FGFR blockade boosts T cell infiltration into triple-negative breast cancer by regulating cancer-associated fibroblasts.

Background: Since T cell exclusion contributes to tumor immune evasion and immunotherapy resistance, how to improve T cell infiltration into solid tumors becomes an urgent challenge. Methods: We employed deep learning to profile the tumor immune microenvironment (TIME) in triple negative breast cancer (TNBC) samples from TCGA datasets and noticed that fibroblast growth factor receptor (FGFR) signaling pathways were enriched in the immune-excluded phenotype of TNBC. Erdafitinib, a selective FGFR inhibitor, was then used to investigate the effect of FGFR blockade on TIME landscape of TNBC syngeneic mouse models by flow cytometry, mass cytometry (CyTOF) and RNA sequencing. Cell Counting Kit-8 (CCK-8) assay and transwell migration assay were carried out to detect the effect of FGFR blockade on cell proliferation and migration, respectively. Cytokine array, western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) were employed to investigate the potential mechanism by which FGFR inhibition enhanced T cell infiltration. Results: Blocking FGFR pathway by Erdafitinib markedly suppressed tumor growth with increased T cell infiltration in immunocompetent mouse models of TNBC. Mechanistically, FGFR blockade inhibited cancer-associated fibroblasts (CAFs) proliferation, migration and secretion of vascular cell adhesion molecule 1 (VCAM-1) by down-regulating MAPK/ERK pathway in CAFs, thus promoting T cell infiltration by breaking physical and chemical barriers built by CAFs in TIME. Furthermore, we observed that FGFR inhibition combined with immune checkpoint blockade therapy (ICT) greatly improved the therapeutic response of TNBC tumor models. Conclusions: FGFR blockade enhanced ICT response by turning immune "cold" tumor into "hot" tumor, providing remarkable implications of FGFR inhibitors as adjuvant agents for combinatorial immunotherapy.

Fibroblast Growth Factor-10 (FGF-10) Mobilizes Lung-resident Mesenchymal Stem Cells and Protects Against Acute Lung Injury.

FGF-10 can prevent or reduce lung specific inflammation due to traumatic or infectious lung injury. However, the exact mechanisms are poorly characterized. Additionally, the effect of FGF-10 on lung-resident mesenchymal stem cells (LR-MSCs) has not been studied. To better characterize the effect of FGF-10 on LR-MSCs, FGF-10 was intratracheally delivered into the lungs of rats. Three days after instillation, bronchoalveolar lavage was performed and plastic-adherent cells were cultured, characterized and then delivered therapeutically to rats after LPS intratracheal instillation. Immunophenotyping analysis of FGF-10 mobilized and cultured cells revealed expression of the MSC markers CD29, CD73, CD90, and CD105, and the absence of the hematopoietic lineage markers CD34 and CD45. Multipotency of these cells was demonstrated by their capacity to differentiate into osteocytes, adipocytes, and chondrocytes. Delivery of LR-MSCs into the lungs after LPS injury reduced the inflammatory response as evidenced by decreased wet-to-dry ratio, reduced neutrophil and leukocyte recruitment and decreased inflammatory cytokines compared to control rats. Lastly, direct delivery of FGF-10 in the lungs of rats led to an increase of LR-MSCs in the treated lungs, suggesting that the protective effect of FGF-10 might be mediated, in part, by the mobilization of LR-MSCs in lungs.

Approaches targeting the FGF-FGFR system: a review of the recent patent literature and associated advanced therapeutic agents.

Fibroblast growth factor receptors (FGFRs) and associated ligands (FGFs) are a family of well-validated targets for therapeutic interventions notably in cancer diseases in relation to their prominent roles in cell growth, survival, differentiation and angiogenesis. This patent review encompasses all different approaches (modulators of FGF or FGFR expression, anti-FGF antibodies, anti-FGFR antibodies, FGF traps, tyrosine-kinase (TK) inhibitors, allosteric modulators) used to block completely or partially the activities of the FGF-FGFR complexes resulting in clinical drug candidates or tool agents. Comparative analysis of biochemical, pharmacological or clinical data will be discussed for each class of molecules together with some perspectives.

Selective E-selectin ligands.

In this issue of Blood, Sreeramkumar and colleagues report that E-selectin ligand-1 (ESL-1) is a highly selective ligand for E-selectin on hematopoietic progenitors with unexpected important contributions to their trafficking.

Coordinated and unique functions of the E-selectin ligand ESL-1 during inflammatory and hematopoietic recruitment in mice.

Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils.

Cisterna-specific localization of glycosylation-related proteins to the Golgi apparatus.

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.

The role of chronic inflammation in cutaneous fibrosis: fibroblast growth factor receptor deficiency in keratinocytes as an example.

Fibrosis is associated with a variety of skin diseases and causes severe aesthetic and functional impairments. Functional studies in rodents, together with clinical observations, strongly suggest a crucial role of chronic injury and inflammation in the pathogenesis of fibrotic diseases. The phenotype of mice lacking fibroblast growth factor (FGF) receptors 1 and 2 in keratinocytes supports this concept. In these mice, a defect in keratinocytes alone initiated an inflammatory response, which in turn caused keratinocyte hyperproliferation and dermal fibrosis. As the mechanism underlying this phenotype, we identified a loss of FGF-induced expression of claudins and occludin, which caused abnormalities in tight junctions with concomitant deficits in epidermal barrier function. This resulted in severe transepidermal water loss and skin dryness. In turn, activation of keratinocytes and epidermal γδ T cells occurred, which produced IL-1 family member 8 and S100A8 and S100A9. These cytokines attracted immune cells and activated fibroblasts, resulting in a double paracrine loop through production of keratinocyte mitogens by dermal cells. In addition, a profibrotic response was induced in fibroblasts. Our results highlight the importance of an intact epidermal barrier for the prevention of inflammation and fibrosis and the role of chronic inflammation in the pathogenesis of fibrotic diseases.

Leukocyte ligands for endothelial selectins: specialized glycoconjugates that mediate rolling and signaling under flow.

Reversible interactions of glycoconjugates on leukocytes with P- and E-selectin on endothelial cells mediate tethering and rolling of leukocytes in inflamed vascular beds, the first step in their recruitment to sites of injury. Although selectin ligands on hematopoietic precursors have been identified, here we review evidence that PSGL-1, CD44, and ESL-1 on mature leukocytes are physiologic glycoprotein ligands for endothelial selectins. Each ligand has specialized adhesive functions during tethering and rolling. Furthermore, PSGL-1 and CD44 induce signals that activate the ß2 integrin LFA-1 and promote slow rolling, whereas ESL-1 induces signals that activate the ß2 integrin Mac-1 in adherent neutrophils. We also review evidence for glycolipids, CD43, L-selectin, and other glycoconjugates as potential physiologic ligands for endothelial selectins on neutrophils or lymphocytes. Although the physiologic characterization of these ligands has been obtained in mice, we also note reported similarities and differences with human selectin ligands.

[Molecular basis and clinical results of radiation therapy combined with molecular targeted therapy].

Molecular target-based drugs combined with radiation therapy have been expected as promising approach in cancer treatment, and clinical trials using combined modalities have been performed to evaluate the feasibility and efficacy of this approach. In order to obtain maximum radiotherapeutic gain, a detailed understanding of the mechanism underlying the interaction between radiation and molecular target-based drugs is indispensable. Among molecular target-based drugs, inhibitors targeting EGFR and its signal transduction pathways have been vigorously investigated, and mechanisms regarding the radiosensitizing effect have been getting clear. In addition, the results of randomized clinical trials demonstrated that radiation therapy combined with cetuximab resulted in improvement of overall and disease-specific survival rate compared with radiation therapy in locally advanced head and neck cancer. In this review, established and potential molecular targets for potentiation of radiation-induced cell killing are summarized, especially focusing on EGFR and its signal transduction pathways; also, clinical trials of combined radiation therapy plus molecular target-based drug therapy are summarized.

Biological implications of glycosaminoglycan interactions with haemopoietic cytokines.

Heparan sulphate (HS) glycosaminoglycans (GAGs) are an integral part of the signalling complex of fibroblast derived growth factor (FGF) family members, HS being regarded as a coreceptor. FGFs are also retained in the tissues by binding to HS structures. Early studies on the contribution of the bone marrow stroma to haemopoiesis suggested that cytokines with a role in haemopoiesis were similarly retained in the stroma through interactions with HS. However, the functional outcomes of these cytokines binding HS were poorly understood. Here the GAG-binding properties of cytokines of the four alpha-helical bundle family and the biological consequences of such binding are reviewed. From this analysis it is apparent that although many of these cytokines do bind GAGs, GAG binding is not a consistent feature, nor is the site of GAG binding conserved among these cytokines. The biological outcome of GAG binding depends, in part, on the location of the GAG-binding site on the cytokine. In some cases GAG binding appears to block signalling, whereas in others signalling is likely to be facilitated by binding. It is postulated that the interactions of these cytokines with their receptor complexes evolved independently of GAG binding, with GAG binding being an additional feature for a subset of this cytokine family. Nevertheless, because GAG binding localizes cytokines to sites within tissues, these interactions are likely to be critically important for the biology of these cytokines.

CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts.

Human NK cells are divided into CD56(bright)CD16(-) cells and CD56(dim)CD16(+) cells. We tested the hypothesis that CD56(bright) NK cells can differentiate into CD56(dim) cells by prospectively isolating and culturing each NK subset in vitro and in vivo. Our results show that CD56(bright) cells can differentiate into CD56(dim) both in vitro, in the presence of synovial fibroblasts, and in vivo, upon transfer into NOD-SCID mice. In vitro, this differentiation was inhibited by fibroblast growth factor receptor-1 Ab, demonstrating a role of the CD56 and fibroblast growth factor receptor-1 interaction in this process. Differentiated CD56(dim) cells had reduced IFN-gamma production but increased perforin expression and cytolysis of cell line K562 targets. Flow cytometric fluorescent in situ hybridization demonstrated that CD56(bright) NK cells had longer telomere length compared with CD56(dim) NK cells, implying the former are less mature. Our data support a linear differentiation model of human NK development in which immature CD56(bright) NK cells can differentiate into CD56(dim) cells.

CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency.

Using noncompetitive methodologies comparing CD43(+/+) and CD43(-/-) mice, it has been reported that CD43(-/-) leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43(-/-) T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43(+/+) vs CD43(-/-) activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43(+/+) and congenic CD43(-/-) mice in either B6 or P-selectin(-/-) recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

T-cell P/E-selectin ligand alpha(1,3)fucosylation is not required for graft-vs-host disease induction.

OBJECTIVE: Recognition of E- and P-selectins on vascular endothelium by their leukocyte glycoprotein counterreceptor P-selectin glycoprotein ligand-1 (PSGL-1) initiates and sustains leukocyte rolling, culminating in extravasation of lymphocytes from blood into organs. PSGL-1 is rendered functional by terminal glycosylation steps, which occur mainly in activated Th1 but not Th2 cells. alpha(1,3)Fucosyltransferases IV and VII control this glycosylation pathway. Mice lacking these transferases (Fuc-TIV(-/-)/Fuc-TVII(-/-)) lack functional E- and P-selectin ligands. We hypothesized that Fuc-TIV(-/-)/Fuc-TVII(-/-) donor T cells might have reduced capacity to roll on vessels of inflamed target tissues and mediate graft-vs-host disease (GVHD). MATERIALS AND METHODS: We compared the ability of Fuc-TIV(-/-)/Fuc-TVII(-/-) and wild-type (WT) C57BL/6 (B6) spleen cells (SPCs) to produce GVHD in lethally irradiated major histocompatibility complex (MHC) haplotype-mismatched B6D2F1 recipients. Clinical GVHD, GVHD pathology in target organs, memory phenotype conversion, proliferation of donor T cells, and tissue and serum cytokine expression were examined. RESULTS: Surprisingly, clinical GVHD was not reduced in lethally irradiated mice receiving full haplotype MHC mismatched or matched Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared to those receiving WT SPCs. GVHD pathology in target organs, memory phenotype conversion, and proliferation of donor T cells were similar in both groups. However, reduced interferon-gamma was detected in liver and lung, and serum levels of tumor necrosis factor-alpha were higher in mice receiving Fuc-TIV(-/-)/Fuc-TVII(-/-) SPCs compared with WT SPCs. CONCLUSIONS: These results suggest that donor T cells, including Th1, are capable of trafficking to GVHD target tissues independently of P- and E- selectin ligand in conditioned hosts.

Targeting the extracellular domain of fibroblast growth factor receptor 3 with human single-chain Fv antibodies inhibits bladder carcinoma cell line proliferation.

PURPOSE: Previous gene expression studies have shown that fibroblast growth factor receptor 3 (FGFR3) is overexpressed in early stages of bladder cancer. To study the potential use of therapeutic antibodies against FGFR3, we have produced a collection of human single-chain Fv (scFv) antibody fragments by using phage display libraries. EXPERIMENTAL DESIGN: Two "naïve" semi-synthetic human scFv libraries were used to select antibodies against the extracellular domain of FGFR3alpha(IIIc). The reactivity of the selected scFvs with a recombinant FGFR3 was characterized by an enzyme immunoassay and surface plasmon resonance analysis and with RT112 bladder carcinoma cells by a fluorescence-activated cell sorter. The capacity of the selected scFvs to block RT112 cell proliferation was determined. RESULTS: We have isolated six human scFv antibody fragments directed against FGFR3. These human scFvs specifically bound FGFR3, but not the homologous molecule FGFR1. Biacore analysis was used to determine the affinity constants, which ranged from 12 to 40 nmol/L. Competition analysis showed that the FGF9 ligand was able to block the binding of two scFvs, 3C and 7D, to FGFR3, whereas FGF1 only blocked 7D. Immunoprecipitation and flow cytometric analysis confirmed the specificity of the antibodies to native membrane FGFR3. Two scFvs, 3C and 7D, gave an strong immunofluorescence staining of RT112 cells. Moreover, they recognized equally well wild-type and mutant FGFR3 containing the activating mutation S249C. Furthermore, they blocked proliferation of RT112 cells in a dose- and FGF-dependent manner. CONCLUSION: Our results suggest that these human anti-FGFR3 scFv antibodies may have potential applications as antitumoral agents in bladder cancer.

Receptor-mediated basic fibroblast growth factor signaling regulates cyclic pressure-induced human endothelial cell proliferation.

Vascular endothelial cells sense and respond to pressure by molecular mechanism(s) which, to date, remain poorly understood. The present study investigated basic fibroblast growth factor (bFGF) signaling as a putative mechanotransduction pathway involved in the proliferative responses of human umbilical vein endothelia cells (HUVECs) to 60/20 mm Hg cyclic pressure at 1 Hz for 24 h. Under these conditions, the enhanced proliferative response of these HUVECs was not associated with an increased synthesis/release of bFGF, but involved rapid (within 30 min from the onset of exposure to pressure) tyrosine phosphorylation of the bFGF receptor, FGFR-2. Furthermore, monoclonal antibodies to either bFGF or FGFR-2 attenuated the increased proliferation of HUVECs exposed to 60/20 mm Hg cyclic pressure. HUVECs proliferation under 60/20 mm Hg at 1 Hz cyclic pressure is, therefore, dependent upon bFGF and involves FGFR-2 activation.

Fibroblast growth factor receptor-3 as a therapeutic target for Achondroplasia--genetic short limbed dwarfism.

Achondroplasia, the most common form of human dwarfism is a sporadic autosomal dominant condition that occurs in approximately 1:20,000 births. The major clinical outcome of Achondroplasia is attenuated growth, rhizomelic shortening of the long bones and craniofacial abnormalities. As of today there is no pharmacological treatment for Achondroplasia. Some improvement in the patients well being and daily function can be achieved by a surgical limb lengthening procedure. Growth hormone treatment seems to have only modest short term success and to lack long term benefits. Achondroplasia results from a single point mutation in Fibroblast Growth Factor Receptor 3 (FGFR3). In 97% of the patients, there is a Glycine to Arginine substitution at position 380 within the FGFR-3 transmembrane domain leading to receptor overactivation. This FGF receptor tyrosine kinase is expressed by chondrocytes in the growth plate of developing long bones and plays a crucial role in bone growth. Genetic disruption of the FGFR-3 gene in mice leads to a remarkable increase in the length of the vertebral column and long bones. This suggests that overaction of FGFR3 signaling may specifically impair chondrocyte function within the epiphyseal growth plates and cause Achondroplasia. Reconstituted normal bone growth may therefore be achieved by attenuation of FGFR3 signaling in the appropriate cells within the growth plate. It is highly conceivable that drug development strategies aimed either towards blocking extracellular ligand binding or towards intracellular checkpoints along the FGF signal transduction cascade, may prove successful in the treatment of Achondroplasia. This review focuses on the possible approaches for developing a drug for Achondroplasia and related skeletal disorders, using chemical, biochemical and molecular strategies.

Unspecific labeling of pancreatic islets by antisera against fibroblast growth factors and their receptors.

Six distinct fibroblast growth factors (FGFs) have been detected in pancreatic islets by immunohistochemistry (IHC) using commercially available antisera. We show here that these antisera are useful for Western blotting but that only two are suited for IHC. By Western blotting, these antisera detect recombinant FGFs. Detection can be eliminated by preabsorption with immunizing peptide but not with irrelevant peptide. By IHC we find specific labeling of islets with anti-FGF1 and anti-FGF2 antisera. Labeling can be abolished by preabsorption with the immunizing peptides. In contrast, prominent staining of islets by anti-FGF4, -FGF5, -FGF7, and -FGF10 antisera is unspecific because the staining cannot be competed by preabsorption with the immunizing peptides.

Possible involvement of keratinocyte growth factor and its receptor in enhanced epithelial-cell proliferation and acquired recurrence of middle-ear cholesteatoma.

Middle-ear cholesteatoma is characterized by enhanced proliferation of epithelial cells and granular tissue formation. However, the molecular mechanism underlying these pathological changes is largely unknown. Keratinocyte growth factor (KGF) is a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation. In the present study, we investigated the possible involvement of KGF and its receptor, KGFR, in the pathogenesis of cholesteatoma using in situ hybridization and immunohistochemistry, respectively. We examined 56 cholesteatoma specimens, and 8 normal skin areas as control. KGF and KGFR expression was examined by immunohistochemistry using rabbit anti-human KGF and anti-human KGFR polyclonal antisera raised in our laboratories against synthetic peptides corresponding to parts of human KGF and KGFR, respectively. KGF protein and mRNA were detected exclusively in stromal fibroblasts and infiltrating T lymphocytes in 80% of cholesteatoma cases, whereas KGFR protein and mRNA were localized in the epithelium in 72% of cases. Assessment of the proliferative activity of cholesteatoma using the labeling index for Ki-67 showed a significantly higher Ki-67 labeling index (66%) in KGF+/KGFR+ cases than other cases. There was a significant correlation between KGF+/KGFR+ expression and recurrence. Our results indicate the possible involvement of both KGF and KGFR in enhanced epithelial cell proliferative activity and recurrence of cholesteatoma.

Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1.

Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.