The synergy of ¹⁷⁷Lu-FAPI-46 with tyrosine kinase inhibitor in a sarcoma patient-derived xenograft mouse model

Abstract

Background

Given the heterogeneity and high mortality associated with metastatic soft tissue sarcoma, this study aims to evaluate the therapeutic efficacy of combining ¹⁷⁷Lu-FAPI-46 with Pazopanib against this malignancy.

Methods

Patient-derived xenograft (PDX)-bearing mice were randomly divided into three groups: the control group, the ¹⁷⁷Lu-FAPI-46 monotherapy group, and the ¹⁷⁷Lu-FAPI-46 combined with Pazopanib therapy group. Therapeutic efficacy was regularly monitored.

Results

The microPET imaging showed a 0.84-fold decrease in the T/M ratio of 68Ga-FAPI-46 on day 7/8 post combination therapy, while the control group exhibited a 1.23-fold increase. Combination therapy significantly inhibited tumor proliferation, as evidenced by reduced Ki-67 and increased caspase 3 expressions. Notably, there was no significant body weight loss observed in any group.

Conclusion

This study successfully demonstrated the reduction in FAP expression and suppression of tumor volume in sarcoma PDX following the combination therapy of ¹⁷⁷Lu-FAPI-46 with Pazopanib.

1. Introduction

Soft tissue sarcoma (STS) is a rare and heterogeneous cancer that can occur in various body parts. With over 50 different types according to WHO classification, it is challenging to establish a standard treatment. Treatment options include surgery, radiation therapy, and chemotherapy. The 5-year survival rate for localized STS is around 80%, dropping sharply to 15% for metastatic STS [1]. The discovery of an effective treatment strategy is imperative.

Despite ¹⁸Ffluorodeoxyglucose (¹⁸F-FDG) being the most commonly used positron emission tomography (PET) tracer, its sensitivity to detect some types of soft tissue sarcomas (STS), especially low-grade sarcomas, is unacceptable, rendering it not recommended for STS diagnosis [2,3]. Fibroblast activation protein (FAP) is a glycoprotein expressed on cancer-associated fibroblasts (CAFs) within the microenvironment of most epithelial tumors [4]. In therapeutic applications, Liu et al. demonstrated that both ¹⁷⁷Lu-FAPI-46 can impede the growth of PANC xenografts and are rapidly excreted from the kidneys [5]. Despite the limited number of patients [6], the therapeutic efficacy of ¹⁷⁷Lu-FAPI-46 for sarcoma patients did not meet expectations, indicating that the failure of a singular radiolabeled FAPI regimen may be attributed to the distinct biological characteristics.

Pazopanib (Votrient), a tyrosine kinase inhibitor (TKI), operates by suppressing the activity of vascular endothelial growth factor receptors (VEGFR)-1, -2, and -3, platelet-derived growth factor (PDGF) receptors α and β, as well as the stem cell factor receptor (c-KIT). This inhibition disrupts downstream proliferation signal transduction, demonstrating notable antitumor efficacy in STS patients [7]. However, TKI resistance commonly develops in cancer patients upon recurrence. Considering that ¹⁷⁷Lu-FAPI-46 can effectively induce CAFs death, we aim to assess the therapeutic efficacy of combining ¹⁷⁷Lu-FAPI-46 with Pazopanib to investigate whether this combination therapy can overcome resistance and address the limited effectiveness observed with ¹⁷⁷Lu-FAPI-46 as a monotherapy.

2. Materials and methods

2.1. The preparation of ⁶⁸Ga- and ¹⁷⁷Lu-FAPI-46

FAPI-46 was obtained from Novartis Co. Ltd. (Switzerland). The synthesis of ⁶⁸Ga-FAPI-46 was conducted following previously published methods [8]. Details regarding ¹⁷⁷Lu labeling are provided in the supplementary material.

2.2. Establishment of a sarcoma patient-derived xenograft (PDX) mouse model

The protocol was derived from the method published by Lin et al. [9]. Details regarding the establishment of PDX are provided in the supplementary material.

2.3. Micro-positron emission tomography (microPET) imaging

Mice with sarcoma xenografts were imaged on days 0, 7/8, and 14 after intravenous injection of ⁶⁸Ga-FAPI-46. The scan was conducted for 20 min after a 30-min uptake period of the tracer (nanoScan PET/CT, Mediso, Hungary). Images were corrected using scattering correction, attenuation correction by CT images, and positron range correction. Radioactivity was quantified using PMOD software version 3.2 (PMOD Technologies LLC., Switzerland). Tumor-to-muscle ratio (T/M) was used to compare the relative uptake of ⁶⁸Ga-FAPI-46 before and after treatment.

2.4. Evaluation of the efficacy of different treatment approaches

Xenograft-bearing mice were randomly divided into three groups: the control group, the ¹⁷⁷Lu-FAPI-46 monotherapy group, and the ¹⁷⁷Lu-FAPI-46 combined with Pazopanib therapy group. In the ¹⁷⁷Lu-FAPI-46 monotherapy and combination therapy groups, ¹⁷⁷Lu-FAPI-46 (∼7.4 MBq/mouse) was administered via the tail vein on the next day of microPET imaging (day 1) and on day 7. Pazopanib was at a dosage of 150 mg/kg/day for 1 week. To evaluate therapeutic efficacy, tumor size and body weight were measured weekly using calipers and a balance. Tumor size (mm³) was calculated using the formula: 0.5 × width² × length.

2.5. Histological and immunohistochemistry analysis

Details regarding staining protocols are provided in the supplementary material.

2.6. Statistical analysis

The results were expressed as MSE. Student's t-test was employed for intergroup comparisons, with statistical significance considered at P < 0.05.

3. Results

3.1. The microPET imaging of ⁶⁸Ga-FAPI-46 in a sarcoma PDX mouse model

MicroPET imaging revealed a significant uptake of ⁶⁸Ga-FAPI-46 in the PDX sarcoma on the right flank of mice following intravenous injection [Fig. 1]. The tumor uptake of ⁶⁸Ga-FAPI-46 without treatment was expressed as T/M, 3.22 ± 1.77 (n = 4). Radioactivity was notably retained in the kidneys and bladder, consistent with the predominant urinary excretion pathway of ⁶⁸Ga-FAPI-46. During the treatment, the level of FAP expression was monitored using ⁶⁸Ga-FAPI-46 microPET imaging. The results indicated a 1.23-fold increase in the T/M ratio without any treatment, while it was 0.75-fold on day 7/8 and then increased to 1.08 on day 14 in the monotherapy group. Additionally, there was a 0.84-fold decrease in the T/M ratio of ⁶⁸Ga-FAPI-46 on day 7/8 after combination therapy, without relapse [Fig. 1].

Fig. 1.

The representative PET/CT images of the control and ¹⁷⁷Lu-FAPI-46 + PAZO treatment groups were evaluated before and after treatment using ⁶⁸Ga-FAPI-46.

3.2. The therapeutic efficacy of combination therapy

In the control group, mice displayed a continuous increase in tumor volume, resulting in the demise of all mice around day 20 [Fig. 2A]. The overall tumor growth curve showed that while ¹⁷⁷Lu-FAPI-46 monotherapy suppressed the tumor proliferation, combination therapy was the most effective. Furthermore, there were no significant changes in the body weight of mice in each group before and after treatment [Fig. 2B], and no apparent adverse reactions were observed.

Fig. 2.

The effectiveness of monotherapy and combination therapy. (A) Relative tumor volume. (B) The body weight of the mice in each group during the treatment. (C) Tumors analysis by HE and IHC staining.

3.3. Histological and immunohistochemistry analysis

The H&E staining of tumors in either monotherapy or combination group revealed necrosis and shrinkage of nuclei boundaries, indicative of therapeutic efficacy [Fig. 2C]. FAP expression within the tumors was pronounced in the control (CTRL) group, whereas tumors treated with ¹⁷⁷Lu-FAPI-46 exhibited reduced FAP expression. Additionally, a marked downregulation of Ki-67 expression was also noticed in the treated tumors. Although there were no significant differences in Ki-67 expression between the ¹⁷⁷Lu-FAPI-46 monotherapy and combination groups, the combination therapy group displayed a substantial increase in caspase-3 expression within the tumor, indicating successful induction of tumor damage by the therapy [Fig. 2C].

4. Discussions

The theranostics concept - integrating diagnostic and therapeutic functions within the same radiopharmaceutical molecular targeting platform, has been the cornerstone of therapeutic nuclear medicine since the introduction for treatment of thyroid disease [10]. It allows precise molecular imaging to reflect unique molecular pathology of tumor cells, and identifies individuals who may benefit tumor molecular phenotype-targeted radionuclide therapy. This diagnostic/therapeutic model had achieved great success in different tumor type, such as neuroendocrine tumors [11] and mCRPC prostate cancer [12]. The current proof-of-concept preclinical experiment using FAPI targeting the stroma of sarcoma, is expected to be another successful model and can bring great impact in this malignant disease.

Approximately 90% of drugs or anti-cancer therapies that owned promising preclinical results are failed in the clinical phase. The main reason may be that the monolayer cell culture and in vivo human xenografts did not tell the “real story” because they showed different phenotypes to adapt to the environment. Regarding the importance of tumor microenvironment in FAPI studies, we applied the PDX model to evaluate the therapeutic efficacy as it can recruit stromal cells that are composed of CAFs, tumor endothelial cells, tumor-associated macrophages and so on [13]. When compared to the conventional tumor model, the PDX model can retain heterogeneity and mutations, which is crucial to TKI studies. In fact, sarcoma does not belong to the five most common PDX models (lung cancer, breast cancer, colorectal cancer, prostate cancer, and gastric cancer). This is one of the reasons why we established this model in the present study.

The primary strength of this study lies in providing proof-of-concept data supporting the rationale for combining ¹⁷⁷Lu-FAPI-46 with Pazopanib in sarcoma treatment. However, it is important to acknowledge several limitations. Firstly, the study primarily assessed the feasibility of combination therapy using a PDX animal model; however, determining tumor volume is challenging due to the inclusion of the original implanted tumor mass, resulting in difficulties in evaluating therapeutic efficacy by caliper measurement. Secondly, we did not elucidate the detailed mechanism for the efficacy of combination therapy. Further studies are needed in this regard.

5. Conclusions

This study effectively showcased reduced FAP expression in sarcoma PDX through combined therapy of ¹⁷⁷Lu-FAPI-46 and Pazopanib, as indicated by ⁶⁸Ga-FAPI-46 imaging. Moreover, the combination therapy notably slowed tumor proliferation, offering valuable insights for future clinical treatments.

Funding

The authors thank the financial support from grants CMRPVVM0101 and CMRPVVN0241.

Declaration of competing interest

None.

Appendix A. Supplementary data

The following is the Supplementary data to this article: