Targeting FAPα-positive lymph node metastatic tumor cells suppresses colorectal cancer metastasis

Lymphatic metastasis is the main metastatic route for colorectal cancer, which increases the risk of cancer recurrence and distant metastasis. The properties of the lymph node metastatic colorectal cancer (LNM-CRC) cells are poorly understood, and effective therapies are still lacking. Here, we found that hypoxia-induced fibroblast activation protein alpha (FAPα) expression in LNM-CRC cells. Gain- or loss-function experiments demonstrated that FAPα enhanced tumor cell migration, invasion, epithelial-mesenchymal transition, stemness, and lymphangiogenesis via activation of the STAT3 pathway. In addition, FAPα in tumor cells induced extracellular matrix remodeling and established an immunosuppressive environment via recruiting regulatory T cells, to promote colorectal cancer lymph node metastasis (CRCLNM). Z-GP-DAVLBH, a FAPα-activated prodrug, inhibited CRCLNM by targeting FAPα-positive LNM-CRC cells. Our study highlights the role of FAPα in tumor cells in CRCLNM and provides a potential therapeutic target and promising strategy for CRCLNM.

Targeting fibroblast activation protein inhibits endothelial-mesenchymal transition by affecting cancer-associated fibroblasts derived exosomes / 中国药理学通报

Aim To investigate whether targeted inhibition of fibroblast activation protein (FAP) can inhibit the endothelial-to-mesenchymal transition (EndMT) of vascular endothelial cells by affecting exosomes (Exo) of cancer-associated fibroblasts (CAFs) and explore the underlying mechanisms. Methods Primary CAFs and peri-tumor fibroblasts (PTFs) were obtained from lung cancer and peri-cancer tissues, and CAFs-exo and PTFs-exo were collected from culture medium, respectively. Exosomes from CAFs treated with specific FAP inhibitor (3.3 nmol • L-

Causes of False-Positive Results in 68Ga-Labeled Fibroblast Activation Protein Inhibitor PET/CT Imaging / 中国医学科学院学报

Objective To investigate the causes of false-positive results in the 68Ga-labeled fibroblast activation protein inhibitor (68Ga-FAPI-04) PET/CT imaging. Methods The imaging data of 547 patients undergoing 68Ga-FAPI-04 PET/CT examination in the Department of Nuclear Medicine of the Affiliated Hospital of Southwest Medical University from September 2020 to May 2021 were retrospectively collected.Two experienced nuclear medicine diagnostic physicians analyzed the clinical data,relevant imaging examinations,laboratory examinations,pathological results and follow-up results of the patients with false-positive results. Results The 68Ga-FAPI-04 PET/CT imaging of 547 patients showed false-positive results in 99 (18.1%) patients,including 56 males and 43 females.The postoperative pathological examination confirmed false-positive results in 13 patients,including 1 patient of thyroiditis,2 patients of pulmonary tuberculosis,1 patient of bone tuberculosis,2 patients of pulmonary inflammatory pseudotumor,1 patient of pulmonary sarcoidosis,1 patient of pulmonary benign fibroma,1 patient of organic pneumonia,2 patients of renal angiomyolipoma,1 patient of mass pancreatitis,and 1 patient of pancreatic mucinous cystadenoma.The medical history,relevant imaging examination,and long-term follow-up confirmed false-positive results in 86 patients.Specifically,the false-positive uptake in the neck,chest,abdomen,bone joint,and skin occurred in 8 (9.3%),13 (15.1%),5 (5.8%),57 (66.3%),and 3 (3.5%) patients,respectively.Inflammation-related uptake appeared in 83 (83.8%) patients with false-positive imaging results,of which arthritis (23 patients) and osteophyte (29 patients) were the most common.Sixteen (16.2%) patients showed the false-positive uptake related to fibroblasts. Conclusion 68Ga-FAPI-04 PET/CT imaging will show non-malignant tumor false-positive results,which are mainly associated with inflammation and fibroblasts.

The FAP α -activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway

Osteosarcoma is a kind of bone tumor with highly proliferative and invasive properties, a high incidence of pulmonary metastasis and a poor prognosis. Chemotherapy is the mainstay of treatment for osteosarcoma. Currently, there are no molecular targeted drugs approved for osteosarcoma treatment, particularly effective drugs for osteosarcoma with pulmonary metastases. It has been reported that fibroblast activation protein alpha (FAPα) is upregulated in osteosarcoma and critically associated with osteosarcoma progression and metastasis, demonstrating that FAPα-targeted agents might be a promising therapeutic strategy for osteosarcoma. In the present study, we reported that the FAPα-activated vinblastine prodrug Z-GP-DAVLBH exhibited potent antitumor activities against FAPα-positive osteosarcoma cells in vitro and in vivo. Z-GP-DAVLBH inhibited the growth and induced the apoptosis of osteosarcoma cells. Importantly, it also decreased the migration and invasion capacities and reversed epithelial-mesenchymal transition (EMT) of osteosarcoma cells in vitro and suppressed pulmonary metastasis of osteosarcoma xenografts in vivo. Mechanistically, Z-GP-DAVLBH suppressed the AXL/AKT/GSK-3β/β-catenin pathway, leading to inhibition of the growth and metastatic spread of osteosarcoma cells. These findings demonstrate that Z-GP-DAVLBH is a promising agent for the treatment of FAPα-positive osteosarcoma, particularly osteosarcoma with pulmonary metastases.

Preclinical evaluation and pilot clinical study of 18FAlF-labeled FAPI-tracer for PET imaging of cancer associated fibroblasts

In recent years, fibroblast activation protein (FAP) has emerged as an attractive target for the diagnosis and radiotherapy of cancers using FAP-specific radioligands. Herein, we aimed to design a novel 18F-labeled FAP tracer ([18F]AlF-P-FAPI) for FAP imaging and evaluated its potential for clinical application. The [18F]AlF-P-FAPI novel tracer was prepared in an automated manner within 42 min with a non-decay corrected radiochemical yield of 32 ± 6% (n = 8). Among A549-FAP cells, [18F]AlF-P-FAPI demonstrated specific uptake, rapid internalization, and low cellular efflux. Compared to the patent tracer [18F]FAPI-42, [18F]AlF-P-FAPI exhibited lower levels of cellular efflux in the A549-FAP cells and higher stability in vivo. Micro-PET imaging in the A549-FAP tumor model indicated higher specific tumor uptake of [18F]AlF-P-FAPI (7.0 ± 1.0% ID/g) compared to patent tracers [18F]FAPI-42 (3.2 ± 0.6% ID/g) and [68Ga]Ga-FAPI-04 (2.7 ± 0.5% ID/g). Furthermore, in an initial diagnostic application in a patient with nasopharyngeal cancer, [18F]AlF-P-FAPI and [18F]FDG PET/CT showed comparable results for both primary tumors and lymph node metastases. These results suggest that [18F]AlF-P-FAPI can be conveniently prepared, with promising characteristics in the preclinical evaluation. The feasibility of FAP imaging was demonstrated using PET studies.

The potential role of long non-coding RNA Dnm3os in the activation of cardiac fibroblasts / 生物医学工程学杂志

Long non-coding RNA (lncRNA) Dnm3os plays a critical role in peritendinous fibrosis and pulmonary fibrosis, but its role in the process of cardiac fibrosis is still unclear. Therefore, we carried out study by using the myocardial fibrotic tissues obtained by thoracic aortic constriction (TAC) in an early study of our group, and the

SP13786 Inhibits the Migration and Invasion of Lung Adenocarcinoma Cell A549 by Supressing Stat3-EMT via CAFs Exosomes / 中国肺癌杂志

BACKGROUND@#Fibroblast activation protein (FAP) is one of the surface markers of cancer-associated fibroblasts (CAFs) and is closely related to the malignant characterization of CAFs. SP13786 is a specific micromolecule inhibitor of FAP and this study is to investigate the effects and mechanism of SP13786 on the migration and invasion of A549 cells through regulating exosomes of CAFs.@*METHODS@#CAFs and paracancerous fibroblasts (PTFs) were isolated and subcultured from freshly resected lung adenocarcinoma tissues and paracancerous normal tissues separately. MTT assay was used to detect the proliferation of CAFs incubated by different concentrations of SP13786; PTFs-exo, CAFs-exo and CAFs+SP13786-exo were extracted by polymer precipitation method. The A549 cells were divided into Ctrl group, PTFs group, CAFs group and SP13786 group and each group was incubated with DMEM, PTFs-exo, CAFs-exo and CAFs+SP13786-exo separately. Laser confocal microscope was used to observe the endocytoses of exosomes by A549 cells. The expression of alpha-smooth muscle actin (α-SMA) and FAP in PTFs and CAFs and the expression of E-cadherin, N-cadherin, Slug, Stat3 and P-Stat3 in A549 cells were detected by immunofluorescence, immunohistochemistry and Western blot. The migration and invasion ability of A549 cells were detected by cell scratch and transwell methods.@*RESULTS@#α-SMA and FAP were expressed much higher in CAFs than that in PTFs which indicate that CAFs and PTFs were successfully obtained from lung adenocarcinoma and paracancerous tissues (P0.05). Finally, WP1066 (a specific inhibitor of Stat3) was used to comfirm whether SP13786 could influence EMT of A549 cells by inhibiting Stat3 phosphorylation via CAFs-Exo. The results showed that when the phosphorylation of Stat3 in CAFs group was inhibited by WP1066, SP13786 could not influence the P-Stat3 expression and EMT of A549 cells anymore (P>0.05).@*CONCLUSIONS@#As a specific micromolecule inhibitor of FAP, SP13786 indirectly inhibits the migration and invasion of A549 cells by affecting exosomes of CAFs. The possible mechanism is to inhibit the phosphorylation of Stat3 and thus affect the EMT of A549 cells.

Expression of fibroblast activating protein in eyelid basal cell carcinoma associated fibroblasts / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the role and significance of carcinoma-associated fibroblasts(CAFs)in the occurrence and development of eyelid basal cell carcinoma(BCC), and study on the biological characteristics and the expression of fibroblast activation protein(FAP)of two kinds of fibroblasts associated with eyelid BCC and normal eyelid skin fibroblasts(normal fibroblasts, NFs).<p>METHODS: CAFs and NFs were obtained by tissue primary cultured. The cell morphology of the 3^rd generation purified cells were observed under an inverted phase contrast microscope and identified them using their biomarker by immunohistochemistry for CK, VIM, α-SMA and FAP. Cell growth and proliferation were measured by MTT assay. The expression of FAP mRNA and protein in cells was detected by RT-qPCR and Western Blot.<p>RESULTS: The CAFs of the eyelid was long fusiform or spindle-shaped, with reduced cytoplasmic processes, more cytoplasmic granules, different cell sizes, disordered arrangement, overlapping growth, and loss of contact inhibition. NFs were in the form of extensive shuttle, radial arrangement, the cytoplasmic particles were rare, there was no overlapping growth phenomenon, and no contact inhibition. The proliferation rate of eyelid CAFs was faster than that of NFs. And the expression of FAP mRNA in CAFs was 3.672±0.221, which was significantly higher than that in NFs(1.034±0.024)(<i>P</i><0.05). In addition, the expression level of FAP protein in CAFs was high, while NFs were not expressed(<i>P</i>< 0.05).<p>CONCLUSION: There were significant differences in the biological characteristics of CAFs and NFs, such as morphological structure, growth and the proliferation, growth factor expression and so on. It was therefore suggested that the tumor microenvironment of eyelid basal cell carcinoma had changed, and further induced the biological characteristics and function of NFs, and finally transformed into CAFs. In addition, eyelid CAFs expressed higher expression of FAP than NFs, indicating that FAP may be involved in the occurrence and development of tumor cells in tumor microenvironment, which is associated with the invasive growth of CAFs in eyelid.

The potential role of calnexin in the activation of cardiac fibroblasts / 生物医学工程学杂志

Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca homeostasis, and Ca signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts , and the cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

The cell cycle inhibitor p21 promote mouse lung fibrosis by activating alveolar macrophages / 药学学报

This study was to determine the expression of the cell cycle inhibitor p21 in alveolar macrophages (AMs) and the role of p21 in activation of AMs in bleomycin (BLM) injury-induced lung fibrosis. The expression of CD206 in AMs was measured by immunofluorescence staining. Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect the expression of macrophage activation markers. The coculture assay for macrophage and fibroblast was employed to explore the effect of macrophage on fibroblast activation. Immunofluorescence staining and western blotting assay were adopted to detect the expression of p21 in fibrotic tissues. AMs were treated with p21 knockdown or overexpression virus, RT-PCR and the co-culture system were used to explore the effect of p21 expression on macrophage activation. The Experimental Animal Welfare Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College approved all of the protocols for this research. Our results showed that the expression of CD206 and macrophage activation markers was increased in AMs from fibrotic mice, indicating that AMs from fibrotic mice were associated with a profibrotic phenotype. Moreover, the expression of p21 was upregulated in AMs after BLM treatment. Depletion of p21 suppressed macrophage activation, while overexpression of p21 promoted the profibrotic phenotype of AMs from healthy mice. In summary, BLM injury causes the progressive accumulation of p21 in AMs, which induces the production of a number of profibrotic factors promoting the development of pulmonary fibrosis.

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective To compare the protein profile of culture supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status. Methods Dermal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h. MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern. Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8%–16%. Results The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78.8 kDa. These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells. Moreover, we found another seven small size (10–19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/Ionomycine stimulated fibroblasts. Most of these proteins expression were down regulated following fibroblast activation. This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death. Conclusions Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation. Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

SACC-83-derived exosomes induce fibroblast activation protein expression in normal salivary gland stromal fibroblasts / 实用口腔医学杂志

Objective:To study the effects of exosomes (EXO) released by adenoid cystic carcinoma SACC-83 cells on the expression of fibroblast activation protein (FAP) in normal human salivary gland stromal fibroblasts (hSGSFs).Methods:ACC exosomes were isolated from SACC-83 cell culture supernatant by using Total Exosome Isolation Reagent.The whole-mount EXO were characterized and assessed by transmission electron microscope and Western Blot.The exosomes were labeled with green fluorescent dye PKH67 and co-cultured with hSGSFs for 48h,followed by staining with Alexa Fluor 594 Phalloidin and DAPI.Mterwards,exsosomes uptake was observed under a laser scanning confocal microscope.After a 48-hour co-culture of SACC-83 exosomes with hSGSFs,the expression of FAP in SACC-83-EXO-treated hSGSFs was investigated by qRT-PCR and Western Blot.Results:The vesicles isolated from SACC-83 cell culture supernatant had the reported size range of 30-100 nm,expressed the exosomal marker CD63 and TSG101.Mter co-culture of hSGSFs with PKH67 labeled SACC-83 exosomes,exosomes were taken up by hSGSFs and FAP expression was elevated in hSGSFs.Conclusion:Exosomes derived from SACC-83 cells can be taken up by hSGSFs and can induce the expression of FAP in hSGSFs.These results suggest that exosomes derived from SACC-83 cells might induce the transformation of normal salivary gland strormal fibroblasts to cancer associated fibroblasts.

Construction of a eukaryotic expression vector of fibroblast activation protein and establishment of its stable over-expression in the oral squamous cell carcinoma / 华西口腔医学杂志

<p><b>OBJECTIVE</b>This study aimed at constructing fibroblast activation protein (FAP) over-expression lentivinus vectors to investigate transfection in SCC9 cell lines and establish a stable FAP over-expression oral squamous cell line.</p><p><b>METHODS</b>The cDNA of FAP gene from an oral squamous cell carcinoma (OSCC) tissue was amplified by polymerase chain reaction (PCR) and subcloned into eukaryotic expression vector pCDH-CMV-MCS-EF1-copGFP. The recombinant plasmid was sequenced and then transfected into an SCC9 cell line. Subsequently, the SCC9 cell line that over-expressed FAP stably was established by fluorescence activated cell sorting (FACS). The expression of green fluorescent protein (GFP) was detected with fluorescence microscopy, and the over-expression of FAP was identified by real-time PCR and Western blot.</p><p><b>RESULTS</b>The FAP gene was amplified by PCR and then cloned into the vector, whose sequence was identical to that in the GenBank. GFP was expressed in the transfected cells. Furthermore, FAP over-expression in the transfected cells was detected by real-time PCR and Western blot.</p><p><b>CONCLUSIONS</b>The recombinant eukaryotic expression vector pCDH-FAP was constructed successfully. This result provides a foundation for further studies on the function of FAP in vitro.</p>

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective:To compare the protein profile of culture supematants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dcrmal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8％-16％.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78,8 kDa.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size (10-19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/lonomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.ConcLusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

Correlations of apparent diffusion coefficient with fibrosis and fibroblast activationprotein scores in pancreatic cancer / 实用放射学杂志

Objective To investigate the correlations of apparent diffusion coefficient (ADC) with fibrosis and fibroblast activation protein (FAP) score in pancreatic cancer .Methods Eighteen patients with pathologically confirmed pancreatic cancer were per‐formed conventional MR imaging ,DWI examinations .ADCs were measured with region of interest method on a “Single slice” .The wax blocks of 18 patients with pancreatic cancer were received Masson staining and FAP immunohistochemical staining .The correla‐tions of ADC with levels of fibrosis and FAP scores of pancreatic cancer were assessed by Pearson correlation analysis .Results The mild negative correlation between ADC value of cancerous foci and fibrosis was not significant (r= -0 .459 ,P=0 .056) .Significant negative correlation was found between ADC values of moderate and high differentiation cancerous foci and fibrosis (r= -0 .564 ,P=0 .044) .Significant negative correlation was found between ADC value and FAP score (r= -0 .497 , P=0 .036) .Conclusion The negative correlations are found between ADC and fibrosis ,FAP score of pancreatic cancer .DWI will be helpful to infer the pathologi‐cal characteristics .

Effect of fibroblast activation protein expression silencing of mouse fibroblast cells on the proliferation of muse pancreatic cancer cells / 中华胰腺病杂志

ObjectiveSmall interfering RNA (siRNA) was used to silence the fibroblast activation protein4 (FAP) expression of mouse pancreatic cancer related fibroblast cells (mPCa-FCs-1212),and to observe the effects of mPCa-FCs-1212 silencing FAP gene on mouse pancreatic cancer cells (mPCa-1212) proliferation and apoptosis.MethodsThe small interfering RNA targeting FAP gene was designed; the recombinant siRNA plasmid siFAP and control plasmid siMOCK was constructed,which were transfected into mPCa-FCs-1212,respectively.The FAP mRNA and protein expression in transfected cells were examined by real-time PCR and Western blotting.The mPCa-1212 and transfected mPCa-FCs-1212 were co-cultured with a 1:1 ratio in vitro.The growth inhibitory rates and apoptosis rates of mPCa-1212 were detected by MTT assay and Annexin V-FTTC/PI staining and FCM assay.ResultsThe mRNA and protein expressions of FAP in siFAP transfected mPCa-FCs-1212 were significantly down-regulated when compared with that in siMOCK transfected mPCa-FCs-1212[0.584 ±0.029vs.1.052±0.281,P=0.0213; (27.18±3.23)％ vs.(61.58±4.72)％,P=0.0317].The mPCa-1212 was co-cultured with the mPCa-FCs-1212 transfected with siFAP or siMOCK for 3 d,and the inhibitory rates of mPCa-1212 were (23.02 ±3.32)％ and (1.11 ±0.23)％,and the apoptosis rates were (42.31 ±5.34)％ and (7.38 ± 2.09)％,the difference between the two groups was statistically significant (P =0.000).ConclusionsmPCa-FCs-1212 silencing FAP gene can inhibit the proliferation of mPCa-1212 in vitro and induce cell apoptosis,and may be a potential new approach to gene therapy.