Podoplanin is a type I transmembrane sialomucin-like glycoprotein that is highly expressed in malignant mesothelioma. The rat-human chimeric antibody NZ-12 has high affinity for human podoplanin and antibody-dependent cellular cytotoxicity and is applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the present study, we evaluated the in vivo and in vitro properties of radiolabeled NZ-12 and the antitumor effect of RIT with

Podoplanin is a small cell-surface mucin-like glycoprotein that plays a crucial role in the development of the alveoli, heart, and lymphatic vascular system. Emerging evidence indicates that it is also involved in the control of mammary stem-cell activity and biogenesis of platelets in the bone marrow, and exerts an important function in the immune response. Podoplanin expression is upregulated in different cell types, including fibroblasts, macrophages, T helper cells, and epithelial cells, during inflammation and cancer, where it plays important roles. Podoplanin is implicated in chronic inflammatory diseases, such as psoriasis, multiple sclerosis, and rheumatoid arthritis, promotes inflammation-driven and cancer-associated thrombosis, and stimulates cancer cell invasion and metastasis through a variety of strategies. To accomplish its biological functions, podoplanin must interact with other proteins located in the same cell or in neighbor cells. The binding of podoplanin to its ligands leads to modulation of signaling pathways that regulate proliferation, contractility, migration, epithelial⁻mesenchymal transition, and remodeling of the extracellular matrix. In this review, we describe the diverse roles of podoplanin in inflammation and cancer, depict the protein ligands of podoplanin identified so far, and discuss the mechanistic basis for the involvement of podoplanin in all these processes.

BACKGROUND: Podoplanin (PDPN) is a mucin-type transmembrane glycoprotein specific to the lymphatic system. PDPN expression has been found in various human tumors and is considered to be a marker of cancer. We had previously shown that PDPN expression contributes to carcinogenesis in the TPC1 papillary thyroid cancer-derived cell line by enhancing cell migration and invasiveness. The aim of this study was to determine the effect of PDPN down-regulation in another thyroid cancer-derived cell line: BcPAP.
METHODS: In order to determine the effects of PDPN on malignant features of BcPAP cells (harboring the BRAFV600E mutated allele) and TPC1 cells (carrying the RET/PTC1 rearrangement), we silenced PDPN in these cells using small interfering RNA (siRNA). The efficacy of PDPN silencing was confirmed by qRT-PCR and Western blotting. Then, we tested the motility and invasiveness of these cells (using scratch test and Transwell assay), their growth capacities F(cell cycle analysis, viability, clonogenic activity) and apoptosis assays), adhesion-independent colony-formation capacities, as well as the effect of PDPN silencing on MMPs expression and activity (zymography).
RESULTS: We found that PDPN-induced cell phenotype depended on the genetic background of thyroid tumor cells. PDPN down-regulation in BcPAP cells was negatively correlated with the migration and invasion, in contrast to TPC1 cells in which PDPN depletion resulted in enhanced migration and invasiveness. Moreover, our results suggest that in BcPAP cells, PDPN may be involved in the epithelial-mesenchymal transition (EMT) through regulating the expression of the ezrin, radixin and moesin (E/R/M) proteins, MMPs 9 and MMP2, remodeling of actin cytoskeleton and cellular protrusions. We also demonstrated that PDPN expression is associated with the MAPK signaling pathway. The inhibition of the MAPK pathway resulted in a decreased PDPN expression, increased E/R/M phosphorylation and reduced cell migration. Additionally, PDPN depleted BcPAP cells treated with inhibitors of MEK1/2 kinases (U0126) or of the BRAF V600E protein (PLX4720) had reduced motility, similar to that previously observed in TPC1 cells after PDPN knock-down.
CONCLUSIONS: Altogether, our data suggest that PDPN may play an important role in the control of invasion and migration of papillary thyroid carcinoma cells in association with the E/R/M, MMPs and MAPK kinases.

INTRODUCTION: Metaplastic breast cancer (MpBC) is a rare but aggressive type of breast cancer accounting for 0.25-1% of all diagnosed invasive breast cancers. Morphologically, it is characterized by differentiation of the neoplastic epithelium into squamous cells and/or mesenchymal-looking tissue.
MATERIAL AND METHODS: We analyzed 13 MpBCs selected from the group of 1122 invasive breast cancers. Histopathological examination and analysis of estrogen (ER), progesterone (PR) and HER2 receptors expression in MpBC patients and their comparison to other types of invasive breast cancer has been performed.
RESULTS: 13 MpBC cases represented 1.16% of the 1122 invasive breast cancers. The MpBC group presented with a significantly larger tumor size (≥T2, 69% versus 49%, p < 0.001) and with higher grade of histological malignancy (G1-G3) (p < 0.001). MpBC group had significantly more cases with no hormone receptors (ER, PR) and HER2 overexpression/gene amplification compared with the other invasive breast cancer types group (ER-, 69% versus 23%, p < 0.001; PR-, 69% versus 28%, p < 0.001; HER2 0/1+, 93% versus 82%, p = 0.019). Most MpBCs (62%) were triple-negative. We found a correlation between hormone receptors expression and lymph node metastasis (p < 0.001). The analysis of the HER2 expression allowed us to find correlation between its expression and tumor histological grade (G1-G3) (p < 0.001), tumor size (T1a-T4) (p < 0.001) and lymph node metastasis (pN0-pN4) (p < 0.001) in MpBCs.
DISCUSSION: MpBCs are usually larger at primary diagnosis and most of MpBCs present with other poor prognostic indicators and show lack of steroid hormone receptors expression as well as HER2. Hormone receptor status and HER2 expression seems to correlate with histological grade of malignancy (G1-G3), tumor size (T1a-T4) and regional lymph node involvement (pN0-pN4) and these features are directly related to MpBC malignancy.

BACKGROUND/AIM: Tumor lymphangiogenesis plays a key role in lymph node (LN) metastasis in oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and their relationship to nodal metastasis and other clinicopathological variables.
PATIENTS AND METHODS: Podoplanin and LYVE-1 expression of the primary tumor and normal tissue were investigated by means of a quantitative real-time PCR assay and immunohistochemistry in samples from 33 cases of OSCC.
RESULTS: The mRNA high expression levels of both genes had a statistically significantly higher rate of LN metastasis (p<0.01) and histological grade (p<0.01 for podoplanin, p<0.05 for LYVE-1). High expression of each gene, as shown by immunohistochemistry, had a statistically significant higher rate of LN metastasis (p<0.01 for podoplanin, p<0.05 for LYVE-1).
CONCLUSION: Podoplanin and LYVE-1 were strongly associated with LN metastasis.

Podoplanin is upregulated in the invasive front of oral squamous cell carcinoma (OSCC). Carcinoma-associated fibroblasts (CAFs) may mediate podoplanin expression. However, the role of podoplanin in OSCC cell and fibroblast interaction remains elusive. In the present study, we found that positive podoplanin expression in OSCC cells correlated with smooth muscle actin (α-SMA) expression in CAFs. Using CAFs and normal mucosal fibroblasts (NFs), we established indirect and direct co-culture systems mimicking the structure of OSCC. Podoplanin-overexpressing OSCC cells promoted NF activation; in direct co-culture, but not in indirect co-culture, podoplanin-overexpressing OSCC cells increased fibroblast invasion via matrix metalloproteinase 2 (MMP-2), MMP-14, and αv/β6 integrin receptor (ITGA5/ITGB6) signaling. CAFs also induced podoplanin expression through the transforming growth factor-β1 (TGF-β1)/Smad pathway. TGF-β1 increased the podoplanin-dependent activation of epidermal growth factor receptor (EGFR), AKT, and extracellular signal-regulated kinase (ERK) signaling. Additionally, CAFs promoted OSCC cell invasion by upregulating MMP-2 and MMP-14 expression in both indirect and direct co-culture. Taken together, our findings indicate that podoplanin regulates the interaction between OSCC cells and CAFs via the mutual paracrine effects of TGF-β1.

BACKGROUND: Induction of podoplanin by transforming growth factor-β (TGF-β) has been shown in a number of lesions but not in odontogenic tumors (OTs). We evaluated the association between these markers in OTs for the first time and compared their expression among the different neoplasms.
METHODS: Immunohistochemistry using monoclonal antibody against podoplanin and TGF-β was performed on 76 odontogenic cysts and tumors. Spearman's correlation coefficient, Kruskal-Wallis, and Mann-Whitney U tests followed by adjustment with Bonferroni were used for statistical analysis (P < .05).
RESULTS: A significant difference in podoplanin expression was found among the lesions consisting of solid ameloblastomas, adenomatoid odontogenic tumors, ameloblastic fibromas, odontogenic myxomas (OMs), odontogenic keratocysts, and calcifying odontogenic cysts. Significant differences were observed only between OMs and each of the other neoplasms. Podoplanin immunostaining in the connective tissue was absent in most lesions. TGF-β was significantly different among the study sample but not between the lesions in paired comparisons. None of the studied OTs showed significant correlations between podoplanin-TGF-β, in either the epithelium or the stroma. These markers were also descriptively reported in calcifying epithelial odontogenic tumors.
CONCLUSIONS: The inductive effect of TGF-β on podoplanin seems to be limited, if any, in odontogenic lesions. Podoplanin appears to play a role in some aspects of OTs with epithelial or mixed origins. Despite the possible participation of podoplanin in tumorigenesis, it may not necessarily be involved in the aggressive behavior of OTs.

Podoplanin (PDPN) is a transmembrane receptor glycoprotein that is upregulated on transformed cells, cancer associated fibroblasts and inflammatory macrophages that contribute to cancer progression. In particular, PDPN increases tumor cell clonal capacity, epithelial mesenchymal transition, migration, invasion, metastasis and inflammation. Antibodies, CAR-T cells, biologics and synthetic compounds that target PDPN can inhibit cancer progression and septic inflammation in preclinical models. This review describes recent advances in how PDPN may be used as a biomarker and therapeutic target for many types of cancer, including glioma, squamous cell carcinoma, mesothelioma and melanoma.

Acute promyelocytic leukemia (APL) is a medical emergency because of associated lethal early bleeding, a condition preventable by prompt diagnosis and therapeutic intervention. The mechanisms underlying the hemostatic anomalies of APL are not completely elucidated. RNA-sequencing-based characterization of APL (n = 30) was performed and compared to that of other acute myeloid leukemia (n = 400) samples and normal promyelocytes. Perturbations in the transcriptome of coagulation and fibrinolysis-related genes in APL extend beyond known culprits and now include Thrombin, Factor X and Urokinase Receptor. Most intriguingly, the Podoplanin (PDPN) gene, involved in platelet aggregation, is aberrantly expressed in APL promyelocytes and is the most distinctive transcript for this disease. Using an antibody panel optimized for AML diagnosis by flow cytometry, we also found that PDPN was the most specific surface marker for APL, and that all-trans retinoic acid therapy rapidly decreases its expression. Functional studies showed that engineered overexpression of this gene in human leukemic cells causes aberrant platelet binding, activation and aggregation. PDPN-expressing primary APL cells, but not PDPN-negative primary leukemias, specifically induce platelet binding, activation and aggregation. Finally, PDPN expression on leukemia cells in a xenograft model was associated with thrombocytopenia and prolonged bleeding time in vivo. Together our results suggest that PDPN may contribute to the hemostatic perturbations found in APL.

Tumor invasion is a critical first step in the organismic dissemination of cancer cells and the formation of metastasis in distant organs, the most important prognostic factor and the actual cause of death in most of the cancer patients. We report herein that the cell surface protein podoplanin (PDPN), a potent inducer of cancer cell invasion, is conspicuously expressed by the invasive front of squamous cell carcinomas (SCCs) of the cervix in patients and in the transgenic human papillomavirus/estrogen mouse model of cervical cancer. Laser capture microscopy combined with gene expression profiling reveals that the expression of interferon-responsive genes is up-regulated in PDPN-expressing cells at the tumor invasive front, which are exposed to CD45-positive inflammatory cells. Indeed, PDPN expression can be induced in cultured SCC cell lines by single or combined treatments with interferon-γ, transforming growth factor-β, and/or tumor necrosis factor-α. Notably, shRNA-mediated ablation of either PDPN or STAT1 in A431 SCC cells repressed cancer cell invasion on s.c. transplantation into immunodeficient mice. The results highlight the induction of tumor cell invasion by the inflammatory cytokine-stimulated expression of PDPN in the outermost cell layers of cervical SCC.

Degree of histological differentiation is an important characteristic of cancers and may be associated with malignant potential. However, in squamous cell carcinomas, a key transcriptional factor regulating tumor differentiation is largely unknown. Chemoradiotherapy (CRT) is a standard treatment for locally advanced esophageal squamous cell carcinoma; however, the survival rate is still below 40%. From microarray data, single-minded 2 (SIM2) was overexpressed in the epithelial subtype. Here, we investigated the correlation between SIM2 expression and its clinical implication, and in vitro and in vivo functions of SIM2 in tumor differentiation and in CRT sensitivity. Although SIM2 was suppressed in cancerous tissues, SIM2-high ESCC showed a favorable prognosis in CRT. Transient SIM2 expression followed by 3D culture induced expression of differentiation markers and suppressed epithelial-mesenchymal transition- and basal-cell markers. Levels of PDPN-high tumor basal cells and of expression of genes for DNA repair and antioxidant enzymes were reduced in stable transfectants, and they showed high CDDP and H

BACKGROUND: Moesin is a member of the ERM (ezrin, radixin and moesin) proteins that participate in cell migration and tumor invasion through transductional signals sent to actin filaments by glycoproteins, such as podoplanin.
METHODS: This study aimed to evaluate the participation of moesin and podoplanin in the invasive tumor front of oral squamous cell carcinomas, and their influence on patients' prognosis. Podoplanin and moesin immunoexpressions were evaluated by a semi-quantitative score method, based on the capture of 10 microscopic fields, at 400X magnification, in the invasive tumor front of oral squamous cell carcinomas. The association of moesin and podoplanin expression with clinicopathological variables was analyzed by the chi-square, or Fisher's exact test. The 5 and 10 years survival rates were calculated by the Kaplan-Meier method and the survival curves were compared by using the log-rank test.
RESULTS: The immunohistochemical expression of moesin in the invasive front of oral squamous cell carcinomas was predominantly strong, homogenously distributed on the membrane and in the cytoplasm of tumor cells. The expression of moesin was not associated with clinical, demographic and microscopic features of the patients. Otherwise, podoplanin expression by malignant epithelial cells was predominantly strong and significantly associated with radiotherapy (p = 0.004), muscular invasion (p = 0.006) and lymph node involvement (p = 0.013). Strong moesin expression was considered an unfavorable prognostic factor for patients with oral squamous cell carcinomas, clinical stage II and III (p = 0.024).
CONCLUSIONS: These results suggested that strong moesin expression by malignant cells may help to determine patients with oral squamous cell carcinoma and poor prognosis.

We report a case of concomitant EML4-ALK and TPM3-ROS1 fusion in non-small cell lung cancer (NSCLC) in a 47-year-old Chinese man and review the clinical characteristics of this type double of fusion. The patient presented with a local tumor of the left upper lobe and underwent thoracoscopy. Postoperative surgical pathologic staging revealed T

BACKGROUND/AIM: As a result of activation of transcription factors engaged in epithelial-mesenchymal transition (EMT), such as Twist, inhibition of epithelial markers and an increased expression of mesenchymal markers are observed. One of the specific markers of cancer-associated fibroblasts is podoplanin (PDPN) - a mucin-type membrane glycoprotein. The aim of this work was to study the localisation and intensity of expression of Twist and PDPN on the mRNA and protein level in cases of invasive ductal breast carcinoma (IDC), and its association with patients' clinico-pathological data.
MATERIALS AND METHODS: The study included archival material in a form of 80 paraffin IDC blocks and 11 IDC fragments frozen in liquid nitrogen. Immunohistochemical expression of Twist and PDPN was evaluated using light microscope and semiquantitative scale for evaluation of nuclear expression or immunoreactive scale (IRS) for evaluation of cytoplasmic expression. Material was isolated from frozen IDC fragments using laser micro-dissection (from cancer and stromal cells, separately) and was used to perform real-time PCR.
RESULTS: Twist expression was higher in stromal cells in comparison to cancer cells. Analysis of patients' survival rate showed, that higher expression of Twist in cancer cells was associated with shorter overall survival time and shorter event-free survival time. The expression of PDPN was also higher in stromal cells in comparison with cancer cells. In addition, positive correlation was observed between expression of Twist and PDPN in stromal cells of IDC (r=0.267; p<0.05).
CONCLUSION: The relationship between the higher expression of Twist in both cancer and stromal cells and shorter patients' survival indicates Twist as a potential useful prognostic marker in IDC. Positive correlation of Twist and PDPN expression may indicate the role of PDPN in EMT in IDC.

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such podoplanin-mediated effects can affect tube formation by endothelial cells and participate in their pathological properties in the tumor context. Our experimental data were supported by clinical studies. First, when IDC and DCIS were analyzed by immunohistochemistry according to the presence of podoplanin-expressing cells, the numbers of cancer-associated fibroblasts with high expression of this glycoprotein were significantly higher in IDC than in DCIS cases. Second, using immunofluorescence, the co-localization of PDPN-positive CAFs with blood vessels stained with antibody directed against CD34 was observed in tumor stroma of IDC samples.

BACKGROUND: During the epithelial tumor development process, changes in tumor cell genes are an important driving factor for tumorigenesis. Recently, however, studies have shown that the tumor microenvironment, especially for cancer-associated fibroblasts (CAFs) and the infiltration of platelets into tumors, plays an essential role in the progression of human malignant disease.
METHODS: In this study, we investigated the presence and prognostic role of podoplanin-expressing CAFs (CAF+), the infiltration of platelets into tumors (CD61+) and platelet count before surgery in a large sample of patients with breast cancer. Podoplanin expression and platelet infiltration were evaluated by immunohistochemistry in 164 patients with breast cancer.
RESULTS: Seventy-two patients (44%) showed CAF+, while fifty-seven patients (35%) showed CD61+. Several strong positive correlations were found, including CD61+ with blood platelet count before surgery (P=0.004), and CAF+ with CD61+ (P=0.048). Patients with CAF+, CD61+ or platelet count >280×10
CONCLUSIONS: CAF+ and CD61+ were found to be good negative prognosis factors for invasive breast cancer patients. CD61+ also had strong positive correlation with blood platelet count before surgery. These targets may be used as strategies for the treatment of breast cancer in the future.

BACKGROUND: Podoplanin (PDPN), a mucin-type transmembrane glycoprotein, is expressed in a variety of human cancer types, and contributes to tumor progression. Our goal was to evaluate PDPN expression in hepatocellular carcinoma (HCC) using both immunohistochemistry (IHC) and RNAscope in situ hybridization.
MATERIALS AND METHODS: Twenty patients with HCC who underwent partial hepatectomy with curative intent were retrospectively analyzed.
RESULTS: IHC gave positive results in 11 cases, while RNAscope assay for PDPN detected amplification in 16 cases. A significant association was noted between PDPN protein expression and histological tumor grade (p=0.036). Four cases that had negative PDPN results by RNAscope were also negative by IHC, while the remaining five cases with negative results by IHC were positive by RNAscope. A positive relationship was found between PDPN mRNA protein expression (p<0.001).
CONCLUSION: Our preliminary results suggest that PDPN contributes to the malignant potential of HCC. RNAscope proved to be a more sensitive and reliable method than IHC in PDPN detection.

Although it has been well-demonstrated that bone marrow mesenchymal stromal cells (MSCs) from CML patients do not belong to the Ph1-positive clone, there is growing evidence that they could play a role in the leukemogenesis process or the protection of leukemic stem cells from the effects of tyrosine kinase inhibitors (TKIs). The aim of the present study was to identify genes differentially expressed in MSCs isolated from CML patients at diagnosis (CML-MSCs) as compared to MSCs from healthy controls. Using a custom gene-profiling assay, we identified six genes over-expressed in CML-MSCs (BMP1, FOXO3, MET, MITF, NANOG, PDPN), with the two highest levels being documented for PDPN (PODOPLANIN) and NANOG. To determine whether this aberrant signature persisted in patients in deep molecular response induced by TKIs, we analyzed MSCs derived from such patients (MR-MSCs). This analysis showed that, despite the deep molecular responses, BMP1, MET, MITF, NANOG, and PDPN mRNA were upregulated in MR-MSCs. Moreover, BMP1, MITF, and NANOG mRNA expressions in MR-MSCs were found to be intermediate between control MSCs and CML-MSCs. These results suggest that CML-MSCs exhibit an abnormal gene expression pattern which might have been established during the leukemogenic process and persist in patients in deep molecular response.

OBJECTIVES: To investigate the relevance of lymphangiogenic gene expression in primary and liver metastasis of colorectal cancer (CRC) and identify determinants of lymphatic invasion.
BACKGROUND: Lymphatic development promoting vascular endothelial growth factor C (VEGFC) is associated with poor outcome in primary CRC. For colorectal liver metastasis (CRLM), intrahepatic lymph invasion and lymph node metastasis are poor prognostic factors. Exact biological factors promoting lymphatic involvement remain elusive, just as the association with molecular subtypes of CRC.
METHODS: We designed a lymphangiogenic gene set (VEGFC, Nrp-2, PDPN, LYVE-1, MRC1, CCL-21) and applied it to large datasets of CRC. Gene expression of the lymphangiogenic signature was assessed in resected CRLM specimens by Rt-QPCR. In vitro experiments were performed with colon cancer cell line Colo320 (high Nrp-2 expression) and human dermal microvascular lymphatic endothelial cells (LECs).
RESULTS: Lymphangiogenic gene expression was associated with poor prognosis in both primary and liver metastasis of CRC. CRLM with high expression of consensus molecular subtype-4 identifier genes also exhibited high lymphangiogenic gene expression. Lymph node recurrence following CRLM resection was associated with high expression of VEGFC and Nrp-2. Blocking Nrp-2 significantly reduced invasion of Colo320 cells through an LEC monolayer.
CONCLUSIONS: Lymphangiogenic gene expression is correlated with worse prognosis and consensus molecular subtype-4 in both primary and liver metastatic CRC. VEGFC and Nrp-2 expression may be predictive of lymph node involvement in recurrence after resection of CRLM. Nrp-2, expressed on both tumor and LECs, may have a mechanistic role in lymphatic invasion and is a potential novel target in CRC.

Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.

PURPOSE: To investigate the role of podoplanin (PDPN) expression in invasive ductal carcinoma of the pancreas (IDCP) in humans.
METHODS: Tumor samples were obtained from 95 patients with IDCP. Immunohistochemical staining was done to evaluate the expression of PDPN in cancer tissues.
RESULTS: PDPN was detected predominantly in stromal fibroblasts, stained with α-smooth muscle actin. The cutoff value of PDPN-positive areas was calculated according to a histogram. There was no significant difference in clinicopathologic factors between patients with high vs. those with low PDPN expression. The high PDPN group showed significantly poorer disease-free and disease-specific survival rates than the low PDPN group. Among patients from the high PDPN group, those with lymph node metastases and those with a tumor larger than 20 cm in diameter had significantly poorer prognoses than similar patients from the low PDPN group. Multivariate Cox proportional hazards analysis indicated that a high expression of PDPN was an independent risk factor for disease-specific survival.
CONCLUSIONS: PDPN expression in cancer-related fibrotic tissues is associated with a poor prognosis, especially in patients with large tumors or lymph node metastases.

Podoplanin and fibroblast growth factor (FGF) 1 have been detected more frequently in lung squamous cell carcinoma (SQCC) compared with lung adenocarcinoma. Furthermore, it has been previous demonstrated that FGF1 is located on the edge of tumor nests in certain lung SQCC sections, which resembles the characteristic expression pattern of podoplanin. Podoplanin and FGF1 have roles in lymphangiogenesis and angiogenesis. Based on their consistently specific expression in lung SQCC and similar localization patterns, the present study aimed to investigate whether the expression of podoplanin in tumor cells is correlated with FGF1 expression in lung SQCC and whether their co‑expression has clinicopathological significance, particularly for lymphangiogenesis/angiogenesis. The correlation between podoplanin and FGF1 expression in tumor cells of 82 lung SQCC cases was investigated by immunohistochemical staining and the association between the co‑expression of podoplanin and FGF1, and clinicopathological factors such as microvessel density (MVD), was examined in these samples. In addition, the prognostic value of co‑expression of podoplanin and FGF1 in tumor cells was determined, and the regulation of FGF1 expression and angiogenesis by podoplanin was examined in vitro in a human lung SQCC cell line. Immunohistochemical analysis demonstrated that there was a significant correlation between podoplanin and FGF1 expression in lung SQCC tumor cells (R=0.591; P<0.0001). Co‑expression of podoplanin and FGF1 was significantly associated with larger primary tumor size, advanced TNM stage and higher intratumoral MVD. Survival analysis demonstrated that cases with podoplanin and FGF1 double‑positive staining had a significantly lower survival rate compared with cases with double‑negative staining. In vitro experiments revealed that podoplanin regulated FGF1 expression and affected tube formation of human umbilical vein endothelial cells. Combined, the results demonstrated that podoplanin was co‑expressed with FGF1 in lung SQCC and this co‑expression was correlated with poor prognosis.

BACKGROUND: Myeloid-derived lymphatic endothelial cells (M-LECP) are induced by inflammation and play an important role in adult lymphangiogenesis. However, the mechanisms driving M-LECP differentiation are currently unclear. We previously showed that activation of Toll-like receptor-4 (TLR4) induces myeloid-lymphatic transition (MLT) of immortalized mouse myeloid cells. Here the goals were to assess the potential of different TLR4 ligands to induce pro-lymphatic reprogramming in human and mouse primary myeloid cells and to identify transcriptional changes regulating this process.
METHODOLOGY/PRINCIPAL FINDINGS: Human and mouse myeloid cells were reprogrammed to the lymphatic phenotype by TLR4 ligands including lipopolysaccharide (LPS), recombinant high mobility group box 1 protein (HMGB1), and paclitaxel. TLR4 induced similar MLT in cells from mice of different strains and immune status. Commonly induced genes were detected by transcriptional profiling in human and mouse myeloid cells from either immunocompetent or immunodeficient mice. Shared trends included: (1) novel expression of lymphatic-specific markers vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and podoplanin (PDPN) largely absent prior to induction; (2) lack of notable changes in blood vessel-specific markers; (3) transient expression of VEGFR-3, but sustained increase of vascular endothelial growth factor-C (VEGF-C) and a variety of inflammatory cytokines; (4) dependency of VEGFR-3 upregulation and other LEC genes on NF-κB; and (5) novel expression of lymphatic-specific (e.g., PROX1) and stem/progenitor (e.g., E2F1) transcription factors known for their roles in adult and embryonic vascular formation. M-LECP generated by TLR4 ligands in vitro were functional in vivo as demonstrated by significantly increased lymphatic vessel density and lymphatic metastasis detected in orthotopic breast cancer models.
CONCLUSIONS/SIGNIFICANCE: We established a novel TLR4-dependent protocol for in vitro production of functionally competent M-LECP from primary human or mouse myeloid cells and identified many potential regulators of this process. This information can be further exploited for research and therapeutic purposes.

Chlamydia trachomatis is the most prevalent infection of the genital tract in women worldwide. C. trachomatis has a tendency to cause persistent infection and induce a state of chronic inflammation, which has been reported to play a role in carcinogenesis. We report that persistent C. trachomatis infection increases the expression of inflammatory tumour cytokines and upregulates molecular biomarkers such as podoplanin, Wilms' tumour gene 1 and osteopontin in primary cultures of mesothelial cells (Mes1) and human mesothelioma cells (NCI). Infection experiments showed that Mes1 and NCI supported the growth of C. trachomatisin vitro, and at an m.o.i. of 4, the inclusion-forming units/cell showed many intracellular inclusion bodies after 3 days of infection. However, after 7 days of incubation, increased proliferative and invasive activity was also observed in Mes1 cells, which was more evident after 14 days of incubation. ELISA analysis revealed an increase in vascular endothelial growth factor, IL-6, IL-8, and TNF-α release in Mes1 cells infected for a longer period (14 days). Finally, real-time PCR analysis revealed a strong induction of podoplanin, Wilms' tumour gene 1 and osteopontin gene expression in infected Mes1 cells. The aim of the present study was to investigate the inflammatory response elicited by C. trachomatis persistent infection and the role played by inflammation in cell proliferation, secretion of proinflammatory cytokines and molecular biomarkers of cancer. The results of this study suggest that increased molecular biomarkers of cancer by persistent inflammation from C. trachomatis infection might support cellular transformation, thus increasing the risk of cancer.

Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG

Several lymphangiogenic factors, such as vascular endothelial growth factors (VEGFs), have been found to drive the development of lymphatic metastasis in bladder cancer (BCa).Here, we have analyzed the gene expression of lymphangiogenic factors in tissue specimens from 12 non-muscle invasive bladder cancers (NMIBC) and 11 muscle invasive bladder cancers (MIBC), considering tumor and tumor-adjacent normal bladder areas obtained from the same organs. We then compared the results observed in patients with those obtained after treating human primary bladder microvascular endothelial cells (MEC) with either direct stimulation with VEGF-A or VEGF-C or by co-culturing (trans-well assay) MEC with bladder cancer cell lines varying in VEGF-A and VEGF-C production based on tumor grade.The genes of three markers of lymphatic endothelial commitment and development (PDPN, LYVE-1 and SLP-76) were significantly overexpressed in tissues of MIBC patients showing positive lymphovascular invasion (LVI+), lymph node metastasis (Ln+) and tumor progression. Their expression was also significantly enhanced either after direct stimulation of MEC by VEGF-A and VEGF-C or in the trans-well assay with each bladder cancer cell line.SLP-76 showed the highest gene expression. Both VEGF-A and VEGF-C also enhanced the expression of SLP-76 protein in MEC. However, a correlation between increase of SLP-76 gene expression and the ability of MEC to migrate could only be seen after induction by VEGF-C.The significant expression of SLP-76 in LVI+/Ln+ progressive MIBC and its overexpression in MEC after VEGF-A and VEGF-C stimulation suggest the need to develop this regulator of developmental lymphangiogenesis as a diagnostic tool in BCa.

The role of podoplanin in hepatocellular carcinoma (HCC) is not clear yet. The aim of our study was to evaluate the expression of podoplanin in HCC and to determine its role in hepatocarcinogenesis. We performed immunohistochemistry with monoclonal D2-40 antibody, on paraffin-embedded tissue sections of 72 patients diagnosed with HCC. Lymphatic vessels density (LVD) was increased in patients who had vascular invasion at the time of diagnosis (P=0.018) and in those with associated cirrhosis (P=0.006). Tumor cells showing podoplanin expression were correlated with histological grade (P=0.040). Podoplanin-expressing cancer associated fibroblasts (CAFs) were correlated with both LVD (P=0.019) and tumor cells (P=0.015). Our results sustain the dual role of podoplanin in HCC by its involvement in both HCC tumorigenesis, lymphatic neovascularization and tumor invasion invasiveness. A possible crosstalk between epithelial and stromal tumor cells in HCC tumor microenvironment may be mediated by podoplanin, but this hypothesis needs further studies to elucidate this interrelation.

The tumour microenvironment is critical for various characteristics of tumour malignancies. Platelets, as part of the tumour microenvironment, are associated with metastasis formation via increasing the rate of tumour embolus formation in microvasculature. However, the mechanisms underlying the ability of tumour cells to acquire invasiveness and extravasate into target organs at the site of embolization remain unclear. In this study, we reported that platelet aggregation-inducing factor podoplanin expressed on tumour cell surfaces were found to not only promote the formation of tumour-platelet aggregates via interaction with platelets, but also induced the epithelial-mesenchymal transition (EMT) of tumour cells by enhancing transforming growth factor-β (TGF-β) release from platelets. In vitro and in vivo analyses revealed that podoplanin-mediated EMT resulted in increased invasiveness and extravasation of tumour cells. Treatment of mice with a TGF-β-neutralizing antibody statistically suppressed podoplanin-mediated distant metastasis in vivo, suggesting that podoplanin promoted haematogenous metastasis in part by releasing TGF-β from platelets that was essential for EMT of tumour cells. Therefore, our findings suggested that blocking the TGF-β signalling pathway might be a promising strategy for suppressing podoplanin-mediated haematogenous metastasis in vivo.

Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

Tumor microenvironment has been recognized as a key determinant of tumor formation and metastasis, but how tumor microenvironment is affected by nanomaterials is essentially unknown. Here, we investigated whether carbon nanotubes (CNTs), a widely used nanomaterial with known carcinogenic potential, can affect cancer-associated fibroblasts (CAFs), which are a key component of tumor microenvironment that provides necessary support for tumor growth. We show for the first time that single-walled CNT and to a lesser extent multi-walled and its COOH-functionalized form induced CAF-like cells, which are non-tumorigenic in animals, but promote tumor growth of human lung carcinoma and CNT-transformed lung epithelial cells. The mechanism by which CNT-induced CAF-like cells promote tumor growth involved the acquisition of cancer stem cells (CSCs) in cancer population. Gene knockdown experiments showed that an expression of podoplanin on CAF-like cells is essential for their effects, indicating the functional role of CAF-like cells and podoplanin in CNT tumorigenic process. Our findings unveil a novel mechanism of CNT-induced carcinogenesis through the induction of CAF-like cells that support CSCs and drive tumor formation. Our results also suggest the potential utility of podoplanin as a mechanism-based biomarker for rapid screening of carcinogenicity of CNTs and related nanomaterials for their safer design.