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Nontransformed cells form heterotypic cadherin junctions with adjacent transformed cells to inhibit tumor cell growth and motility. Transformed cells must override this form of growth control, called "contact normalization", to invade and metastasize during cancer progression. Heterocellular cadherin junctions between transformed and nontransformed cells are needed for this process. However, specific mechanisms downstream of cadherin signaling have not been clearly elucidated. Here, we utilized a β-catenin reporter construct to determine if contact normalization affects Wnt signaling in transformed cells. β-catenin driven GFP expression in Src transformed mouse embryonic cells was decreased when cultured with cadherin competent nontransformed cells compared to transformed cells cultured with themselves, but not when cultured with cadherin deficient nontransformed cells. We also utilized a layered culture system to investigate the effects of oncogenic transformation and contact normalization on gene expression and oncogenic Src kinase mediated phosphorylation events. RNA-Seq analysis found that cadherin dependent contact normalization inhibited the expression of 22 transcripts that were induced by Src transformation, and increased the expression of 78 transcripts that were suppressed by Src transformation. Phosphoproteomic analysis of cells expressing a temperature sensitive Src kinase construct found that contact normalization decreased phosphorylation of 10 proteins on tyrosine residues that were phosphorylated within 1 h of Src kinase activation in transformed cells. Taken together, these results indicate that cadherin dependent contact normalization inhibits Wnt signaling to regulate oncogenic kinase activity and gene expression, particularly PDPN expression, in transformed cells in order to control tumor progression. [ABSTRACT FROM AUTHOR]

Abstract Nontransformed cells form heterotypic cadherin junctions with adjacent transformed cells to inhibit tumor cell growth and motility. Transformed cells must override this form of growth control, called “contact normalization”, to invade and metastasize during cancer progression. Heterocellular cadherin junctions between transformed and nontransformed cells are needed for this process. However, specific mechanisms downstream of cadherin signaling have not been clearly elucidated. Here, we utilized a β-catenin reporter construct to determine if contact normalization affects Wnt signaling in transformed cells. β-catenin driven GFP expression in Src transformed mouse embryonic cells was decreased when cultured with cadherin competent nontransformed cells compared to transformed cells cultured with themselves, but not when cultured with cadherin deficient nontransformed cells. We also utilized a layered culture system to investigate the effects of oncogenic transformation and contact normalization on gene expression and oncogenic Src kinase mediated phosphorylation events. RNA-Seq analysis found that cadherin dependent contact normalization inhibited the expression of 22 transcripts that were induced by Src transformation, and increased the expression of 78 transcripts that were suppressed by Src transformation. Phosphoproteomic analysis of cells expressing a temperature sensitive Src kinase construct found that contact normalization decreased phosphorylation of 10 proteins on tyrosine residues that were phosphorylated within 1 h of Src kinase activation in transformed cells. Taken together, these results indicate that cadherin dependent contact normalization inhibits Wnt signaling to regulate oncogenic kinase activity and gene expression, particularly PDPN expression, in transformed cells in order to control tumor progression.

Objectives: The aim of this study is to identify and analyze the existing literature on the utility of podoplanin to predict the risk of malignancy development (MD) in patients previously diagnosed with oral leukoplakia (OL). Methods: A systematic review and meta‐analysis (SRMA) was performed though a search strategy using several electronic databases and a combination of keywords related to podoplanin and MD of OL, until 15 May, 2022 (PROSPERO CRD42022329326). Evaluation of the risk of bias (ROB) was performed using the Quality in Prognosis Studies Tool. The meta‐analyses were estimated using fixed‐effect models. Results: From 421 articles, 6 studies were finally included, that enrolled 546 patients with OL, of whom 125 presented with an oral cancer during follow‐up (32 to 90 months). Some limitations regarding the ROB were identified mostly related to small sample sizes, short follow‐up times, lack of information on covariables in the included studies and lack of accuracy (including sensitivity and specificity). Meta‐analysis of 6 studies reveal that high expression of podoplanin carries a pooled hazard ratio (HR) of 3.72 (95% CI, 2.40–5.76; p < 0.00001) for MD without statistical heterogeneity (I2 = 0%, p = 0.53). Conclusion: The results of this SRMA support the role of podoplanin immunohistochemical expression as a potential predictive biomarker to assess the risk of malignancy development in oral leukoplakia. [ABSTRACT FROM AUTHOR]

Aims: Glioblastoma is the most frequent and aggressive primary brain tumor, characterized by rapid disease course and poor treatment responsiveness. The abundance of immunosuppressive macrophages in glioblastoma challenges the efficacy of novel immunotherapy. Methods: Bulk RNA‐seq and single‐cell RNA‐seq of glioma patients from public databases were comprehensively analyzed to illustrate macrophage infiltration patterns and molecular characteristics of podoplanin (PDPN). Multiplexed fluorescence immunohistochemistry staining of PDPN, GFAP, CD68, and CD163 were performed in glioma tissue microarray. The impact of PDPN on macrophage immunosuppressive polarization was investigated using a co‐culture system. Bone marrow‐derived macrophages (BMDMs) and OT‐II T cells isolated from BALB/c and OT‐II mice respectively were co‐cultured to determine T‐cell adherence. Pathway alterations were probed through RNA sequencing and western blot analyses. Results: Our findings demonstrated that PDPN is notably correlated with the expression of CD68 and CD163 in glioma tissues. Additionally, macrophages phagocytosing PDPN‐containing EVs (EVsPDPN) from GBM cells presented increased CD163 expression and augmented secretion of immunoregulatory cytokine (IL‐6, IL‐10, TNF‐α, and TGF‐β1). PDPN within EVs was also associated with enhanced phagocytic activity and reduced MHC II expression in macrophages, compromising CD4+ T‐cell activation. Conclusions: This investigation underscores that EVsPDPN derived from glioblastoma cells contributes to M2 macrophage‐mediated immunosuppression and is a potential prognostic marker and therapeutic target in glioblastoma. [ABSTRACT FROM AUTHOR]

Abstract Background Podoplanin (PDPN) is a highly conserved, mucin-type protein specific to the lymphatic system. Overexpression of PDPN is associated with the progression of various solid tumors, and plays an important roles in the tumor microenvironment by regulating the immune system. However, the role of PDPN-mediated signal activation in the progression of melanoma is still unknown. Methods PDPN expression was first analyzed in 112 human melanoma tissue microarrays and melanoma cell lines. Functional experiments including proliferation, clone formation, migration, and metastasis were utilized to identify the suppressive effects of PDPN. The Ph.D.TM-12 Phage Display Peptide Library was used to obtain a PDPN antagonist peptide, named CY12-RP2. The immunofluorescence, SPR assay, and flow cytometry were used to identify the binding specificity of CY12-RP2 with PDPN in melanoma cells. Functional and mechanistic assays in vivo and in vitro were performed for discriminating the antitumor and immune activation effects of CY12-RP2. Results PDPN was overexpressed in melanoma tissue and cells, and inhibited melanoma cells proliferation, migration, and metastasis by blocking the EMT and Wnt/β-catenin pathway. PDPN antagonistic peptide, CY12-RP2, could specifically bind with PDPN, suppressing melanoma various functions inducing apoptosis in both melanoma cells and 3D spheroids. CY12-RP2 also enhanced the anti-tumor capacity of PBMC, and inhibited melanoma cells growth both in xenografts and allogeneic mice model. Moreover, CY12-RP2 could inhibit melanoma lung metastasis, and abrogated the immunosuppressive effects of PDPN by increasing the proportion of CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD49b + Granzyme B + NK cells, and CD11b + CD86 + M1-like macrophages and the levels of IL-1β, TNF-α, and IFN-γ. Conclusions This study has demonstrated the important role of PDPN in the progression of melanoma and formation of immunosuppressive environment, and provided a potential approach of treating melanoma using the novel CY12-RP2 peptide. Graphical Abstract In melanoma, PDPN is overexpressed in the cancer cells, and promotes melanoma cells growth and metastasis through activating the Wnt/β-catenin pathway. Treatment with the PDPN antagonistic peptide CY12-RP2 could not only inhibit the melanoma growth and metastasis both in vitro and in vivo through Wnt/β-catenin pathway blockade, but also abrogate the immunosuppressive effects of PDPN through modulating immune cells.

Immunohistochemistry staining is an essential method in pathological diagnoses. Podoplanin (PDPN) is a specific maker of alveolar epithelium, lymphatic vessels, and glomeruli. In this study, we established a novel anti-giraffe PDPN (girPDPN) mAb, PMab-301, using the Cell-Based Immunization and Screening (CBIS) method. PMab-301 (mouse IgG1, kappa) detected girPDPN in various applications, such as flow cytometry, western blot, and immunohistochemistry. PMab-301 specifically stained type-I alveolar cells using formalin-fixed paraffin-embedded giraffe lung tissues. Our findings suggest the potential usefulness of PMab-301 for the pathophysiological analyses of giraffe tissues. [ABSTRACT FROM AUTHOR]

ObjectiveThe aim of this study was to compare the clinical outcomes of spinal cord stimulation (SCS) and dorsal root ganglion stimulation (DRG-S) in the treatment of painful diabetic peripheral neuropathy (PDPN).MethodsIn this prospective cohort study, 55 patients received dorsal column spinal cord stimulation (SCS group) and 51 patients received dorsal root spinal cord stimulation (DRG-S group). The primary outcome was a Numerical Rating Scale (NRS) remission rate of ≥50%, and secondary outcomes included the effects of SCS and DRG-S on quality of life scores (EQ-5D-3L), nerve conduction velocity, and HbA1c, respectively.ResultsThe percentage of NRS remission rate ≥ 50% at 6 months was 80.43 vs. 79.55%, OR (95% CI): 1.06 (0.38–2.97) in the SCS and DRG-S groups, respectively, and the percentage of VAS remission rate ≥ 50% at 12 months was 79.07 vs. 80.95%, OR (95% CI): 0.89 (0.31–2.58). Compared with baseline, there were significant improvements in EQ-5D and EQ-VAS at 6 and 12 months (p 0.05). Nerve conduction velocities of the common peroneal, peroneal, superficial peroneal, and tibial nerves were significantly improved at 6 and 12 months compared with the preoperative period in both the SCS and PND groups (p 0.05).ConclusionBoth SCS and DRG-S significantly improved pain, quality of life, and lower extremity nerve conduction velocity in patients with PDPN, and there was no difference between the two treatments at 12 months.

Abstract Gastric cancer (GC) is a frequent malignant disease and the main cause of cancer‐related death in the world. Podoplanin (PDPN) has been proved to be involved in the progression of various cancers. However, the role and biological mechanism of PDPN in GC are still vague. In our study, we detected the expression of PDPN in GC tissues and cell lines using RT‐qPCR, western blot and datasets. The overall survival of GC patients was analysed with a Kaplan–Meier plot. The effects of PDPN overexpression and silencing on GC cell progression were assessed by Cell Counting Kit‐8, flow cytometry and a wound healing assay. Besides, the modulation of PDPN on ezrin activation was investigated. We further explored the role of PDPN in the crosstalk between GC cells and cancer associated fibroblasts (CAFs). Results showed that PDPN was upregulated in GC tissues and cell lines. High expression of PDPN was correlated with poor prognosis of GC patients. PDPN positively regulated the viability, migration and invasion, but inhibited apoptosis, of GC cells by mediating the activation of ezrin. Meanwhile, the change in PDPN in GC cells activated CAFs and promoted the production of cytokines secreted by CAFs, which induced the progression of GC cells. These findings may provide a novel target for GC therapy.

New Findings: What is the central question of this study?To reveal the role and biological mechanism of PDPN in the progression of gastric cancer.What is the main finding and its importance?This study focused on a prognostic predictor, PDPN, which acted as a promoter in the progression of gastric cancer through the activation of Ezrin expression and CAFs. This finding may expand a new route for the gene‐targeted therapy in gastric cancer. Gastric cancer (GC) is a frequent malignant disease and the main cause of cancer‐related death in the world. Podoplanin (PDPN) has been proved to be involved in the progression of various cancers. However, the role and biological mechanism of PDPN in GC are still vague. In our study, we detected the expression of PDPN in GC tissues and cell lines using RT‐qPCR, western blot and datasets. The overall survival of GC patients was analysed with a Kaplan–Meier plot. The effects of PDPN overexpression and silencing on GC cell progression were assessed by Cell Counting Kit‐8, flow cytometry and a wound healing assay. Besides, the modulation of PDPN on ezrin activation was investigated. We further explored the role of PDPN in the crosstalk between GC cells and cancer associated fibroblasts (CAFs). Results showed that PDPN was upregulated in GC tissues and cell lines. High expression of PDPN was correlated with poor prognosis of GC patients. PDPN positively regulated the viability, migration and invasion, but inhibited apoptosis, of GC cells by mediating the activation of ezrin. Meanwhile, the change in PDPN in GC cells activated CAFs and promoted the production of cytokines secreted by CAFs, which induced the progression of GC cells. These findings may provide a novel target for GC therapy. [ABSTRACT FROM AUTHOR]

Golden (Syrian) hamster (Mesocricetus auratus) is a small animal model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Pathological analyses of the tissues are required to understand the pathogenesis of SARS-CoV-2 and the evaluation of therapeutic modalities, including neutralizing monoclonal antibodies (mAbs). However, mAbs that recognize the golden hamster-derived antigens and distinguish specific cell types, such as the pneumocytes, are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-Chinese hamster (Cricetulus griseus) PDPN mAb PMab-281 (IgG3, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. A defucosylated mouse IgG2a version of PMab-281 (281-mG2a-f) was also developed. The 281-mG2a-f strongly recognized both the Chinese hamster and the golden hamster PDPN using flow cytometry and could detect lung type I alveolar epithelial cells, lymphatic endothelial cells, and Bowman's capsules in the kidney from the golden hamster using immunohistochemistry. These results suggest the usefulness of 281-mG2a-f for analyzing the golden hamster-derived tissues and cells for SARS-CoV-2 research. [ABSTRACT FROM AUTHOR]

Painful diabetic peripheral neuropathy is characterized by burning, stabbing, or electric shock–type pain, which severely impacts day‐to‐day functioning and quality of life. Here, we report the results of 3 phase I studies with NRD135S.E1 (referred to as NRD.E1), a new, orally available chemical entity, presently developed for the treatment of painful diabetic peripheral neuropathy. The first study was a first‐in‐human, randomized, placebo‐controlled, single‐ascending‐dose study, where NRD.E1 was administered to healthy male subjects in single dosages ranging from 300 to 1200 mg. The second study was a randomized, placebo‐controlled multiple‐dose study, where healthy male subjects received 300 mg of NRD.E1 once daily for 5 consecutive days. The third study was an open‐label food interaction study in healthy men and women following a crossover design, where NRD.E1 was administered under fed and fasted conditions at 40 mg. The studies revealed dose‐dependent absorption, increased exposure to NRD.E1 when administered with food, and no relevant accumulation after once‐daily administration. All 3 phase I studies consistently showed rapid absorption of orally administered NRD.E1 followed by fast elimination, mainly via metabolization (glucuronidation), and small secondary increases in plasma concentrations. NRD.E1 was well tolerated, with no subject discontinuation due to treatment‐emergent adverse events in any study. [ABSTRACT FROM AUTHOR]

Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target. [ABSTRACT FROM AUTHOR]

Background: Streptococcus pneumoniae is the major cause of life‐threatening infections. Toll‐like receptors (TLRs) and NOD‐like receptors (NLRs) could recognise S. pneumoniae and regulate the production of pro‐inflammatory cytokines. UGRP1, highly expressed in lung, is predominantly secreted in airways. However, the function of UGRP1 in pneumonia is mainly unknown. Methods and results: We showed that upon TLR2/TLR4/NOD2 agonists stimulation or S. pneumoniae infection, treatment with UGRP1 could promote phosphorylation of p65 and enhance IL‐6, IL‐1β and TNFα production in macrophages. We further elucidated that after binding with cell‐surface receptor PDPN, UGRP1 could activate RhoA to enhance interaction of IKKγ and IKKβ, which slightly activated NF‐κB to improve expression of TLR2, MyD88, NOD2 and NLRP3. Deletion of UGRP1 or blocking UGRP1 interaction with PDPN protected mice against S. pneumoniae‐induced severe pneumococcal pneumonia, and activating RhoA with agonist in UGRP1‐deficient mice restored the reduced IL‐6 production. Conclusion: We demonstrated that UGRP1–PDPN–RhoA signaling could activate NF‐κB to promote expression of TLR2, MyD88, NOD2 and NLRP3, which enhanced inflammatory cytokines secretion during S. pneumoniae infection. Antibodies, which could interrupt interaction of UGRP1 and PDPN, are potential therapeutics against S. pneumoniae. [ABSTRACT FROM AUTHOR]

Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

Abstract Background Streptococcus pneumoniae is the major cause of life‐threatening infections. Toll‐like receptors (TLRs) and NOD‐like receptors (NLRs) could recognise S. pneumoniae and regulate the production of pro‐inflammatory cytokines. UGRP1, highly expressed in lung, is predominantly secreted in airways. However, the function of UGRP1 in pneumonia is mainly unknown. Methods and results We showed that upon TLR2/TLR4/NOD2 agonists stimulation or S. pneumoniae infection, treatment with UGRP1 could promote phosphorylation of p65 and enhance IL‐6, IL‐1β and TNFα production in macrophages. We further elucidated that after binding with cell‐surface receptor PDPN, UGRP1 could activate RhoA to enhance interaction of IKKγ and IKKβ, which slightly activated NF‐κB to improve expression of TLR2, MyD88, NOD2 and NLRP3. Deletion of UGRP1 or blocking UGRP1 interaction with PDPN protected mice against S. pneumoniae‐induced severe pneumococcal pneumonia, and activating RhoA with agonist in UGRP1‐deficient mice restored the reduced IL‐6 production. Conclusion We demonstrated that UGRP1–PDPN–RhoA signaling could activate NF‐κB to promote expression of TLR2, MyD88, NOD2 and NLRP3, which enhanced inflammatory cytokines secretion during S. pneumoniae infection. Antibodies, which could interrupt interaction of UGRP1 and PDPN, are potential therapeutics against S. pneumoniae.

Based on the concept of "Evolutionary Traps", targeting survival essential genes obtained during tumor drug resistance can effectively eliminate resistant cells. While, it still faces limitations. In this study, lapatinib-resistant cells were used to test the concept of "Evolutionary Traps" and no suitable target stand out because of the identified genes without accessible drug. However, a membrane protein PDPN , which is low or non-expressed in normal tissues, is identified as highly expressed in lapatinib-resistant tumor cells. PDPN CAR-T cells were developed and showed high cytotoxicity against lapatinib-resistant tumor cells in vitro and in vivo , suggesting that CAR-T may be a feasible route for overcoming drug resistance of tumor based on "Evolutionary Trap". To test whether this concept is cell line or drug dependent, we analyzed 21 drug-resistant tumor cell expression profiles reveal that JAG1, GPC3, and L1CAM, which are suitable targets for CAR-T treatment, are significantly upregulated in various drug-resistant tumor cells. Our findings shed light on the feasibility of utilizing CAR-T therapy to treat drug-resistant tumors and broaden the concept of the "Evolutionary Trap". [Display omitted] [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep, and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reacted with type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman's capsules from kidney, and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN. [ABSTRACT FROM AUTHOR]

Cancer tissues are not just simple masses of malignant cells, but rather complex and heterogeneous collections of cellular and even non-cellular components, such as endothelial cells, stromal cells, immune cells, and collagens, referred to as tumor microenvironment (TME). These multiple players in the TME develop dynamic interactions with each other, which determines the characteristics of the tumor. Platelets are the smallest cells in the bloodstream and primarily regulate blood coagulation and hemostasis. Notably, cancer patients often show thrombocytosis, a status of an increased platelet number in the bloodstream, as well as the platelet infiltration into the tumor stroma, which contributes to cancer promotion and progression. Thus, platelets function as one of the important stromal components in the TME, emerging as a promising chemotherapeutic target. However, the use of traditional antiplatelet agents, such as aspirin, has limitations mainly due to increased bleeding complications. This requires to implement new strategies to target platelets for anti-cancer effects. In oral squamous cell carcinoma (OSCC) patients, both high platelet counts and low tumor-stromal ratio (high stroma) are strongly correlated with increased metastasis and poor prognosis. OSCC tends to invade adjacent tissues and bones and spread to the lymph nodes for distant metastasis, which is a huge hurdle for OSCC treatment in spite of relatively easy access for visual examination of precancerous lesions in the oral cavity. Therefore, locoregional control of the primary tumor is crucial for OSCC treatment. Similar to thrombocytosis, higher expression of podoplanin (PDPN) has been suggested as a predictive marker for higher frequency of lymph node metastasis of OSCC. Cumulative evidence supports that platelets can directly interact with PDPN-expressing cancer cells via C-type lectin-like receptor 2 (CLEC2), contributing to cancer cell invasion and metastasis. Thus, the platelet CLEC2-PDPN axis could be a pinpoint target to inhibit interaction between platelets and OSCC, avoiding undesirable side effects. Here, we will review the role of platelets in cancer, particularly focusing on CLEC2-PDPN interaction, and will assess their potentials as therapeutic targets for OSCC treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. Results ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR

Cancer tissues are not just simple masses of malignant cells, but rather complex and heterogeneous collections of cellular and even non-cellular components, such as endothelial cells, stromal cells, immune cells, and collagens, referred to as tumor microenvironment (TME). These multiple players in the TME develop dynamic interactions with each other, which determines the characteristics of the tumor. Platelets are the smallest cells in the bloodstream and primarily regulate blood coagulation and hemostasis. Notably, cancer patients often show thrombocytosis, a status of an increased platelet number in the bloodstream, as well as the platelet infiltration into the tumor stroma, which contributes to cancer promotion and progression. Thus, platelets function as one of the important stromal components in the TME, emerging as a promising chemotherapeutic target. However, the use of traditional antiplatelet agents, such as aspirin, has limitations mainly due to increased bleeding complications. This requires to implement new strategies to target platelets for anti-cancer effects. In oral squamous cell carcinoma (OSCC) patients, both high platelet counts and low tumor-stromal ratio (high stroma) are strongly correlated with increased metastasis and poor prognosis. OSCC tends to invade adjacent tissues and bones and spread to the lymph nodes for distant metastasis, which is a huge hurdle for OSCC treatment in spite of relatively easy access for visual examination of precancerous lesions in the oral cavity. Therefore, locoregional control of the primary tumor is crucial for OSCC treatment. Similar to thrombocytosis, higher expression of podoplanin (PDPN) has been suggested as a predictive marker for higher frequency of lymph node metastasis of OSCC. Cumulative evidence supports that platelets can directly interact with PDPN-expressing cancer cells via C-type lectin-like receptor 2 (CLEC2), contributing to cancer cell invasion and metastasis. Thus, the platelet CLEC2-PDPN axis could be a pinpoint target to inhibit interaction between platelets and OSCC, avoiding undesirable side effects. Here, we will review the role of platelets in cancer, particularly focusing on CLEC2-PDPN interaction, and will assess their potentials as therapeutic targets for OSCC treatment.

The invasiveness of late‐stage cutaneous squamous cell carcinoma (cSCC) is associated with poor patients' prognosis and linked to strong upregulation of the glycoprotein Podoplanin (PDPN) in cancer cells. However, the function of PDPN in these processes in cSCC carcinogenesis has not been characterized in detail yet. Employing a CRISPR/Cas9‐based loss‐of‐function approach on murine cSCC cells, we show that the loss of Pdpn results in decreased migration and invasion in vitro. Complementing these in vitro studies, labelled murine control and Pdpn knockout cells were injected orthotopically into the dermis of nude mice to recapitulate the formation of human cSCC displaying a well‐differentiated morphology with a PDPN‐positive reaction in fibroblasts in the tumor stroma. Smaller tumors were observed upon Pdpn loss, which is associated with reduced tumor cell infiltration into the stroma. Utilizing Pdpn mutants in functional experiments in vitro, we provide evidence that both the intra‐ and extracellular domains are essential for cancer cell invasion. These findings underline the critical role of PDPN in cSCC progression and highlight potential therapeutic strategies targeting PDPN‐dependent cancer cell invasion, especially in late‐stage cSCC patients. [ABSTRACT FROM AUTHOR]

Background: It has been demonstrated that activated islet stellate cells (ISCs) play a critical role in islet fibrogenesis and significantly contribute to the progression of type 2 diabetes mellitus. However, the key molecules responsible for ISCs activation have not yet been determined. This study aimed to identify the potential key genes involved in diabetes-induced activation of ISCs. Method: Stellate cells were isolated from three 10-week-old healthy male Wistar rats and three Goto-Kakizaki (GK) rats. Cells from each rat were primary cultured under the same condition. A Genome-wide transcriptional sequence of stellate cells was generated using the Hiseq3000 platform. The identified differentially expressed genes were validated using quantitative real-time PCR and western blotting in GK rats, high fat diet (HFD) rats, and their controls. Results: A total of 204 differentially expressed genes (DEGs) between GK. ISCs and Wistar ISCs (W.ISCs) were identified, accounting for 0.58% of all the 35,362 genes detected. After the Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses, the mRNA levels of these genes were further confirmed by real-time PCR in cultured ISCs. We then selected Fos , Pdpn , Bad as the potential key genes for diabetes-induced activation of ISCs. Finally, we confirmed the protein expression levels of FOS, podoplanin, and Bad by western blotting and immunofluorescence in GK rats, HFD rats, and their controls. The results showed that the expression level of FOS was significantly decreased, while podoplanin and Bad were significantly increased in GK.ISCs and HFD rats compared with controls, which were consistent with the expression of α-smooth muscle actin. Conclusions: A total of 204 DEGs were found between the GK.ISCs and W.ISCs. After validating the expression of potential key genes from GK rats and HFD rats, Fos , Pdpn , and Bad might be potential key genes involved in diabetes-induced activation of ISCs. [ABSTRACT FROM AUTHOR]

BackgroundIt has been demonstrated that activated islet stellate cells (ISCs) play a critical role in islet fibrogenesis and significantly contribute to the progression of type 2 diabetes mellitus. However, the key molecules responsible for ISCs activation have not yet been determined. This study aimed to identify the potential key genes involved in diabetes-induced activation of ISCs.MethodStellate cells were isolated from three 10-week-old healthy male Wistar rats and three Goto-Kakizaki (GK) rats. Cells from each rat were primary cultured under the same condition. A Genome-wide transcriptional sequence of stellate cells was generated using the Hiseq3000 platform. The identified differentially expressed genes were validated using quantitative real-time PCR and western blotting in GK rats, high fat diet (HFD) rats, and their controls.ResultsA total of 204 differentially expressed genes (DEGs) between GK. ISCs and Wistar ISCs (W.ISCs) were identified, accounting for 0.58% of all the 35,362 genes detected. After the Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses, the mRNA levels of these genes were further confirmed by real-time PCR in cultured ISCs. We then selected Fos, Pdpn, Bad as the potential key genes for diabetes-induced activation of ISCs. Finally, we confirmed the protein expression levels of FOS, podoplanin, and Bad by western blotting and immunofluorescence in GK rats, HFD rats, and their controls. The results showed that the expression level of FOS was significantly decreased, while podoplanin and Bad were significantly increased in GK.ISCs and HFD rats compared with controls, which were consistent with the expression of α-smooth muscle actin.ConclusionsA total of 204 DEGs were found between the GK.ISCs and W.ISCs. After validating the expression of potential key genes from GK rats and HFD rats, Fos, Pdpn, and Bad might be potential key genes involved in diabetes-induced activation of ISCs.

The development of specific antibodies is essential to understand a wide variety of biological phenomena and pathophysiological analyses. Podoplanin (PDPN), a type I transmembrane glycoprotein, is known as a diagnostic marker. Anti-PDPN monoclonal antibodies (mAbs) against many species, such as human, mouse, rat, rabbit, dog, bovine, cat, tiger, horse, pig, goat, alpaca, Tasmanian devil, bear, whale, and sheep, have been established in recent studies. However, sensitive and specific mAbs against elephant PDPN (elePDPN) have not been established. Thus, this study established a novel mAb against African savanna elephant (Loxodonta africana) PDPN using the Cell-Based Immunization and Screening method. elePDPN-overexpressed Chinese hamster ovary-K1 (CHO/elePDPN) cells were immunized, and mAbs were screened against elePDPN using flow cytometry. One of the mAbs, PMab-265 (IgM, κ), specifically detected CHO/elePDPN cells by flow cytometry. These findings suggested the potential usefulness of PMab-265 for the functional analyses of elePDPN. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN) plays an important role in the development of many normal tissues and is expressed in various cancers. We have previously developed multiple monoclonal antibodies (mAbs) against PDPNs from a variety of animal species and characterized each of these PDPNs using the anti-PDPN mAbs. In this study, we evaluated whether these anti-PDPN mAbs possess cross-reactivity with ferret PDPN (ferPDPN) using flow cytometry. Comprehensive analysis using 17 differing anti-PDPN mAbs available for immunohistochemistry use, demonstrated that the anti-bear PDPN mAb (clone PMab-241) strongly cross-reacts with ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells. Immunohistochemistry analysis demonstrated intense PMab-241 staining within Bowman's capsules and glomeruli of the ferret kidney, and lymphatic endothelial cells of the ferret lung. These results demonstrate that PMab-241 is suitable for the detection of PDPN in ferret tissues. [ABSTRACT FROM AUTHOR]

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent. • Maackia amurensis seed lectin (MASL) is a potential anti-cancer agent. • MASL subunits form disulfide dimers while some other M. amurensis lectins do not. • MASL subunits have the same protein sequence but different glycosylation sites. • MASL and soluble human PDPN inhibit human OSCC cell growth and motility. • MASL targets human podoplanin with binding kinetics in the nanomolar range. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Patients with cancer are at a high risk of venous thromboembolism (VTE), studies have shown that high expression of podoplanin (PDPN) in tumors is associated with increased risk of VTE. Two human malignant cell lines (NCI-H226 and C8161) expressing high levels of PDPN were selected to explore the role of platelet in cancer-associated venous thrombosis in vitro and in vivo. Immunohistochemical staining using anti-PDPN antibody was performed in the pulmonary carcinoma patients. Both NCI-H226 and C8161 cells expressing high PDPN triggered platelet activation via CLEC-2 in vitro , which was abrogated by an anti-PDPN antibody SZ-168. Furthermore, the in vivo study revealed that injection of CHO-PDPN or C8161 in two mouse model of venous thrombosis activated platelets, increased platelet counts and enhanced thrombosis. More importantly, PDPN-enhanced thrombosis was reduced in mice treated with SZ168. A total of 63.3% tumor specimens stained positive for PDPN. High PDPN expression was associated with an increased risk of VTE and poor prognosis. PDPN expression in tumors induced platelet activation and was related to a high risk of VTE via platelet activation. SZ168 inhibited PDPN-induced platelet activation in vitro and decreased the incidence of VTE in mice. • PDPN expression in tumors induces VTE via platelet activation. • The PDPN antibody SZ168 inhibits PDPN-induced platelet activation in vitro. • SZ168 decreases the incidence of VTE in mice with PDPN-expressing tumors. • High PDPN level is associated with a high risk of VTE in cancer patients. • Targeting hemITAM signaling may serve as a potential therapeutic strategy for cancer-associated venous thrombosis. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to compare the clinical outcomes of spinal cord stimulation (SCS) and dorsal root ganglion stimulation (DRG-S) in the treatment of painful diabetic peripheral neuropathy (PDPN)., Methods: In this prospective cohort study, 55 patients received dorsal column spinal cord stimulation (SCS group) and 51 patients received dorsal root spinal cord stimulation (DRG-S group). The primary outcome was a Numerical Rating Scale (NRS) remission rate of ≥50%, and secondary outcomes included the effects of SCS and DRG-S on quality of life scores (EQ-5D-3L), nerve conduction velocity, and HbA1c, respectively., Results: The percentage of NRS remission rate ≥ 50% at 6 months was 80.43 vs. 79.55%, OR (95% CI): 1.06 (0.38-2.97) in the SCS and DRG-S groups, respectively, and the percentage of VAS remission rate ≥ 50% at 12 months was 79.07 vs. 80.95%, OR (95% CI): 0.89 (0.31-2.58). Compared with baseline, there were significant improvements in EQ-5D and EQ-VAS at 6 and 12 months ( p  < 0.05), but there was no difference in improvement between the SCS and DRG-S groups ( p  > 0.05). Nerve conduction velocities of the common peroneal, peroneal, superficial peroneal, and tibial nerves were significantly improved at 6 and 12 months compared with the preoperative period in both the SCS and PND groups ( p < 0.05). However, at 6 and 12 months, there was no difference in HbA1c between the two groups ( p  > 0.05)., Conclusion: Both SCS and DRG-S significantly improved pain, quality of life, and lower extremity nerve conduction velocity in patients with PDPN, and there was no difference between the two treatments at 12 months., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Han and Cong.)

Podoplanin (PDPN) is a cell-surface mucin-like glycoprotein that plays a critical role in tumor development and normal development of the lung, kidney, and lymphatic vascular systems. PDPN is overexpressed in several tumors and is involved in their malignancy. PDPN induces platelet aggregation through binding to platelet receptor C-type lectin-like receptor 2. Furthermore, PDPN modulates signal transductions that regulate cell proliferation, differentiation, migration, invasion, epithelial-to-mesenchymal transition, and stemness, all of which are crucial for the malignant progression of tumor. In the tumor microenvironment (TME), PDPN expression is upregulated in the tumor stroma, including cancer-associated fibroblasts (CAFs) and immune cells. CAFs play significant roles in the extracellular matrix remodeling and the development of immunosuppressive TME. Additionally, PDPN functions as a co-inhibitory molecule on T cells, indicating its involvement with immune evasion. In this review, we describe the mechanistic basis and diverse roles of PDPN in the malignant progression of tumors and discuss the possibility of the clinical application of PDPN-targeted cancer therapy, including cancer-specific monoclonal antibodies, and chimeric antigen receptor T technologies.

Podoplanin (PDPN), a 36-kDa type I transmembrane O-glycoprotein, is expressed in normal cells, including renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, and in cancer cells, including brain tumors and squamous cell lung carcinomas. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and PDPN/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced an anti-human PDPN monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda) and its rat-human chimeric mAbs (NZ-8/NZ-12), which neutralize PDPN/CLEC-2 interactions and inhibit platelet aggregation and cancer metastasis. In this study, we first developed a humanized anti-human PDPN mAb, named as NZ-27. We further produced a core-fucose-deficient version of NZ-27, named as P1027 and a core-fucose-deficient version of NZ-12, named as NZ-12f. We investigated the binding affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of P1027 and NZ-12f. We demonstrated that the binding affinities of P1027 and NZ-12f against LN319 (a human glioblastoma cell line) are 1.1 × 10−8 and 3.9 × 10−9 M, respectively. ADCC reporter assays demonstrated that NZ-12f shows 1.5 times higher luminescence than P1027. Furthermore, NZ-12f showed 2.2 times higher ADCC than P1027, whereas both P1027 and NZ-12f showed high CDC activities against LN319 cells. Using LN319 xenograft models, P1027 and NZ-12f significantly reduced tumor development in an LN319 xenograft model compared with control human IgG. Treatment with P1027 and NZ-12f may be a useful therapy for patients with PDPN-expressing cancers. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is a paediatric tumour that shows great biomolecule and clinical heterogeneity, and patients with NB often develop various neurological complications. Currently, the disease is mainly treated by surgery and still lacks specific therapeutic drugs; therefore, targets are urgently needed. Makorin ring finger protein 2 (MKRN2) is an E3 ligase whose effects on neuroblastoma have not been illustrated. shRNAs for MKRN2 have been designed, and MKRN2-knockdown human neuroblastoma SHSY5Y cells were established. MKRN2 knockdown promotes the proliferation and migration of SHSY5Y cells. Because MKRN2 is an E3 ligase, we performed a series of experiments, and Insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was identified as a new substrate for MKRN2. IGF2BP3 is an RNA-binding protein that regulates the stability of many mRNAs, including CD44 and PDPN, and our study demonstrated that MKRN2 regulates the expression of CD44 and PDPN in an IGF2BP3-dependent manner. These results suggest that MKRN2 might be a potential therapeutic target for neuroblastoma. • The ablation of MKRN2 promotes the proliferation and migration of human neuroblastoma SHSY5Y cells. • MKRN2 interacts and ubiquitylates IGF2BP3. • MKRN2 regulates the expression of cancer-related gene, CD44 and PDPN, in an IGF2BP3-dependent manner. [ABSTRACT FROM AUTHOR]

Background: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. Results: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions. Conclusion: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN. [ABSTRACT FROM AUTHOR]

Background/Aim: Mammary neoplasms are very common tumours in female dogs. Cancer-associated fibroblasts (CAFs) play an important role in the oncogenesis process. One of the useful proteins used in the diagnostics of CAFs cells is podoplanin (PDPN). The aim of our study was to assess the expression of PDPN in mammary cancer in female dogs. Materials and Methods: Our study cohort included 61 cancers and 21 adenomas of the mammary tumour in bitches. Expression of podoplanin, Ki-67 and HER2 was determined using the Immunohistochemical (IHC) method. PDPN expression at the mRNA level was determined using real-time PCR. Results: Expression of PDPN in CAFs was observed in 22.9% of cases of mammary cancers in bitches, with no PDPN expression in adenomas. A positive correlation was found between the expression of PDPN in CAFs and the grade of histological malignancy and expression of Ki-67. Conclusion: PDPN plays a significant role during the process of carcinogenesis of mammary tumours in female dogs. [ABSTRACT FROM AUTHOR]

Sensitive and specific monoclonal antibodies (mAbs) against not only human but also mouse, rat, rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. However, anti-goat PDPN (gPDPN) has not been established yet. PDPN has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses; therefore, mAbs for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues are needed. Although we recently demonstrated that an anti-bovine PDPN mAb, PMab-44 cross-reacted with gPDPN, PMab-44 did not detect lymphatic endothelial cells in immunohistochemistry. In this study, we immunized mice with gPDPN-overexpressing Chinese hamster ovary (CHO)–K1 (CHO/gPDPN) cells, and screened mAbs against gPDPN using flow cytometry. One of the mAbs, PMab-235 (IgG1, kappa), specifically detected CHO/gPDPN cells by flow cytometry. Furthermore, PMab-235 strongly detected lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells of colon by immunohistochemistry. These findings suggest that PMab-235 may be useful as a lymphatic endothelial cell marker for goat tissues.

Podoplanin (PDPN)/T1alpha is a type I transmembrane sialoglycoprotein, which is expressed on podocytes of the kidneys and type I alveolar cells of the lungs. PDPN is also known as Aggrus, a platelet aggregation-inducing factor, which comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains (PLDs) in the middle of the PDPN protein. We have previously established a mouse anti-bear PDPN (bPDPN) monoclonal antibody (mAb) clone, PMab-247 using the Cell-Based Immunization and Screening (CBIS) method. PMab-247 is very useful in flow cytometry, Western blotting, and immunohistochemical (IHC) analyses; however, the binding epitope of PMab-247 has not been elucidated. In this study, we aimed to investigate the epitope of PMab-247 using enzyme-linked immunosorbent assay and IHC analyses. The results revealed that the critical epitopes of PMab-247 are Asp76, Arg78, Glu80, and Arg82 of bPDPN. The Glu80 and Arg82 are included in PLD of bPDPN. The findings of our study can be applied to the production of more functional anti-bPDPN mAbs. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, and podocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an anti-goat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In this study, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75, Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the production of more functional anti-gPDPN mAbs. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on lymphatic endothelial cells, podocytes of the kidneys, and type I alveolar cells of the lungs. PDPN, a platelet aggregation-inducing factor, comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported a mouse antipig PDPN (pPDPN) monoclonal antibody (mAb) clone, PMab-213, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-213 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-213, which was developed by CBIS method, remains to be elucidated. Therefore, this study aimed to investigate the epitope of PMab-213 using enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analyses. The results revealed that the critical epitopes of PMab-213 are Pro53, Arg54, Arg56, and Tyr60 of pPDPN. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, and immunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung and podocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN), also known as T1alpha, has been used as a lung type I alveolar cell marker in the pathophysiological condition. Although we have established several monoclonal antibodies (mAbs) against mammalian PDPNs, mAbs against tiger PDPN (tigPDPN), which are useful for immunohistochemical analysis, remain to be developed. In this study, we immunized mice with tigPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/tigPDPN) and screened hybridomas producing mAbs against tigPDPN using flow cytometry. One of the mAbs, PMab-231 (IgG2a, kappa), specifically detected CHO/tigPDPN cells using flow cytometry as well as recognized tigPDPN protein using western blotting. In addition, PMab-231 was found to cross-react with cat PDPN (cPDPN). The dissociation constants (KD) of PMab-231 for CHO/tigPDPN and CHO/cPDPN cells were determined to be 1.2 × 10−8 and 1.9 × 10−8, respectively, indicating moderate affinity for CHO/tigPDPN and CHO/cPDPN cells. PMab-231 stained type I alveolar cells of the feline lungs and podocytes of the feline kidneys using immunohistochemistry. Our findings suggest the potential usefulness of PMab-231 for the functional analyses of tigPDPN and cPDPN. [ABSTRACT FROM AUTHOR]

Podoplanin (PDPN) is expressed in type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. We have characterized the PDPNs of various animal species using specific anti-PDPN monoclonal antibodies (mAbs). In this study, we investigated whether these anti-PDPN mAbs cross-react with goat PDPN (gPDPN). Flow cytometry demonstrated that the anti-bovine PDPN mAb PMab-44 (IgG1, kappa) reacts with gPDPN, which is overexpressed in CHO-K1 cells. Using immunohistochemical analysis, type I alveolar cells of goat lung were strongly detected by PMab-44. These results indicate that PMab-44 is useful for investigating gPDPN. [ABSTRACT FROM AUTHOR]

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs. [ABSTRACT FROM AUTHOR]