Research On The Correlation Between The Expression Of FGFR1 In NSCLC And FDG PET/CT And The Corresponding Molecular Imaging Based On A FGFR1-targeted Peptide

Malignant tumors are a major public health problem and pose a serious threat to human health worldwide.Patient stratification and individualized precision therapy strategies rely on the identification of molecular alterations that may be causative agents in tumorigenesis and tumor growth.Understanding of fibroblast growth factor receptor（FGFR）signaling is rapidly evolving and is a primary research topic currently under investigation.The FGFR family comprises 4 highly conserved transmembrane tyrosine kinase receptors（RTKs）,which interact with 18 soluble fibroblast growth factors（FGFs）.Oncogenic FGFR-encoding gene alterations-including genetic amplification,Translocation or rearrangements-have been identified in multiple cancer types,including lung,bladder,breast,glioblastoma,ovarian and prostate cancer.FGF/FGFR signaling has critical functions in tumorigenesis,invasion,angiogenesis,metastasis,recurrence,epithelial mesenchymal transition（EMT）and survival with higher heterogeneity than classical c-ros oncogene 1 receptor tyrosine kinase（ROS1）,mouse sarcoma virus oncogene homolog B1（BRAF）,anaplastic lymphoma kinase（ALK）,or epidermal growth factor receptor（EGFR）signaling.The activation of FGF/FGFR has been considered a potent inducer of acquired resistance about radiotherapy,chemotherapy and targeted therapy in multiple tumours.In addition,the FGF/FGFR signaling are now suggested as another resistance mechanism to immune checkpoint treatment,which could inhibited the IFNγ-stimulated JAK/STAT signaling pathway,decreasing the expression of B2M,CXCL10,and PD-L1.Since FGFR is important in cancer,abundant efforts have been dedicated to its efficient inhibition,leading to an almost simultaneous release of competing targeting peptides and drug candidates.Erdafitinib（JNJ42756493）,Infigratinib（BGJ398）,Rogaratinib（BAY1163877）and Pemigatinib（INCB54828）are now in Phase 3 Clinical Trail.All FGFR TKIs are targeted to FGFR1-3,except Erdafitinib is targeted to FGFR1-4.It is worthwhile to mention that FGFR inhibitors have offered new hope for patients.FGFR1,the first member of the FGFR family,has been investigated predominantly in the process of human tumori-genesis.Dynamical monitoring of the FGFR1 expression is valuable for screening out the patients may benefit from the FGFR targeted therapy,predicting the efficacy of the radiotherapy,chemotherapy,targeted therapy and immune checkpoint treatment.Currently,to assess FGFR1 mutation status,the collection of tissue specimens via invasive procedures such as surgery or biopsy is generally required.These approaches may be limited by heterogeneity,tumor stage,an invasive procedure,long processing time and the function of multiple patient organs.Consequently,molecular imaging has emerged as a noninvasive method to detect FGFR1 expression in tumors at the cellular or subcellular level.The ideal target for molecular imaging was high expressed in tumor cells,neovascularization,microenvironment and no or little expressed in normal tissue.Our reaserch explored the feasibility of the FGFR1 targeted for molecular imaging,through the evaluation of FGFR1 expression in NSCLC patients.At the same time,appraising the connection between the level of FGFR1 expression and maximum standard uptake value（SUVmax）,the metabolic parameters of ¹⁸F-Fluorodeoxyglucose(¹⁸F-FDG)of positron emission tomography/computed tomography（PET/CT）.It is nesserary to develop the FGFR1 targeted molecular imaging probe.Then our reaserch studied on the character of the ¹⁸F labeled FGFR1-peptide in vitro and in vivo,analyzied the effect of Micro-PET/CT imaging.With that in mind,to monitor the FGFR1 expression is valuable for screening out the patients may benefit from the FGFR targeted therapy,predicting the efficacy of the TKI targeted therapy and immune checkpoint treatment.In order to explore the feasibility of the ¹⁸F-FGFR1-HER2 bispecific heterodimers molecular probe in tumor diagnosis,HER2 affinity was synthesized and then labeled with ¹⁸F.Evaluating the characteristics of[¹⁸F]Al F-NOTA-HER2 affinity,comparing to[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide,lay a foundation for the subsequent research and the development of ¹⁸F-FGFR1-HER2 bispecific heterodimers molecular probe.Part one FGFR1 expression level and the prediction value of ¹⁸F-FDG PET/CT imaging metabolic parameter SUVmax in patients with NSCLCObjective:The purpose of this part was to evaluate FGFR1 expression in NSCLC patients.At the same time,appraising the connection between the level of FGFR1 expression and maximum standard uptake value（SUVmax）,the metabolic parameters of ¹⁸F-FDG of PET/CT.Evaluating the prediction value of ¹⁸F-FDG PET/CT imaging metabolic parameter SUVmax to FGFR1expression level in patients with NSCLC.Methods:Retrospectively analyzed total of 86 patients diagnosed with NSCLC by postoperative pathological examination and underwent preoperative PET/CT examination in the Fourth Hospital of Hebei Medical University from January2017 to November 2021.The major pathological pattern including squamous carcinoma（n=40）and adenocarcinoma（n=46）.Immunohistochemical staining was performed on postoperative pathological wax samples in order to determine FGFR1 expression.Meanwhile,region of interests（ROIs）of ¹⁸F-FDG PET/CT imaging were delineated by workstation image processing system.SUVmax was calculated automatically.The correlation between FGFR1 expression level and SUVmax was analyzed between different groups at the same time point.Results:A total of 86 patients were enrolled,55 patients were FGFR1-positive,while 31 patients were FGFR1-negative.The FGFR1 positive rate was93.48%（43/46）in lung adenocarcinoma and 30%（12/40）in lung squamous cell carcinoma（P=0.000）.There was no correlation between the expression level of FGFR1 in age,gender or SUVmax.Summary:1.In our study,the FGFR1 expression in lung adenocarcinoma was significantly higher than in lung squamous（93.48%vs.30%）.2.The SUVmax of ¹⁸F-FDG PET/CT would be no obviously valuable for the level of FGFR1 expression.Part two Synthesis and Targeting of a Novel ¹⁸F-FGFR1 Molecular ProbeObjective:In patients with NSCLC,there was 93.48%lung adenocarcinoma patients showed postive expression of FGFR1.To achieve the aim of the PET diagnosis and treatment,FGFR1 could be recognized as a potential target for molecular imaging.Designed and assessed the stability of a positron-emitting probe,¹⁸F-FGFR1 targeting peptide([¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide)was designed to assess the feasibilityMethods:1.The NOTA-（PEG2）-FGFR1-peptide was synthesized by solid-phase synthesis and was labeled with ¹⁸F via manually labeled method.In the in vitro stability test,the labeled tracer samples were mixed with sterile saline and fresh human serum and then kept at 37°C for 60min,120min and 4 h.The radiochemical purity of the final compounds was determined by RP-HPLC.2.To verify the radiotracer’s targeting efficiency,five cell lines,the human bladder cancer cell line RT-112,the lung squamous carcinoma cell line NCI-H520,the lung adenocarcinoma cell lines A549 and Calu-3 and gastric carcinoma cell line SNU-16 were screened out and studied the FGFR subtype expression by western blot analysis.3.Cells were cultured under standard conditions.In cellular uptake experiments,[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide was added into those different cell lines.In order to determine the difference of the internalization rate of the five cell lines,acetate buffer（p H 2.5）was added to the wells to remove surface-bound radioactivity.To determine the specific binding of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide with FGFR1 on the cell surface,200-fold of unlabeled peptide were added to the cells with the highest uptake rate.Saturation binding assay was designed to determine the affitiny of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide to FGFR1.Results:1.The preparation process of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide was simple.The labeling efficiency without decay-correction was about 16.67%and the radiochemical purity was more than 98%.The radiochemical purity of the mixtures was greater than 90%,as verified by analytical RP-HPLC analysis.2.Western blot analysis determined that the degrees of FGFR subtypes,FGFR1-4 expression varied among the individual cell lines.FGFR1expression was significantly higher in RT-112 cell line.The over expression of FGFR2 proteins was shown in SNU-16 cell line.FGFR3 mainly expressed in Calu-3 and RT-112 cell lines.The cell lines Calu-3 and A549 stably expressed FGFR4.3.The cellular uptake rate of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide were consistently higher in RT-112 cell line than in other cells（P=0.000）.The internalization rate of the five cell lines in each time point did not show the significantly difference.The uptake of the RT-112 cell lines could be successfully blocked by 200-flod of the unlabeled peptide.Saturation binding assay showed the high affitiny of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide to FGFR1.Summary:1.The preparation process of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide was simple.The novel radiotracer showed a good character in the labeling efficiency,the radiochemical purity and the stability.2.The FGFR subtypes expressed different in the five cell lines.FGFR1expression was significantly higher in RT-112 cell line.3.The results of the cellular uptake experiments declared that[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide showed the highest affinity to FGFR1.The cellular internalization experiments did not show the connection between the expression level of FGFR1 and cell lines’internalization rate.The uptake of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide could improve the specifity of the cellular uptake.Saturation binding assay showed the high affitiny of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide to FGFR1.Part three The Micro-PET/CT imaging and the biodistribution of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide in xenograft miceObjective:The human bladder cancer cell line RT-112,the lung squamous carcinoma cell line NCI-H520,the lung adenocarcinoma cell lines A549 and Calu-3 and gastric carcinoma cell line SNU-16,five cell lines（2×10⁷cells/nude mouse）were injected into the right forelimb to induce solid tumors.Assessed the effects of Micro-PET/CT imaging and the in vivo stability of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide.Methods:1.BALB/c nude mice were used for this study.RT-112,NCI-H520,A549,Calu-3 and SNU-16,all five cell lines were subcutaneously injected into the right forelimb to induce solid tumors.The indication for Micro-PET/CT imaging was xenografts with a tumor of the suitable volume.30min,60min and 120min after the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide was injected through the tail vein,Micro-PET/CT imaging was performed on a Micro-PET/SPECT/CT machine2.According to the Micro-PET/CT imaging,five mice were selected randomly from the positive group.Tumor-bearing mice of this group were injected tracer which was mixed with excess unlabeled peptide to saturate the receptors in the tumors.To compare the accumulation of the tumors between the blocked and unblocked group,whole-body scan was performed at 30min,60min and 120min after tracer administration.3.The tumor to muscle（T/M）ratio,tumor to liver（T/L）ratio and tumor to kidney（T/K）ratio in the model were calculated by manually outlining the regions of interest（ROIs）at each time point.4.Urine samples were obtained from xenograft nude mice 60min after injection of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide for the in vivo radiochemical stability experiment and were analyzed by RP-HPLC.Results:1.After administration of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide,RT-112 xenograft mice had highest tumor uptake,followed by NCI-H520tumor bearing mice.No significant tumor uptake was found in the A549,SNU-16 and Calu-3 xenograft mice.2.Significant reductions of the RT-112 tumor accumulation were detected after excess unlabeled peptide blocked.3.The biodistribution analysis data indicated that[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide metabolized mainly through the kidney.In the RT-112 cell line xenograft mice,the novel radiotracer,[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide showed the highest up-taken with the highest T/M,T/L and T/K ratio..4.Urine samples were detected by RP-HPLC,which revealed no peak corresponded to free ¹⁸F.Summary:1.[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide is selectively accumulated in RT-112 cell induceed tumors with FGFR1 overexpressed.The uptake of the¹⁸F-NOTA-（PEG2）-FGFR1-peptide could be blocked by the excess unlabeled peptide-blocked,which proved the specificity of the novel radiotracer.2.[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide showed good stability and metabolite features over time.3.[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide is a potential targeted molecular imaging probe for imaging of cancer patients with FGFR1 high expression.Part four Preliminary study on design of bispecific FGFR1-HER2heterodimers for tumor imagingObjective:HER2 affinity was synthesized and then labeled with ¹⁸F.Evaluating the characteristics of[¹⁸F]Al F-NOTA-HER2 affinity,comparing to[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide,lay a foundation for the subsequent research and the development of ¹⁸F-FGFR1-HER2 bispecific heterodimers molecular probe.Methods:1.The NOTA-HER2 affibody was synthesized by solid-phase synthesis and was labeled with ¹⁸F via manually labeled method.2.Cells were cultured under standard conditions.Compared the results of cellular uptake,internalization,blocking and saturation binding assay to these of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide.3.BALB/c nude mice were used for this study.Human gastric cancer NCI-N87 and MKN74 cells were subcutaneously injected into the right forelimb to induce solid tumors.The indication for PET/CT imaging was xenografts with a tumor of the suitable volume.30min,60min and 120min after the[¹⁸F]Al F-NOTA-HER2 affibody was injected through the tail vein,PET/CT imaging was performed.The tumor to muscle（T/M）ratio was calculated by manually outlining the ROIs at each time point.Then compared the results to these of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide.Results:1.The preparation process of the[¹⁸F]Al F-NOTA-HER2 affibody was simple.The labeling efficiency without decay-correction was about 32.69%and the radiochemical purity was more than 98%.2.[¹⁸F]Al F-NOTA-HER2 affibody could bind to HER2 specifically.The affitiny of[¹⁸F]Al F-NOTA-HER2 affibody to HER2 were higher than these of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide to FGFR1.3.PET/CT imaging results showed that the molecular imaging probe[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide and[¹⁸F]Al F-NOTA-HER2 affibody could bind specifically to the target receptor and obtain good imaging effects,while the optimal imaging time window of the two was different.Summary:1.The preparation process of the[¹⁸F]Al F-NOTA-HER2 affibody was simple.The novel radiotracer showed a good character in the labeling efficiency and the radiochemical purity.The[¹⁸F]Al F-NOTA-HER2 affibody could bind to HER2 specifically.2.[¹⁸F]Al F-NOTA-HER2 affibody and[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide could bind specifically to the target receptor,laying a foundation for the development of bispecific FGFR1-HER2 heterodimers for tumor imaging.Conclusions:The conclusions that can be drawn for this study are the following.1.FGFR1 positive expression was more common in adenocarcinoma patients.The SUVmax of PET/CT parameters had no predictive value for the level of FGFR1 expression.2.The preparation process of the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide was simple.The novel radiotracer showed a good character in the labeling efficiency,the radiochemical purity and the stability,which could specifically targeted to FGFR1.3.[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide showed good stability over time and accumulated in tumors with FGFR1 overexpressed selectively.The excretion of[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide was via the urinary system.In normal organs,the novel radiotracer,[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide is no or very low up taken.It was performed that the[¹⁸F]Al F-NOTA-（PEG2）-FGFR1-peptide could be a potential targeted molecular imaging prob4.The development of bispecific FGFR1-HER2 heterodimers molecular imaging probe has certain feasibility in tumor imaging diagnosis...