Probes for INS

The hormone fibroblast growth factor 23 (FGF23) is secreted from bone and is involved in phosphorus (P) metabolism. FGF23 mainly binds the FGF receptor, which interacts with αKlotho in the kidney or parathyroid and regulates Na-dependent phosphate co-transporter type IIa (NaPi-IIa) and type IIc (NaPi-IIc) expression, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity, and parathyroid hormone (PTH) secretion. In this study, we utilized hemi-nephrectomized rats fed a high-P diet (HP Nx), rats subjected to a partial nephrectomy (PN) and rats with doxorubicin-induced renal failure (DXR) as chronic kidney disease (CKD) animal models and analyzed the P metabolism and FGF23 expression in the kidneys in each CKD model. We cultured HK2 cells with a high level of P, 1,25(OH)2D3 or transforming growth factor-β1 (TGFβ1) to investigate the FGF23 expression mechanism. In both the HP Nx and PN rats, the blood FGF23 and PTH levels were increased. However, the 1,25(OH)2D3 level was increased in the HP Nx rats and decreased in the PN rats. In all three animal models, the mRNA expression of αKlotho, NaPi-IIa and NaPi-IIc was decreased, and the mRNA expression of TGFβ1, collagen1a1, osteopontin and FGF23 was elevated in the kidney. FGF23 protein and mRNA were expressed at high levels in the extended tubule epithelium, which was an osteopontin-positive region in the HP and PN rats. FGF23 and osteopontin mRNAs were expressed in HK2 cells incubated with TGFβ1; however, these levels were not altered in HK2 cells incubated with 1,25(OH)2D3 and high P levels in vitro. Altogether, FGF23 is expressed in the kidneys in CKD model rats. Following stimulation with TGFβ1, the injured renal tubular epithelial cells are strongly suspected to express both FGF23 and osteopontin. FGF23 produced in the kidney might contribute to P metabolism in subjects with CKD.

The CXCL12-CXCR4 axis is proposed to mediate metastasis formation. In this study, we examined CXCL12, CXCR4 and the relative CXCL12-CXCR4 expression as prognostic factors in two cohorts of colon cancer patients. Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to study CXCR4, CXCL12 and relative CXCL12-CXCR4 expression in tissue microarrays. Our study included totally 596 patients, 290 in cohort 1 and 306 in cohort 2. For tumour, node, metastasis (TNM) stage III, low nuclear expression of CXCR4 was a positive prognostic factor for 5-year disease-free survival (DFS) in cohort 1 (P = 0.007) and cohort 2 (P = 0.023). In multivariate analysis for stage III, nuclear expression of CXCR4 in cohort 1 was confirmed as a prognostic factor for DFS (hazard ratio (HR), 0.27; 95 % CI, 0.09 to 0.77). For TNM stage III, high cytoplasmic expression of CXCL12 was associated with better 5-year DFS in both cohorts (P = 0.006 and P = 0.006, respectively). We further validated the positive prognostic value of CXCL12 expression for 5-year DFS in stage III with ISH (P = 0.022). For TNM stage III, the relative CXCL12-CXCR4 expression (CXCL12 > CXCR4 vs CXCL12 = CXCR4 vs CXCL12 < CXCR4) was a prognostic factor for 5-year DFS in cohort 1 (92 % vs 46 % vs 31 %, respectively; P < 0.001) and cohort 2 (92 % vs 66 % vs 30 %, respectively; P = 0.006). In conclusion, CXCL12 and relative CXCL12-CXCR4 expression are independent prognostic factors for 5-year DFS in TNM stage III colon cancer.

Fibroblast growth factor 23 (FGF23) secreted by osteocytes is a circulating factor essential for phosphate homeostasis. High plasma FGF23 levels are associated with cardiovascular complications and mortality. Increases of plasma FGF23 in uremia antedate high levels of phosphate, suggesting a disrupted feedback regulatory loop or an extra-skeletal source of this phosphatonin. Since induction of FGF23 expression in injured organs has been reported we decided to examine the regulation of FGF23 gene and protein expressions in the kidney and whether kidney-derived FGF23 contributes to the high plasma levels of FGF23 in uremia. FGF23 mRNA was not detected in normal kidneys, but was clearly demonstrated in injured kidneys, already after four hours in obstructive nephropathy and at 8 weeks in the remnant kidney of 5/6 nephrectomized rats. No renal extraction was found in uremic rats in contrast to normal rats. Removal of the remnant kidney had no effect on plasma FGF23 levels. Well-known regulators of FGF23 expression in bone, such as parathyroid hormone, calcitriol, and inhibition of the FGF receptor by PD173074, had no impact on kidney expression of FGF23. Thus, the only direct contribution of the injured kidney to circulating FGF23 levels in uremia appears to be reduced renal extraction of bone-derived FGF23. Kidney-derived FGF23 does not generate high plasma FGF23 levels in uremia and is regulated differently than the corresponding regulation of FGF23 gene expression in bone.

High-grade serous ovarian cancers (HGSOC) have been subdivided into molecular subtypes. The mesenchymal HGSOC subgroup, defined by stromal-related gene signatures, is invariably associated with poor patient survival. We demonstrate that stroma exerts a key function in mesenchymal HGSOC. We highlight stromal heterogeneity in HGSOC by identifying four subsets of carcinoma-associated fibroblasts (CAF-S1-4). Mesenchymal HGSOC show high content in CAF-S1 fibroblasts, which exhibit immunosuppressive functions by increasing attraction, survival, and differentiation of CD25+FOXP3+ T lymphocytes. The beta isoform of the CXCL12 chemokine (CXCL12β) specifically accumulates in the immunosuppressive CAF-S1 subset through a miR-141/200a dependent-mechanism. Moreover, CXCL12β expression in CAF-S1 cells plays a crucial role in CAF-S1 immunosuppressive activity and is a reliable prognosis factor in HGSOC, in contrast to CXCL12α. Thus, our data highlight the differential regulation of the CXCL12α and CXCL12β isoforms in HGSOC, and reveal a CXCL12β-associated stromal heterogeneity and immunosuppressive environment in mesenchymal HGSOC.

The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.

Peripheral enhanced complement activation has long been considered as one of the major pathogenesis of immune thrombocytopenia. Impaired bone marrow microenvironment, especially the dysfunction of mesenchymal stem cells, has been observed in patients with immune thrombocytopenia. However, the potential role of the complement system involved in impaired bone marrow microenvironment remains poorly understood. Here, bone marrow samples of patients were divided into the MSC-ITP-C+ and MSC-ITP-C- groups based on the deposition of the complement components on the surfaces of mesenchymal stem cells. Reduced and dysfunctional mesenchymal stem cells, characterized by reduced proliferation capacity, increased apoptosis as well as abnormal secretion of interleukin-1β and C-X-C motif chemokine ligand 12, were observed in the MSC-ITP-C+ group. In vitro treatment with all-trans retinoic acid quantitatively and functionally improved MSC-ITP-C+ by upregulating DNA hypermethylation of the interleukin-1β promoter. In vivo studies showed that all-trans retinoic acid could rescue the impaired mesenchymal stem cells to support the thrombopoietic niche in both patients and the murine model with immune thrombocytopenia. Taken together, these results indicate that deficient mesenchymal stem cells mediated by the complement-IL-1β loop play a role in the pathogenesis of immune thrombocytopenia. All-trans retinoic acid represents a promising therapeutic approach in patients with immune thrombocytopenia by repairing impaired mesenchymal stem cells.

Phosphaturic mesenchymal tumors typically cause paraneoplastic osteomalacia, chiefly as a result of FGF23 secretion. In a prior study, we identified FN1-FGFR1 fusion in 9 of 15 phosphaturic mesenchymal tumors. In this study, a total of 66 phosphaturic mesenchymal tumors and 7 tumors resembling phosphaturic mesenchymal tumor but without known phosphaturia were studied. A novel FN1-FGF1 fusion gene was identified in two cases without FN1-FGFR1 fusion by RNA sequencing and cross-validated with direct sequencing and western blot. Fluorescence in situ hybridization analyses revealed FN1-FGFR1 fusion in 16 of 39 (41%) phosphaturic mesenchymal tumors and identified an additional case with FN1-FGF1 fusion. The two fusion genes were mutually exclusive. Combined with previous data, the overall prevalence of FN1-FGFR1 and FN1-FGF1 fusions was 42% (21/50) and 6% (3/50), respectively. FGFR1 immunohistochemistry was positive in 82% (45/55) of phosphaturic mesenchymal tumors regardless of fusion status. By contrast, 121 cases of potential morphologic mimics (belonging to 13 tumor types) rarely expressed FGFR1, the main exceptions being solitary fibrous tumors (positive in 40%), chondroblastomas (40%), and giant cell tumors of bone (38%), suggesting a possible role for FGFR1 immunohistochemistry in the diagnosis of phosphaturic mesenchymal tumor. With the exception of one case reported in our prior study, none of the remaining tumors resembling phosphaturic mesenchymal tumor had either fusion type or expressed significant FGFR1. Our findings provide insight into possible mechanisms underlying the pathogenesis of phosphaturic mesenchymal tumor and imply a central role of the FGF1-FGFR1 signaling pathway. The novel FN1-FGF1 protein is expected to be secreted and serves as a ligand that binds and activates FGFR1 to achieve an autocrine loop. Further study is required to determine the functions of these fusion proteins.