A novel synthetic smoothened antagonist transiently inhibits pancreatic adenocarcinoma xenografts in a mouse model

Background

Hedgehog (Hh) signaling is over-activated in several solid tumors where it plays a central role in cell growth, stroma recruitment and tumor progression. In the Hh signaling pathway, the Smoothened (SMO) receptor comprises a primary drug target with experimental small molecule SMO antagonists currently being evaluated in clinical trials.

Principal Findings

Using Shh-Light II (Shh-L2) and alkaline phosphatase (AP) based screening formats on a “focused diversity” library we identified a novel small molecule inhibitor of the Hh pathway, MS-0022 (2-bromo-N-(4-(8-methylimidazo[1,2-a]pyridin-2-yl)phenyl)benzamide). MS-0022 showed effective Hh signaling pathway inhibition at the level of SMO in the low nM range, and Hh pathway inhibition downstream of Suppressor of fused (SUFU) in the low µM range. MS-0022 reduced growth in the tumor cell lines PANC-1, SUIT-2, PC-3 and FEMX in vitro. MS-0022 treatment led to a transient delay of tumor growth that correlated with a reduction of stromal Gli1 levels in SUIT-2 xenografts in vivo.

Significance

We document the in vitro and in vivo efficacy and bioavailability of a novel small molecule SMO antagonist, MS-0022. Although MS-0022 primarily interferes with Hh signaling at the level of SMO, it also has a downstream inhibitory effect and leads to a stronger reduction of growth in several tumor cell lines when compared to related SMO antagonists.     

Prognostic impacts of angiopoietins in NSCLC tumor cells and stroma: VEGF-A impact is strongly associated with Ang-2

Introduction

Angiopoietins and their receptor Tie-2 are, in concert with VEGF-A, key mediators in angiogenesis. This study evaluates the prognostic impact of all known human angiopoietins (Ang-1, Ang-2 and Ang-4) and their receptor Tie-2, as well as their relation to the prognostic expression of VEGF-A.

Methods

335 unselected stage I-IIIA NSCLC-patients were included and tissue samples of respective tumor cells and stroma were collected in tissue microarrays (TMAs). Immunohistochemistry (IHC) was used to semiquantitatively evaluate the expression of markers in duplicate tumor and stroma cores.

Principal Findings

In univariate analyses, low tumor cell expression of Ang-4 (P = 0.046) and low stromal expressions of Ang-4 (P = 0.009) and Ang-2 (P = 0.017) were individually associated with a poor survival. In the multivariate analysis, low stromal Ang-2 (HR 1.88; CI 95% 1.15-3.08) and Ang-4 (HR 1.47, CI 95% 1.02–2.11, P = 0.04) expressions were independently associated with a poor prognosis. In patients with high tumor cell expression of Ang-2, a concomitantly high tumor VEGF-A expression mediated a dramatic survival reduction (P<0.001). In the multivariate analysis of patients with high Ang-2 expression, high tumor VEGF-A expression appeared an independent poor prognosticator (HR 6.43; CI 95% 2.46–16.8; P<0.001).

Conclusions

In tumor cells, only Ang-4 expression has prognostic impact in NSCLC. In tumor stroma, Ang-4 and Ang-2 are independently associated with survival. The prognostic impact of tumor cell VEGF-A in NSCLC appears strongly associated with a concomitantly high tumor cell expression of Ang-2.     

Differential bone marrow homing capacity of VLA-4 and CD38 high expressing chronic lymphocytic leukemia cells

Background

VLA-4 and CD38 predict a poor clinical outcome in chronic lymphocytic leukemia (CLL). We used CLL samples with discordant VLA-4/CD38 risk to address their individual roles in human bone marrow infiltration (BM), CLL cell homing to murine BM, and in supportive CLL cell-stromal cell interactions.

Methods

VLA-4, CD38, and Ki-67 expression was measured in CLL cells from peripheral blood (PB) and bone marrow (BM) aspirates. CLL BM infiltration rates, routinely determined by Pathology, were correlated to VLA-4 and CD38 expression. Short-term homing capacity of CLL cells was evaluated by adoptive transfer experiments. CLL cell viability and adhesion in stromal cell co-culture was determined.

Results

About 20% of CLL samples in our cohort displayed discordant VLA-4 and CD38 risk, with either high VLA-4 and low CD38 risk or vice versa. Using particularly such samples, we observed that VLA-4, and not CD38, was responsible for recirculation of CLL cells to murine BM. Human BM infiltration was also significantly higher in patients with high VLA-4 risk but not high CD38 risk. However, both molecules acted as independent prognostic markers. While both VLA-4 and CD38 expression were increased in BM-derived CLL cells, and VLA-4+ and CD38+ subpopulations showed enriched Ki-67 expression, VLA-4 did not contribute to CLL cell protection by stromal cells in vitro.

Conclusions

Our data argue for a prominent role of VLA-4 but not CD38 expression in the homing of CLL cells to BM niches and in human BM infiltration,but only a limited role in their protection by stromal cells.     

Bone marrow cells in murine colitis: multi-signal analysis confirms pericryptal myofibroblast engraftment without epithelial involvement

Background

The contribution of bone marrow-derived cells to epithelial tissues in the inflamed gut remains controversial. Recent reports have suggested that cell fusion between bone marrow-derived cells and the intestinal epithelium takes place in inflammatory conditions.

Methods

In attempts to confirm this, we have undertaken gender mis-matched bone marrow (BM) transplants from male Swiss Webster (SWR) mice to B and T cell-deficient female Rag2 KO mice which, 4 weeks later, were given 5% dextran sodium sulphate in drinking water to induce acute colitis. A further BM-treated group of animals with a graft versus host-like condition was also studied. We developed a new method to combine up to three brightfield or fluorescent lectin- or immuno-histochemical signals with fluorescent in situ hybridisation for the Y and X chromosomes to enable us unequivocally to identify BM-derived male cells which presented as different cell types in the gastrointestinal tract.

Principal Findings

In rolled preparations of whole intestines we scanned around 1.5 million crypts at many tissue levels. In no instance did we see a Y chromosome-positive cell in the epithelial compartment, which was not also CD45-positive. We saw no evidence of cell fusion, based on combined X and Y chromosome analysis. Levels of CD45-positive stromal and lymphoid cells and pericryptal myfibroblasts (positive for α-smooth muscle actin) increased with time up to a plateau, which resembled the level seen in untreated control grafted animals. We saw very few Y chromosome-positive endothelial cells in intestinal stromal vessels.

Conclusions

We conclude that whole BM transplantation does not result in intestinal epithelial engraftment in this model. Our new methods can usefully assist in multi-signal analyses of cell phenotypes following BM transplant and in models of chimaerism and regenerative medicine.     

Carcinoembryonic Antigen-Related Cell Adhesion Molecules (CEACAM) 1, 5 and 6 as Biomarkers in Pancreatic Cancer

Background

Aim of this study was to assess the biological function in tumor progression and metastatic process carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1, 5 and 6 in pancreatic adenocarcinoma (PDAC).

Experimental Design

CEACAM knock down cells were established and assessed in vitro and in a subcutaneous and intraperitoneal mouse xenograft model. Tissue and serum expression of patients with PDAC were assessed by immunohistochemistry (IHC) and by enzyme linked immunosorbent assays.

Results

Presence of lymph node metastasis was correlated with CEACAM 5 and 6 expression (determined by IHC) and tumor recurrence exclusively with CEACAM 6. Patients with CEACAM 5 and 6 expression showed a significantly shortened OS in Kaplan-Meier survival analyses. Elevated CEACAM6 serum values showed a correlation with distant metastasis and. Survival analysis revealed a prolonged OS for patients with low serum CEACAM 1 values. In vitro proliferation and migration capacity was increased in CEACAM knock down PDAC cells, however, mice inoculated with CEACAM knock down cells showed a prolonged overall-survival (OS). The number of spontaneous pulmonary metastasis was increased in the CEACAM knock down group.

Conclusion

The effects mediated by CEACAM expression in PDAC are complex, though overexpression is correlated with loco-regional aggressive tumor growth. However, loss of CEACAM can be considered as a part of epithelial-mesenchymal transition and is therefore of rather importance in the process of distant metastasis.     

Clinical,Molecular and Genetic Validation of a Murine Orthotopic Xenograft Model of Pancreatic Adenocarcinoma Using Fresh Human Specimens

Background

Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC.

Methods

Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression.

Results

Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines.

Conclusions

The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.     

Adult Stromal Cells Derived from Human Adipose Tissue Provoke Pancreatic Cancer Cell Death both In Vitro and In Vivo

Background

Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation.

Principal Findings

Co-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth.

Conclusion

These data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.     

Hedgehog signaling antagonist promotes regression of both liver fibrosis and hepatocellular carcinoma in a murine model of primary liver cancer

Objective

Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2^−/− mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC.

Methods

Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2^−/− mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging.

Results

Unlike controls, Mdr2^−/− mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality.

Conclusions

Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.     

Granulocyte Colony-Stimulating Factor Reduces Fibrosis in a Mouse Model of Chronic Pancreatitis

Background

Chronic pancreatitis (CP) is a necroinflammatory process resulting in extensive pancreatic fibrosis. Granulocyte colony-stimulating factor (G-CSF), a hematopoietic stem cell mobilizer, has been shown to exert an anti-fibrotic effect partly through the enrichment of bone marrow (BM) cells in fibrotic organ. We aimed to test the effect of G-CSF on fibrosis in a mouse model of CP.

Methods

CP was induced in C57Bl/6J mice by consecutive cerulein injection (50 µg/kg/day, 2 days a week) for 6 weeks. Mice were then treated with G-CSF (200 µg/kg/day, 5 day a week) or normal saline for 1 week, and sacrificed at week 7 or week 9 after first cerulein injection. Pancreatic histology, pancreatic matrix metallopeptidase 9 (MMP-9), MMP-13 and collagen expression were examined. Pancreatic myofibroblasts were isolated and cultured with G-CSF. Collagen, MMP-9 and MMP-13 expression by myofibroblasts was examined. The BM-mismatched mice model was used to examine the change of BM-derived myofibroblasts and non-myofibroblastic BM cells by G-CSF in the pancreas.

Results

The pancreatic collagen expression were significantly decreased in the G-CSF-treated group sacrificed at week 9. While collagen produced from myofibroblasts was not affected by G-CSF, the increase of MMP13 expression was observed in vitro. There were no effect of G-CSF in the number of myofibroblasts and BM-derived myofibroblasts. However, the number of non-myofibroblastic BM cells and macrophages were significantly increased in the pancreata of cerulein- and G-CSF-treated mice, suggesting a potential anti-fibrotic role of non-myofibroblastic BM cells and macrophages stimulated by G-CSF.

Conclusions

Our data indicated that G-CSF contributed to the regression of pancreatic fibrosis. The anti-fibrotic effects were possibly through the stimulation of MMP-13 from myofibroblasts, and the enhanced accumulation of non-myofibroblastic BM cells and macrophages in the pancreas.     

Stromal Cell-Derived Factor-1/CXCL12 Contributes to MMTV-Wnt1 Tumor Growth Involving Gr1+CD11b+ Cells

Background

Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors.

Methodology/Principal Findings

Here, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12) but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1⁺ myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1⁺ cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1⁺ cells.

Conclusions/Significance

We demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1⁺ myeloid cells to facilitate growth and/or angiogenesis.     

Recombinant human endostatin normalizes tumor vasculature and enhances radiation response in xenografted human nasopharyngeal carcinoma models

Background

Hypoxic tumor cells can reduce the efficacy of radiation. Antiangiogenic therapy may transiently “normalize” the tumor vasculature to make it more efficient for oxygen delivery. The aim of this study is to investigate whether the recombinant human endostatin (endostar) can create a “vascular normalization window” to alleviate hypoxia and enhance the inhibitory effects of radiation therapy in human nasopharyngeal carcinoma (NPC) in mice.

Methodology/Principal Findings

Transient changes in morphology of tumor vasculature and hypoxic tumor cell fraction in response to endostar were detected in mice bearing CNE-2 and 5–8F human NPC xenografts. Various treatment schedules were tested to assess the influence of endostar on the effect of radiation therapy. Several important factors relevant to the angiogenesis were identified through immunohistochemical staining. During endostar treatment, tumor vascularity decreased, while the basement membrane and pericyte coverage associated with endothelial cells increased, which supported the idea of vessel normalization. Hypoxic tumor cell fraction also decreased after the treatment. The transient modulation of tumor physiology caused by endostar improved the effect of radiation treatment compared with other treatment schedules. The expressions of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14 decreased, while the level of pigment epithelium-derived factor (PEDF) increased.

Conclusions

Endostar normalized tumor vasculature, which alleviated hypoxia and significantly sensitized the function of radiation in anti-tumor in human NPC. The results provide an important experimental basis for combining endostar with radiation therapy in human NPC.     

Heterogeneity of Tumor Vasculature and Antiangiogenic Intervention: Insights from MR Angiography and DCE-MRI

Purpose

Solid tumor vasculature is highly heterogeneous, which presents challenges to antiangiogenic intervention as well as the evaluation of its therapeutic efficacy. The aim of this study is to evaluate the spatial tumor vascular changes due to bevacizumab/paclitaxel therapy using a combination approach of MR angiography and DCE-MRI method.

Experimental Design

Tumor vasculature of MCF-7 breast tumor mouse xenografts was studied by a combination of MR angiography and DCE-MRI with albumin-Gd-DTPA. Tumor macroscopic vasculature was extracted from the early enhanced images. Tumor microvascular parameters were obtained from the pharmacokinetic modeling of the DCE-MRI data. A spatial analysis of the microvascular parameters based on the macroscopic vasculature was used to evaluate the changes of the heterogeneous vasculature induced by a 12 day bevacizumab/paclitaxel treatment in mice bearing MCF-7 breast tumor.

Results

Macroscopic vessels that feed the tumors were not affected by the bevacizumab/paclitaxel combination therapy. A higher portion of the tumors was within close proximity of these macroscopic vessels after the treatment, concomitant with tumor growth retardation. There was a significant decrease in microvascular permeability and vascular volume in the tumor regions near these vessels.

Conclusion

Bevacizumab/paclitaxel combination therapy did not block the blood supply to the MCF-7 breast tumor. Such finding is consistent with the modest survival benefits of adding bevacizumab to current treatment regimens for some types of cancers.     

hSulf-1 gene exhibits anticancer efficacy through negatively regulating VEGFR-2 signaling in human cancers

Background

Human sulfatase 1 (hSulf-1) is a heparin-degrading endosulfatase that desulfates cell surface heparan sulfate proteoglycans (HSPGs) in extracellular matrix and negatively modulates heparin-binding growth factor and cytokine signaling in cell proliferation. But hSulf-1 function is more complicated, and its molecular mechanism has not been well known.

Principal Findings

To further investigate the functions of hSulf-1 gene in regulating the vascular endothelial growth factor receptor (VEGFR) signaling, a series of vectors expressing hSulf-1, hSulf-1 small hairpin RNA (shRNA) and VEGFR-2 shRNA were generated. hSulf-1 re-expression could downregualte the VEGFR-2 phosphorylation and inhibit cancer cell proliferation both in ovarian and hepatocellular cancer cell lines. Knockdown of hSulf-1 expression by hSulf-1 shRNA enhanced the recovery of high levels of phosphorylated VEGFR-2, and knockdown of VEGFR-2 expression by VEGFR-2 shRNA inhibited the proliferation activity of cancer cells in vitro to some extent. In human cancer xenografts in nude mice, tumor growth was inhibited markedly after injections of adenovirus expressing hSulf-1, with the tumor inhibition rates of 46.19% and 49.56% in ovarian and hepatocellular tumor models, respectively. hSulf-1 expression significantly reduced tumor microvessel density.

Conclusions

The results demonstrated that hSulf-1 re-expression both in ovarian and hepatocellular cancer cells induces antitumor efficacy by attenuating the phosphorylation of VEGFR-2 and suppressing angiogenesis. Therefore, hSulf-1-mediated antiproliferation and antiangiogenesis could be a reasonable approach for cancer therapy.     

Angiopoietin-2 and Angiopoietin-2/Angiopoietin-1 Ratio as Indicators of Potential Severity of Plasmodium vivax Malaria in Patients with Thrombocytopenia

Introduction

Angiogenic factors such as angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2) are biomarkers produced during activation and dysfunction of the vascular endothelium in several infectious diseases. The aim of this study was to determine the serum levels of Ang-1 and Ang-2 and to establish their relationship with the main indicators of worst-case prognosis in patients with P. vivax malaria.

Methods

This is a retrospective case-control study nested within a cohort of symptomatic malaria patients. A potentially severe case was defined as a patient that presented at least one of the main indicators of the worst-case prognosis for falciparum malaria, as established by the World Health Organization. Ang-2 and Ang-1 and the Ang-2/Ang-1 ratio were used to analyze the role of angiopoietins as biomarkers in signaling potentially severe vivax malaria. ROC curves were generated to identify a cut-off point discriminating between the angiopoietin concentrations that were most strongly associated with potential infection severity.

Results

The serum levels of Ang-2 and the Ang-2/Ang-1 ratio were higher in the case group. In contrast, the serum levels of Ang-1 were lower in the cases than in the control patients. The blood count for platelets showed a positive correlation with Ang-1 and a negative correlation with Ang-2 and with the Ang-2/Ang-1 ratio. The area under the ROC curve (AUC) for serum angiopoietins, as an indicator of worst-case prognosis in a potentially severe P. vivax malarial infection, was larger in the subgroup of patients with platelet counts <75,000/µL.

Conclusion

This study showed that patients with predictors of worst-case prognoses for P. vivax malaria have lower Ang-1 and higher Ang-2 serum levels (and higher values for the Ang-2/Ang-1 ratio) than controls. Elevated serum levels of Ang-2 and high values for the Ang-2/Ang-1 ratio may potentially be used as predictors of worst-case prognoses for P. vivax malaria, especially in patients with thrombocytopenia.     

Overexpression of COL11A1 by Cancer-Associated Fibroblasts: Clinical Relevance of a Stromal Marker in Pancreatic Cancer

Background

The collagen11A1 (COL11A1) gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available.

Methods and findings

A total of 54 pancreatic ductal adenocarcinomas (PDAC), 23 chronic pancreatitis (CP) samples, and cultured peritumoral stromal cells of PDAC (passages 3-6) were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program.1) Validation of COL11A1 gene overexpression by q-RT-PCR. Findings: the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples.2) Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. Findings: anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs) of PDAC but it does not stain chronic benign condition (chronic pancreatitis) stromal cells, epithelial cells, or normal fibroblasts.3) Evaluation of the discrimination ability of the antibody. Findings: anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981).4) Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. Findings: ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype) in different proportions.

Conclusions/Significance

Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for CAFs, and thus, anti-proCOL11A1 is a powerful new tool for cancer research and clinical diagnostics.     

Interleukin-22 suppresses the growth of A498 renal cell carcinoma cells via regulation of STAT1 pathway

Background

Renal cell carcinoma (RCC) is one of the most common kidney cancers and is highly resistant to chemotherapy. Accumulating evidence suggests that interleukin-22 (IL-22) may mediate host defense against varietal pathogens as a proinflammatory and anti-inflammatory cytokine. The purpose of this study is to assess the inhibitory effects of IL-22 on human RCC cell line A498 and to investigate the possible mechanisms underlying the anti-tumor effects of this cytokine.

Methodology

A498 cells, a RCC cell line, were used to assess the inhibitory growth effects of IL-22 using the MTT assay and flow cytometric analysis in vitro. BALB/C nude mice bearing A498 cell xenografts were used to examine the antitumor efficacy of IL-22 in vivo. Western blotting assay was performed to detect the regulation of the intracellular signaling pathway of IL-22.

Principal Findings

We found that IL-22 suppressed the growth of A498 cells in a dose-dependent manner and inhibited the growth of A498 xenografts. We also observed that IL-22 produced a dose-dependent inhibitory effect on A498 cells that involved the induction of G2/M cell cycle arrest without cell apoptosis. Moreover, we showed that the phosphorylation of STAT1 was increased and the phosphorylation of ERK1/2 was attenuated in A498 cells exposed to IL-22. The growth inhibition of A498 cells was partially revised after IL-22 treatment as the expression of STAT1 was knocked down. And inflammatory cytokines, interferon-α and tumor necrosis factor-α (TNF-α) were barely involved in the suppression of A498 cell xenografts treated with IL-22.

Conclusions

IL-22 dose-dependently suppresses RCC cell line A498 cells in vitro and induces growth inhibition of A498 cell-bearing mouse xenografts. These results suggest that the anti-RCC effects of IL-22 are at least partially mediated through regulation of STAT1 signaling pathways and G2/M cell cycle arrest, rather than by inducing apoptosis and inflammatory cytokines.     

Sequestration and homing of bone marrow-derived lineage negative progenitor cells in the lung during pneumococcal pneumonia

Background

Bone marrow (BM)-derived progenitor cells have been shown to have the potential to differentiate into a diversity of cell types involved in tissue repair. The characteristics of these progenitor cells in pneumonia lung is unknown. We have previously shown that Streptococcus pneumoniae induces a strong stimulus for the release of leukocytes from the BM and these leukocytes preferentially sequester in the lung capillaries. Here we report the behavior of BM-derived lineage negative progenitor cells (Lin- PCs) during pneumococcal pneumonia using quantum dots (QDs), nanocrystal fluorescent probes as a cell-tracking technique.

Methods

Whole BM cells or purified Lin- PCs, harvested from C57/BL6 mice, were labeled with QDs and intravenously transfused into pneumonia mice infected by intratracheal instillation of Streptococcus pneumoniae. Saline was instilled for control. The recipients were sacrificed 2 and 24 hours following infusion and QD-positive cells retained in the circulation, BM and lungs were quantified.

Results

Pneumonia prolonged the clearance of Lin- PCs from the circulation compared with control (21.7 ± 2.7% vs. 7.7 ± 0.9%, at 2 hours, P < 0.01), caused preferential sequestration of Lin- PCs in the lung microvessels (43.3 ± 8.6% vs. 11.2 ± 3.9%, at 2 hours, P < 0.05), and homing of these cells to both the lung (15.1 ± 3.6% vs. 2.4 ± 1.2%, at 24 hours, P < 0.05) and BM as compared to control (18.5 ± 0.8% vs. 9.5 ± 0.4%, at 24 hours, P < 0.01). Very few Lin- PCs migrated into air spaces.

Conclusion

In this study, we demonstrated that BM-derived progenitor cells are preferentially sequestered and retained in pneumonic mouse lungs. These cells potentially contribute to the repair of damaged lung tissue.