pancreatic, colon, and breast cancer. Cancer-associated fibroblasts feature high expression of fibroblast activation protein (FAP), which is not detectable in adult normal tissue but is associated with a poor prognosis in cancer patients. Methods: We developed an iodinated and a DOTA-coupled radiotracer based on a FAP-specific enzyme inhibitor (FAPI) and evaluated them in vitro using uptake, competition, and efflux studies as well as confocal microscopy of a fluorescence-labeled variant. Furthermore, we performed imaging and biodistribution studies on tumor-bearing animals. Finally, proof of concept was realized by imaging patients with ⁶⁸Ga-labeled FAPI. Results: Both FAPIs showed high specificity, affinity, and rapid internalization into FAP-expressing cells in vitro and in vivo. Biodistribution studies on tumor-bearing mice and on the first cancer patients demonstrated high intratumoral uptake of the tracer and fast body clearance, resulting in high-contrast images and negligible exposure of healthy tissue to radiation. A comparison with the commonly used radiotracer ¹⁸F-FDG in a patient with locally advanced lung adenocarcinoma revealed that the new FAP ligand was clearly superior. Conclusion: Radiolabeled FAPIs allow fast imaging with very high contrast in tumors having a high stromal content and may therefore serve as pantumor agents. Coupling of these molecules to DOTA or other chelators allows labeling not only with ⁶⁸Ga but also with therapeutic isotopes such as ¹⁷⁷Lu or ⁹⁰Y.

Keywords: FAP; PET; activated fibroblasts; radiopharmaceuticals; small molecule; tumor.

FIGURE 1.

(A) Binding of radiolabeled FAPI-01 and FAPI-02 to the 4 human cancer cell lines and to the HT-1080-FAP, HEK-muFAP, and HEK-CD26 cell lines after 60 min of incubation. (B) Internalization of radiolabeled FAPI-01 and FAPI-02 into HT-1080-FAP cells after incubation for 10 min to 24 h. Internalized fraction is gray or black, and extracellularly bound fraction is white. (C) Competitive binding of radiolabeled FAPI-01 and FAPI-02 to HT-1080-FAP cells after adding increasing concentrations of unlabeled FAPI-01 and Lu-FAPI-02. (D) Efflux kinetics of FAPI-01 and FAPI-02 after 1 h of incubation of HT-1080-FAP cells with radiolabeled compounds, followed by incubation with compound-free medium for 1–24 h. All data are %ID normalized to 1 million (mio) cells.

FIGURE 2.

Internalization of FAPI-02 into HT-1080-FAP cells, HEK-muFAP cells, and HEK-CD26 cells after incubation for 2 h. Blue indicates 4′,6-diamidino-2-phenylindole, and green indicates FAPI-02-Atto488.

FIGURE 3.

⁶⁸Ga-FAPI-02 PET in mice bearing FAP-negative (Capan-2) (A) and human FAP-expressing (HT-1080-FAP) (B) xenografts. Images were obtained at the indicated times after injection and show rapid uptake in tumor (arrows), no accumulation in noncancerous tissue, and rapid elimination via kidneys and bladder. Quantification of PET images shows solid clearance from cardiovascular system and constant uptake into tumor.

FIGURE 4.

(A) Blocking of ⁶⁸Ga-FAPI-02 tumor accumulation by coadministration of 30 nmol of unlabeled FAPI-02 in mice bearing HT-1080-FAP tumors. (B) Time–activity curves for ⁶⁸Ga-FAPI-02 in selected organs after administration with and without unlabeled FAPI-02 as competitor.

FIGURE 5.

(A) Ex vivo biodistribution of ¹⁷⁷Lu-FAPI-02 after administration to mice bearing HT-1080-FAP tumors. Tumor uptake is highest after 2 h (4.7 %ID/g). (B) Pharmacokinetic profile of ¹⁷⁷Lu-FAPI-02 up to 24 h after administration.

FIGURE 6.

PET/CT maximum-intensity projections of patient with metastasized pancreatic cancer (A) and patient with breast cancer (C). (B) Maximum uptake of ⁶⁸Ga-FAPI-02 at 10 min, 1 h, and 3 h after administration to breast cancer patient. Me = metastases.

FIGURE 7.

PET/CT maximum-intensity projections (top) and transaxial views (bottom) 1 h after administration of ¹⁸F-FDG (A) and ⁶⁸Ga-FAPI-02 (B) to patient with locally advanced lung adenocarcinoma. ⁶⁸Ga-FAPI-02 is seen to selectively accumulate in FAP-expressing tissue and to be significantly higher than ¹⁸F-FDG in malignant lesions. Unlike ¹⁸F-FDG, ⁶⁸Ga-FAPI-02 shows no uptake in brain, spleen, or liver.