Fibroblast Activation Protein Expression Research Articles

Impact of 68Ga-FAPI PET/CT Imaging on the Therapeutic Management of Primary and Recurrent Pancreatic Ductal Adenocarcinomas.

Pancreatic ductal carcinoma (PDAC) is a highly lethal cancer, and early detection and accurate staging are critical to prolonging survival. PDAC typically has a prominent stroma including cancer-associated fibroblasts that express fibroblast activation protein (FAP). FAP is a new target molecule for PET imaging of various tumors. In this retrospective study, we describe the clinical impact of PET/CT imaging using 68Ga-labeled FAP-inhibitors (68Ga-FAPI PET/CT) in 19 patients with PDAC (7 primary, 12 progressive/recurrent). Methods: All patients underwent contrast-enhanced CT (ceCT) for TNM staging before 68Ga-FAPI PET/CT imaging. PET scans were acquired 60 min after administration of 150-250 MBq of 68Ga-labeled FAP-specific tracers. To characterize 68Ga-FAPI uptake over time, additional scans after 10 or 180 min were acquired in 6 patients. SUVmax and SUVmean values of PDAC manifestations and healthy organs were analyzed. The tumor burden according to 68Ga-FAPI PET/CT was compared with TNM staging based on ceCT and changes in oncologic management were recorded. Results: Compared with ceCT, 68Ga-FAPI PET/CT results led to changes in TNM staging in 10 of 19 patients. Eight of 12 patients with recurrent/progressive disease were upstaged, 1 was downstaged, and 3 had no change. In newly diagnosed PDAC, 1 of 7 patients was upstaged, and the staging of 6 patients did not change. Changes in oncologic management occurred in 7 patients. Markedly elevated uptake of 68Ga-FAPI in PDAC manifestations after 1 h was seen in most cases. Differentiation from pancreatitis based on static imaging 1 h after injection was challenging. With respect to imaging after multiple time points, PDAC and pancreatitis showed a trend for differential uptake kinetics. Conclusion:68Ga-FAPI PET/CT led to restaging in half of the patients with PDAC and most patients with recurrent disease compared with standard of care imaging. The clinical value of 68Ga-FAPI PET/CT should be further investigated.

Improved cancer detection in Waldeyer\u2019s tonsillar ring by 68Ga-FAPI PET/CT imaging

PurposeIn cancer of unknown primary (CUP), positron emission tomography/computed tomography (PET/CT) with the glucose analog [18F]FDG represents the standard imaging approach for localization of the malignant primary. Frequently, however, [18F]FDG PET/CT cannot precisely distinguish between small occult tumors and chronic inflammation, especially in Waldeyer’s tonsillar ring. To improve the accuracy for detecting primary tumors in the Waldeyer’s tonsillar ring, the novel PET tracer [68Ga]Ga-FAPI-4 for specific imaging of fibroblast activation protein (FAP) expression was used as a more specific target for cancer imaging.MethodsEight patients with suspicion of a malignant tumor in Waldeyer’s tonsillar ring or a CUP syndrome were examined. PET/CT scans with [18F]-FDG and [68Ga]Ga-FAPI-4 were performed for pre-operative tumor localization. After surgical resection, histopathological and immunohistochemical results were compared to PET/CT findings.ResultsHistopathology revealed a palatine or lingual tonsil carcinoma in all patients. In case of lymph node metastases smaller than 7 mm in size, the [18F]FDG PET/CT detection rate of cervical lymph node metastases was higher than that of [68Ga]FAPI PET/CT, while both tracers identified the primary tumors in all eight cases. The size of the primary and the lymph node metastases was directly correlated to the respective FAP expression, as detected by immunohistochemistry. The mean SUVmax for the primary tumors was 21.29 ± 7.97 for 18F-FDG and 16.06 ± 6.29 for 68Ga-FAPI, respectively (p = 0.2). The mean SUVmax for the healthy contralateral tonsils was 8.38 ± 2.45 for [18F]FDG and 3.55 ± 0.47 for [68Ga]FAPI (p < 0.001). The SUVmax ratio of [68Ga]FAPI was significantly different from [18F] FDG (p = 0.03). Mean TBRmax for the [68Ga]Ga-FAPI-4 tracer was markedly higher in comparison to [18F]FDG (10.90 vs. 4.11).ConclusionNon-invasive imaging of FAP expression by [68Ga]FAPI PET/CT resulted in a better visual detection of the malignant primary in CUP, as compared to [18F]FDG imaging. However, the detection rate of lymph node metastases was inferior, presumably due to low FAP expression in small metastases. Nevertheless, by offering a detection method for primary tumors with the potential of lower false positive rates and thus avoiding biopsies, patients with CUP syndrome may benefit from [68Ga]FAPI PET/CT imaging.

Expression of fibroblast-activation protein in keloid tissue and its clinical significance

Expression of fibroblast-activation protein in keloid tissue and its clinical significance

Abstract 4973: Comparison of tumor immune microenvironment in triple negative breast cancer (TNBC) of African American versus that of white patients

Abstract Introduction: Although there are many possible causes, it has been observed that Triple Negative Breast Cancer (TNBC) has a worse overall outcome in African American (AA) than white patients. Recently, there is also evidence that the patients response to the tumor, or the biological response expressed as the tumor immune microenvironment, impacts outcome. The goal of this study was to compare the Tumor Microenvironment (TME) of TNBC in the two mentioned groups in order to seek molecular parameters that are different in TNBC in AA patients as compared to whites. Methods: We selected N=50 AA and N=50 white TNBC samples from the Yale Pathology archives that were matched by diagnosis date. We measured CD45, CD3, CD8, CD20, CD14, CD68, PD-L1, THY1, aSMA and Fibroblast Activation Protein (FAP) expression in both the tumor and stromal compartments in whole slides from formalin fixed paraffin embedded (FFPE) tissues using multiplexed quantitative immunofluorescence (QIF). We also assessed the activation status of CD3-positive cells using Ki67 and Granzyme B markers. Median of each marker expression was calculated for each case by using quantitation from all Fields of View (FOV) that contained at least 10% tumor area. Results: AA tumors contained a significantly higher CD45-positive cells in overall, within tumor and stromal compartments (p = 0.0102, 0.0007 and 0.0280 respectively). Being more specific about CD45 marker, AA also showed to have a significantly higher level of overall lymphocyte population, measured by quantitating CD45+ CD14- cells (p =0.0081). AA tumors also had a significantly higher level of CD8 expression (p &amp;lt;0.0001). Even though we could not find any differences in CD3 expressions between the two groups, AA tumors contained a significantly higher level of activated T-cells (p =0.0432), where activation is defined as CD3 cells with high Ki67 or Granzyme B. AA tumors contained a significantly higher level of CD68 positive cells (p &amp;lt; 0.0001) and PD-L1 expression in CD68+ cells (p =0.0101). All other markers showed no difference between the two groups. Conclusion: Significant differences are seen in the TME of AA vs. white TNBCs. Further characterization of macrophages, fibroblasts, regulatory T-cells and Extracellular Matrix are underway to more specifically define TME and determine the relationship between TME features and outcome. Citation Format: Vesal Yaghoobi, Vasiliki Pelekanou, Tess O'Meara, Andrea Silber, Lajos Pusztai, David Rimm, Kim Blenman. Comparison of tumor immune microenvironment in triple negative breast cancer (TNBC) of African American versus that of white patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4973.

Targeted photodynamic therapy selectively kills activated fibroblasts in experimental arthritis

ObjectiveIn RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX.MethodsAfter conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis.Results28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT.ConclusionHere we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.

1896-P: Adipocyte-Derived Fibroblast Growth Factor 21 (FGF21) Contributes to Circulating FGF21 Pools in Diet-Induced Obese Mice

Fibroblast growth factor 21 (FGF-21) is a hormone derived from hepatocytes and adipocytes that plays a role in energy homeostasis, and promotes weight loss when given pharmacologically. Nutritional stress such as fasting increases liver-derived FGF-21 expression, whereas over-nutrition (i.e., obesity) promotes adipocyte-derived FGF-21 (Ad-FGF-21) production. Despite the current dogma that Ad-FGF-21 acts only in a paracrine manner, obesity is associated with elevated circulating FGF-21 levels which correlate with adiposity and adipose tissue FGF-21 mRNA expression. To date, a concise role for Ad-FGF-21 in obesity-associated energy balance has not been identified. To determine whether Ad-FGF-21 plays a pathological role in obesity, and whether it contributes to circulating FGF-21 levels, we generated mice with Ad-FGF-21 deficiency (Ad-FGF-21 KO) and placed them on an obesogenic high fat high sucrose diet for 18 weeks. Body composition, energy expenditure, glucose and insulin tolerance were assessed. Male and female Ad-FGF-21 KO mice were protected from obesity, gained less body fat, and exhibited improved glucose metabolism over wild type (WT) controls. Circulating FGF-21 levels were higher in obese WT mice than age-matched chow-fed WT or obese Ad-FGF-21 KO mice with no changes in hepatic FGF-21, suggesting that adipocytes are a source of FGF-21 in obesity. Moreover, adipose tissue explants from obese WT mice secreted higher levels of FGF-21 than explants from chow-fed mice, an effect that was blunted in Ad-FGF-21 KO mice. Finally, expression of fibroblast activation protein (FAP), an enzyme that cleaves FGF-21 to blunt its activity, was reduced from obese adipose tissue, providing a potential mechanism by which adipocyte-derived FGF-21 circulates in obesity. In summary, adipocyte-derived FGF-21 may contribute to obesity-associated increases in circulating FGF-21, with the potential to influence the pathology of obesity. Disclosure S. Wang: None. L. Goodspeed: None. K. Ogimoto: None. L. den Hartigh: None. Funding National Institutes of Health (HL092969)

Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs.

Primary tumor-derived factors (TDFs) act upon normal cells to generate a pre-metastatic niche, which promotes colonization of target organs by disseminated malignant cells. Here we report that TDFs-induced activation of the p38α kinase in lung fibroblasts plays a critical role in the formation of a pre-metastatic niche in the lungs and subsequent pulmonary metastases. Activation of p38α led to inactivation of type I interferon signaling and stimulation of expression of fibroblast activation protein (FAP). FAP played a key role in remodeling of the extracellular matrix as well as inducing the expression of chemokines that enable lung infiltration by neutrophils. Increased activity of p38 in normal cells was associated with metastatic disease and poor prognosis in human melanoma patients whereas inactivation of p38 suppressed lung metastases. We discuss the p38α-driven mechanisms stimulating the metastatic processes and potential use of p38 inhibitors in adjuvant therapy of metastatic cancers.

Fibroblast activation protein inhibitor (FAPI) PET for diagnostics and advanced targeted radiotherapy in head and neck cancers

PurposeCancer-associated fibroblasts (CAFs) expressing fibroblast activation protein (FAP) have been associated with the aggressive nature of head and neck cancers (HNCs). These tumours grow diffusely, leading to extremely challenging differentiation between tumour and healthy tissue. This analysis aims to introduce a novel approach of tumour detection, contouring and targeted radiotherapy of HNCs using visualisation of CAFs: PET-CT with 68Ga-radiolabeled inhibitors of FAP (FAPI).MethodsFAPI PET-CT was performed without complications prior to radiotherapy in addition to contrast enhanced CT (CE-CT) and MRI on 14 patients with HNC. First, for tissue biodistribution analysis, volumes of interest were defined to quantify SUVmean and SUVmax in tumour and healthy parenchyma. Secondly, using four thresholds of three-, five-, seven- and tenfold increase of FAPI enhancement in the tumour as compared with normal tissue, four different gross tumour volumes (FAPI-GTV) were created automatically. These were compared with GTVs created conventionally with CE-CT and MRI (CT-GTV).ResultsThe biodistribution analysis revealed high FAPI avidity within tumorous lesions (e.g. primary tumours, SUVmax 14.62 ± 4.44; SUVmean 7.41 ± 2.39). In contrast, low background uptake was measured in healthy tissues of the head and neck region (e.g. salivary glands: SUVmax 1.76 ± 0.31; SUVmean 1.23 ± 0.28). Considering radiation planning, CT-GTV was of 27.3 ml, whereas contouring with FAPI resulted in significantly different GTVs of 67.7 ml (FAPI × 3, p = 0.0134), 22.1 ml (FAPI × 5, p = 0.0419), 7.6 ml (FAPI × 7, p = 0.0001) and 2.3 ml (FAPI × 10, p = 0.0001). Taking these significant disparities between the GTVs into consideration, we merged FAPI-GTVs with CT-GTVs. This resulted in median volumes, that were, as compared to CT-GTVs, significantly larger with FAPI × 3 (54.7 ml, + 200.5% relative increase, p = 0.0005) and FAPI × 5 (15.0 ml, + 54.9%, p = 0.0122). Furthermore, FAPI-GTVs were not covered by CE-CT-based planning target volumes (CT-PTVs) in several cases.ConclusionWe present first evidence of diagnostic and therapeutic potential of FAPI ligands in head and neck cancer. Larger studies with histopathological correlation are required to validate our findings.

Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21.

Recent studies indicate that cancer-associated fibroblasts (CAFs) are phenotypically and functionally heterogeneous. However, little is known about CAF subtypes, the roles they play in cancer progression, and molecular mediators of the CAF "state." Here, we identify a novel cell surface pan-CAF marker, CD49e, and demonstrate that two distinct CAF states, distinguished by expression of fibroblast activation protein (FAP), coexist within the CD49e+ CAF compartment in high-grade serous ovarian cancers. We show for the first time that CAF state influences patient outcomes and that this is mediated by the ability of FAP-high, but not FAP-low, CAFs to aggressively promote proliferation, invasion and therapy resistance of cancer cells. Overexpression of the FAP-low-specific transcription factor TCF21 in FAP-high CAFs decreases their ability to promote invasion, chemoresistance, and in vivo tumor growth, indicating that it acts as a master regulator of the CAF state. Understanding CAF states in more detail could lead to better patient stratification and novel therapeutic strategies.

Deletion of fibroblast activation protein provides atheroprotection.

Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice. Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries. Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies.

FAP-specific PET signaling shows a moderately positive correlation with relative CBV and no correlation with ADC in 13 IDH wildtype glioblastomas

ObjectivesTargeting Fibroblast Activation Protein (FAP) is a new approach for glioblastoma imaging. In a recent pilot study glioblastomas showed elevated tracer uptake with high intratumoral heterogeneity in projection on the corresponding T2w/FLAIR and contrast enhanced MRI lesions. In this study, we correlated FAP-specific signaling with apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) signals in MRI to further characterize the significance of FAP uptake. MethodsClinical PET/CT scans of 13 glioblastoma patients were performed post i. v. administration of 68Ga-labelled-FAP-specific tracer molecules. PET- and corresponding MRI-scans were co-registrated. 3d volumetric segmentations were performed of T2w/FLAIR lesions and contrast enhancing lesions within co-registrated MRI slides. Signal intensity values of FAP-specific PET signaling, ADC and rCBV were analyzed for their pixel wise correlation in each patient. Pooled estimates of the correlation coefficients were calculated by using the Fisher z-transformation. ResultsFAP-specific PET signals showed a moderately positive correlation with rCBV values which is more pronounced within the T2w/FLAIR lesion (pooled correlation 0,229) than in the contrast enhancing tumor region (pooled correlation 0.09). FAP-specific PET signals showed no correlation with ADC values. ConclusionsThe moderately positive correlation of FAP-specific signals with rCBV values in MRI indicates that FAP-signaling is not independent from perfusion, but also does not only reflect intratumoral perfusion differences. The missing correlation of FAP-specific signals with ADC indicates that FAP-specific imaging does not reflect cell density, but the spot-like expression of FAP in glioblastomas. The clinical value of FAP-specific imaging needs further investigation.

Immunohistochemical study of fibroblast activation protein and α-smooth muscle actin expression and distribution in triple-negative breast cancer

Background: Triple-negative breast cancer constitutes 10-20% of all breast cancers, and are more frequent in younger patients with a poor prognostic outcome. The tumor microenvironment is formed of activated fibroblasts (FAP) which are known as cancer-associated fibroblasts, which can be detected by fibroblast activation protein [FAP] and α-smooth muscle actin [α-SMA]. Aim: The aim of the work is the evaluation of the expression and distribution of FAP and α-SMA in triple-negative breast cancer. Material and Methods: This study was carried on 100 paraffin blocks from pathologically proved triple-negative invasive breast cancer patients and subjected to immunostaining of SMA and FAP antibodies, Evaluation of antibodies expression according to its distribution in tumor margin and in the tumor center. Results: There were significant differences between FAP as well as SMA expression in the tumor center and tumor margin, where FAP and SMA expression in the tumor center was positively correlated with tumor size and grade. In contrast to tumor margin, FAP and SMA expression were negatively correlated with the tumor size and lymph node metastasis. Conclusion: We speculated that high FAP expression and α-SMA expression in the tumor center, but not the tumor margin, is correlated with poor patients clinicopathological parameters.

The prognostic significance of fibroblast activation protein-α in human lung adenocarcinoma.

BackgroundFibroblast activation protein (FAP) is a type II cell surface-bound integral serine protease, which is an important biomarker of cancer-associated fibroblasts. FAP-α performs several biological activities, including remolding extracellular matrix and acting as an immunosuppressor in the tumor microenvironment. However, the proliferation role of FAP-α in human lung adenocarcinoma has not been fully elucidated.MethodsThe expression of FAP-α in 94-paired human lung adenocarcinoma tissues was identified by immunohistochemistry test. The effect of FAP on cell proliferation was examined by CCK-8 assay. RNA-sequencing and bioinformatics analysis were utilized to investigate the underlying mechanism. Western blot analysis, quantitative polymerase chain reaction (qPCR), and nude mice experiments, were also conducted for further validation.ResultsThe proliferation rates of human fibroblast strains FAP-HFF and FAP-BJ, and human lung adenocarcinoma cell line FAP-SPC-A-1 were higher than those of controls. The nude mice experiment also showed that FAP could promote the proliferation of SPC-A-1 cell line in vivo. qPCR and Western blot analysis indicated that CCNB1 was upregulated by the overexpression of FAP in the lung adenocarcinoma cell line. The expression of FAP-α was higher in both the cytoplasm and stroma of lung adenocarcinoma than in adjacent normal tissues. Survival analysis indicated that patients with higher expression of FAP-α in tumor stroma had a poor prognosis (P=0.019). The Cancer Genome Atlas Program (TCGA) data also showed that the expression of FAP within tumor tissues was higher (in both cytoplasm and stroma) compared with that in normal tissues (P<0.05).ConclusionsOur study indicates that FAP-α could facilitate the proliferation of lung adenocarcinoma cells and can be a prognostic marker in human lung adenocarcinoma.

Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Introduction Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated a high response rate, the durable response rate is around 30%. Most patients lose their CART cells, and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in this field. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and immunosuppression. Objective CAFs from MM patients lead BCMA CART cell dysfunction and targeting both MM cells and CAFs can overcome this resistance. Methods CAFs were isolated from the bone marrow (BM) of MM patients and purified using anti fibroblast beads. MM-TME mouse model was established by injecting a 1:1 ratio of BCMA+ OPM2 (MM cell line) to CAFs. Five different CART cells were tested in this study. For single transduced CAR: BCMA, CS1, and fibroblast activation protein (FAP). For dual transduced CAR: BCMA-CS1 and BCMA-FAP CART. These were generated through the double transduction of two lentiviral vectors. Results BCMA CART cell proliferation assay showed inhibition of CART expansion in the presence of CAFs, and this was mediated by TGF-β (Fig A). MM-TME mouse model revealed a significant acceleration of MM progression (Fig B). To identify targets, we verified FAP expression on CAFs. Interestingly, there was a low level of CS1, but not BCMA expressed on CAFs (Fig C). Then, we evaluated the impact of CAFs on functions of BCMA CART cells compared to dual CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of CAFs, the proliferation of BCMA CART cells, but not the dual CART cells was significantly inhibited (Fig A). Similarly, in the presence of CAFs, the production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, we studied the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells was able to eradicate MM (Fig E). Then, the MM-TME model was used to assess the effect of targeting CAFs. Mice were treated with 1) control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A low dose (1 × 106 iv) of CART cell was used to induce relapse. BCMA CART cells led to a transient antitumor activity in this model (mice died within 2 weeks), while dual CART cells resulted in durable remissions, a higher number of circulating T cells, and long term survival (Fig F). Conclusion We demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in MM.

Expression of fibroblast activation protein and the clinicopathological relevance in distal cholangiocarcinoma

Objectives: The current survival of patients with distal cholangiocarcinoma (dCCA) is poor. There is a need to develop new prognostic and predictive biomarkers to improve the survival of patients. Fibroblast activation protein (FAP) expression has been associated with survival in several solid malignancies. The goal of this study was to evaluate the expression pattern and prognostic significance of FAP in dCCA.Materials and methods: FAP expression was examined in 57 resected dCCA specimens and 28 paired lymph node metastasis specimens, as well as 10 benign bile ducts using immunohistochemistry. FAP expression was scored in the epithelial and stromal component of the dCCA specimens. The association between FAP expression and prognosis was evaluated using univariable and multivariable statistical modeling.Results: FAP expression was absent in the benign controls. FAP expression was evident in the epithelial 43 (75%) and stromal compartment 34 (60%) of dCCA. There was no association between epithelial or stromal FAP expression and clinicopathological factors. Epithelial FAP expression (HR 0.4 95% CI 0.20–0.78; p=.007) but not stromal FAP expression was significantly associated with better survival in univariable and multivariable analysis.Conclusions: FAP overexpression is evident in dCCA. There was a positive association between epithelial FAP expression and better survival which merits further evaluation.

Design and validation of fibroblast activation protein alpha targeted imaging and therapeutic agents

Background: Cancer-associated fibroblasts (CAFs) comprise a major cell type in the tumor microenvironment where they support tumor growth and survival by producing extracellular matrix, secreting immunosuppressive cytokines, releasing growth factors, and facilitating metastases. Because tumors with elevated CAFs are characterized by poorer prognosis, considerable effort is focused on developing methods to quantitate, suppress and/or eliminate CAFs. We exploit the elevated expression of fibroblast activation protein (FAP) on CAFs to target imaging and therapeutic agents selectively to these fibroblasts in solid tumors.Methods: FAP-targeted optical imaging, radioimaging, and chemotherapeutic agents were synthesized by conjugating FAP ligand (FL) to either a fluorescent dye, technetium-99m, or tubulysin B hydrazide. In vitro and in vivo studies were performed to determine the specificity and selectivity of each conjugate for FAP in vitro and in vivo.Results: FAP-targeted imaging and therapeutic conjugates showed high binding specificity and affinity in the low nanomolar range. Injection of FAP-targeted 99mTc into tumor-bearing mice enabled facile detection of tumor xenografts with little off-target uptake. Optical imaging of malignant lesions was also readily achieved following intravenous injection of FAP-targeted near-infrared fluorescent dye. Finally, systemic administration of a tubulysin B conjugate of FL promoted complete eradication of solid tumors with no evidence of gross toxicity to the animals.Conclusion: In view of the near absence of FAP on healthy cells, we conclude that targeting of FAP on cancer-associated fibroblasts can enable highly specific imaging and therapy of solid tumors.

Endothelial, pericyte and tumor cell expression in glioblastoma identifies fibroblast activation protein (FAP) as an excellent target for immunotherapy.

ObjectivesTargeted immunotherapies such as chimeric antigen receptor (CAR)‐T cells are emerging as attractive treatment options for glioblastoma, but rely on identification of a suitable tumor antigen. We validated a new target antigen for glioblastoma, fibroblast activation protein (FAP), by undertaking a detailed expression study of human samples.MethodsGlioblastoma and normal tissues were assessed using immunostaining, supported by analyses of published transcriptomic datasets. Short‐term cultures of glioma neural stem (GNS) cells were compared to cultures of healthy astrocytes and neurons using flow cytometry. Glioblastoma tissues were dissociated and analysed by high‐parameter flow cytometry and single‐cell transcriptomics (scRNAseq).ResultsCompared to normal brain, FAP was overexpressed at the gene and protein level in a large percentage of glioblastoma tissues, with highest levels of expression associated with poorer prognosis. FAP was also overexpressed in several paediatric brain cancers. FAP was commonly expressed by cultured GNS cells but absent from normal neurons and astrocytes. Within glioblastoma tissues, the strongest expression of FAP was around blood vessels. In fact, almost every tumor vessel was highlighted by FAP expression, whereas normal tissue vessels and cultured endothelial cells (ECs) lacked expression. Single‐cell analyses of dissociated tumors facilitated a detailed characterisation of the main cellular components of the glioblastoma microenvironment and revealed that vessel‐localised FAP is because of expression on both ECs and pericytes.ConclusionFibroblast activation protein is expressed by multiple cell types within glioblastoma, highlighting it as an ideal immunotherapy antigen to target destruction of both tumor cells and their supporting vascular network.

Multiple Myeloma Bone Marrow Mesenchymal Stromal Cells Inhibit CD8+ T Cell Function in a Process that May Implicate Fibroblast Activation Protein α.

Multiple myeloma (MM) is a malignant plasma cell proliferative disorder with limited immunotherapy treatment because of T cell dysfunction. To investigate the immunomodulatory function of bone marrow mesenchymal stromal cells (MM-BMSCs) on CD8+ T cells. Proliferation and cytotoxicity were detected by cell counting kit-8 assay. Cell cycle was detected by flow cytometry, and p16 expression was detected by PCR. The expression of fibroblast activation protein α (FAPα) was evaluated by immunohistochemistry. Co-culture of CD8+ T cells with MM-BMSCs decreased the cell survival rate and increased the killing rate (p=0.03, p=0.001, respectively), the percentage of cells in G0/G1 phase and p16 expression (p<0.001). FAPα was mainly in the mesenchymal matrix of the MM microenvironment and elevated in MM derived bone marrow compared to healthy donors (p<0.001). The FAPα inhibitor PT-100, increased survival and the killing rate (p<0.001, p=0.043, respectively), and decreased the percentage of cells in G0/G1 phase and p16 expression (p=0.024, p=0.004, respectively). Therefore, MM-BMSCs inhibit the proliferation and cytotoxicity of CD8+ T cells, significantly block the cell cycle and increase p16 expression in co-cultured CD8+ T cells in a cell-cell contact-dependent manner.

Abstract AP17: MOLECULAR AND FUNCTIONAL HETEROGENEITY OF CANCER ASSOCIATED FIBROBLASTS IN HIGH-GRADE SEROUS OVARIAN CANCER

Abstract High grade serous ovarian cancer (HGSC) is typically diagnosed at an advanced stage and the vast majority of patients relapse and die within 5 years of diagnosis. Significant clinical problems in HGSC include wide-spread abdominal dissemination of disease and chemotherapy resistance. Cancer-associated fibroblasts (CAFs) have been shown to play a role in promotion of cancer cell proliferation and invasion, and mediation of chemotherapy resistance. To interrogate the molecular properties of CAFs in HGSC we used fluorescence activated cell sorting to isolate CAFs directly from primary tumor samples and performed gene expression profiling. We found that patients stratify into two classes based on their CAF gene signatures: One with high expression of Fibroblast Activation Protein (FAP-High; FH) and one with low expression of FAP (FAP-Low; FL). FH CAFs express classical CAF genes whereas FL CAFs possesses a preadipocyte-like molecular signature. The FL phenotype has remained largely unnoticed as it is generally out-competed in vitro by FH cells when grown under classical CAF culture conditions. Patients from The Cancer Genome Atlas (TCGA), as well as from our own institute, can be stratified into FH and FL subtypes; in both cohorts patients with FH CAFs have a significantly shorter disease-free and overall survival. In vitro and in vivo functional assays performed with isolated CAFs of both types indicate that FH CAFs aggressively promote proliferation, invasion and therapy resistance of cancer cells, whereas FL CAFs do not. Finally, we identified TCF21, a transcriptional repressor, as a FL-specific transcription factor. Analysis of published TCF21 ChIP-Seq data indicates that TCF21 targets a large number of genes specific to FH CAFs. Overexpression of TCF21 in FH CAFs partially reversed their ability to promote cancer cell invasion and tumor growth. Our discovery of CAF heterogeneity in HGSC highlights the need to personalize patient treatment with respect to both cancer and stromal phenotypes. FH patients may benefit from inhibition of cancer-stroma interactions or from epigenetic modulators that reprogram cancer-promoting FH CAFs into the non-supportive FL state. Citation Format: Ali Hussain, Veronique Voisin, Stephanie Poon, Jalna Meens, Julia Dmytryshyn, Josh Paterson, Marcus Bernardini3, Gary Bader, Benjamin G Neel, Laurie E Ailles. MOLECULAR AND FUNCTIONAL HETEROGENEITY OF CANCER ASSOCIATED FIBROBLASTS IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP17.

Theranostics Targeting Fibroblast Activation Protein in the Tumor Stroma: 64Cu- and 225Ac-Labeled FAPI-04 in Pancreatic Cancer Xenograft Mouse Models.

Fibroblast activation protein (FAP), which promotes tumor growth and progression, is overexpressed in cancer-associated fibroblasts of many human epithelial cancers. Because of its low expression in normal organs, FAP is an excellent target for theranostics. In this study, we used radionuclides with relatively long half-lives, 64Cu (half-life, 12.7 h) and 225Ac (half-life, 10 d), to label FAP inhibitors (FAPIs) in mice with human pancreatic cancer xenografts. Methods: Male nude mice (body weight, 22.5 ± 1.2 g) were subcutaneously injected with human pancreatic cancer cells (PANC-1, n = 12; MIA PaCa-2, n = 8). Tumor xenograft mice were investigated after the intravenous injection of 64Cu-FAPI-04 (7.21 ± 0.46 MBq) by dynamic and delayed PET scans (2.5 h after injection). Static scans 1 h after the injection of 68Ga-FAPI-04 (3.6 ± 1.4 MBq) were also acquired for comparisons using the same cohort of mice (n = 8). Immunohistochemical staining was performed to confirm FAP expression in tumor xenografts using an FAP-α-antibody. For radioligand therapy, 225Ac-FAPI-04 (34 kBq) was injected into PANC-1 xenograft mice (n = 6). Tumor size was monitored and compared with that of control mice (n = 6). Results: Dynamic imaging of 64Cu-FAPI-04 showed rapid clearance through the kidneys and slow washout from tumors. Delayed PET imaging of 64Cu-FAPI-04 showed mild uptake in tumors and relatively high uptake in the liver and intestine. Accumulation levels in the tumor or normal organs were significantly higher for 64Cu-FAPI-04 than for 68Ga-FAPI-04, except in the heart, and excretion in the urine was higher for 68Ga-FAPI-04 than for 64Cu-FAPI-04. Immunohistochemical staining revealed abundant FAP expression in the stroma of xenografts. 225Ac-FAPI-04 injection showed significant tumor growth suppression in the PANC-1 xenograft mice, compared with the control mice, without a significant change in body weight. Conclusion: This proof-of-concept study showed that 64Cu-FAPI-04 and 225Ac-FAPI-04 could be used in theranostics for the treatment of FAP-expressing pancreatic cancer. α-therapy targeting FAP in the cancer stroma is effective and will contribute to the development of a new treatment strategy.