Synthesis of fluorinated analogues of sphingosine-1-phosphate antagonists as potential radiotracers for molecular imaging using positron emission tomography

Sphingosine-1-phosphate (S1P) receptors play major roles in cardiovascular, immunological and neurological diseases. The recent approval of the sphingolipid drug Fingolimod (Gilenya®), a sphingosine-1-phosphate agonist for relapsing multiple sclerosis, in 2010 exemplifies the potential for targeting sphingolipids for the treatment of human disorders. Moreover, non-invasive in vivo imaging of S1P receptors that are not available till now would contribute to the understanding of their role in specific pathologies and is therefore of preclinical interest. Based on fluorinated analogues of the S1P₁ receptor antagonist W146 showing practically equal in vitro potency as the lead structure, the first S1P receptor antagonist [¹⁸F]-radiotracer has been synthesized and tested for in vivo imaging of the S1P₁ receptor using positron emission tomography (PET). Though the tracer is serum stable, initial in vivo images show fast metabolism and subsequent accumulation of free [¹⁸F]fluoride in the bones.     

Near-infrared fluorescence-labeled anti-PD-L1-mAb for tumor imaging in human colorectal cancer xenografted mice

The expression of programmed death ligand-1 (PD-L1) in tumor has been used as a biomarker to predict the anti-PD-L1 immunotherapy response. To develop a noninvasive imaging technique to monitor the dynamic changes in PD-L1 expression in colorectal cancer (CRC), we labeled an anti-PD-L1 monoclonal antibody with near-infrared (NIR) dye and tested the ability of the NIR-PD-L1-mAb probe to monitor the PD-L1 expression in CRC-xenografted mice by performing optical imaging. Consistent with the expression levels of PD-L1 protein in three CRC cell lines in vitro by flow cytometry and Western blot analyses, our in vivo imaging showed the highest fluorescence signal of the xenografted tumors in mice bearing SW620 CRC cells, followed by tumors derived from SW480 and HCT8 cell lines. We detected the highest fluorescent intensity of the tumor at 120 hours after injection of NIR-PD-L1-mAb. The highest fluorescence intensity was seen in the tumor, followed by the spleen and the liver in SW620 xenografted mice. In SW480 and HCT8 xenografted mice, however, the highest fluorescent signals were detected in the spleen, followed by the liver and the tumor. Our findings indicate that SW620 cells express a higher level of PD-L1, and the NIR-PD-L1-mAb binding to PD-L1 on the surface of CRC cells was specific. The technique was safe and could provide valuable information on PD-L1 expression of the tumor for development of a therapeutic strategy of personized targeted immunotherapies as well as treatment response of patients with CRC.     

Evaluation of 111In-DOTA-F56 peptide targeting VEGFR1 for potential non-invasive gastric cancer xenografted tumor mice Micro-SPECT imaging

Non-invasive imaging of vascular endothelial growth factor receptor 1 (VEGFR1) remains a great challenge in the early diagnosis of tumors, especially in gastric cancer. Here, we designed and evaluated a novel ¹¹¹In-DOTA-F56 peptide as a radioactive analogue of F56 (peptide WHSDMEWWYLLG) to bind VEGFR1. It was obtained by radiolabeling DOTA-F56 with ¹¹¹InCl₃ with 98% radiochemical purity and 1.4 ± 0.4 GBq/µmol specific activity. ¹¹¹In-DOTA-F56 was obtained by the reaction of DOTA-F56 (10 µg) with ¹¹¹InCl₃ in pH 4.0 sodium acetate buffer at 85 °C for 20 min. ¹¹¹In-DOTA-F56 shows good stability in 0.01 M Phosphate Buffered Saline (PBS) and 5% Human Serum Albumin (HSA). ¹¹¹In-DOTA-F56 has a high binding affinity for human gastric cancer BGC-823 cells. Bio-distribution studies of ¹¹¹In-DOTA-F56 were performed in nude mice xenografted with human gastric cancer BGC-823 cells and the results revealed tumor uptake accumulation. A blocking dose of DOTA-F56 significantly reduced the tumor uptake of ¹¹¹In-DOTA-F56. Tumors were observed with Micro-SPECT images, and the uptake in the tumor increased with time from 4 h to 24 h. The MIP of the Micro-SPECT also showed that the excess DOTA-F56 can specifically block ¹¹¹In-DOTA-F56 in a mouse tumor model. We successfully synthesized the ¹¹¹In-DOTA-F56 VEGFR1-targeted peptide as a non-invasive molecule with fine radiochemical properties. Micro-SPECT indicates tumor uptake, which can be further blocked by excess of the F56 peptide, indicating that ¹¹¹In-DOTA-F56 peptide has potential for early detection of VEGFR1 positive gastric cancer and is worthy of further clinical investigations.     

Regulation of PD-L1 expression by matrix stiffness in lung cancer cells

Expression of programmed death-ligand 1 (PD-L1) in tumor cells such as lung cancer cells plays an important role in mechanisms underlying evasion of an immune check point system. Lung cancer tissue with increased deposition of extracellular matrix is much stiffer than normal lung tissue. There is emerging evidence that the matrix stiffness of cancer tissue affects the phenotypes and properties of cancer cells. Nevertheless, the effects of substrate rigidity on expression of PD-L1 in lung cancer cells remain elusive. We evaluated the effects of substrate stiffness on PD-L1 expression in HCC827 lung adenocarcinoma cells by using polyacrylamide hydrogels with stiffnesses of 2 and 25?kPa. Expression of PD-L1 protein was higher on the stiffer substrates (25?kPa gel and plastic dish) than on the soft 2?kPa gel. PD-L1 expression was reduced by detachment of cells adhering to the substrate. Interferon-γ enhanced expression of PD-L1 protein cultured on stiff (25?kPa gel and plastic dishes) and soft (2?kPa gel) substrates and in the cell adhesion-free condition. As the stiffness of substrates increased, formation of actin stress fiber and cell growth were enhanced. Transfection of the cells with short interfering RNA for PD-L1 inhibited cell growth without affecting stress fiber formation. Treatment of the cells with cytochalasin D, an inhibitor of actin polymerization, significantly reduced PD-L1 protein levels. Taken together, a stiff substrate enhanced PD-L1 expression via actin-dependent mechanisms in lung cancer cells. It is suggested that stiffness as a tumor environment regulates PD-L1 expression, which leads to evasion of the immune system and tumor growth.     

Synthesis, [18F] radiolabeling,and evaluation of poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors for in vivo imaging of PARP-1 using positron emission tomography

Imaging of poly (ADP-ribose) polymerase-1 (PARP-1) expression in vivo is a potentially powerful tool for developing PARP-1 inhibitors for drug discovery and patient care. We have synthesized several derivatives of benzimidazole carboxamide as PARP-1 inhibitors, which can be ¹⁸F-labeled easily for positron emission tomographic (PET) imaging. Of the compounds synthesized, 12 had the highest inhibition potency for PARP-1 (IC₅₀ = 6.3 nM). [¹⁸F]12 was synthesized under conventional conditions in high specific activity with 40–50% decay-corrected yield. MicroPET studies using [¹⁸F]12 in MDA-MB-436 tumor-bearing mice demonstrated accumulation of [¹⁸F]12 in the tumor that was blocked by olaparib, suggesting that the uptake of [¹⁸F]12 in the tumor is specific to PARP-1 expression.     

In situ positron emission tomography monitoring of endothelial cells embedded in perfused fibrin gels

In tissue engineering, the continuous monitoring of cell and tissue cultures in vitro is crucial to assess their functional status over time. However, these constructs can be large, thick and non-transparent. Medical imaging techniques can allow real-time in situ monitoring of cell and tissue cultures in thick solid scaffolds. Here, human endothelial cells were embedded in fibrin gels that were continuously perfused by a culture medium. Positron emission tomography (PET) imaging was used to assess cell viability non-destructively over periods extending up to a few weeks. PET imaging protocols were adapted and validated to measure culture perfusion and cell metabolism using [¹⁸F]-fluorodeoxyglucose (¹⁸FDG). Cell densities down to 100,000 cells/mL were detectable after 12 h of culture and cell structures were localized within the fibrin gels after 1–2 weeks of culture. PET is a promising tool to investigate a wide range of cellular properties and reveal information on tissue development.     

Synthesis and evaluation of radiolabeled AGI-5198 analogues as candidate radiotracers for imaging mutant IDH1 expression in tumors

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79?±?6% yield (n?=?9), and ¹⁸F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6?±?1.6% (n?=?5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with ¹⁸F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme.     

Early tumor response prediction for lung cancer patients using novel longitudinal pattern features from sequential PET/CT image scans

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PD-1/PD-L1 immune checkpoint: Potential target for cancer therapy

Recent studies show that cancer cells are sometimes able to evade the host immunity in the tumor microenvironment. Cancer cells can express high levels of immune inhibitory signaling proteins. One of the most critical checkpoint pathways in this system is a tumor-induced immune suppression (immune checkpoint) mediated by the programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1). PD-1 is highly expressed by activated T cells, B cells, dendritic cells, and natural killer cells, whereas PD-L1 is expressed on several types of tumor cells. Many studies have shown that blocking the interaction between PD-1 and PD-L1 enhances the T-cell response and mediates antitumor activity. In this review, we highlight a brief overview of the molecular and biochemical events that are regulated by the PD-1 and PD-L1 interaction in various cancers.     

In vivo monitoring of the therapeutic efficacy of a CXCR1/2 inhibitor with 18F-FDG PET/CT imaging in experimental head and neck carcinoma: A feasibility study

The chemokine receptors CXCR1/2 play a key role in the aggressiveness of several types of cancers including head and neck squamous cell carcinomas (HNSCCs). In HNSCCs, CXCR1/2 signaling promotes cell proliferation and angiogenesis leading to tumor growth and metastasis. The competitive inhibitor of CXCR1/2, C29, inhibits the growth of experimental HNSCCs in mice. However, a non-invasive tool to monitor treatment response is essential to implement the use of C29 in clinical practices. ¹⁸F-FDG PET/CT is a gold-standard tool for the staging and the post-therapy follow-up of HNSCCs patients. Our study aimed to perform the first in vivo monitoring of C29 efficacy by non-invasive ¹⁸F-FDG PET/CT imaging. Mice bearing experimental HNSCCs (CAL33) were injected with ¹⁸F-FDG (T0) and thereafter treated (n = 7 mice, 9 tumors, 50 mg/kg by gavage) or not (n = 7 mice, 10 tumors) with C29 for 4 consecutive days. Final ¹⁸F-FDG-tumor uptake was determined at day 4 (TF). The average relative change (TF-T0) in ¹⁸F-FDG tumor uptake was +25.85 ± 10.93 % in the control group vs ?5.72 ± 10.07 % in the C29-treated group (p < 0.01). These results were consistent with the decrease of the tumor burden and with the decrease of tumor proliferating Ki67+ cells. These results paved the way for the use of ¹⁸F-FDG to monitor tumor response following C29 treatment.     

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3⁺, CD4⁺ and CD8⁺ T cells in spleens and lower PD-1 expression in CD4⁺ and CD8⁺ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.     

Combined multimodal ctDNA analysis and radiological imaging for tumor surveillance in Non-small cell lung cancer

BackgroundRadiology is the current standard for monitoring treatment responses in lung cancer. Limited sensitivity, exposure to ionizing radiations and related sequelae constitute some of its major limitation. Non-invasive and highly sensitive methods for early detection of treatment failures and resistance-associated disease progression would have additional clinical utility.MethodsWe analyzed serially collected plasma and paired tumor samples from lung cancer patients (61 with stage IV, 48 with stages I-III disease) and 61 healthy samples by means of next-generation sequencing, radiological imaging and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays.ResultsA 62% variant concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing was observed between baseline liquid and tissue biopsies in stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial circulating tumor DNA (ctDNA) mutation analysis by means of ddPCR revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, serial ctDNA analyses only recapitulated computed tomography (CT) -monitored tumor dynamics of some, but not all lesions within the same patient. To complement ctDNA variant analysis we devised a ctDNA methylation assay (^methcfDNA) based on methylation-sensitive restriction enzymes. cfDNA methylation showed and area under the curve (AUC) of > 0.90 in early and late stage cases. A decrease in ^methcfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status.ConclusionTaken together, our data support the use of cfDNA sequencing for unbiased characterization of the molecular tumor architecture, highlights the impact of tumor architectural heterogeneity on ctDNA-based tumor surveillance and the added value of complementary approaches such as cfDNA methylation for early detection and monitoring     

Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues

Low expression of the oxidative stress sensor Keap1 is thought to be involved in carcinogenesis. However, the mechanisms responsible for inactivation of the Keap1 gene remain unknown. We investigated Keap1 expression using RT-PCR and found that it was downregulated in lung cancer cell lines and tissues when compared with a normal bronchial epithelial cell line. Treatment with 5-Aza-2′-deoxycytidine restored Keap1 expression in lung cancer cell lines, indicating the silencing mechanism to be promoter methylation. Moreover, we evaluated cytosine methylation in the Keap1 promoter and demonstrated that the P1 region, including 12 CpG sites, was highly methylated in lung cancer cells and tissues, but not in normal cells. Importantly, we found evidence that three specific CpG sites (the 3rd, 6th, and 10th CpGs of P1) might be binding sites for proteins that regulate Keap1 expression. Thus, our results suggest for the first time that Keap1 expression is regulated by an epigenetic mechanism in lung cancer.     

不同检测策略在预测非小细胞肺癌的PD1/PD-L1抑制剂临床疗效中的应用

近10年来,程序性死亡因子1(programmed death-1,PD-1)及其配体(programmed death ligand-1,PD-L1)的抑制剂在非小细胞肺癌(non-small cell lung cancer,NSCLC)的临床治疗中取得了重大突破,有望改变晚期NSCLC的治疗方式。然而,PD-1/PD-L1抑制剂在对NSCLC的治疗中需要借助有效的生物标志物以寻找受益人群(约20%～40%)。目前,临床上主要的判断标准是PD-L1的表达水平。本文综述了近年来在NSCLC中,与预测PD-1/PD-L1抑制剂疗效的PD-L1表达相关的检测方法,包括免疫组化、基于DNA/RNA水平检测、可溶性PD-L1的检测、正电子发射断层显像(positron emission tomography,PET)技术、多重免疫组化技术、流式细胞术和液体活检技术等,着重探讨了不同检测策略在评价PD-L1表达上的最新进展及应用前景,从而推动其在NSCLC免疫治疗中的临床应用。     

Down-regulation of RCC1 sensitizes immunotherapy by up-regulating PD-L1 via p27kip1/CDK4 axis in non-small cell lung cancer

In recent years, although Immune Checkpoint Inhibitors (ICIs) significantly improves survival both in local advanced stage and advanced stage of non-small cell lung cancer (NSCLC), the objective response rate of ICI monotherapy is still only about 20%. Thus, to identify the mechanisms of ICI resistance is critical to increase the efficacy of ICI treatments. By bioinformatics analysis, we found that the expression of regulator of chromosome condensation 1 (RCC1) in lung adenocarcinoma was significantly higher than that in normal lung tissue in TCGA and Oncomine databases. The survival analysis showed that high expression RCC1 was associated with the poor prognosis of NSCLC. And the expression of RCC1 was inversely related to the number of immune cell infiltration. In vitro, knockdown of RCC1 not only significantly inhibited the proliferation of lung adenocarcinoma cells but also increased the expression levels of p27^kip1 and PD-L1, and decreased the expression level of CDK4 and p-Rb. In vivo, knockdown of RCC1 significantly slowed down the growth rate of tumour, and further reduced the volume and weight of tumour model after treated by PD-L1 monoclonal antibody. Therefore, RCC1 could up-regulate the expression level of PD-L1 by regulating p27^kip1/CDK4 pathway and decrease the resistance to ICIs. And this study might provide a new way to increase the efficacy of PD-L1 monoclonal antibody by inhibiting RCC1.     

One-step radiosynthesis and initial evaluation of a small molecule PET tracer for PD-L1 imaging

Programmed cell death protein-ligand 1 (PD-L1) is a crucial biomarker in immunotherapy and its expression level plays a key role in the guidance of anti-PD-L1 therapy. It had been reported that PD-L1 was quantified by noninvasive imaging with more developed radiotracers. In our study, a novel [¹⁸F]fluoride labeled small molecule inhibitor, [¹⁸F]LN was designed for positron emission tomography (PET) imaging in both PD-L1 transfected (A375-hPD-L1) and non-transfected (A375) melanoma-bearing mice. LN showed the specificity (IC₅₀ = 50.39 ± 2.65 nM) to PD-L1 confirmed by competitive combination and cell flow cytometry (FACS) analysis. The radiotracer [¹⁸F]LN was obtained via ¹⁸F-¹⁹F isotope exchange from precursor LN. After radiosynthesis, [¹⁸F]LN was achieved with a high radiochemical purity (RCP) above 95% and got a favorable molar activity of 36.34 ± 5.73 GBq/μmol. [¹⁸F]LN displayed the moderate affinity (K_d = 65.27 ± 3.47 nM) to PD-L1 by specific binding assay. And it showed 1.3-fold higher uptake in A375-hPD-L1 cells than that in A375 cells. PET imaging revealed that [¹⁸F]LN could enter into PD-L1 expressing tumor site and visualize the outline of tumor. And tumor uptake (1.96 ± 0.27 %ID/g) reached the maximum at 15 min in the positive group, showed 2.2-fold higher than the negative (0.89 ± 0.31 %ID/g) or the blocked (1.07 ± 0.26 %ID/g) groups. Meanwhile, biodistribution could slightly distinguish the positive from the negative. The results indicated [¹⁸F]LN would become an efficient tool for evaluating PD-L1 expression with further optimization.     

Synthesis and evaluation of 18F-labeled CJ-042794 for imaging prostanoid EP4 receptor expression in cancer with positron emission tomography

The potent and selective prostanoid EP4 receptor antagonist CJ-042794 was radiolabeled with ¹⁸F, and evaluated for imaging EP4 receptor expression in cancer with positron emission tomography (PET). The fluorination precursor, arylboronic acid pinacol ester 4, was prepared in 4 steps with 42% overall yield. ¹⁸F-CJ-042794 was synthesized via a copper-mediated ¹⁸F-fluorination reaction followed by base hydrolysis, and was obtained in 1.5 ± 1.1% (n = 2) decay-corrected radiochemical yield. PET/CT imaging and biodistribution studies in mice showed that ¹⁸F-CJ-042794 was excreted through both renal and hepatobiliary pathways with significant retention in blood. The EP4-receptor-expressing LNCaP prostate cancer xenografts were clearly visualized in PET images with 1.12 ± 0.08%ID/g (n = 5) uptake value and moderate tumour-to-muscle contrast ratio (2.73 ± 0.22) at 1 h post-injection. However, the tumour uptake was nonspecific as it could not be blocked by co-injection of cold standard, precluding the application of ¹⁸F-CJ-042794 for PET imaging of EP4 receptor expression in cancer.     

Programmed cell death ligand-1 expression and survival in a cohort of patients with non-small cell lung cancer receiving first-line through third-line therapy in Denmark

BackgroundPD-L1 expression on tumor cells (TCs) or immune cells (ICs) may be used as a prognostic marker for survival in patients with NSCLC. We characterized PD-L1 expression on TCs or ICs in a patient cohort with NSCLC to determine associations between PD-L1 expression and overall survival (OS), according to EGFR and KRAS mutation status.MethodsDanish patients aged >18 years diagnosed with NSCLC before 2014 on first- (N = 491), second- (N = 368), or third-line (N = 498) therapy were included. Data were extracted from population-based medical registries. Tumor samples from pathology archives were tested for biomarkers. High PD-L1 expression was defined as expression on ≥25 % of TCs or ICs based on first diagnostic biopsy or surgical resection. KRAS and EGFR mutation status were tested using PCR-based assays. Cox regression analysis was used to compute adjusted HRs and associated 95 % CIs.ResultsPD-L1 TC and IC ≥ 25 % were observed in 24.3 %–31.0 % and 11.7–14.7 % of patients, respectively. EGFR and KRAS mutations were detected in 4.7 %–8.8 % and 26.5 %–30.7 % of patients, respectively. PD-L1 TC ≥ 25 % was not associated with survival advantage in first- (HR = 0.96, 95 % CI: 0.75–1.22), second- (1.08, 0.81–1.42), or third-line (0.94, 0.74–1.20) therapy. PD-L1 IC ≥ 25 % was associated with survival advantage in second-line (HR = 0.56, 95 % CI: 0.36–0.86) and third-line (0.69, 0.49–0.97) but not first-line (1.00, 0.70–1.41) therapy.ConclusionNo association was observed between PD-L1 TC ≥ 25 % and OS in any therapy line. PD-L1 IC ≥ 25 % may confer survival benefit among some patients who reach second-line therapy.