High CXCL12 Expression Research Articles

Instructive Interaction between Myelodysplastic Hematopoiesis and the Bone Marrow Microenvironment at Single Cell Level

Myelodysplastic Neoplasms (MDS) are hypothesized to re-model their bone marrow (BM) microenvironment to re-inforce conditions for their propagation. In this study, we investigated interactions between MDS cells and the BM niche at single cell level in a MDS patient-derived xenograft model (PDX) as well as primary human MDS and healthy (HY) bone marrow (BM samples in order to identify cues of disturbed crosstalk between MDS hematopoiesis and the BM microenvironment. In the PDX model, we analyzed >13,000 cells of n=15 different murine niche cell populations after long-term (>24 weeks) exposure to n=3 MDS patient samples versus n=2 age-matched HY human grafts. Subsequently, we analyzed over 24,000 primary human BM cells enriched for the non-hematopoietic compartment by using whole bone fragments from n=8 MDS patients and n=7 HY age-matched donors. All cells were analyzed with combined 10x single cell RNA sequencing and human “Cellular Indexing of the Transcriptome and Epitopes by Sequencing” (CITE Seq). Results were validated by flow cytometry and gene expression analyses. Mesenchymal cell (MSC) subpopulations from PDX mice exposed to MDS versus HY hematopoiesis could be subdivided into distinct “Cxcl12+ abundant reticular” (CAR) cells with osteogenic and adipogenic differentiation trajectories. Various CAR cell populations displayed an increased frequency in MDS transplanted mice as compared to PDX transplanted with HY grafts ( Panel A). Upon contact with hematopoietic MDS cells, CAR cell populations overexpressed numerous genes involved in extracellular matrix reorganization and hematopoietic supporting factors, such as Cxcl12, Il7, Lox and Loxl1 as compared to mice bearing HY grafts. In situ, BM cells from MDS patients showed highly heterogeneous MSC subpopulations with similar differentiation trajectories to the ones observed in our PDX models on a patient-individual level. We detected two non-hematopoietic cell populations, designated as endothelial cells (ECs) and MSCs characterized by expression of CXCL12, IGFBP5 or COL1A2 and quantification with epitope sequencing (CITE Seq) with mutually exclusive detection of CD31 and CD271 expression (n=581 cells in total, Panel B). In n=3 MDS patients, we identified an MDS-specific MSC subpopulation with high IGFPB4 and CXCL12 expression as well as overexpression of KITLG and IL7. This suggests that this altered specialized secretory mesenchymal state is present in a subset of MDS patients and may support the malignant hematopoietic clones by overexpression of key hematopoietic factors. Interactome analyses using spatial receptor:ligand bioinformatic tools such as CellphoneDB, RNAMagnet and omnipath further confirmed that stromal cells alter their cellular profile and secretory behavior in response to MDS hematopoiesis. In conclusion, we show that murine BM stroma is re-programmed by exposure to human MDS xenografts to upregulate extracellular matrix (ECM) re-modeling and HSC supporting factors. In native human MDS BM in vivo, rare primary mesenchymal cells are re-programmed to increase hematopoietic support in a patient-individual manner pointing to altered MSC subpopulations with increased growth factor expression profiles in a subgroup of MDS patients.

High density of CXCL12-positive immune cell infiltration predicts chemosensitivity and recurrence-free survival in ovarian carcinoma

BackgroundOvarian carcinoma is the most lethal gynecologic malignancy because of its late diagnosis, extremely high recurrence rate, and limited curative treatment options. In clinical practice, high-grade serous carcinoma (HGSC) predominates due to its frequency, high aggressiveness, and rapid development of drug resistance. Recent evidence suggests that CXCL12 is an important immunological factor in ovarian cancer progression. Therefore, we investigated the predictive and prognostic significance of the expression of this chemokine in tumor and immune cells in patients with HGSC.MethodsWe studied a cohort of 47 primary high-grade serous ovarian carcinomas and their associated recurrences. A tissue microarray was constructed to evaluate the CXCL12 immunostained tumor tissue. CXCL12 expression was evaluated and statistically analyzed to correlate clinicopathologic data, overall survival, and recurrence-free survival.ResultsA high proportion of CXCL12 + positive immune cells in primary ovarian serous carcinoma correlated significantly with chemosensitivity (p = 0.005), overall survival (p = 0.021), and longer recurrence-free survival (p = 0.038). In recurrent disease, high expression of CXCL12 was also correlated with better overall survival (p = 0.040). Univariate and multivariate analysis revealed that high CXCL12 + tumor-infiltrating immune cells (TICs) (HR 0.99, p = 0.042, HR 0.99, p = 0.023, respectively) and combined CXCL12 + /CD66b + infiltration (HR 0.15, p = 0.001, HR 0.13, p = 0.001, respectively) are independent favorable predictive markers for recurrence-free survival.ConclusionA high density of CXCL12 + TICs predicts a good response to chemotherapy, leading to a better overall survival and a longer recurrence-free interval. Moreover, with concomitant high CXCL12/CD66b TIC density, it is an independent favorable predictor of recurrence-free survival in patients with ovarian carcinoma.

Exploring potential targets of HPV&BC based on network pharmacology and urine proteomics

BackgroundBladder cancer (BC) caused by Human papillomavirus (HPV) infection remains a complex public health problem in developing countries. Although the HPV vaccine effectively prevents HPV infection, it does not benefit patients with BC who already have HPV. MethodsFirstly, the differential genes of HPV-related BC patients were screened by transcriptomics, and then the prognostic and clinical characteristics of the differential genes were analyzed to screen out the valuable protein signatures. Furthermore, the compound components and targets of Astragali Radix (AR) were analyzed by network pharmacology, and the intersection targets of drug components and HPV_BC were screened out for pathway analysis. In addition, the binding ability of the compound to the Astragali-HPV_BC target was verified by molecular docking and virtual simulation. Finally, to identify potential targets in BC patients through urine proteomics and in vitro experiments. ResultsEleven HPV_BC-related protein signatures were screened out, among which high expression of EGFR, CTNNB1, MYC, GSTM1, MMP9, CXCR4, NOTCH1, JUN, CXCL12, and KRT14 had a poor prognosis, while low expression of CASP3 had a poor prognosis. In the analysis of clinical characteristics, it was found that high-risk scores, EGFR, MMP9, CXCR4, JUN, and CXCL12 tended to have higher T stage, pathological stage, and grade. Pharmacological and molecular docking analysis identified a natural component of AR (Quercetin) and it corresponding core targets (EGFR). The OB of the natural component was 46.43, and the DL was 0.28, respectively. In addition, EGFR-Quercetin has high affinity. Urine proteomics and RT-PCR showed that EGFR was expressed explicitly in BC patients. Mechanism analysis revealed that AR component targets might affect HPV_BC patients through Proteoglycans in the cancer pathway. ConclusionAR can target EGFR through its active component (Quercetin), and has a therapeutic effect on HPV_BC patients.

Integrative multi-omics analyses unravel the immunological implication and prognostic significance of CXCL12 in breast cancer.

CXCL12 is a vital factor in physiological and pathological processes, by inducing migration of multiple cells. We aimed to comprehensively detect the role of CXCL12 in breast cancer, and explore novel CXCL12-related biomarkers through integrative multi-omics analyses to build a powerful prognostic model for breast cancer patients. Immunohistochemistry analysis of the tissue microarray was performed to evaluate the correlation between CXCL12 expression levels and breast cancer patient outcomes. Combined single-nucleus and spatial transcriptomics data was used to uncover the expression distribution of CXCL12 in breast cancer microenvironment. CXCL12-related genes were identified by WGCNA analysis. Univariate Cox and LASSO regression analyses were then conducted to screen prognostic genes from above CXCL12-related genes, followed by the construction of the CXCL12-related prognostic signature, identification of risk groups, and external validation of the prognostic signature. Analyses of biological function, mutation landscape, immune checkpoint genes and immune cells, were performed to further reveal the differences between high/low-risk groups. Paired single-cell RNA-seq and bulk RNA-seq were analyzed to further disclose the association between the risk score and the complex tumor immune microenvironment. To screen potential therapeutic agents for breast cancer patients, analyses of gene-drug correlation and sensitivity to immunotherapy were conducted. High expression of CXCL12 was linked with a prolonged survival in breast cancer. A total of 402 genes were identified by WGCNA analysis and 11 genes, covering VAT1L, TMEM92, SDC1, RORB, PCSK9, NRN1, NACAD, JPH3, GJA1, BMP8B and ADAMTS2, were screened as the candidate prognostic genes. Next, the prognostic signature was built and validated using these genes to predict the outcomes of breast cancers. The high-risk group patients exhibited significantly inferior prognoses. The combination of the risk score and tumor mutational burden (TMB) had remarkably improved performance in predicting patient outcomes. Besides, high-risk group patients showed higher infiltration of M2-like macrophages. Finally, several potential anticancer drugs were identified. The high-risk group patients were more sensitive to immunotherapy but resistant to docetaxel. CXCL12 has important immunological implication and prognostic significance in breast cancer. The CXCL12-related prognostic model could well predict the prognosis and treatment response of breast cancers.

Prognostic Value of CXCL12 in Non-Small Cell Lung Cancer Patients Undergoing Tumor Resection

Adjuvant chemotherapy is commonly indicated in lung cancer patients undergoing surgical therapy because tumor recurrence is frequent. A biomarker that can predict tumor recurrence in the postoperative period is currently unavailable. CXCR4 receptor and its ligand CXCL12 play important roles in metastasis. This study investigated the value of tumor CXCL12 expression to predict prognosis and indicate adjuvant chemotherapy in non-small cell lung cancer patients. This study enrolled 82 non-small cell lung cancer patients. The expression of CXCL12 was evaluated by immunohistochemistry. The degree of CXCL12 expression was assessed using the Allred score system. Among all subjects, the progression-free survival and overall survival were significantly prolonged in cancer patients with low tumor expression of CXCL12 compared to patients with high tumor expression. Multivariate analysis showed that the increased level of CXCL12 is a significant predictor of progression-free survival and overall survival in NSCLC patients. Among subjects with high tumor CXCL12 expression, progression-free survival and overall survival were significantly improved in patients treated with adjuvant chemotherapy compared to untreated patients. These results suggest the potential value of tumor CXCL12 expression as a marker to predict prognosis and to indicate adjuvant chemotherapy after surgical tumor resection in non-small cell lung cancer patients.

Loss of bone morphogenetic protein signaling in fibroblasts results in CXCL12-driven serrated polyp development

Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling–CXCL12 interaction could have a role in the etiology of serrated polyp formation.

Smooth muscle cell specific ablation of CXCL12 downregulates endothelial CXCR7 leading to defective coronary arteries and cardiac hypertrophy

Abstract Aims The chemokine CXCL12 plays a fundamental role in cardiovascular development, cell trafficking, and myocardial repair. Human genome-wide association studies even have identified novel loci downstream of the CXCL12 gene locus associated with coronary artery disease and myocardial infarction. Nevertheless, cell and tissue specific effects of CXCL12 are barely understood. Since we detected high expression of CXCL12 in smooth muscle (SM) cells, we generated a SM22-alpha-Cre driven mouse model to ablate CXCL12 (SM-CXCL12−/−). Methods and results SM-CXCL12−/− mice revealed high embryonic lethality (50%) with developmental defects, including aberrant topology of coronary arteries. Postnatally, SM-CXCL12−/− mice developed severe cardiac hypertrophy associated with fibrosis, apoptotic cell death, impaired heart function, and severe coronary vascular defects characterized by thinned and dilated arteries. Transcriptome analyses showed specific upregulation of pathways associated with hypertrophic cardiomyopathy, collagen protein network, heart-related proteoglycans, and downregulation of the M2 macrophage modulators. CXCL12 mutants showed endothelial downregulation of the CXCL12 co-receptor CXCR7. Treatment of SM-CXCL12−/− mice with the CXCR7 agonist TC14012 attenuated cardiac hypertrophy associated with increased pERK signaling. Conclusion Our data suggest a critical role of smooth muscle-specific CXCL12 in arterial development, vessel maturation, and cardiac hypertrophy. Pharmacological stimulation of CXCR7 might be a promising target to attenuate adverse hypertrophic remodeling. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): FWF-Austria

243 PROGNOSTIC IMPACT OF CXCR7 AND CXCL12 EXPRESSION IN PATIENTS WITH ESOPHAGEAL ADENOCARCINOMA

Abstract Chemokines are major regulators of cell trafficking and adhesion. The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, have recently been reported as biomarkers in various cancers, including esophageal squamous cell carcinoma; however, there are few studies of these chemokines in esophageal adenocarcinoma (EAC). In this study, we investigated the relationship between expression of CXCL12, CXCR4 and CXCR7, and prognosis in patients with EAC. Methods This study examined 55 patients with EAC who were treated in Toronto General Hospital from 2001 to 2010. Tissue microarray immunohistochemistry was used to evaluate the expression of CXCL12, CXCR4 and CXCR7. Evaluation of immunohistochemistry was performed by a pathologist without knowledge of patients’ information and scored based on a semiquantitative scoring system. The average score from multiple cancer tissues on the microarray was utilized and patients were divided into high or low expression groups using the median score as a cutoff point. These results were compared with the patients’ clinicopathological features and survival. Results The score of CXCR7 was positively correlated with that of CXCL12 (r = 0.3154). High CXCR7 expression was significantly associated with lymphatic invasion (present vs absent, P = 0.005), higher number of lymph node metastases (pN0–1 vs pN2–3, P = 0.0014) and TNM stage (Stage I-II vs III-IV, P = 0.0168). Patients with high CXCR7 (n = 23) expression was associated with worse overall (OS) and disease-free survival (DFS) (P = 0.0221, 0.0090, respectively), and patients with high CXCL12 (n = 24) tended to have worse OS and DFS (P = 0.1091, 0.1477, respectively). High expression of both CXCR7 and CXCL12 was an independent prognostic factor for DFS on multivariable analysis (HR0.3, 95%CI: 0.1–0.8, P = 0.0115). Conclusion High CXCR7 expression was associated with poor prognosis in patients with EAC, and high expression of CXCR7 and its ligand CXCL12, had a stronger association on prognosis. Further study of this potential biomarker using whole tissue samples and larger sample size is warranted.

Bioinformatics integrated analysis to investigate candidate biomarkers and associated metabolites in osteosarcoma

BackgroundThis study hoped to explore the potential biomarkers and associated metabolites during osteosarcoma (OS) progression based on bioinformatics integrated analysis.MethodsGene expression profiles of GSE28424, including 19 human OS cell lines (OS group) and 4 human normal long bone tissue samples (control group), were downloaded. The differentially expressed genes (DEGs) in OS vs. control were investigated. The enrichment investigation was performed based on DEGs, followed by protein–protein interaction network analysis. Then, the feature genes associated with OS were explored, followed by survival analysis to reveal prognostic genes. The qRT-PCR assay was performed to test the expression of these genes. Finally, the OS-associated metabolites and disease-metabolic network were further investigated.ResultsTotally, 357 DEGs were revealed between the OS vs. control groups. These DEGs, such as CXCL12, were mainly involved in functions like leukocyte migration. Then, totally, 38 feature genes were explored, of which 8 genes showed significant associations with the survival of patients. High expression of CXCL12, CEBPA, SPARCL1, CAT, TUBA1A, and ALDH1A1 was associated with longer survival time, while high expression of CFLAR and STC2 was associated with poor survival. Finally, a disease-metabolic network was constructed with 25 nodes including two disease-associated metabolites cyclophosphamide and bisphenol A (BPA). BPA showed interactions with multiple prognosis-related genes, such as CXCL12 and STC2.ConclusionWe identified 8 prognosis-related genes in OS. CXCL12 might participate in OS progression via leukocyte migration function. BPA might be an important metabolite interacting with multiple prognosis-related genes.

The CXCL12/CXCR4 Pathway As a Potential Target of Tipifarnib in Acute Myeloid Leukemia and Myelodysplastic Syndromes

BackgroundCXCL12 is a chemokine that is essential for homing of myeloid cells in the bone marrow (BM). It has been postulated that a CXCL12 receptor present in myeloid cells, CXCR4, signals in part through HRAS, a protein that is uniquely dependent on farnesylation for activity. Tipifarnib is a potent and selective inhibitor of the enzyme farnesyltransferase (FT) that had been shown to provide clinical benefit in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS); however, no molecular target that could explain the activity of tipifarnib in AML and MDS has been identified. Based on initial observations in T cell lymphoma (Witzig EHA 2017), we investigated a role for CXCL12 and myeloid cell homing in BM as biomarkers of the activity of tipifarnib in AML and MDS studies.MethodsCTEP20 was a phase 2, multicenter, open-label study that investigated the efficacy and safety of tipifarnib in 158 older adults with previously untreated, poor-risk AML. AML301 was a phase 3 (N=457), multicenter, open-label study that evaluated the efficacy and safety of tipifarnib compared with best supportive care (BSC), including hydroxyurea, as first-line therapy in elderly patients with newly diagnosed, de novo, or secondary AML. INT28 was a multicenter phase 2 study that evaluated tipifarnib in 82 pts with poor-risk MDS. Global gene expression data generated using the Affymetrix U133A gene chip from 34 pretreatment BM samples collected from CTEP20 pts (GSE8970) was analyzed with respect to pretreatment hematological parameters and study outcomes. The Kaplan-Meier method was employed to estimate survival and progression free survival (PFS) and the analysis of prognostic variables. Clinical trial information: NCT00027872, NCT00093990, NCT00050154.ResultsNine complete responses (CR) and 13 best responses of progression of disease (PD) were observed in 34 pts with available BM gene expression data in study CTEP20. Pts in the 3rd (upper) tertile of CXCL12 expression experienced 6 CRs and 2 PDs whereas 1 CR and 8 PDs were observed in the 1st (lower) tertile (p=0.015). BM homing of AML blasts correlated with CXCL12 expression: Median percentage of BM blasts (BMB) and peripheral blasts (PB) were, respectively, 45% and 0% in the upper CXCL12 tertile, and 30% and 26.5% in the lower tertile (p<0.001) (Figure, A). Pts with high tumor burden in BM (BMB blasts>40%) and low circulating blasts (PB<1%) (N=5) had high CXCL12 expression in BM (3423 vs 742 counts, P<0.001, N=27) and experienced prolonged clinical benefit, with 5 CRs and a median PFS of 431 days (431 vs 35 days, HR=0.23, P<0.001, N=27) (Figure, B). The effect of BM homing was then investigated in the phase 3 AML301 trial of tipifarnib vs BSC. While the overall study (N=457) failed to identify an increased efficacy of tipifarnib compared with BSC (median survival of 109 vs 107 days, hazards ratio, HR=1.02, ns), median survival approximately doubled in the tipifarnib am compared with BSC in the subset of patients with high BMB (>40%) and low PB (<10%) while no effect was observed in the control arm (201 vs 109 days, HR=0.58, p=0.02, N=82) (Figure, B). Since CXCL12 is also a potent chemotactic factor for normal neutrophils and syndromes of hyperactivation of the CXCL12/CXCR4 pathway present with neutropenia, we investigated whether neutropenia could be a surrogate of tipifarnib activity. In AML301 pts with WBC counts>LLN (no myelosuppression), increasing neutropenia correlated with increasing clinical benefit from tipifarnib that was due in part to a poorer prognosis of BSC in neutropenic pts (113 vs 80 days median OS, HR=0.81, p=0.036, N=109, for pts with baseline neutrophils<1 giga/L). Finally, we tested the effect of neutropenia on the activity of tipifarnib in INT28. Sixty seven of the 82 MDS pts were transfusion dependent (TD) at study entry. Overall conversion to transfusion independence (TI) and response rate (ORR: CR, CRp, PR) were respectively 16% and 7.5% in the TD pts. Pts with >1 giga/L neutrophils at study entry experienced 5% TI and no responses (N=37) while 30% TI, 2 CRs, 2 CRp and 1 PR were observed in the subset of neutropenic pts.ConclusionCXCL12 expression and BM homing constitute potential biomarkers of the activity of tipifarnib in AML and MDS supporting the hypothesis that the CXCL12 pathway is a potential target of FT inhibitors. [Display omitted] DisclosuresGualberto:Kura Oncology: Employment, Equity Ownership, Other: Chief Medical Officer, Patents & Royalties. Scholz:Kura Oncology: Employment, Equity Ownership, Patents & Royalties. Janes:Wellspring Biosciences: Employment. Kessler:Kura Oncology: Employment. Raza:Kura Oncology: Research Funding; Syros Pharmaceuticals: Research Funding; Novartis: Speakers Bureau; Celgene Inc.: Research Funding; Genoptix: Speakers Bureau; Onconova Therapeutics: Research Funding, Speakers Bureau; Janssen R&D: Research Funding.

The CXCL12/CXCR4 Pathway As a Potential Target of Tipifarnib: Preliminary Results from an Open-Label, Phase II Study in Relapsed or Refractory Peripheral T-Cell Lymphoma

BackgroundTipifarnib is a potent and selective inhibitor of the enzyme farnesyltransferase (FT). FT catalyzes post-translational attachment of farnesyl groups required for localization of signaling molecules to the inner cell membrane. CXCL12 is a chemokine that is essential for T cell homing to lymphoid organs and the bone marrow, and for the maintenance of immune cell progenitors. CXCL12 has been postulated to signal in part through HRAS, a signaling protein that is uniquely dependent on farnesylation for activity. We report herein the identification of CXCL12 as a marker of sensitivity to FT inhibition by tipifarnib in PTCL patients (pts).MethodsThis Phase 2 study is a multi-institutional, single-arm, open-label, two-stage (11+7) study designed to determine the efficacy, safety and biomarker correlates of tipifarnib in pts with relapsed/refractory (R/R) PTCL. Based on initial findings, the study has been extended to include a cohort of angioimmunoblastic T-cell lymphoma (AITL) pts (N=12). Pts with R/R PTCL after prior cytotoxic systemic therapy, aged ≥ 18 years old, and with a performance status of 0-2 were eligible. Pts enrolled in stages 1 and 2 of the study were treated with tipifarnib 600 mg administered orally twice daily on days 1-7 and 15-21 of 28-day treatment cycles until progression of disease or unacceptable toxicity. Pts enrolled in the AITL cohort are treated with tipifarnib 300 mg administered orally twice daily on days 1-21 of 28-day treatment cycles until progression of disease or unacceptable toxicity. The primary endpoint of the study was overall response rate. Biomarker studies included gene expression profiling of pre-treatment tumor biopsies by RNASeq and DNA next-generation sequencing (NGS). Clinical trial information: NCT02464228.ResultsAt data cut-off (July 2017), 19 pts (3 AITL, 1 ALK- ALCL, 15 PTCL-NOS) were treated with tipifarnib. The most common treatment-related AE (grade ≥ 3) was myelosuppression, including neutropenia (83%), thrombocytopenia (61%) and anemia (33%). Of 18 evaluable pts at data cut-off, 3 pts achieved a partial response (PR, 2 AITL; 1 PTCL-NOS) and 3 pts experienced stable disease (SD) with -38%, -45% and -47% tumor size reductions. One additional pt experienced a -63% tumor size reduction and splenomegaly. Median duration of response (PR/SD) was 4 months with treatment ongoing in 2 subjects at cycles 10 and 15. Tumor gene expression data were available for 12 pts. Five of those pts experienced tumor size reductions and prolonged (>6 mo) median time to progression that was associated with elevated CXCL12 expression (p<0.001). Two pts with AITL histology expressed high levels of CXCL12 and experienced PRs. One of the AITL pts overexpressed CXCL13 and moderate VCAM1 while the other one overexpressed VCAM1 and other vascular markers (PDGFRA, ANGPT1, TEK), pointing towards two known mechanisms of CXCL12 upregulation (Mir23A downregulation by CXCL13 in stroma cells and vascular endothelial cell production of CXCL12). In contrast, the 7 pts with tumors of low CXCL12 expression experienced a best response of disease progression (PD). Low CXCL12 expression and PD were significantly associated (p=0.04, P<0.001, respectively) with the presence of single nucleotide variations (SNV) in the 3'UTR of the CXCL12 gene, including rs2839695 and two novel SNVs identified by NGS. Studies investigating the interaction between plasma CXCL12 and the activity of tipifarnib as well as the prognostic value of CXCL12 expression and gene variation in tumor series of PTCL patients treated with standard therapy are ongoing. Enrollment continues in the AITL cohort.ConclusionEncouraging activity of tipifarnib was observed in PTCL pts, particularly in those with tumors of AITL histology and high CXCL12 expression. DisclosuresWitzig:Acerta: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kura: Research Funding; Novartis: Research Funding. Advani:Sutro: Consultancy; Regeneron: Research Funding; Spectrum: Consultancy; Nanostring: Consultancy; Agensys: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pharmacyclics: Consultancy; Janssen: Research Funding; Kura: Research Funding; Infinity: Research Funding; Celgene: Research Funding; Gilead: Consultancy; Genentech: Research Funding; Seattle Genetics: Research Funding; Juno Therapeutics: Consultancy; Merck: Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; FortySeven: Research Funding; Bayer Healthcare Pharmaceuticals: Research Funding; Cell Medica: Research Funding. Foss:celgene: Honoraria; immune design: Research Funding; spectrum: Honoraria, Speakers Bureau; Eisai: Honoraria; seattle genetics: Speakers Bureau. Mondejar:Kura Oncology: Research Funding. Piris:Kura Oncology: Research Funding. Bolognese:Kura Oncology: Consultancy. Burrows:Kura Oncology: Employment, Equity Ownership. Kessler:Kura Oncology: Employment. Bracken:Kura Oncology: Employment. Mishra:Kura Oncology: Employment, Equity Ownership. Scholz:Kura Oncology: Employment, Equity Ownership, Patents & Royalties. Gualberto:Kura Oncology: Employment, Equity Ownership, Other: Chief Medical Officer, Patents & Royalties.

Smooth Muscle Specific Ablation of CXCL12 in Mice Downregulates CXCR7 Associated with Defective Coronary Arteries and Cardiac Hypertrophy.

The chemokine CXCL12 plays a fundamental role in cardiovascular development, cell trafficking, and myocardial repair. Human genome-wide association studies even have identified novel loci downstream of the CXCL12 gene locus associated with coronary artery disease and myocardial infarction. Nevertheless, cell and tissue specific effects of CXCL12 are barely understood. Since we detected high expression of CXCL12 in smooth muscle (SM) cells, we generated a SM22-alpha-Cre driven mouse model to ablate CXCL12 (SM-CXCL12−/−). SM-CXCL12−/− mice revealed high embryonic lethality (50%) with developmental defects, including aberrant topology of coronary arteries. Postnatally, SM-CXCL12−/− mice developed severe cardiac hypertrophy associated with fibrosis, apoptotic cell death, impaired heart function, and severe coronary vascular defects characterized by thinned and dilated arteries. Transcriptome analyses showed specific upregulation of pathways associated with hypertrophic cardiomyopathy, collagen protein network, heart-related proteoglycans, and downregulation of the M2 macrophage modulators. CXCL12 mutants showed endothelial downregulation of the CXCL12 co-receptor CXCR7. Treatment of SM-CXCL12−/− mice with the CXCR7 agonist TC14012 attenuated cardiac hypertrophy associated with increased pERK signaling. Our data suggest a critical role of smooth muscle-specific CXCL12 in arterial development, vessel maturation, and cardiac hypertrophy. Pharmacological stimulation of CXCR7 might be a promising target to attenuate adverse hypertrophic remodeling.

Identification of CXCL12‐abundant reticular cells in human adult bone marrow

SummaryA population of mesenchymal stem cells, termed CXC chemokine ligand (CXCL)12‐abundant reticular (CAR) cells or leptin receptor‐expressing cells, are the major cellular component of niches for haematopoietic stem cells (HSCs) in murine bone marrow. CAR cells are characterized by several salient features, including much higher expression of CXCL12, stem cell factor (SCF), forkhead box C1 (FOXC1) and early B‐cell factor 3 (EBF3), which are essential for HSC maintenance, than other cells. However, the human counterpart of CAR cells has not been fully described. Here, we show the presence of cells expressing much higher CXCL12 than other cells in human adult bone marrow using a flow cytometry‐based in situ technique that enables high‐throughput detection of mRNA at single‐cell resolution. Most CXCL12hi cells expressed high levels of SCF, FOXC1 and EBF3 and had the potential to differentiate into adipocytes and osteoblasts. Histologically, the nuclei of CXCL12hi cells were identified and quantified by EBF3 expression in fixed marrow sections. CXCL12hi cells sorted from residual bone marrow aspirates of chronic myeloid leukaemia patients expressed reduced levels of CXCL12, SCF, FOXC1 and EBF3 in correlation with increased leukaemic burden. Together, we identified the human counterpart of CAR cells, enabling the evaluation of their alterations in various haematological disorders by flow cytometric and histological analyses.

Prognostic Impact of CXCR7 and CXCL12 Expression in Patients with Esophageal Adenocarcinoma.

Chemokines are major regulators of cell trafficking and adhesion. The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, have been reported as biomarkers in various cancers, including esophageal cancer; however, there are few studies in esophageal adenocarcinoma (EAC). In this study, we investigated the relationship between expression of CXCL12, CXCR4, and CXCR7, and prognosis in patients with EAC. This study examined 55 patients with EAC who were treated in Toronto General Hospital from 2001 to 2010. Tissue microarray immunohistochemistry was used to evaluate the expression of CXCL12, CXCR4, and CXCR7. Evaluation of immunohistochemistry was performed by a pathologist without knowledge of patients' information and results were compared with the patients' clinicopathological features and survival. High CXCR7 expression was significantly associated with lymphatic invasion (present vs absent, P = 0.005) and higher number of lymph node metastases (pN0-1 vs pN2-3, P = 0.0014). Patients with high CXCR7 expression (n = 23) were associated with worse overall (OS) and disease-free survival (DFS) (P = 0.0221, P = 0.0090, respectively), and patients with high CXCL12 (n = 24) tended to have worse OS and DFS (P = 0.1091, P = 0.1477, respectively). High expression of both CXCR7 and CXCL12 was an independent prognostic factor for OS and DFS on multivariate analysis (HR = 0.3, 95% CI: 0.1-0.9, P = 0.0246, HR = 0.3, 95% CI: 0.1-0.8, P = 0.0134, respectively). High CXCR7 expression was associated with poor prognosis in patients with EAC, and high expression of CXCR7 with its ligand CXCL12 had a stronger association with prognosis. Further study of this potential biomarker using whole tissue samples and a larger sample size is warranted.

CXCL12 and CD3E as Indicators for Tumor Microenvironment Modulation in Bladder Cancer and Their Correlations With Immune Infiltration and Molecular Subtypes.

Bladder cancer (BLCA) represents the ninth most common malignant tumor in the world and is characterized by high recurrence risk. Tumor microenvironment (TME) plays an important role in regulating the progression of BLCA. Immunotherapy, including Bacillus Calmette-Guerin (BCG) and programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1), is closely associated with TME and is widely used for treating BLCA. But parts of BLCA patients have no response to these treatment ways, thus a better understanding of the complex TME of BLCA is still needed. We downloaded the gene expression profile and corresponding clinical information of 414 BLCA patients from the TCGA database. Via the ESTIMATE and CIBERSORT algorithm, we identified the two hub genes (CXCL12 and CD3E) and explored their correlations with immune infiltration. We found that BLCA patients with higher expression of CXCL12 and lower expression of CD3E had prolonged survival. Gene set enrichment analysis (GSEA) revealed that both CXCL12 and CD3E were enriched in immune-related pathways. We also discovered that stromal score and the level of CXCL12 were higher in luminal subtype, and immune score and the level of CD3E were higher in the basal subtype. Furtherly, we found that CXCL12 was associated with naive B cells, resting mast cell, M2 macrophages, follicular helper T cells, and dendritic cells. CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), and macrophages were correlated with CD3E. In conclusions, we found that CXCL12 and CD3E might serve as indicators of TME modulation in BLCA. Therapy targeting CXCL12 and CD3E had the potential as novel therapeutic strategy.

Cancer-associated fibroblasts in gastric cancer affect malignant progression via the CXCL12-CXCR4 axis.

Background: Cancer-associated fibroblasts (CAFs) are principal constituents of the tumor microenvironment (TME) and play a critical role in tumor progression. The CXCL12/CXCR4 axis regulates multiple facets of the TME. The aim of this study was to determine the relationship between CXCL12 expression in CAFs and the malignant progression of gastric cancer (GC).Methods: In the GEO (Gene Expression Omnibus) database, we performed transcriptome analysis on paired gastric cancer RNA sequencing samples, and scRNA analysis was performed on advanced malignant GC samples from the scRNA sequencing data set. Fibroblast cells were co-cultured with GC cells, and invasion, migration, epithelial-mesenchymal transformation (EMT) were determined. After blocking the expression of fibroblast CXCL12, cells were co-cultured with a GC cell line. Detection of GC cell line invasion, migration, EMT and CXCR4, Wnt5a and β-Catenin expression levels was performed. Primary CAFs and gastric normal fibroblasts were isolated and CXCL12 mRNA and protein expression were determined. In addition, a cohort of 285 GC cases was established, protein expression was evaluated immunohistochemically, and prognostic results were analyzed.Results: GC transcriptome analysis suggested that cytokine-cytokine receptor interaction and the Wnt signaling pathway in GC tissues were significantly up-regulated. scRNA analysis of advanced malignant GC samples showed that severe intestinal metaplasia (SIM) in GC specimens of different malignant grades had obvious fibroblast clusters compared to non-atrophic gastritis (NAG) and early gastric cancer (EGC). In the SIM group, fibroblast cluster, CXCL12, CXCR4, and Wnt5a were overexpressed. Co-culturing with fibroblast cells significantly increased the invasion, migration, and EMT of GC cells, and blocking CXCL12 in CAFs disturbed the expression of Wnt5a and β-catenin. In our cohort of GC patients, high CXCL12 expression in CAFs significantly correlated with histological grade (P = 0.012) and TNM stage (P = 0.014), as well as with poor overall survival (p = 0.0107).Conclusion: High expression of CXCL12 in CAFs in a GC microenvironment can affect the migration, invasion, and EMT of GC cells. Furthermore, it can cause poor prognosis in patients with GC.

Expression of chemokine receptor CXCR4 in tumor cells and content of CXCL12+-fibroblasts in endometrioid carcinoma of endometrium.

The expression of the CXCL12chemokine and its receptor CXCR4in the stromal component of the tumor plays an important role in tumor cell migration, proliferation, inhibition of apoptosis and determination of invasive and metastatic potential of malignant neoplasms of various genesis. The significance of CXCL12and CXCR4expression in endometrial tumor cells for cancer progression is not fully understood. To evaluate the content of CXCL12+-fibroblasts and expression of CXCL12and CXCR4in endometrial cancer cells, depending on the tumor stage. Surgical material of 45patients with endometrioid carcinoma of the endometrium (ECE) of the stages I-II and III was studied using morphological and immunohistochemical methods. In ECE of stage I-II CXCR4expression was lower (43.3± 4.2%) while CXCL12expression was higher (33.6± 2.4%) compared with the corresponding indices​​ in ECE of stage III (63.6± 3.5%, 24.5± 1.9%, respectively, p< 0.05). In ECE of stage III, high expression of CXCR4 (> Me) and low CXCL12 (< Me) was observed in 80% of samples; these tumors invaded more than 1/2of the myometrium. There was a positive correlation between the depth of tumor invasion in the myometrium and the presence of metastases and CXCR4expression in tumor cells (R= 0.5and R= 0.4, respectively, p< 0.05) and the negative correlation with the expression of CXCL12 (R= -0.6and R= -0.3, respectively, p< 0.05). In tumors that deeply invaded the myometrium, a high number of the CXCL12+-fibroblasts (> Me) (14.9± 1.3%) was detected. The obtained data reflect the communication of the immunosuppressive factor of the tumor microenvironment, i.e. CXCL12+-fibroblasts and CXCR4expressing tumor cells. We suggest that the aggressiveness of ECE is determined by the combined effect of these two factors.

KRT8 and KRT19, associated with EMT, are hypomethylated and overexpressed in lung adenocarcinoma and link to unfavorable prognosis.

Background: Lung adenocarcinoma (LUAD) is the most common histological type of lung cancer. To date, the prognosis of patients with LUAD remains dismal. Methods: Three datasets were downloaded from the GEO database. Differentially expressed genes (DEGs) were obtained. FunRich was used to perform pathway enrichment analysis. Protein–protein interaction (PPI) networks were established and hub genes were obtained by Cytoscape software. GEPIA was utilized to conduct correlation and survival analysis. Upstream miRNAs of DEGs were predicted via miRNet database, and methylation status of promoters of DEGs was determined through UALCAN database. Results: A total of 375 DEGs, including 105 and 270 up-regulated and down-regulated genes in LUAD, were commonly appeared in three datasets. These DEGs were significantly enriched in mesenchymal-to-epithelial transition (MET) and epithelial-to-mesenchymal transition (EMT). About 8 up-regulated and 5 down-regulated DEGs were commonly appeared in EMT/MET-related gene set and the top 50 hub gene set. Among the 13 genes, increased expression of KRT8 and KRT19 indicated unfavorable prognosis whereas high expression of DCN and CXCL12 suggested favorable prognosis in LUAD. Correlation analysis showed that KRT8 (DCN) expression was linked to KRT19 (CXCL12) expression. Further analysis displayed that KRT8 and KRT19 could jointly forecast poor prognosis in LUAD. About 42 and 2 potential miRNAs were predicted to target KRT8 and KRT19, respectively. Moreover, methylation level analysis demonstrated that KRT8 and KRT19 were significantly hypomethylated in LUAD compared with normal controls. Conclusions: All these findings suggest that KRT8 and KRT19 are hypomethylated and overexpressed in LUAD and associated with unfavorable prognosis.

Mining TCGA database for tumor mutation burden and their clinical significance in bladder cancer.

Background: Bladder cancer is the ninth most-common cancer worldwide and it is associated with high morbidity and mortality. Tumor mutational burden (TMB) is an emerging biomarker in cancer characterized by microsatellite instability. TMB has been described as a powerful predictor of tumor behavior and response to immunotherapy.Methods: A total of 443 bladder cancer samples obtained from The Cancer Genome Atlas (TCGA) were analyzed for mutation types, TMB values, and prognostic value of TMB. Differentially expressed genes (DEGs) were identified from the TMB groupings. Functional analysis was performed to assess the prognostic value of the first 30 core genes. CIBERSORT algorithm was used to determine the correlation between the immune cells and TMB subtypes.Results: Single nucleotide polymorphism (SNP) and C>T were reported as the most common missense mutations and we also identified a high rate of mutations in TP53, TTN, KMT2D. Bladder cancer patients with high TMB showed a better prognosis. Enrichment analysis of the DEGs revealed that they were involved in the regulation of the P13K-Akt signaling pathway, cytokine–cytokine receptor interaction, and Ras signaling pathway. The high expression of hub genes ADRA2A, CXCL12, S1PR1, ADAMTS9, F13A1, and SPON1 was correlated with poor overall survival. Besides, significant differences in the composition of the immune cells of T cells CD8, T cells CD4 memory activated, NK cells resting and Mast cells resting were observed.Conclusions: The present study provides a comprehensive and systematic analysis of the prediction of TMB in bladder cancer and its clinical significance. Also, the study provides additional prognostic information and opportunities for immunotherapy in bladder cancer.

High hepatocyte growth factor expression in primary tumor predicts better overall survival in male breast cancer

BackgroundBreast cancer is rare in men, but management is focused on tumor characteristics commonly found in female breast cancer. The tumor microenvironment of male breast cancer is less well understood, and insight may improve male breast cancer management. The hepatocyte growth factor (HGF)/c-MET axis and the stromal cell-derived factor-1 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis are prognostic in women with breast cancer. We aimed to investigate these factors in male breast cancer and correlate them with patient survival.MethodsFrom 841 Dutch males with breast cancer who were enrolled in the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program (NCT01101425) and diagnosed between 1990 and 2010, archival primary tumor samples were collected. Tissue microarrays were constructed with 3 cores per sample and used for immunohistochemical analysis of HGF, c-MET, CXCL12, and CXCR4. Overall survival (OS) of the patients without metastases (M0) was analyzed using the Kaplan-Meier method. The value of the markers regarding OS was determined using univariable and multivariable Cox regression analyses, providing hazard ratios (HRs) and 95% confidence intervals (95% CIs).ResultsOf 720 out of 841 patients, sufficient tissue was available for analysis; 487 out of 720 patients had M0 disease. Patients with high HGF expression and high CXCL12 expression had a superior OS (low vs high expression of both markers, 7.5 vs 13.0 years, hazard ratio [HR] 0.64, 95% CI 0.49–0.84, P = 0.001 [HGF]; 9.1 vs 15.3 years, HR 0.63, 95% CI 0.45–0.87, P = 0.005 [CXCL12]). Multivariate analysis identified HGF as an independent predictor for OS (HR 0.64, 95% CI 0.47–0.88, P = 0.001).ConclusionsHGF and CXCL12 tumor expression appear to identify male breast cancer patients with a relatively good prognosis. Possibly, this could support male breast cancer-specific management strategies in the future.