(1) Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally. Cancer-associated fibroblasts (CAFs) are major components of CRC\'s tumour microenvironment (TME), but their biological background and interplay with the TME remain poorly understood. This study investigates CAF biology and its impact on CRC progression. (2) The cohort comprises 155 cases, including CRC, with diverse localizations, adenomas, inflammations, and controls. Digital gene expression analysis examines genes associated with signalling pathways (MAPK, PI3K/Akt, TGF-β, WNT, p53), while next-generation sequencing (NGS) determines CRC mutational profiles. Immunohistochemical FAP scoring assesses CAF density and activity. (3) FAP expression is found in 81 of 150 samples, prevalent in CRC (98.4%), adenomas (27.5%), and inflammatory disease (38.9%). Several key genes show significant associations with FAP-positive fibroblasts. Gene set enrichment analysis (GSEA) highlights PI3K and MAPK pathway enrichment alongside the activation of immune response pathways like natural killer (NK)-cell-mediated cytotoxicity via CAFs. (4) The findings suggest an interplay between CAFs and cancer cells, influencing growth, invasiveness, angiogenesis, and immunogenicity. Notably, TGF-β, CDKs, and the Wnt pathway are affected. In conclusion, CAFs play a significant role in CRC and impact the TME throughout development.

Cell sheet (CS) engineering using mesenchymal stromal cells (MSC) draws significant interest for regenerative medicine and this approach translates to clinical use for numerous indications. However, little is known of factors that define the timing of CS assembly from primary cultures. This aspect is important for planning CS delivery in autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors\' (n = 14) adipose-derived MSCs and evaluated the impact of healthy subject\'s sex, MSC culture features (population doubling time and lag-phase), and extracellular matrix (ECM) composition along with factors related to connective tissue formations (α-SMA and FAP-α) on CS assembly duration. Using qualitative and quantitative analysis methods, we found that, in seeded MSCs, high contents of collagen I and collagen IV had a direct correlation with longer CS assembly duration. We found that short lag-phase cultures faster turned to a ready-to-use CS, while age, sex, fibronectin, laminin, α-SMA, and FAP-α failed to provide a significant correlation with the timing of assembly. In detachable CSs, FAP-α was negatively correlated with the duration of assembly, suggesting that its concentration rose over time and contributed to MSC activation, transitioning to α-SMA-positive myofibroblasts and ECM turnover. Preliminary data on cell density and collagen I deposition suggested that the TGF-β1 signaling axis is of pivotal importance for ECM composition and construct maturation.

UNASSIGNED: The purpose of this study is to compare the application value of 68Ga-FAPI and 18F-FDG PET/CT in primary and metastatic lesions of abdominal and pelvic malignancies (APMs).
UNASSIGNED: The search, limited to the earliest available date of indexing through 31 July 2022, was performed on PubMed, Embase, and Cochrane Library databases using a data-specific Boolean logic search strategy. We calculated the detection rate (DR) of 68Ga-FAPI and 18F-FDG PET/CT in the primary staging and recurrence of APMs, and pooled sensitivities/specificities based on lymph nodes or distant metastases.
UNASSIGNED: We analyzed 473 patients and 2775 lesions in the 13 studies. The DRs of 68Ga-FAPI and 18F-FDG PET/CT in evaluating the primary staging and recurrence of APMs were 0.98 (95% CI: 0.95-1.00), 0.76 (95% CI: 0.63-0.87), and 0.91(95% CI: 0.61-1.00), 0.56 (95% CI: 0.44-0.68), respectively. The DRs of 68Ga-FAPI and 18F-FDG PET/CT in primary gastric cancer and liver cancer were 0.99 (95% CI: 0.96-1.00), 0.97 (95% CI: 0.89-1.00) and 0.82 (95% CI: 0.59-0.97), 0.80 (95% CI: 0.52-0.98), respectively. The pooled sensitivities of 68Ga-FAPI and 18F-FDG PET/CT in lymph nodes or distant metastases were 0.717(95% CI: 0.698-0.735) and 0.525(95% CI: 0.505-0.546), and the pooled specificities were 0.891 (95% CI: 0.858-0.918) and 0.821(95% CI: 0.786-0.853), respectively.
UNASSIGNED: This meta-analysis concluded that 68Ga-FAPI and 18F-FDG PET/CT had a high overall diagnostic performance in detecting the primary staging and lymph nodes or distant metastases of APMs, but the detection ability of 68Ga-FAPI was significantly higher than that of 18F-FDG. However, the ability of 68Ga-FAPI to diagnose lymph node metastasis is not very satisfactory, and is significantly lower than that of distant metastasis.
UNASSIGNED: https://www.crd.york.ac.uk/prospero/, identifier CRD42022332700.

BACKGROUND: Cancer-associated fibroblasts are found in the stroma of epithelial tumors. They are characterized by overexpression of the fibroblast activation protein (FAP), a serine protease which was already proven as attractive target for chelator-based theranostics. Unfortunately, the value of gallium-68 labeled tracers is limited by their batch size and the short nuclide half-life. To overcome this drawback, radiolabeling with aluminum fluoride complexes and 6-fluoronicotinamide derivatives of the longer-lived nuclide fluorine-18 was established. The novel compounds were tested for their FAP-specific binding affinity. Uptake and binding competition were studied in vitro using FAP expressing HT-1080 cells. HEK cells transfected with the closely related dipeptidyl peptidase-4 (HEK-CD26) were used as negative control. Small animal positron emission tomography imaging and biodistribution experiments were performed in HT-1080-FAP xenografted nude mice. [18F]AlF-FAPI-74 was selected for PET/CT imaging in a non-small cell lung cancer (NSCLC) patient.
RESULTS: In vitro, 18F-labeled FAPI-derivatives demonstrated high affinity (EC50 = < 1 nm to 4.2 nm) and binding of up to 80% to the FAP-expressing HT1080 cells while no binding to HEK-CD26 cells was observed. While small animal PET imaging revealed unfavorable biliary excretion of most of the 18F-labeled compounds, the NOTA bearing compounds [18F]AlF-FAPI-74 and -75 achieved good tumor-to-background ratios, as a result of their preferred renal excretion. These two compounds showed the highest tumor accumulation in PET imaging. The organ distribution values of [18F]AlF-FAPI-74 were in accordance with the small animal PET imaging results. Due to its less complex synthesis, fast clearance and low background values, [18F]AlF-FAPI-74 was chosen for clinical imaging. PET/CT of a patient with metastasized non-small cell lung cancer (NSCLC), enabled visualization of the primary tumor and its metastases at the hepatic portal and in several bones. This was accompanied by a rapid clearance from the blood pool and low background in healthy organs.
CONCLUSIONS: [18F]AlF-labeled FAPI derivatives represent powerful tracers for PET. Owing to an excellent performance in PET imaging, FAPI-74 can be regarded as a promising precursor for [18F]AlF-based FAP-imaging.

This study aimed to compare the performance of 68Ga-labelled fibroblast activating protein inhibitor (FAPI) PET and 18F-FDG PET for imaging of hepatic tumours.
We prospectively assessed 20 patients with suspected intrahepatic lesions. Tumour radiological features, pathology, or follow-up examinations were assessed as ground truth in correlation with PET scans. Semiquantitative analysis was additionally performed by measuring the standardised uptake value (SUV). Tumour-to-liver background ratios (TBR) were calculated and compared between 68Ga-FAPI PET and 18F-FDG PET. FAPI expression was assessed by immunochemistry in samples obtained from 7 patients with hepatocellular carcinomas (HCC)/intrahepatic cholangiocarcinoma (ICC) or granulomas.
Primary intrahepatic tumours, including 16 HCC in 14 patients and 4 ICC in 3 patients with extrahepatic metastases, were determined by histology (n = 14) and clinical examinations (n = 3). Based on visual analysis, 17 patients presented elevated 68Ga-FAPI uptake (sensitivity: 100%, specificity: 100%), while 7 patients presented 18F-FDG avid tumours (sensitivity: 58.8%, specificity: 100%). 68Ga-FAPI PET/CT identified 17 extrahepatic metastases vs. 13 in 18F-FDG PET/CT in 2 ICC patients. Three benign liver nodules in three patients showed negligible uptake in dual-PET scans. The SUVmax_HCC = 8.47 ± 4.06 and TBRmax_HCC = 7.13 ± 5.52, and SUVmax_ICC = 14.14 ± 2.20 TBRmax_ICC = 26.46 ± 4.94 in 68Ga-FAPI-04 PET/CT were significantly higher than the 18F-FDG uptake presenting SUVmax_HCC = 4.86 ± 3.58 and TBRmax_HCC = 2.39 ± 2.21, and SUVmax_ICC = 9.19 ± 3.60 and TBRmax_ICC = 2.39 ± 2.21 (all p values < 0.05). ICC patients showed higher levels of FAPI uptake in the primary hepatic lesions compared to extrahepatic metastases, TBRmax_ICC = 15.18 ± 5.80 (p = 0.04).
68Ga-FAPI PET-CT has superior potential in the detection of primary hepatic malignancy compared to 18F-FDG.

68Ga-FAPI-04 is a rapidly evolving PET tracer for whole-body imaging in a variety of cancers. We aimed to evaluate the diagnostic performance of 68Ga-FAPI-04 for detecting and characterizing hepatic nodules in patients with suspected carcinoma.
Twenty-five patients showing suspicious hepatic lesions for malignancy underwent 68Ga-FAPI-04 PET. The maximum and mean standardised uptake values (SUVmax, SUVmean) were measured for all detected lesions and normal hepatic tissues, respectively. The target-to-background ratio (TBR) was calculated by dividing the lesion SUVmax with the SUVmean of non-tumour liver tissue. Lesion uptake value was correlated with the in vitro hepatic FAP expression determined by immunohistochemistry (IHC).
In total, 17 patients who underwent surgery or biopsy were recruited for the final analysis. A total of 28 intrahepatic malignant lesions were detected in 16 patients; the mean SUVmax was 8.36 ± 4.21 (range 2.21 to 15.86), and mean TBR was 13.15 ± 9.48 (range 2.79 to 38.12) in all detected lesions (n = 28). One benign patient showed negligible hepatic uptake (SUVmax = 0.47), whereas 75% of the primary intrahepatic hepatocellular carcinoma (HCC) lesions (n = 6) showed prominent FAP expression, 12.5% of the lesions (n = 1) showed moderate expression in stromal cells, and one showed negligible expression.
68Ga-FAPI-04 showed high sensitivity in detecting hepatic malignancies, particularly in poorly differentiated forms with concordantly elevated FAP expression.

Most epithelial tumors recruit fibroblasts and other nonmalignant cells and activate them into cancer-associated fibroblasts. This often leads to overexpression of the membrane serine protease fibroblast-activating protein (FAP). It has already been shown that DOTA-bearing FAP inhibitors (FAPIs) generate high-contrast images with PET/CT scans. Since SPECT is a lower-cost and more widely available alternative to PET, 99mTc-labeled FAPIs represent attractive tracers for imaging applications in a larger number of patients. Furthermore, the chemically homologous nuclide 188Re is available from generators, which allows FAP-targeted endoradiotherapy. Methods: For the preparation of 99mTc-tricarbonyl complexes, a chelator was selected whose carboxylic acids can easily be converted into various derivatives in the finished product, enabling a platform strategy based on the original tracer. The obtained 99mTc complexes were investigated in vitro by binding and competition experiments on FAP-transfected HT-1080 (HT-1080-FAP) or on mouse FAP-expressing (HEK-muFAP) and CD26-expressing (HEKCD26) HEK cells and characterized by planar scintigraphy and organ distribution studies in tumor-bearing mice. Furthermore, a first-in-humans application was done on 2 patients with ovarian and pancreatic cancer, respectively. Results: 99mTc-FAPI-19 showed specific binding to recombinant FAP-expressing cells with high affinity. Unfortunately, liver accumulation, biliary excretion, and no tumor uptake were observed on planar scintigraphy for a HT-1080-FAP-xenotransplanted mouse. To improve the pharmacokinetic properties, hydrophilic amino acids were attached to the chelator moiety of the compound. The resulting 99mTc-labeled FAPI tracers revealed excellent binding properties (≤45% binding; >95% internalization), high affinity (half-maximal inhibitory concentration, 6.4-12.7 nM), and significant tumor uptake (≤5.4% injected dose per gram of tissue) in biodistribution studies. The lead candidate 99mTc-FAPI-34 was applied for diagnostic scintigraphy and SPECT of patients with metastasized ovarian and pancreatic cancer for follow-up to therapy with 90Y-FAPI-46. 99mTc-FAPI-34 accumulated in the tumor lesions, as also shown on PET/CT imaging using 68Ga-FAPI-46. Conclusion: 99mTc-FAPI-34 represents a powerful tracer for diagnostic scintigraphy, especially when PET imaging is not available. Additionally, the chelator used in this compound allows labeling with the therapeutic nuclide 188Re, which is planned for the near future.

Fibroblast activating protein (FAP) is a membrane serine protease expressed by activated fibroblasts, particularly tumour associated fibroblasts (TAFs). FAP expression has not been reported in canine mast cell tumours (MCTs). The objective of this study was to evaluate the expression of FAP in TAFs and its correlation with histological grade, mitotic index and Ki67 expression in canine MCTs. FAP expression was evaluated by immunohistochemistry (IHC) in 30 canine MCTs. Twenty-eight (90%) of the MCTs expressed FAP in the stroma, 16 cases showed low to intermediate FAP score and 14 cases had a high FAP score. FAP was correlated positively with both Patnaik (P = 0.007) and Kiupel (P = 0.008) grading systems, mitotic index (P = 0.0008) and Ki67 expression (P = 0.009). High stromal FAP expression could be a potential negative prognostic factor in canine MCTs.

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