68Ga-FAPI-04 PET Distinguishes Malignancy from Inflammatory Lesions

doi:10.21203/rs.3.rs-1216321/v1

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⁶⁸Ga-FAPI-04 PET Distinguishes Malignancy from Inflammatory Lesions

https://doi.org/10.21203/rs.3.rs-1216321/v1

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Background

The ⁶⁸Ga-labelled FAPI provides new oncology imaging option other than ¹⁸F-FDG-PET. However, it's unclear about whether the FAPI-PET distinguishes malignancy from benign lesions.

Methods

We established an AOM/DSS-induced rat colorectal tumor model. A double PET/CT tracer of ⁶⁸Ga-FAPI-04 and ¹⁸F-FDG was used in the rat colorectal tumor model. Histological examination, immunohistochemistry staining, and radioautography were performed in this study.

Results

⁶⁸Ga-FAPI PET imaging distinguishes neoplasia from inflammatory lesions in an AOM/DSS-induced rat colorectal tumor model, and FAPI accumulation gradually increases along with tumor progression. An inflammatory lesion did not interfere with ⁶⁸Ga-FAPI PET imaging.

Conclusion

The ⁶⁸Ga-FAPI-04 PET distinguishes malignant tumors from inflammatory lesions by detecting FAP in a rat colorectal tumor model, suggesting that ⁶⁸Ga-FAPI-04 PET is a better diagnostic tool than ¹⁸F-FDG PET, at least to colorectal cancer patients.

Nuclear Medicine & Medical Imaging

PET

fibroblast activation protein

18F-FDG

68Ga-FAPI-04

Colitis-Associated Colorectal Cancer

AOM/DSS

¹⁸F-fluorodeoxyglucose (FDG) positron emission tomography/computer tomography (PET/CT) is frequently applied in cancer diagnosis and therapeutic response monitoring. However, FDG high-uptake lesions indicate carcinoma and benign lesions, such as inflammatory diseases [1, 2]. Colorectal cancer (CRC) is one of the most common malignancies worldwide. The increased uptake of FDG in the benign lesions of the colon/rectum has been described in several studies [3] [4]. Identifying malignant colorectal tumors from high FDG uptake lesions is a clinical challenge.

Fibroblast activation protein (FAP) is highly expressed in the stroma of cancer entities, including colorectal carcinomas [5–7]. A quinoline-based FAP-specific inhibitor (FAPI) was developed to target FAP as cancer-targeting molecules[8–10] and was highly promising as an imaging probe [11, 12]. In our clinical study, we have shown that ⁶⁸Ga-FAPI-PET provides better imaging than ¹⁸F-FDG-PET.

In this study, we further evaluate the capacity of FAPI PET in distinguishing colorectal cancer diagnosis from inflammatory lesions in a rat colorectal tumor model.

AOM/DSS-induced colon cancer

Six-week-old male F344 rats were used for the AOM/DSS-induced colon cancer model. All animals were administered AOM (Sigma) by intraperitoneal injection at a 10 mg/kg dose on the first day of the first week. After one week, rats were given 2% (w/v) DSS (MP Biomedicals, Solon, OH, USA) dissolved in drinking water on the second and fourth weeks. AOM/DSS-induced colon lesions were verified by micro-enteroscopy, ¹⁸F-FDG / ⁶⁸Ga-FAPI-04 micro-PET/CT, and pathological examination.

In Vivo PET/CT imaging on rats

AOM/DSS-induced rats were subjected to ¹⁸F-FDG and ⁶⁸Ga-FAPI-04 micro-PET/CT analysis, performed on an Inveon MM Platform (Siemens Preclinical Solutions, Knoxville, Tennessee, USA). Rats were anesthetized with 2% isoflurane for ¹⁸F-FDG (a single injection of 8-10 MBq/0.1 mL ¹⁸F-FDG via tail vein) and ⁶⁸Ga-FAPI-04 (a single injection of 10 MBq/0.1 mL ⁶⁸Ga-FAPI-04 via tail vein) injection within three days. Following an uptake period of 40 mins (¹⁸F-FDG) and 20 mins (⁶⁸Ga-FAPI-04), rats were placed on the PET scanner bed and maintained under continuous anesthesia during the study. The Inveon Acquisition Workplace (Siemens) was used for scanning. 3D regions of interest were drawn over the entire tumor guided by CT images, and tracer uptake was measured using Inveon Research Workplace software. The SUV was calculated as decay-corrected activity (kBq) per milliliter of tissue volume / injected tracer activity (¹⁸F-FDG/ ⁶⁸Ga-FAPI-04) (kBq) per gram of body weight.

Digital autoradiography

⁶⁸Ga-FAPI-04 was injected via tail vein (a single injection of 10 MBq/0.1 mL) 20 min before animal euthanasia. The tumors were immediately embedded with a tissue-freezing medium (Leica, Wetzlar, Germany) and frozen after animal sacrifice. Five contiguous tissue sections of 5/10 µm thick were carried out by a 3050S cryostat microtome (Leica) for autoradiography and immunohistochemistry analysis. The tumor sections were placed in a cassette against an imaging film (Fuji, Tokyo, Japan). A Typhoon 5 (GE Healthcare, CA, USA) read the exposed plate with a Phosphor reader scanning system. The same tumor section was used for autoradiography, and the adjacent sections were used for histological assays.

Immunohistochemistry

Immunohistochemistry of FAP and HK2 was performed using the tumor sections obtained from induced rats' colorectal lesions. Slides containing the sections were stained with antibodies against HK2 (2867S, Cell Signaling Technology) or FAP (ab207178, Abcam). After microwaving for 5 mins, 3 times, endogenous peroxidase was neutralized with 3% hydrogen peroxidase in methanol for 15 mins at room temperature, and primary antibodies were applied at 4°C overnight. Anti-rabbit IgG biotinylated secondary antibody was applied for 30 mins at 37°C, followed by SA-HRP for 30 mins at 37°C. The sections were visualized using DAB for 5 minutes and counterstained in Mayer's hematoxylin for 20 minutes.

Statistical analysis

Data were presented as mean ± SD. The student t-test was used to compare two variables. A p-value of 0.05 or less was considered to be significant. Statistical analysis was performed using SPSS Statistics software (version 24.0.0; IBM).

⁶⁸ Ga-FAPI PET only reveals neoplasia in a rat colorectal tumor model

Since the AOM/DSS could spontaneously induce inflammation-associated carcinogenesis in the colorectum, we established an AOM/DSS-induced rat colorectal tumor model to test whether FAPI-PET distinguishes malignancy from benign lesions. This model perfectly represents the pathological changes of human colorectal carcinomas (CRC) from normal-aberrant crypt foci–adenoma-carcinoma. The SUVmax of ⁶⁸Ga-FAPI-04 and ¹⁸F-FDG PET were synchronously analyzed on rats administered with the AOM/DSS.

The FDG uptake increased in the colorectal region of all rats (n=8). The localization of the ¹⁸F-FDG was diffuse in the distant colon, proximal colon, and even intestine. In FAPI PET-positive cases, the localization of the ⁶⁸Ga-FAPI was almost always in the distant colon. The SUVmax of ¹⁸F-FDG in the colorectal region varied from 1.6 to 4.8 in the fourth month, while the SUVmax of ⁶⁸Ga-FAPI ranged from 0.3 to 4.5 (Fig. 3A). After the dissection, some of the rats were nodular hyperplasia and bowel wall invasion, and the others were only congestion and edema of the bowel (Fig. 3B).

Eight rats were divided into two groups, the neoplasia group and the inflammation group without neoplasia (Fig. 2B), which were verified by pathological examination (Fig. 3C). The expression of FAP and hexokinase2 (HK2) was detected by IHC in the neoplasia or the inflammatory tissue. HK2 is a key glycolysis enzyme. As shown in Fig. 3C, the HK2 expression was upregulated in both the inflammatory lesion and the neoplasia. In contrast, the FAP expression was increased merely in neoplasia. This observation was highly consistent with the double tracer imaging of FDG-PET and FAPI-PET (Fig. 3D). The SUVmax of ¹⁸F-FDG was 2.325 ± 0.64 and 2.250 ± 1.74 in the neoplasia group and in the inflammation group (p = 0.9380), while the SUVmax of ⁶⁸Ga-FAPI-04 was 3.475 ± 1.07 in the neoplasia group and 0.675 ± 0.33 in the inflammation group (p = 0.0024), suggesting that ⁶⁸Ga-FAPI PET imaging distinguishes malignancy from inflammation.

⁶⁸ Ga-FAPI PET distinguishes malignancy from inflammatory lesions

Moreover, the ¹⁸F-FDG PET and ⁶⁸Ga-FAPI-04 PET were performed on one rat with invasive neoplasia and polyp to avoid background interference. As shown in Fig. 3E, the invasive neoplasia was ¹⁸F-FDG-positive and ⁶⁸Ga-FAPI-04 positive, while the polyp was only ¹⁸F-FDG positive. Two lesions were located in different parts of the colorectum (Fig. 3F). Consistent with the report, the AOM/DSS-induced tumors were more often in the distal colorectum [13]. After dissection, the slices from two segments were analyzed by the autoradiography of ⁶⁸Ga-FAPI-04 and the FAP staining. As shown in the lower panel of Fig. 3F, the radiation intensity of the distal slice was much higher than the proximal one. Consistent with the autoradiography, only the distal sections were FAP-positive. This malignant lesion was further confirmed by pathological examination (Fig. 3G).

⁶⁸ Ga-FAPI PET signal increases along with colorectal cancer progression

To determine whether ⁶⁸Ga-FAPI-04 PET can distinguish tumors from premalignant lesions, we synchronously analyzed the SUVmax of ⁶⁸Ga-FAPI-04 and ¹⁸F-FDG PET at the indicated time points after AOM/DSS administration in a rat colorectal tumor model. After three months of administering AOM/DSS, ¹⁸F-FDG and ⁶⁸Ga-FAPI-04 PET imaging (n=3) was synchronously performed monthly. As shown in Fig. 4A, the uptake of FDG was already high in the third month. The SUVmax of ¹⁸F-FDG was 3.5 ± 0.5 at the third month, while the SUVmax of ⁶⁸Ga-FAPI-04 was only 0.227 ± 0.07. However, the ⁶⁸Ga-FAPI-04 signal progressively increased since the third month. The SUVmax of ⁶⁸Ga-FAPI-04 reached 1.867 ± 0.47 in the sixth month. However, FDG SUVmax was still 4.63 ± 1.06 at the same time point (Fig. 4B). The change fold of the SUVmax of ⁶⁸Ga-FAPI-04 was 8.67 ± 2.68 at the sixth month compared with the third month (p < 0.05), while the change fold of the SUVmax of ¹⁸F-FDG was 1.32 ± 0.13. There were no significant changes in rat weight, which was 1.09 ± 0.14 (Fig. 4B). This observation further supported the above finding that non-malignant inflammation does not affect ⁶⁸Ga-FAPI PET imaging, distinguishing neoplasia from inflammation.

High uptake of FDG in the intestine is nonspecific as the ¹⁸F-FDG is known to accumulate in acute inflammatory lesions. Therefore, high uptake of FDG could present in various colon and rectum diseases, including tumor, polyp, Crohn's disease, or colitis [14, 15]. However, no optimal management algorithm is helpful for patients with incidental colorectal ¹⁸F-FDG PET-avid lesions. Thus, it's challenging to distinguish a malignant tumor from inflammatory diseases based on the FDG uptake [3].

This study found that FAPI-04 was uptaken by the AOM/DSS-induced rat CRC tumor, where the washout was slower than the other organs except the kidney and bladder. Also, it has a high tumor-to-background contrast ratio in vivo. Moreover, the increased accumulation of FAPI was only in colorectal carcinomas. The ⁶⁸Ga-FAPI-04 signal was negative or low at the inflammatory lesions, where ¹⁸F-FDG uptake was markedly increased. Therefore, the specificity of ⁶⁸Ga-FAPI PET is more than ¹⁸F-FDG PET, at least to colorectal carcinomas.

This study has demonstrated that ⁶⁸Ga-FAPI-04 PET distinguishes malignant tumors from inflammatory lesions by detecting fibroblast activation protein (FAP) in a rat colorectal tumor model, suggesting that ⁶⁸Ga-FAPI-04 PET is a better diagnostic method than ¹⁸F-FDG PET, at least to colorectal cancer patients.

Ethics approval and consent to participate

This study received animal ethics board approval at Shanghai Jiao Tong University School of Medicine.

Competing interests

The authors state that no conflict of interest to disclose.

ACKNOWLEDGMENTS

We are sincerely grateful to our colleagues for their valuable suggestions and technical assistance for this study.

This study was supported by grants from the Ministry of Science and Technology of the People's Republic of China (2018YFC1313205), the Shanghai Committee of Science and Technology (11DZ2260200) (20JC1410100), and the National Natural Science Foundation of China (81572300) (81872342) to Dr. Mi, the National Natural Science Foundation of China (81801725) to Dr. Shangguan.

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Download PDF

Version 1

posted

You are reading this latest preprint version

⁶⁸Ga-FAPI-04 PET Distinguishes Malignancy from Inflammatory Lesions

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

AOM/DSS-induced colon cancer

In Vivo PET/CT imaging on rats

Digital autoradiography

Immunohistochemistry

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Status:

Version 1