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Background Interactions between cancer cells and surrounding cancer-associated fibroblasts (CAFs) play an important role in cancer progression. Invasive ductal carcinoma (IDC) of the pancreas is characterized by abundant fibrous connective tissue called desmoplasia. Podoplanin (PDPN) is a lymphatic vessel marker (D2-40), and expression of PDPN by stromal CAFs has been reported to be a prognostic indicator in various types of cancer. Methods Expression of PDPN in pancreatic IDCs was assessed by immunohistochemical examination in 105 patients who underwent pancreatic resection. Primary CAFs were established from pancreatic cancer tissue obtained by surgery. Quantitative reverse transcription-polymerase chain reaction and flow cytometric analysis were performed to investigate PDPN expression in CAFs. We sorted CAFs according to PDPN expression, and analyzed the functional differences between PDPN+ CAFs and PDPN– CAFs using indirect co-culture with pancreatic cancer cell lines. We also investigated the culture conditions to regulate PDPN expression in CAFs. Results PDPN expression in stromal fibroblasts was associated with lymphatic vessel invasion (P = 0.0461), vascular invasion (P = 0.0101), tumor size ≥3 cm (P = 0.0038), histological grade (P = 0.0344), Union for International Cancer Control classification T stage (P = 0.029), and shorter survival time (P in vitro. Moreover, migration and invasion of pancreatic cancer cell lines (PANC-1 and SUIT-2) were associated with PDPN expression in CAFs (P Conclusions PDPN-expressing CAFs enhance the progression of pancreatic IDC, and a high ratio of PDPN-expressing CAFs is an independent predictor of poor outcome. Understanding the regulation of the tumor microenvironment is an important step towards developing new therapeutic strategies.

Podoplanin (PDPN) is a mucin like type-1 transmembrane protein consisting of 162 amino acids, which comprises a highly glycosylated extracellular domain and a nine amino acid intracellular domain. The specific expression of PDPN on lymphatic but not on blood vascular endothelial cells has established application as a histological marker for lymphangiogenesis and lymphatic vessels in different species. PDPN is highly expressed in tumors of different origin, including squamous cell cancers, germinal neoplasia and tumors of the central nervous system. Recently, we found strong overexpression of PDPN in human astrocytic brain tumors, specifically in primary glioblastoma multiforme (GB). Regulation of PDPN expression involves increased PI3 kinase activation, subsequent activation of protein kinaseB/Akt and the downstream transcription factor AP-1. Activation of this pathway is mediated by loss of PTEN function, consistently, mice lacking Pten exhibit elevated Pdpn protein levels in the subventricular zone of the brain tissue. Additionally, PDPN expression is subject to negative transcriptional regulation by promoter methylation in human GB and in glioma cell lines. In patients high expression of PDPN is associated with poor prognosis. Silencing of PDPN results in a decrease in proliferation, migration and invasion of glioma cell lines in vitro and slows down the migration in orthotopic brain slices. Consistently, tumor formation after injection of PDPN knock-down cells into SCID mice is significantly reduced compared to tumors after injection of parental cells. These data highlight a functional role of PDPN in glioma progression and suggest targeting of PDPN expression as a promising strategy for the treatment of patients with primary GB. Citation Format: Heike Peterziel, Julia Müller, Danner Andreas, Sebastian Barbus, Haikun Liu, Philipp Pfenning, Patrick N. Harter, Michel Mittelbronn, Wolfgang Wick, Günther Schuetz, Bernhard Radlwimmer, Peter Lichter, Jochen Hess, Peter Angel. Regulation and function of the mucin-like glycoprotein podoplanin in glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3797. doi:10.1158/1538-7445.AM2013-3797

Head and neck squamous cell carcinoma (HNSCC) is characterised by an elevated capacity for cell invasion and lymph node metastasis. The cause remains to be determined. Recent studies suggest that microRNAs can regulate the evolution of malignant behaviours by regulating multiple target genes. In this study, we first confirmed that miR-363 is down-regulated in HNSCC tissues with lymph node metastasis and cell lines with highly invasive capacity. We also found that podoplanin (PDPN) is up-regulated in the metastatic HNSCC tissues, and ectopic over-expression of miR-363 suppressed the metastatic behaviour of HNSCC cells by directly targeting PDPN. These data support a key role of miR-363-PDPN in head and neck cancer metastasis by regulating cellular invasion and migration. Citation Format: Wantao Chen. Dysregulated miR-363 affects head and neck cancer invasion and metastasis by targeting PDPN gene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5295. doi:10.1158/1538-7445.AM2013-5295

Alesia Sadosky,1 Caroline Schaefer,2 Rachael Mann,3 Felicia Bergstrom,2 Rebecca Baik,2 Bruce Parsons,1 Srinivas Nalamachu,4 Edward Nieshoff,5 Brett R Stacey,6 Alan Anschel,7 Michael Tuchman81Pfizer Inc, New York, NY, 2Covance Market Access Services Inc, Gaithersburg, MD, 3Covance Market Access Services Inc, San Diego, CA, 4International Clinical Research Institute, Overland Park, KS, 5Rehabilitation Institute of Michigan/Wayne State University, Detroit, MI, 6Oregon Health and Science University, Portland, OR, 7Rehabilitation Institute of Chicago, Chicago, IL, 8Palm Beach Neurological Center, Palm Beach Gardens, FL, USABackground: The purpose of this study was to characterize the burden of illness among adult subjects with painful diabetic peripheral neuropathy (pDPN) seeking treatment in the US.Methods: This observational study recruited 112 subjects with pDPN during routine visits from general practitioner and specialist sites. Subjects completed a one-time questionnaire, which included demographics, symptom duration, health care resource use, out-of-pocket costs, employment status, and validated measures that assessed pain, functioning, sleep, anxiety and depression, health status, and productivity. Investigators completed a case report form based on a 6-month retrospective chart review to capture clinical information, pDPN-related treatments, and other pDPN-related health care resource use over the past 6 months. Annualized costs were extrapolated based on reported 6-month health care resource use.Results: The mean age of the subjects was 61.1 years, 52.7% were female, and 17.9% were in paid employment. The most common comorbid conditions were sleep disturbance/insomnia (43.8%), depressive symptoms (41.1%), and anxiety (35.7%). The mean pain severity score was 5.2 (0–10 scale), and 79.5% reported moderate or severe pain. The mean pain interference with function score was 5.0 (0–10 scale) overall, with 2.0 among mild, 5.1 among moderate, and 7.0 among severe. The mean Medical Outcomes Study sleep problems index score was 48.5 (0–100 scale). The mean health state utility score was 0.61. Among subjects employed for pay, mean overall work impairment was 43.6%. Across all subjects, mean overall activity impairment was 52.3%. In total, 81.3% were prescribed at least one medication for their pDPN; 50.9% reported taking at least one nonprescription medication. Adjusted mean annualized total direct and indirect costs per subject were $4841 and $9730, respectively. Outcomes related to pain interference with function, sleep, health status, activity impairment, prescription medication use, and direct and indirect costs were significantly worse among subjects with more severe pain (P < 0.0020).Conclusion: Subjects with pDPN exhibited high pain levels, which were associated with poor sleep, function, and productivity. Health care resource utilization in pDPN was prevalent and costs increased with greater pain severity. The burden of pDPN was greater among subjects with greater pain severity.Keywords: painful diabetic peripheral neuropathy, pain assessment, burden of illness, quality of life, treatment patterns, health care resource use, costs, productivity

Abstract 908 There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software. Results: TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.

Better understanding the mechanisms underlying the metastatic process is essential to developing novel targeted therapeutics. Recently, invadopodia have been increasingly recognized as important drivers of local invasion in metastasis. Invadopodia are actin-rich structures that have the ability to degrade the underlying extracellular matrix (ECM) and promote cell invasion. However, many questions concerning the mechanisms of invadopodia formation, regulation and function remain open to debate. Meanwhile, invasion of cells into the surrounding tissue and destruction of normal tissue architecture are two hallmarks of malignant tumors. Morphologically, two patterns of tumor invasion can be distinguished; single cell and collective cell invasion. The invasion of single cells and collective cells is often correlated with dramatic changes in the expression and function of adhesive and regulatory proteins. These changes are reminiscent of early developmental processes when cells acquire a migratory, mesenchymal phenotype. Currently, the role of invadopodia in collective cancer invasion is completely unknown. In the present study, we reveal that insuline-like growth factor-II mRNA-binding protein-3 (IMP-3) associated with invadopodial infiltration and collective cancer invasion through podoplanin (PDPN) mRNA stabilization as a component of invadopodia, suggesting that invadopodia plays a pivotal role in invasive front of collective cancer invasion and thereby involved the cancer metastasis. This finding, combined with other investigations into the mechanisms of invadopodia formation, reveal clinically-relevant targets for intervention in invadopodia, including IMP-3 and PDPN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4326. doi:1538-7445.AM2012-4326

Umbilical cord matrix stem cells (UCMSC) represent a promising source of therapeutics for various diseases including cancer. We have shown that naïve rat UCMSC significantly attenuate Fisher 344 rat-derived mammary adenocarcinoma (Mat B III) cell proliferation both in vivo and in vitro. To study the underlying mechanisms and genes involved in the Mat B III growth attenuation, we investigated gene expression profiles of naive rat UCMSC alone co-cultured with Mat BIII cells by cDNA microarray analysis. Total RNA was extracted from the naïve rat UCMSC alone and those co-cultured with Mat B III in transwell culture dishes. The comparison of gene expression profiles between untreated and co-cultured rat UCMSC identified five up-regulated (Follistatin (FST), Sulfatase1 (SULF-1), Glucose phosphate isomerase (GPI), HtrA serine peptidase (HTRA1), and Adipocyte differentiation-related protein (ADRP)) and two down-regulated (Transforming growth factor, beta-induced (TGFBI), Podoplanin (PDPN)) candidate genes based upon the following screening criteria: 1) candidate gene expression should show at least a 1.5 fold change in rat UCMSC co-cultured with Mat B III cells; 2) candidate genes encode secretory proteins; and 3) candidate genes encode cell growth-related proteins. Following confirmation of gene expression by real time-PCR, ADRP, SULF-1 and GPI were selected for further analysis. Addition of specific neutralizing antibodies against these three gene products individually or all together in the co-culture of Mat B III cells and 1/20 cell number of rat UCMSC significantly increased [3H] thymidine uptake in the Mat B III cells implying that these gene products are functionally produced under the co-cultured condition and attenuate cell growth. Immunoprecipitation followed by Western Blot analysis demonstrated that these proteins are indeed secreted into the culture medium. We are investigating the siRNA-dependent down regulation of these genes in rat UCMSC to confirm their tumor suppressor function. These results suggest that ADRP, SULF-1 and GPI act as tumor suppressor genes and these genes might be involved in rat UCMSC-dependent growth attenuation of rat mammary tumor. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH P20 RR017686, p20RR1556, R21CA135599, Kansas Bioscience Authority research fund and by the Intramural Research Program of the NIH, National Institute on Aging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3355. doi:10.1158/1538-7445.AM2011-3355

The majority of patients with malignant gliomas, the most common and most lethal brain tumor, develop disease recurrence despite aggressive, multi-modality therapies. This treatment resistance has been attributed to a small population of tumor-initiating glioma cancer stem cells (GSCs). We recently described a new marker of GSCs, the transmembrane glycoprotein podoplanin (pdpn), based on analysis of gene expression profiling data and have found it to be more accurately identify the stem cell population than other described markers. Pdpn expression correlates with poor patient survival and decreased response to radiotherapy. Pdpn expressing cells form neurospheres at high rates in vitro, are more resistant to ionizing radiation, and initiate tumors in vivo. Silencing of pdpn leads to increased radiosensitivity. To identify the mechanism of radiation resistance in pdpn-expressing GSCs, we screened for putative binding partners and identified target of myb-like-1 (tom1l1), a known regulator of Src-pathway activation. We hypothesized that modulation of pdpn and/or tom1l1 expression would lead to alternations in the response of GSCs to radiation via src signaling. Mechanisms of radiation resistance in GSCs are poorly understood to date and the role of src family kinases in modulating radiosensitivity has not previously been reported in gliomas. Pdpn binding of tom1l1 was confirmed by co-immunoprecipation and src activation monitored by immunoblot. Pdpn and tom1l1 expression was downregulated by RNA interference and dsDNA damage and repair assayed by neutral comet assay and by scoring of γH2AX foci. Silencing of pdpn in primary culture, neurophere forming GSC-like cells resulted in increased DNA damage following 2 and 6 Gy ionizing radiation. We similarly examined effects of decreased tom1l1 to determine if the effects on DNA repair mediating by pdpn were acting via the src pathway. In summary, our data suggest that pdpn expression correlates with increased dsDNA repair following ionizing radiation and presents a possible mechanism by which GSCs resist treatment and regrow the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2467. doi:10.1158/1538-7445.AM2011-2467

Glioblastomas (GBMs) are among the most common and most lethal primary brain tumors, characterized by a high cellular proliferation and invasion into brain parenchyma. Glioma stem-like cells (GSCs) have been proposed as being responsible for tumor initiation and recurrence following conventional therapy with chemoradiation. Attempts to identify a single cell surface marker which specifically identifies GSCs within human tumors have had limited success. In an effort to refine GSC identification through the use of a panel of cell surface markers, we identified the glycoprotein CD58 as a marker which more selectively identifies GSCs when added to established markers, such as CD133, podoplanin (pdpn), or CD15. To characterize the frequency of CD58 expression in neurosphere forming primary culture cell lines and freshly obtained surgical specimens, cells were subjected to flow cytometry using an anti-CD58 monoclonal antibody. In vitro analysis of self-renewal, a principle feature of GSCs, was performed with the neurosphere assay. Treatment resistance was compared by radiating single cell suspensions with 2 Gy ionizing radiation prior to neurosphere formation. Cell cycle was measured by flow cytometry and S-phase by incorporation of EdU. Survival analysis was performed on archival tumor specimens following immunohistochemistry with an anti-CD58 monoclonal antibody. Using a panel of 14 GSC primary cultures, CD58 expression was found in all lines, with a median expression of 85.4% of cells, ranging from 8.7% to 100%. CD58 expression was found in 67% of fresh GBM specimens, with expression ranging from 5% to 89% of tumor cells (median 9.4). Culturing of cells beyond 5 passages led to further enrichment for CD58 expressing cells. Neurosphere assays demonstrated an increase in self-renewal in cells expressing CD58 compared to pdpn/CD133/CD15 alone. To investigate whether this difference was a result of increased proliferation of CD58 cells, we performed cell cycle analysis and found a significantly higher percentage of CD58 expressing cells in S-phase (mean of 23.2% vs. 7.9% for CD58 expressing vs. non-expressing, respectively). Immunofluorescence of archival specimens with MIB1/Ki-67 revealed preferential staining in CD58 expression cells. In cell mixing experiments, we observed that CD58 expressing cells rapidly outgrew CD58 non-expressing, highlighting the differences in proliferative capacity. Analysis of patients based on CD58 expression in their tumors revealed that patients with CD58 expression tumors had a poorer survival compared to patients with low or non-expressing tumors (median survival 64 vs 43 weeks, 2yr survival 3% vs 25%, p=0.0192, logrank). In summary, CD58 expression identifies a highly proliferative subpopulation of GSCs, improves upon established markers of these cells, and is an independent predictor of survival based on expression in patient tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3313. doi:10.1158/1538-7445.AM2011-3313

Infiltrating gliomas are the most frequently occurring adult brain tumor and, among high-grade forms, typically result in poor patient survival. These tumors tend to be refractory to most treatments, including aggressive surgical resection and chemoradiation. This treatment resistance is hypothesized to be the result of a small population of cells within the tumor which are responsible for tumor initiation, called glioma cancer stem cells (GCSCs). As is true in other stem cell systems, such as hematopoiesis, multiple cell-surface protein markers are needed to accurately define the true stem cell population. We and others have previously identified the glycoprotein podoplanin (pdpn) as a marker of GCSC, which improves upon the established marker, CD133. To refine the GCSC marker profile beyond these two proteins, we examined a number of candidate markers in combination with pdpn that have been proposed as stem cell markers in gliomas or other tumors or tissues. Candidate markers were analyzed by flow cytometry using primary glioma cell lines maintained as neurosphere cultures and fresh tumor specimens. Markers identifying a unique cell population either alone or in combination with pdpn were further analyzed by in vitro neurosphere formation assay and clonogenic survival assay following 2 Gy ionizing radiation. Candidates showing enrichment for stem cells based on these assays were examine for in vivo tumor initiation using flow sorted subpopulations of cells with and without the expression of the candidate protein. As expression of putative stem cell markers will likely predict for poor patient survival, candidate markers were also examined in human tumors by immunohistochemsitry. Among the most promosing candidates were CD97, CXCR4, CD58 and CD44. Flow cytometry revealed CD97 expression in 75%, CD58 in 80%, CXCR4 in 100%, and CD44 in 100% of tumors examined to date. Examination of primary neurosphere cultures, found CD97 in 80%, CXCR4 in 83%, CD58 in 100%, and CD44 in 100% of lines examined. Both CD44+ and CD58+ cells exhibited increased neurophere formation rates and in vivo tumor initiation. These stem-like characteristics were enhanced in those subpopulations also expressing pdpn. RNAs collected from these subpopulations were used for expression profiling to determine if these subpopulation express a unique gene expression. Results thus far indicate that pdpn expression is needed for the stem-like phenotype and that this is enhanced in populations co-expressing CD44, CD15, and/or CD58. Further refinement of this profile will aide in the isolation of the “true” GCSC and allow for improved therapeutic targeting of these resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4305.

The homeobox transcription factor Prox1 is related to the Drosophila prospero gene, which mediates cell fate decisions of neuroblasts. Prox1 is a regulator of lymphangiogenesis but it is also expressed in the developing central nervous system. Homeobox proteins are known to play essential role in the determination of cell fate and the development of the body plan. It has been shown in Drosophila that prospero-mediated transcriptional repression of stem cell genes and activation of differentiation genes prevents tumorigenic growth, suggesting that prospero is in fact a tumor suppressor. Due to its homology to prospero, we speculated that prox1 may play a role in glioma tumorigensis. We previously identified the transmembrane glycoprotein podoplanin (pdpn) as a prognostic marker in infiltrating gliomas, the most common and lethal of adult brain tumors, by analyses of gene expression profiling data. Subsequent functional studies revealed that pdpn identified a subpopulation of cells from primary neurophere cultures and fresh tumors that have characteristics of glioma cancer stem cells (GSCs), such as radiation resistance and tumor initiation, and that the protein played a functional role in maintaining the stem cell phenotype. Like prox1, pdpn is expressed both during lymphangiogenesis and neural development and mice null for either protein have a similar phenotype, including lymphedema and the lack of terminal alveoli formation in the lung. While other transcription factors have been implicated in cancer stem cells, such as Bmi-1, prox1 has note previously been implicated to have a role in this tumor-initiating, treatment resistant cell population. We therefore hypothesized that prox1 transcriptionally regulates PDPN expression in GSCs. To test this, we examined gene expression profiling data from a large glioma dataset for correlations between PDPN and PROX1 expression. No correlation was seen between expression of the two genes; however, PROX1 expression was predictive of patient survival independent of tumor grade (p=.0333). Next, we screened primary neurosphere cell lines for levels of prox1 and pdpn by immunoblot. Co-expression with pdpn was observed in all cell lines expressing prox1. To examine the role of prox1 in PDPN expression, we compared the levels of PDPN in cells for whom prox1 expression had been silenced. In preliminary results, real-time PCR for PDPN showed significant decreases in expression in the presence of PROX1-targeting siRNA. Ongoing promotor and functional analyses will validate the role of prox1 in the transcriptional activation of PDPN. Comparison of the expression profiles of isogenic clones differing only in their expression of PROX1 identifies additional transcriptional targets. In summary, we have identified prox1 as a candidate transcription factor in the regulation of the glioma stem cell phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5147.

The majority of patients with malignant gliomas, the most common and most lethal brain tumor, develop disease recurrence despite aggressive, multi-modality therapies. This treatment resistance has been attributed to a small population of tumor-initiating glioma cancer stem cells (GSCs). We recently described a new marker of GSCs, podoplanin (pdpn), based on analyses of gene expression profiling data and have found it to more accurately identify the stem cell population than other described markers, such as CD133, and to play a functional role stem cell phenotype. Pdpn expressing cells formed neurospheres at high rates in vitro, were more resistant to ionizing radiation, and initiated tumors in vivo. Pdpn belongs to a mesenchymal gene signature that is characteritic of poor patients survival from malignant gliomas. To identify the mechasism of pdpn within GSCs, we evaluated putuative binding partners to this transmembrane glycoprotein. One such partner, target of myb-like-1 (tom1l1) has been shown to regulate the Src oncogene pathway. The cytoplasmic tyrosine kinases of the Src family (SFK) play important roles in signal transduction induced by growth factors leading to DNA synthesis, cytoskeletal rearrangement, and receptor endocytosis. We therefore hypothesized that pdpn's role GSCs is due to its interaction with the Src pathway. Our aim for this study was to analyze downstream signaling events of Src in association with pdpn and its functional significance in glioma stem cell maintanence, invasion, and chemoradiation resistance. Using primary GSC and established glioma cell lines engineered to either overexpress or silence PDPN, we screened for SFK protein activation (phosphorylation) by immunblot. Changes in Src activation were correlated to in vitro invasion assays, in vivo tumor initiation, and radiation resistance. Preliminary data confirms binding of tom1l1 to pdpn and regulation of SFK activation. Engineered clones with shRNA targeting pdpn and/or tom1l1 were examined by expression profiling for downstream transcriptional changes. In summary, our data suggest a role of SFKs in the regulation of stemness by pdpn. These data integrate well with ongoing trials using Src-kinase inhibitors which may directly target the pdpn-expressing, GSC population in tumors and perhaps overcome the treatment resistance of these critical cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5157.

SummaryWithin secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTβR) ligands were critical mediators, and LTβR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTβR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases.

SummaryThe expression of podoplanin, a small mucin-like protein, is upregulated in the invasive front of a number of human carcinomas. We have investigated podoplanin function in cultured human breast cancer cells, in a mouse model of pancreatic β cell carcinogenesis, and in human cancer biopsies. Our results indicate that podoplanin promotes tumor cell invasion in vitro and in vivo. Notably, the expression and subcellular localization of epithelial markers are unaltered, and mesenchymal markers are not induced in invasive podoplanin-expressing tumor cells. Rather, podoplanin induces collective cell migration by filopodia formation via the downregulation of the activities of small Rho family GTPases. In conclusion, podoplanin induces an alternative pathway of tumor cell invasion in the absence of epithelial-mesenchymal transition (EMT).

Objective This study aims to evaluate the reliability and validity of neuropathic pain assessment tools among Chinese patients with painful diabetic peripheral neuropathy (PDPN). Methods One hundred patients with PDPN and 70 patients with non-neuropathic pain were recruited from five grade III general hospitals in Guangzhou. Pain was assessed using the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS), Douleur Neuropathique 4 questionnaire (DN4), and Brief Pain Inventory for Painful Diabetic Peripheral Neuropathy (BPI-DPN). Reliability was evaluated by internal consistency of the Cronbach's α coefficient and Guttman split-half. Construct validity was analyzed by factor analysis and Spearman correlation coefficients. Sensitivity and specificity were also assessed. Results The Cronbach's α coefficients of the LANSS, DN4, and BPI-DPN were 0.735, 0.750, and 0.898, respectively. The Guttman split-half coefficients of the LANSS, DN4, and BPI-DPN were 0.660, 0.726, and 0.849, respectively. The cumulative contributions of the LANSS, DN4, and BPI-DPN to the total variance were 61.945%, 57.010%, and 66.056%, respectively. The items of the LANSS, DN4, and BPI-DPN presented high factorial loads, ranging from 0.387 to 0.841, 0.137 to 0.948, and 0.487 to 0.953, respectively. The LANSS and DN4 exhibited sensitivities of 58.0% and 82.7%, respectively, and specificity of 97.1%. Conclusions The LANSS or DN4 can be used to detect neuropathic pain in Chinese patients with PDPN. The BPI-DPN can be employed to monitor the effectiveness of pain intervention.

Podoplanin (Pdpn), a brain-tumor-related glycoprotein identified in humans and animals, is endogenously expressed in several organs critical for life support such as kidney, lung, heart and brain. In the brain, Pdpn has been identified in proliferative nestin-positive adult neural progenitor cells and in neurons of the neurogenic hippocampal dentate gyrus (DG), a structure associated to anxiety, critical for learning and memory functions and severely damaged in people with Alzheimer's Disease (AD). The in vivo role of Pdpn in adult neurogenesis and anxiety-like behavior remained however unexplored. Using mice with disrupted Pdpn gene as a model organism and applying combined behavioral, molecular biological and electrophysiological assays, we here show that the absence of Pdpn selectively impairs long-term synaptic depression in the neurogenic DG without affecting the CA3-Schaffer's collateral-CA1 synapses. Pdpn deletion also enhanced the proliferative capacity of DG neural progenitor cells and diminished survival of differentiated neuronal cells in vitro. In addition, mice with podoplanin gene disruption showed increased anxiety-like behaviors in experimentally validated behavioral tests as compared to wild type littermate controls. Together, these findings broaden our knowledge on the molecular mechanisms influencing hippocampal synaptic plasticity and neurogenesis in vivo and reveal Pdpn as a novel molecular target for future studies addressing general anxiety disorder and synaptic depression-related memory dysfunctions.

Growing evidence supports a key role for inflammation in the onset and progression of tendinopathy. However, the effect of the inflammatory infiltrate on tendon cells is poorly understood. We investigated stromal fibroblast activation signatures in tissues and cells from patients with tendinopathy. Diseased tendons were collected from well-phenotyped patient cohorts with supraspinatus tendinopathy before and after sub-acromial decompression treatment. Healthy tendons were collected from patients undergoing shoulder stabilisation or anterior cruciate ligament repair. Stromal fibroblast activation markers including podoplanin (PDPN), CD106 (VCAM-1) and CD248 were investigated by immunostaining, flow cytometry and RT-qPCR. PDPN, CD248 and CD106 were increased in diseased compared to healthy tendon tissues. This stromal fibroblast activation signature persisted in tendon biopsies in patients at 2–4 years post treatment. PDPN, CD248 and CD106 were increased in diseased compared to healthy tendon cells. IL-1β treatment induced PDPN and CD106 but not CD248. IL-1β treatment induced NF-κB target genes in healthy cells, which gradually declined following replacement with cytokine-free medium, whilst PDPN and CD106 remained above pre-stimulated levels. IL-1β-treated diseased cells had more profound induction of PDPN and CD106 and sustained expression of IL6 and IL8 mRNA compared to IL-1β-treated healthy cells. We conclude that stromal fibroblast activation markers are increased and persist in diseased compared to healthy tendon tissues and cells. Diseased tendon cells have distinct stromal fibroblast populations. IL-1β treatment induced persistent stromal fibroblast activation which was more profound in diseased cells. Persistent stromal fibroblast activation may be implicated in the development of chronic inflammation and recurrent tendinopathy. Targeting this stromal fibroblast activation signature is a potential therapeutic strategy.

// Anna Tejchman 1, 2 , Nathalie Lamerant-Fayel 1 , Jean-Claude Jacquinet 3 , Aleksandra Bielawska-Pohl 2 , Katarzyna Mleczko-Sanecka 1 , Catherine Grillon 1 , Salem Chouaib 4 , Maciej Ugorski 2 and Claudine Kieda 1, 5 1 Centre for Molecular Biophysics, UPR 4301 CNRS affiliated to Orleans University and INSERM, Orleans, France 2 Laboratory of Glycobiology and Intercellular Interactions, Institute of Immunology and Experimental Therapy, PAN, Wroclaw, Poland 3 ICOA, UMR CNRS 7311, University of Orleans, Orleans, France 4 INSERM U1186, Gustave Roussy Institute, Villejuif, France 5 Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland Correspondence to: Claudine Kieda, email: claudine.kieda@cnrs-orleans.fr Keywords: adhesion, cancer associated fibroblasts, CCL21, hypoxia, podoplanin Received: June 29, 2016 Accepted: October 21, 2016 Published: March 17, 2017 ABSTRACT Podoplanin (PDPN), an O-glycosylated, transmembrane, mucin-type glycoprotein, is expressed by cancer associated fibroblasts (CAFs). In malignant transformation, PDPN is subjected to changes and its role is yet to be established. Here we show that it is involved in modulating the activity of the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. In the present model, breast cancer MDA-MB-231 cells and NKL3 cells express the surface CCR7 receptor for CCL21 chemokine which is a potent chemoattractant able to bind to PDPN. The impact of the CCL21/CCR7 axis in the molecular mechanism of the adhesion of NKL3 cells and of MDA-MB-231 breast cancer cells was reduced in a hypoxic tumor environment. In addition to its known effect on migration, CCL21/CCR7 interaction was shown to allow NK cell adhesion to endothelial cells (ECs) and its reduction by hypoxia. A PDPN expressing model of CAFs made it possible to demonstrate the same CCL21/CCR7 axis involvement in the tumor cells to CAFs recognition mechanism through PDPN binding of CCL21. PDPN was induced by hypoxia and its overexpression undergoes a reduction of adhesion, making it an anti-adhesion molecule in the absence of CCL21, in the tumor. CCL21/CCR7 modulated NK cells/ECs and MDA-MB-231 cells/CAF PDPN-dependent interactions were further shown to be linked to hypoxia-dependent microRNAs as miRs: miR-210 and specifically miR-21, miR-29b which influence PDPN expression.

Background There has been debate about the existence of lymphatic vessels in placenta. Lymphatic endothelial cell (LEC) markers such as LYVE-1 and podoplanin/D2–40 have been found, although PROX1 has not been detected. The most reliable marker for LECs is the double staining for CD31 and PROX1, which has not been performed yet. Methods We studied three term placentas and dissected them into three areas: i.) basal plate area, ii.) intermediate area, and iii.) chorionic plate area. We used immunofluorescence single and double staining with antibodies against CD31, PROX1, LYVE-1, VEGFR-3, D2–40/PDPN, CD34, CCBE-1, and vimentin, as well as nested PCR, qPCR, Western blot and transmission electron microscopy (TEM). Results At TEM level we observed structures that have previously mistakenly been interpreted as lymphatics, however, we did not find any CD31/PROX1 double-positive cells in placenta. Absence of PROX1 was also noted by nested PCR, qPCR and Western blot. Also, LEC marker VEGFR-3 was expressed only in a small number of scattered leukocytes but was absent from vessels. The LEC marker D2–40/PDPN was expressed in most stromal cells, and the LEC marker LYVE-1 was found in a considerable number of stromal cells, but not in endothelial cells, which were positive for CD31, CD34, CCBE-1 and vimentin. Additionally, vimentin was found in stromal cells. Conclusions Our studies clearly show absence of lymphatics in term placenta. We also show that the functional area of the mother’s endometrium is not penetrated by lymphatics in term pregnancy.

Introduction Acute respiratory distress syndrome (ARDS) is a devastating pulmonary condition in the critically ill patient. A therapeutic intervention is yet to be found that can prevent progression to ARDS. We recently demonstrated that the interaction between podoplanin expressed on inflammatory alveolar macrophages (iAMs) and its endogenous ligand, platelet C-type lectin-like 2 (CLEC-2), protects against exaggerated lung inflammation during a mouse model of ARDS. In this study, we aim to investigate the therapeutic use of a crosslinking/activating anti-podoplanin antibody (α-PDPN, clone 8.1.1) during lipopolysaccharide (LPS)-induced lung inflammation in mice. Methods Intravenous administration of α-PDPN was performed 6 hours after intratracheal LPS in wildtype, C57Bl/6 mice. Lung function decline was measured by pulse oximetry as well as markers of local inflammation including bronchoalveolar lavage neutrophilia and cytokine/chemokine expression. In parallel, alveolar macrophages were isolated and cultured in vitro from haematopoietic-specific podoplanin-deficient mice (Pdpn fl/fl VAV1cre + ) and floxed-only controls treated with or without LPS in the presence or absence of α-PDPN. Results Lung function decline as well as alveolar neutrophil recruitment was significantly decreased in mice treated with the crosslinking/activating α-PDPN in vivo. Furthermore, we demonstrate that, in vitro, activation of podoplanin on iAMs regulates their secretion of proinflammatory cytokines and chemokines. Conclusions These data confirm the importance of the CLEC-2–podoplanin pathway during intratracheal (IT)-LPS and demonstrate the beneficial effect of targeting podoplanin during IT-LPS in mice possibly via modulation of local cytokine/chemokine expression. Moreover, these data suggest that podoplanin-targeted therapies may have a beneficial effect in patients at risk of developing ARDS.

Podoplanin (PDPN) is a glycoprotein that is expressed by various cell types, including keratinocytes, fibroblasts, and lymphatic endothelial cells. We found that PDPN is expressed in the hair follicle (HF) keratinocyte region and HF stem cell area during the late anagen phase but not during the telogen phase in mice. Importantly, keratinocyte-specific PDPN deletion in mice (K5-Cre;PDPNflox/flox) promoted anagen HF growth after depilation-induced HF regeneration as compared to control mice. RNA sequencing, followed by gene ontology analysis, showed down-regulation of focal adhesion and extracellular matrix interaction pathways in HF stem cells isolated from K5-Cre;PDPNflox/flox mice as compared to control mice. Furthermore, HF keratinocytes isolated from K5-Cre;PDPNflox/flox mice exhibited a decreased ability to interact with collagen type I in cell adhesion assays. Taken together, these results show that PDPN deletion promotes HF cycling, possibly via reduced focal adhesion and concomitantly enhanced migration of HF stem cells towards the bulb region. They also indicate potential new therapeutic strategies for the treatment of conditions associated with hair loss.

Patients with acute promyelocytic leukemia (APL) often present with potentially life-threatening hemorrhagic diathesis. The underlying pathomechanisms of APL-associated coagulopathy are complex. However, two pathways considered to be APL-specific had been identified: 1) annexin A2 (ANXA2)-associated hyperfibrinolysis and 2) podoplanin (PDPN)-mediated platelet activation and aggregation. In contrast, since disseminated intravascular coagulation (DIC) is far less frequent in patients with non-APL acute myeloid leukemia (AML), the pathophysiology of AML-associated hemorrhagic disorders is not well understood. Furthermore, the potential threat of coagulopathy in non-APL AML patients may be underestimated. Herein, we report a patient with non-APL AML presenting with severe coagulopathy with hyperfibrinolysis. Since his clinical course resembled a prototypical APL-associated hemorrhagic disorder, we hypothesized pathophysiological similarities. Performing multiparametric flow cytometry (MFC) and immunofluorescence imaging (IF) studies, we found the patient’s bone-marrow mononuclear cells (BM-MNC) to express ANXA2 - a biomarker previously thought to be APL-specific. In addition, whole-exome sequencing (WES) on sorted BM-MNC (leukemia-associated immunophenotype (LAIP)1: ANXAlo, LAIP2: ANXAhi) demonstrated high intra-tumor heterogeneity. Since ANXA2 regulation is not well understood, further research to determine the coagulopathy-initiating events in AML and APL is indicated. Moreover, ANXA2 and PDPN MFC assessment as a tool to determine the risk of life-threatening DIC in AML and APL patients should be evaluated.

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (P

Background We investigated two distinct synovial fibroblast populations that were located preferentially in the lining or sub-lining layers and defined by their expression of either podoplanin (PDPN) or CD248, and explored their ability to undergo self-assembly and transmigration in vivo.

This 12-week study evaluated the efficacy and safety of capsaicin 8% patch versus placebo patch in painful diabetic peripheral neuropathy (PDPN). Patients aged 18 years or older with PDPN were randomized (1:1) to one 30-minute treatment (capsaicin 8% patch or placebo patch) to painful areas of the feet. Overall, 369 patients were randomized (capsaicin 8% patch, n = 186; placebo patch, n = 183). Percentage reduction in average daily pain score from baseline to between weeks 2 through 8 (the primary end point) was statistically significant for capsaicin 8% patch versus placebo (−27.4% vs −20.9%; P = .025); improvements in pain were observed from week 2 onward. Versus placebo, patients treated with capsaicin 8% patch had a shorter median time to treatment response (19 vs 72 days) and modest improvements in sleep interference scores from baseline to between weeks 2 through 8 ( P = .030) and weeks 2 through 12 ( P = .020). Apart from application site reactions, treatment-emergent adverse events were similar between groups. No indications of deterioration in sensory perception of sharp, cold, warm, or vibration stimuli were observed. In patients with PDPN, capsaicin 8% patch treatment provided modest pain relief and sleep quality improvements versus a placebo patch, similar in magnitude to other treatments with known efficacy, but without systemic side effects or sensory deterioration. Perspective To our knowledge, this is the first study of the capsaicin 8% patch versus placebo in patients with PDPN to show that one 30-minute capsaicin treatment provides modest improvements in pain and sleep quality. Results confirm the clinical utility of the capsaicin 8% patch in the diabetic population.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Podoplanin (PDPN) is a transmembrane glycoprotein that plays crucial roles in embryonic development, the immune response, and malignant progression. Here, we report that cells ectopically or endogenously expressing PDPN release extracellular vesicles (EVs) that contain PDPN mRNA and protein. PDPN incorporates into membrane shed microvesicles (MVs) and endosomal-derived exosomes (EXOs), where it was found to colocalize with the canonical EV marker CD63 by immunoelectron microscopy. We have previously found that expression of PDPN in MDCK cells induces an epithelial-mesenchymal transition (EMT). Proteomic profiling of MDCK-PDPN cells compared to control cells shows that PDPN-induced EMT is associated with upregulation of oncogenic proteins and diminished expression of tumor suppressors. Proteomic analysis of exosomes reveals that MDCK-PDPN EXOs were enriched in protein cargos involved in cell adhesion, cytoskeletal remodeling, signal transduction and, importantly, intracellular trafficking and EV biogenesis. Indeed, expression of PDPN in MDCK cells stimulated both EXO and MV production, while knockdown of endogenous PDPN in human HN5 squamous carcinoma cells reduced EXO production and inhibited tumorigenesis. EXOs released from MDCK-PDPN and control cells both stimulated in vitro angiogenesis, but only EXOs containing PDPN were shown to promote lymphatic vessel formation. This effect was mediated by PDPN on the surface of EXOs, as demonstrated by a neutralizing specific monoclonal antibody. These results contribute to our understanding of PDPN-induced EMT in association to tumor progression, and suggest an important role for PDPN in EV biogenesis and/or release and for PDPN-EXOs in modulating lymphangiogenesis., This work was supported by grants SAF2013-46183-R from the Spanish Ministry of Economy and Competitiveness and S2010/BMD-2359 (SkinModel) from the Community of Madrid (to MQ). Further support from the National Health and Medical Research Council of Australia Program, grant 487922 and project grant 1057741 (to RJS). PC-R is funded by the Spanish FPI (Formación de Personal Investigador) program. SKG is supported by La Trobe University Postgraduate Scholarship, Australia. EM-V is the recipient of a postdoctoral research contract from the scientific foundation of AECC (Asociación Española Contra el Cáncer).