Fibroblast Activation Protein Expression Research Articles

Fibroblast Activation Protein-Directed Imaging Outperforms 18F-FDG PET/CT in Malignant Mesothelioma: A Prospective, Single-Center, Observational Trial.

The fibroblast activation protein (FAP) is highly expressed in tumor and stromal cells of mesothelioma and thus is an interesting imaging and therapeutic target. Previous data on PET imaging with radiolabeled FAP inhibitors (FAPIs) suggest high potential for superior tumor detection. Here, we report the data of a large malignant pleural mesothelioma cohort within a 68Ga-FAPI46 PET observational trial (NCT04571086). Methods: Of 43 eligible patients with suspected or proven malignant mesothelioma, 41 could be included in the data analysis of the 68Ga-FAPI46 PET observational trial. All patients underwent 68Ga-FAPI46 PET/CT, contrast-enhanced CT, and 18F-FDG PET/CT. The primary study endpoint was the association of 68Ga-FAPI46 PET uptake intensity and histopathologic FAP expression. Furthermore, secondary endpoints were detection rate and sensitivity, specificity, and positive and negative predictive values as compared with 18F-FDG PET/CT. Datasets were interpreted by 2 masked readers. Results: The primary endpoint was met, and the association between 68Ga-FAPI46 SUVmax or SUVpeak and histopathologic FAP expression was significant (SUVmax: r = 0.49, P = 0.037; SUVpeak: r = 0.51, P = 0.030).68Ga-FAPI46 and 18F-FDG showed similar sensitivity by histopathologic validation on a per-patient (100.0% vs. 97.3%) and per region (98.0% vs. 95.9%) basis. Per-region analysis revealed higher 68Ga-FAPI46 than 18F-FDG specificity (81.1% vs. 36.8%) and positive predictive value (87.5% vs. 66.2%). Conclusion: We confirm an association of 68Ga-FAPI46 uptake and histopathologic FAP expression in mesothelioma patients. Additionally, we report high sensitivity and superior specificity and positive predictive value for 68Ga-FAPI46 versus 18F-FDG.

Heterogeneity of fibroblast activation protein expression in the microenvironment of an intracranial tumor cohort: head-to-head comparison of gallium-68 FAP inhibitor-04 (68Ga-FAPi-04) and fluoride-18 fluoroethyl-L-tyrosine (18F-FET) in positron emission tomography-computed tomography imaging.

Cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) can interact with tumor parenchymal cells to promote tumor growth and migration. Fibroblast activation protein (FAP) expressed by CAFs can be targeted with positron emission tomography (PET) tracers, but studies on FAP expression patterns in intracranial tumors remain scarce. We aimed to evaluate FAP expression patterns in intracranial tumors with gallium-68 FAP inhibitor-04 (68Ga-FAPi-04) and immunohistochemical staining and to observe the interactions between CAFs and tumor cells with a head-to-head comparison of 68Ga-FAPi-04 and fluoride-18 fluoroethyl-L-tyrosine (18F-FET) for PET quantification analysis. We prospectively enrolled 22 adult patients with intracranial mass lesions. 68Ga-FAPi-04 and 18F-FET PET-computed tomography (PET/CT) brain imaging were applied before surgery. Maximal tumor-to-brain ratio (TBRmax), metabolic tumor volume (MTV), and total lesion tracer uptake (TLU) was obtained, and different thresholds were used for 68Ga-FAPi-04-positive lesion delineation owing to the lack of relevant guidelines. The MTV and TLU ratios of both tracers were calculated. Linear regression was applied to observe the differential efficacy of semiquantitative PET parameters. A total of 22 patients with a mean age of 50±13 years (range, 27-69 years) were enrolled. Heterogeneous patterns of 68Ga-FAPi-04 uptake [median of maximal standardized uptake value (SUVmax) =3.8; range, 0.1-19.1] were found. More malignant tumors, including brain metastasis, glioblastoma, and medulloblastoma, generally exhibited more significant 68Ga-FAPi-04 uptake than did the less malignant tumors, while the SUVmax and TBRmax exhibited nonsignificant differences across three intracranial lesion groups of primary brain tumor, brain metastasis, and noncancerous disease (SUVmax: P=0.092; TBRmax: P=0.189). Immunohistochemistry staining showed different stromal FAP expression status in various intracranial lesions. In 15 patients with positive 68Ga-FAPi-04 intracranial tumor uptake, the MTVFAPi:MTVFET ratio had differential efficacy in various types of intracranial tumors [95% confidence interval (CI): 0.572-7.712; P=0.027], and further quantification analyses confirmed the differential ability of the MTVFAPi:MTVFET ratio (95% CI: -0.045 to 11.013, P=0.052; 95% CI: 0.044-17.903, P=0.049; 95% CI: -1.131 to 30.596, P=0.065) with different isocontour volumetric thresholds. This head-to-head study demonstrated heterogeneous FAP expression in intracranial tumors. The FAP expression volume percentage in tumor parenchyma may therefore offer benefit with respect to differentiating between intracranial tumor types.

64Cu]Cu-FAP-NOX, a N-oxalyl modified cyclic peptide for FAP PET imaging with a flexible imaging time window.

The aim of the present study was to develop a novel 64Cu-labeled cyclic peptide ([64Cu]Cu-FAP-NOX) that targets fibroblast activation protein (FAP) and may offer advantages in terms of image contrast, imaging time window, and low uptake in normal tissues. The novel cyclic peptide featuring with a N-oxalyl modified tail was constructed and conjugated to NOTA for 64Cu labeling. Biochemical and cellular assays were performed with A549.hFAP cells. The performance of [64Cu]Cu-FAP-NOX was compared to that of two established tracers ([64Cu]Cu-FAPI-04 and [68Ga]Ga-FAP-2286) and three different NOTA-conjugates in HEK-293T.hFAP xenograft mice using micro-PET imaging. Ex vivo biodistribution studies were performed to confirm the FAP specificity and to validate the PET data. Furthermore, a first-in-human study of this novel tracer was conducted on one patient with lung cancer. Compared to [64Cu]Cu-FAPI-04, [64Cu]Cu-FAP-NOX demonstrated faster and higher rates of cellular uptake and internalization in A549.hFAP cells, but lower rates of cellular efflux. All six radiotracers were rapidly taken up by the tumor within the first 4h post-injection. However, [64Cu]Cu-FAP-NOX had more intense tumor accumulation and slower washout from the target. The ratios of the tumor to normal tissue (including kidneys and muscles) increased significantly over time, with [64Cu]Cu-FAP-NOX reaching the highest ratio among all tracers. In the patient, [64Cu]Cu-FAP-NOX PET showed a comparable result to FDG PET in the primary malignant lesion while exhibiting higher uptake in pleural metastases, consistent with elevated FAP expression as confirmed by immunohistochemistry. [64Cu]Cu-FAP-NOX is a promising FAP-targeted tracer with a highly flexible imaging time window, as evidenced by preclinical evaluation encompassing biodistribution and micro-PET studies, along with a successful patient application. Furthermore, [64Cu]Cu-FAP-NOX showed enhanced image contrast and favorable pharmacokinetic properties for FAP PET imaging, warranting translation into large cohort studies.

Mechanistic Characterization of Cancer-associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.

Non-invasive visualization of liver fibrosis with [68Ga]Ga-DOTA-FAPI-04 PET from preclinical insights to clinical translation.

The reversibility of early liver fibrosis highlights the need for improved early detection and monitoring techniques. Fibroblast activation protein (FAP) is a promising theranostics target significantly upregulated during fibrosis. This preclinical and preliminary clinical study investigated a FAP-targeted probe, gallium-68-labeled FAP inhibitor 04 ([68Ga]Ga-DOTA-FAPI-04), for its capability to visualize liver fibrosis. The preclinical study employed [68Ga]Ga-DOTA-FAPI-04 micro-positron emission tomography (PET)/computed tomography (CT) on carbon tetrachloride-induced mice model (n = 34) and olive oil-treated control group (n = 26), followed by validation of the probe's biodistribution. Hepatic uptake was correlated with fibrosis and inflammation levels, quantified through histology and serum assays. FAP and α-smooth muscle actin expression were determined by immunohistochemistry, as well as immunofluorescence. The subsequent clinical trial enrolled 26 patients with suspected or confirmed liver fibrosis to undergo [68Ga]Ga-DOTA-FAPI-04 PET/magnetic resonance imaging or PET/CT. Key endpoints included correlating [68Ga]Ga-DOTA-FAPI-04 uptake with histological inflammation grades and fibrosis stages, and evaluating its diagnostic and differential efficacy compared to established serum markers and liver stiffness measurement (LSM). [68Ga]Ga-DOTA-FAPI-04 mean uptake in mice livers was notably higher than incontrol mice, increasing from week 6 [0.70 ± 0.11 percentage injected dose per cubic centimeter (%ID/cc)], peaking at week 10 (0.97 ± 0.15%ID/cc) and slightly reducing at week 12 (0.89 ± 0.28%ID/cc). The hepatic biodistribution and FAP expression showed a consistent trend. In the patient cohort, hepatic [68Ga]Ga-DOTA-FAPI-04 uptake presented moderate correlations with inflammation grades (r = 0.517 to 0.584, all P < 0.05) and fibrosis stages (r = 0.653 to 0.698, all P < 0.01). The average SUVmax to background ratio in theliver showed superior discriminative ability, especially between stage 0 and stage 1, outperforming LSM (area under curve 0.984 vs. 0.865). [68Ga]Ga-DOTA-FAPI-04 PET shows significant potential for non-invasive visualization and dynamic monitoring of liver fibrosis in both preclinical experiment and preliminary clinical trial, especially outperforming other common clinical indicators in the early stage. NCT04605939. Registered October 25, 2020, https://clinicaltrials.gov/study/NCT04605939.

FAP expression dynamics and role in silicosis: Insights from epidemiological and experimental models

Prolonged exposure to free silica leads to the development of silicosis, wherein activated fibroblasts play a pivotal role in its pathogenesis and progression. Fibroblast Activation Protein (FAP), as a biomarker for activated fibroblasts, its expression pattern and role in key aspects of silicosis pathogenesis remain unclear. This study elucidated the expression pattern and function of FAP through population-based epidemiological investigations, establishment of mouse models of silicosis, and in vitro cellular models. Results indicated a significant elevation of FAP in plasma from silicosis patients and lung tissues from mouse models of silicosis. In the cellular model, we observed a sharp increase in FAP expression early in the differentiation process, which remained high expression. Inhibition of FAP suppressed fibroblast differentiation, while overexpression of FAP produced the opposite effect. Moreover, fibroblast-derived FAP can alter the phenotype and function of neighboring macrophages. In summary, we revealed a high expression pattern of FAP in silicosis and its potential mechanistic role in fibrosis, suggesting FAP as a potential therapeutic target for silicosis.

Interleukin-17A educated hepatic stellate cells promote hepatocellular carcinoma occurrence through fibroblast activation protein expression.

Liver cancer is typified by a complex inflammatory tumor microenvironment, where an array of cytokines and stromal cells orchestrate a milieu that significantly influences tumorigenesis. Interleukin-17A (IL-17A), a pivotal pro-inflammatory cytokine predominantly secreted by Th17 cells, is known to play a substantial role in the etiology and progression of liver cancer. However, the precise mechanism by which IL-17A engages with hepatic stellate cells (HSCs) to facilitate the development of hepatocellular carcinoma (HCC) remains to be fully elucidated. This investigation seeks to unravel the interplay between IL-17A and HSCs in the context of HCC. An HCC model was established in male Sprague-Dawley rats using diethylnitrosamine to explore the roles of IL-17A and HSCs in HCC pathogenesis. In vivo overexpression of Il17a was achieved using adeno-associated virus. A suite of molecular techniques, including RT-qPCR, enzyme-linked immunosorbent assays, Western blotting, cell counting kit-8 assays and colony formation assays, was employed for in vitro analyses. The study findings indicate that IL-17A is a key mediator in HCC promotion, primarily through the activation of hepatic progenitor cells (HPCs). This pro-tumorigenic influence appears to be mediated by HSCs, rather than through a direct effect on HPCs. Notably, IL-17A-induced expression of fibroblast activation protein (FAP) in HSCs emerged as a critical factor in HCC progression. Silencing Fap in IL-17A-stimulated HSCs was observed to reverse the HCC-promoting effects of HSCs. The collective evidence from this study implicates the IL-17A/FAP signaling axis within HSCs as a contributor to HCC development by enhancing HPC activation. These findings bolster the potential of IL-17A as a diagnostic and preventative target for HCC, offering new avenues for therapeutic intervention.

Increased perivascular fibrosis and pro-fibrotic cellular transition in intramyocardial blood vessels in patients with myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): China Scholarship Council Background Myocardial infarction (MI) is pathologically characterized by irreversible myocardial necrosis caused by ischemia. Intramyocardial vascular dysfunction is believed to increase the risk of MI. In this study, we investigated intramyocardial blood vessels in deceased patients during the early and chronic phases of MI to explore the involvement of perivascular fibrosis and pro-fibrotic cellular transitions in the pathological process of MI. Methods Left ventricular tissue (LV) was obtained from the infarction area of autopsied patients with early-phase MI (3-6 hours old; n=23), chronic-phase MI (5-14 days old; n=12), and from non-infarcted controls (n=14). We quantified perivascular fibrosis using EVG staining and fibroblast activation protein (FAP) expression using immunohistochemistry. Co-localization of smooth muscle actin (SMA) or CD31 with fibroblast marker S100A4 indicative of transition of smooth muscle cells (SMC) and endothelial cells (EC) toward fibroblast-like cells in intramyocardial vessels was quantified using immunofluorescent microscopy. Results Compared to controls (61.03%), perivascular fibrosis was significantly elevated in MI patients (early-phase 77.20%; chronic-phase 82.58%)(p&amp;lt;0.0001), with the highest level observed in the infarct cores of chronic-phase patients (95.18%). Compared to controls, FAP expression was 3.5-fold higher in early-phase- and 15.3-fold higher in chronic-phase MI patients (p&amp;lt;0.05), wherein FAP expression again was highest in the infarct cores. Furthermore, in both early- and chronic-phase MI, SMC- and EC fibroblast-like cellular transition was increased. The fraction of SMA+S100A4+ vessels was significantly higher in early-phase MI (31.96%) and chronic-phase MI (21.9%: border zone; 37.25%: infarct core) compared to controls (8.95%)(p&amp;lt;0.05). Similarly, the fraction of CD31+S100A4+ area was significantly increased in early-phase MI (10.14%) and chronic-phase MI (8.31%: border zone 10.15%: infarct core) compared to controls (2.67%)(p&amp;lt;0.05). Conclusion We observed increased perivascular fibrosis, fibroblast activation and fibroblast-like cellular transition toward a pro-fibrotic phenotype of vascular cells in intramyocardial blood vessels in MI patients, that may contribute to the development of MI. The further increase of FAP expression and perivascular fibrosis, particularly in the infarct core, in the chronic-phase suggest that MI itself can exacerbate fibroblast activation and perivascular fibrosis, theoretically increasing the risk of reinfarction.FAP expression and perivascular fibrosispro-fibrotic vascular cellular transitio

Expression of fibroblast activation protein in intrathoracic solitary fibrous tumor

PurposeFibroblast activation protein (FAP) is a serine integral membrane protease, the expression of which has been confirmed in various cancer types. Solitary fibrous tumors of the pleura (SFTP) are rare mesenchymal fibroblastic neoplasms. We present a case of 18F-labeled FAP inhibitor ([18F]FAPI-74) PET imaging and its correlation with histological FAP expression and review an SFTP series at our institution in relation to the extent of FAP expression. MethodsThis retrospective study included 13 patients who underwent surgery between March 2011 and December 2022 at our institute. One of the patients also underwent [18F]FAPI-74 PET imaging. We semi-quantitatively evaluated FAP expression in SFTPs using immunohistochemical staining and H-scores. ResultsNine of the 13 patients were male, with a median age of 64 years (range, 28–79 years). The median tumor size was 6.6 cm (1.1, 16 cm). In the pathological findings, expression levels of Ki67 were 1–5% in 12 of 13 cases. Furthermore, FAP expression was observed in all patients, and the median H-score was 160 (range, 10–280). The H-score of FAP expression in two of the 13 patients was low (10 in both), and that in two of the 13 patients was high (240 and 280). The SUVmax value of [18F]FAPI-74 PET was 3.57 in a patient in whom the H-score of FAP expression was 180. ConclusionsSFTPs expressed FAP to varying degrees in different patients and the [18F]FAPI-74 PET results in one patient reflected FAP expression in the tumor tissue.

FAP Serves as a Prognostic Biomarker in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) poses significant challenges with poor survival rates and limited therapeutic strategies. Our study, using The Cancer Genome Atlas (TCGA) data, assesses cancer-associated fibroblast (CAF) gene signatures' clinical relevance. In our analysis across TCGA tumor types, differential gene expression analysis revealed that fibroblast activation protein (FAP) is upregulated in tumor tissues and associated with poorer survival rates in HNSCC. Furthermore, mechanistic studies employing gene-silencing techniques substantiated that FAP knockout led to a significant decrease in cellular proliferation, invasion, and migration in HNSCC cell lines. Through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we established that high FAP expression correlates with vital biological processes such as extracellular matrix organization, angiogenesis, and cellular motility. Importantly, FAP was found to regulate these processes by promoting the expression of key proteins involved in epithelial-mesenchymal transition-related pathways. Additionally, our analysis revealed a significant correlation between FAP expression and the expression profiles of immune checkpoint molecules, underscoring its potential role in immune modulation. Collectively, our findings illuminate FAP's pivotal role in HNSCC pathogenesis and its potential as a prognostic biomarker and therapeutic target. This research lays the groundwork for understanding the multifaceted roles and regulatory mechanisms of CAFs in HNSCC, thereby offering valuable perspectives for the development of targeted therapeutic strategies aimed at improving patient outcomes.

Prognostic Implications of 68Ga-FAPI-46 PET/CT-Derived Parameters on Overall Survival in Various Types of Solid Tumors.

Tumoral fibroblast activation protein expression is associated with proliferation and angiogenesis and can be visualized by PET/CT. We examined the prognostic value of [68Ga]Ga-fibroblast activation protein inhibitor (FAPI) (68Ga-FAPI)-46 PET/CT for different tumor entities in patients enrolled in 2 prospective imaging studies (NCT05160051, n = 30; NCT04571086, n = 115). Methods: Within 4 wk, 145 patients underwent 68Ga-FAPI-46 and [18F]FDG (18F-FDG) PET/CT. The association between overall survival (OS) and sex, age, tumor entity, total lesion number, highest SUVmax, and the presence of each nodal, visceral, and bone metastasis was tested using univariate Cox regression analysis. Multivariate analyses were performed for prognostic factors with P values of less than 0.05. Results: In the univariate analysis, shorter OS was associated with total lesion number and the presence of nodal, visceral, and bone metastases on 68Ga-FAPI-46 PET/CT (hazard ratio [HR], 1.06, 2.18, 1.69, and 2.05; P < 0.01, < 0.01, = 0.04, and = 0.02, respectively) and 18F-FDG PET/CT (HR, 1.05, 2.31, 1.76, and 2.30; P < 0.01, < 0.01, = 0.03, and < 0.01, respectively) and with SUVmax on 68Ga-FAPI-46 PET/CT (HR, 1.03; P = 0.03). In the multivariate analysis, total lesion number on 68Ga-FAPI-46 PET/CT was an independent risk factor for shorter OS (HR, 1.05; P = 0.02). In patients with pancreatic cancer, shorter OS was associated with total lesion number on 68Ga-FAPI-46 PET/CT (HR, 1.09; P < 0.01) and bone metastases on 18F-FDG PET/CT (HR, 31.39; P < 0.01) in the univariate analysis and with total lesion number on 68Ga-FAPI-46 PET/CT (HR, 1.07; P = 0.04) in the multivariate analyses. In breast cancer, total lesion number on 68Ga-FAPI-46 PET/CT (HR, 1.07; P = 0.02), as well as bone metastases on 18F-FDG PET/CT (HR, 9.64; P = 0.04), was associated with shorter OS in the univariate analysis. The multivariate analysis did not reveal significant prognostic factors. In thoracic cancer (lung cancer and pleural mesothelioma), the univariate and multivariate analyses did not reveal significant prognostic factors. Conclusion: Disease extent on 68Ga-FAPI-46 PET/CT is a predictor of short OS and may aid in future risk stratification by playing a supplemental role alongside 18F-FDG PET/CT.

68Ga]Ga-FAPI-04 PET/MR imaging strategy in management of Krukenberg tumors (KTs) from gastric signet-ring-cell carcinoma: to overcome limitation of [68Ga]Ga-FAPI-04 PET imaging in KTs.

To compare performance of whole-body [68Ga]Ga-FAPI-04 and [18F]FDG PET imaging in the detection of Krukenberg tumors (KTs), primary site and extra-ovarian metastases of gastric signet-ring-cell carcinoma (GSRCC), and evaluate the value of [68Ga]Ga-FAPI-04 PET/MR imaging strategy and its potential impact on the management of KTs from GSRCC. Twelve patients with twenty-three KTs from GSRCC, who underwent both [68Ga]Ga-FAPI-04 pelvic PET/MR and whole-body [68Ga]Ga-FAPI-04 and [18F]FDG PET imaging were retrospectively analyzed. [68Ga]Ga-FAPI-04 and [18F]FDG uptakes were compared by using Wilcoxon signed-rank test or paired t test. McNemar's test was used to compare lesion detectability between two modalities. Two-tailed P<0.05 was considered statistically significant. Immunohistochemistry staining was utilized to analyze the fibroblast activation protein (FAP) expression in KTs. A total of 12 patients with 23 KTs from GSRCC (8 synchronous and 4 metachronous) were evaluated. [68Ga]Ga-FAPI-04 was superior to [18F]FDG PET in detecting primary sites of GSRCC (100% [11/11] vs. 18.2% [2/11], p = 0.002), involved lymph nodes (90.9% [10/11] vs. 54.5% [6/11], p = 0.046) and peritoneal metastases (100% [12/12] vs. 41.7% [5/12], p = 0.008), with higher SUVmax and TBR (all p < 0.005). Both tracers had limited value in identifying KTs, with 100% false negative rate on [68Ga]Ga-FAPI-04 PET and a low detection rate of 8.7% on [18F]FDG PET. Fap immunohistochemistry showed negative or slight FAP expression in neoplastic signet ring cells and ovarian stroma. [68Ga]Ga-FAPI-04 PET/MR imaging strategy greatly improved the detection rate of Krukenberg tumors (87%, 20/23). After adding diffusion-weighted imaging (DWI), the detection rate was further improved (87.5% vs. 100%, p = 0.083). [68Ga]Ga-FAPI-04 PET/MR imaging strategy either upgraded TNM staging or changed treatment management in twelve patients. [68Ga]Ga-FAPI-04 PET outperformed [18F]FDG PET in detecting primary site and most extra-ovarian metastases of GSRCC, but both tracers had limited value in identifying Krukenberg tumors. Pelvis MRI should be applied to compensate the limitation of [68Ga]Ga-FAPI-04 PET imaging to identify Krukenberg tumours. The [68Ga]Ga-FAPI-04 PET/MR imaging strategy has the potential to impact treatment decisions for GSRCC patients with KTs.

Fibrillar extracellular matrix produced by pericyte-like cells facilitates glioma cell dissemination.

Gliomagenesis induces profound changes in the composition of the extracellular matrix (ECM) of the brain. In this study, we identified a cellular population responsible for the increased deposition of collagen I and fibronectin in glioblastoma. Elevated levels of the fibrillar proteins collagen I and fibronectin were associated with the expression of fibroblast activation protein (FAP), which is predominantly found in pericyte-like cells in glioblastoma. FAP+ pericyte-like cells were present in regions rich in collagen I and fibronectin in biopsy material and produced substantially more collagen I and fibronectin in vitro compared to other cell types found in the GBM microenvironment. Using mass spectrometry, we demonstrated that 3D matrices produced by FAP+ pericyte-like cells are rich in collagen I and fibronectin and contain several basement membrane proteins. This expression pattern differed markedly from glioma cells. Finally, we have shown that ECM produced by FAP+ pericyte-like cells enhances the migration of glioma cells including glioma stem-like cells, promotes their adhesion, and activates focal adhesion kinase (FAK) signaling. Taken together, our findings establish FAP+ pericyte-like cells as crucial producers of a complex ECM rich in collagen I and fibronectin, facilitating the dissemination of glioma cells through FAK activation.

Cancer-associated fibroblast activation predicts progression, metastasis, and prognosis of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. Cancer-associated fibroblasts (CAFs) promote progression of cancer, but their role in cSCC is largely unknown. We examined the potential of CAF markers in the assessment of metastasis risk and prognosis of primary cSCC. We utilized multiplexed fluorescence immunohistochemistry for profiling CAF landscape in metastatic and non-metastatic primary human cSCCs, in metastases, and in premalignant epidermal lesions. Quantitative high-resolution image analysis was performed with two separate panels of antibodies for CAF markers and results were correlated with clinical and histopathological parameters including disease-specific mortality. Increased stromal expression of fibroblast activation protein (FAP), α-smooth muscle actin, and secreted protein acidic and rich in cysteine (SPARC) were associated with progression to invasive cSCC. Elevation of FAP and platelet-derived growth factor receptor-β (PDGFRβ) expression was associated with metastasis risk of primary cSCCs. High expression of PDGFRβ and periostin correlated with poor prognosis. Multimarker combination defined CAF subset, PDGFRα-/PDGFRβ+/FAP+, was associated with invasion and metastasis, and independently predicted poor disease-specific survival. These results identify high PDGFRβ expression alone and multimarker combination PDGFRα-/PDGFRβ+/FAP+ by CAFs as potential biomarkers for risk of metastasis and poor prognosis.

Circulating soluble fibroblast activation protein (FAP) levels are independent of cardiac and extra-cardiac FAP expression determined by targeted molecular imaging in patients with myocardial FAP activation

IntroductionTissue Fibroblast Activation Protein alpha (FAP) is overexpressed in various types of acute and chronic cardiovascular disease. A soluble form of FAP has been detected in human plasma, and low circulating FAP concentrations are associated with increased risk of death in patients with acute coronary syndrome. However, little is known about the regulation and release of FAP from fibroblasts, and whether circulating FAP concentration is associated with tissue FAP expression. This study characterizes the release of FAP in human cardiac fibroblasts (CF) and analyzes the association of circulating FAP concentrations with in vivo tissue FAP expression in patients with acute (ST-segment elevation myocardial infarction, STEMI) and chronic (severe aortic stenosis, AS) myocardial FAP expression. Methods and resultsFAP was released from CF in a time- and concentration-dependent manner. FAP concentration was higher in supernatant of TGFβ-stimulated CF, and correlated with cellular FAP concentration. Inhibition of metallo- and serine-proteases diminished FAP release in vitro. Median FAP concentrations of patients with acute (77 ng/mL) and chronic (75 ng/mL, p = 0.50 vs. STEMI) myocardial FAP expression did not correlate with myocardial nor extra-myocardial nor total FAP volume (P ≥ 0.61 in all cases) measured by whole-body FAP-targeted positron emission tomography. ConclusionWe describe a time- and concentration dependent, protease-mediated release of FAP from cardiac fibroblasts. Circulating FAP concentrations were not associated with increased in vivo tissue FAP expression determined by molecular imaging in patients with both chronic and acute myocardial FAP expression. These data suggest that circulating FAP and tissue FAP expression provide complementary, non-interchangeable information.

68Ga-DOTA-D-Alanine-BoroPro Radiotracer for Imaging of the Fibroblast Activation Protein in Malignant and Non-Malignant Diseases.

Recently, we reported a new fibroblast activation protein (FAP) inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-HYNIC-D-Alanine-BoroPro)(99mTc-HYNIC-iFAP) structure for tumor microenvironment SPECT imaging. This research aimed to synthesize 68Ga-[2,2',2″,2‴-(2-(4-(2-(5-(((S)-1-((S)-2-boronopyrrolidin-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carbothioamido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid] (68Ga-DOTA-D-Alanine-BoroPro)(68Ga-iFAP) as a novel radiotracer for PET imaging and evaluate its usefulness for FAP expression in malignant and non-malignant tissues. The coupling of p-SCN-benzene DOTA with HYNIC-iFAP was used for the chemical synthesis and further labeling with 68Ga. Radiochemical purity was verified by radio-HPLC. The specificity of 68Ga-iFAP was evaluated in HCT116 cells, in which FAP expression was verified by immunofluorescence and Western blot. Biodistribution and biokinetic studies were performed in murine models. 68Ga-iFAP uptake at the myocardial level was assessed in mice with induced infarction. First-in-human images of 68Ga-iFAP in healthy subjects and patients with myocardial infarction, glioblastoma, prostate cancer, and breast cancer were also obtained. DOTA-D-Alanine BoroPro was prepared with a chemical purity of 98% and was characterized by UPLC mass spectroscopy, FT-IR, and UV-vis. The 68Ga-iFAP was obtained with a radiochemical purity of >95%. In vitro and in vivo studies demonstrated 68Ga-iFAP-specific recognition for FAP, rapid renal elimination, and adequate visualization of the glioblastoma, breast tumor, prostate cancer, and myocardial infarction sites. The results of this research justify further dosimetry and clinical trials to establish the specificity and sensitivity of 68Ga-iFAP PET for FAP expression imaging.

Progressive changes in the protein expression profile of alveolar septa in early-stage lung adenocarcinoma.

Adenocarcinomas show a stepwise progression from atypical adenomatous hyperplasia (AAH) through adenocarcinoma in situ (AIS) to invasive adenocarcinoma (IA). Immunoglobulin superfamily containing leucine-rich repeat (ISLR) is a marker of tumor-restraining cancer-associated fibroblasts (CAFs), which are distinct from conventional, strongly α-smooth muscle actin (αSMA)-positive CAFs. Fibroblastactivation protein (FAP) has been focused on as a potential therapeutic and diagnostic target of CAFs. We investigated the changes in protein expression during adenocarcinoma progression in the pre-existing alveolar septa by assessing ISLR, αSMA, and FAP expression in normallung, AAH, AIS, and IA. Fourteen AAH, seventeen AIS, and twenty IA lesions were identified and randomly sampled. Immunohistochemical analysis was performed to evaluate cancer-associated changes and FAP expression in the pre-existing alveolar structures. Normal alveolar septa expressed ISLR. The ISLR level in the alveolar septa decreased in AAH and AIS tissues when compared with that in normal lung tissue. The αSMA-positive area gradually increased from the adjacent lung tissue (13.3% ± 15%) to AIS (87.7% ± 14%), through AAH (70.2% ± 21%). Moreover, the FAP-positive area gradually increased from AAH (1.69% ± 1.4%) to IA (11.8% ± 7.1%), through AIS (6.11% ± 5.3%). Protein expression changes are a feature of CAFs in the pre-existing alveolar septa that begin in AAH. These changes gradually progressed from AAH to IA through AIS. FAP-positive fibroblasts may contribute to tumor stroma formation in early-stage lung adenocarcinoma, and this could influence the development of therapeutic strategies targeting FAP-positive CAFs for disrupting extracellular matrix formation.

Diagnostic Potential of Supplemental Static and Dynamic 68Ga-FAPI-46 PET for Primary 18F-FDG-Negative Pulmonary Lesions.

PET using 68Ga-labeled fibroblast activation protein (FAP) inhibitors (FAPIs) holds high potential for diagnostic imaging of various malignancies, including lung cancer (LC). However, 18F-FDG PET is still the clinical gold standard for LC imaging. Several subtypes of LC, especially lepidic LC, are frequently 18F-FDG PET-negative, which markedly hampers the assessment of single pulmonary lesions suggestive of LC. Here, we evaluated the diagnostic potential of static and dynamic 68Ga-FAPI-46 PET in the 18F-FDG-negative pulmonary lesions of 19 patients who underwent surgery or biopsy for histologic diagnosis after PET imaging. For target validation, FAP expression in lepidic LC was confirmed by FAP immunohistochemistry. Methods: Hematoxylin and eosin staining and FAP immunohistochemistry of 24 tissue sections of lepidic LC from the local tissue bank were performed and analyzed visually. Clinically, 19 patients underwent static and dynamic 68Ga-FAPI-46 PET in addition to 18F-FDG PET based on individual clinical indications. Static PET data of both examinations were analyzed by determining SUVmax, SUVmean, and tumor-to-background ratio (TBR) against the blood pool, as well as relative parameters (68Ga-FAPI-46 in relation to18F-FDG), of histologically confirmed LC and benign lesions. Time-activity curves and dynamic parameters (time to peak, slope, k 1, k 2, k 3, and k 4) were extracted from dynamic 68Ga-FAPI-46 PET data. The sensitivity and specificity of all parameters were analyzed by calculating receiver-operating-characteristic curves. Results: FAP immunohistochemistry confirmed the presence of strongly FAP-positive cancer-associated fibroblasts in lepidic LC. LC showed markedly elevated 68Ga-FAPI-46 uptake, higher TBRs, and higher 68Ga-FAPI-46-to-18F-FDG ratios for all parameters than did benign pulmonary lesions. Dynamic imaging analysis revealed differential time-activity curves for LC and benign pulmonary lesions: initially increasing time-activity curves with a decent slope were typical of LC, and steadily decreasing time-activity curve indicated benign pulmonary lesions, as was reflected by a significantly increased time to peak and significantly smaller absolute values of the slope for LC. Relative 68Ga-FAPI-46-to-18F-FDG ratios regarding SUVmax and TBR showed the highest sensitivity and specificity for the discrimination of LC from benign pulmonary lesions. Conclusion: 68Ga-FAPI-46 PET is a powerful new tool for the assessment of single 18F-FDG-negative pulmonary lesions and may optimize patient stratification in this clinical setting.

Targeting CXCR4/CXCL12 axis via [177Lu]Lu-DOTAGA.(SA.FAPi)2 with CXCR4 antagonist in triple-negative breast cancer

PurposeRadiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC).MethodsPublic database was first interrogated to reveal the correlation between CAFs’ scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen.ResultsCAFs’ scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12.ConclusionThe combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.

Cancer-associated fibroblasts expressing fibroblast activation protein and podoplanin in non-small cell lung cancer predict poor clinical outcome.

Cancer-associated fibroblasts (CAFs) are a dominant cell type in the stroma of non-small cell lung cancer (NSCLC). Fibroblast heterogeneity reflects subpopulations of CAFs, which can influence prognosis and treatment efficacy. We describe the subtypes of CAFs in NSCLC. Primary human NSCLC resections were assessed by flow cytometry and multiplex immunofluorescence for markers of fibroblast activation which allowed identification of CAF subsets. Survival data were analysed for our NSCLC cohort consisting of 163 patients to understand prognostic significance of CAF subsets. We identified five CAF populations, termed CAF S1-S5. CAF-S5 represents a previously undescribed population, and express FAP and PDPN but lack the myofibroblast marker αSMA, whereas CAF-S1 populations express all three. CAF-S5 are spatially further from tumour regions then CAF-S1 and scRNA data demonstrate an inflammatory phenotype. The presence of CAF-S1 or CAF-S5 is correlated to worse survival outcome in NSCLC, despite curative resection, highlighting the prognostic importance of CAF subtypes in NSCLC. TCGA data suggest the predominance of CAF-S5 has a poor prognosis across several cancer types. This study describes the fibroblast heterogeneity in NSCLC and the prognostic importance of the novel CAF-S5 subset where its presence correlates to worse survival outcome.