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Loss of phosphatase and tensin homolog (PTEN) has been linked to an immunosuppressive tumor microenvironment, but its underlying mechanisms remain largely enigmatic. Here, we report that PTEN can be secreted by the transmembrane emp24 domain-containing protein 10 (TMED10)-channeled protein secretion pathway. Inhibiting PTEN secretion from tumor cells contributes to immunosuppression and impairs the tumor-suppressive role of PTEN, while intratumoral injection of PTEN protein promotes antitumor immunity and suppresses tumor growth in mice. Mechanistically, extracellular PTEN binds to the plexin domain-containing protein 2 (PLXDC2) on macrophages, triggering subsequent activation of JAK2-STAT1 signaling, which switches tumor-associated macrophages (TAMs) from the immunosuppressive to inflammatory phenotype, leading to enhanced activation of CD8+ T and natural killer cells. Importantly, PTEN treatment also enhances the therapeutic efficacy of anti-PD-1 treatment in mice and reverses the immune-suppressive phenotype of patient-derived primary TAMs. These data identify a cytokine-like role of PTEN in immune activation and tumor suppression and demonstrate the therapeutic potential for extracellular administration of PTEN in cancer immunotherapy. [Display omitted] • PTEN is secreted via the TMED10-channeled protein secretion pathway • Cytokine-like PTEN binds with its receptor PLXDC2 on macrophages • PTEN-PLXDC2 engagement drives antitumor immunity by reprogramming macrophages • Extracellular PTEN synergizes with PD-1 blockade to suppress tumor growth Zhang et al. show that PTEN is secreted as a soluble protein by the TMED10-mediated unconventional protein secretion pathway into the extracellular space, where it exerts a cytokine-like role by binding with PLXDC2 on macrophages to sequentially activate JAK2-STAT1 signaling, reprogram macrophages, and drive antitumor immunity. [ABSTRACT FROM AUTHOR]

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.

Objective To investigate the role of plexin-domain containing 2 (PLXDC2) in regulating the invasion and metastasis of gastric cancer (GC) and explore its mechanism. Methods The data of GC cases were collected from The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) database, and the relationship between PLXDC2 expression and the clinicopathological parameters as well as the prognosis of GC patients were statistically analyzed. PLXDC2 knockdown and overexpression GC cell models were constructed respectively, then Matrigel-transwell invasion assay and mouse peritoneal metastasis model were performed to evaluate the effect of PLXDC2 on the invasion and metastasis of GC cells. Western blotting and immunofluorescence assay were also adopted to investigate the potential mechanism of PLXDC2 in GC. Results PLXDC2 was highly expressed in GC tissues (P < 0.05), and its expression level was positively correlated with neoplasm histological grade (P < 0.01), TNM stage (P < 0.05) and T stage (P < 0.05), while negatively with the prognosis of patients (P < 0.05). Cox regression analysis identified PLXDC2 as an independent risk factor for prognosis (P < 0.05). Silencing PLXDC2 significantly inhibited the invasion ability of GC cells in vitro (P < 0.01) and metastasis in vivo (P < 0.01), whereas overexpression of PLXDC2 obtained the opposite results. Moreover, the changes of PLXDC2 level correspondingly affected the expression of Cdc42 and the formation of filopodia in GC cells. Conclusion PLXDC2 promotes the invasion and metastasis of GC cells by regulating the formation of filopodia, and may act as a potential prognostic biomarker and therapeutic target for GC.

Plexin-domain containing 2 (PLXDC2) has been reported as an oncoprotein in several human malignancies. However, its expression and roles in gastric cancer remain largely unclear. In this study, we found that PLXDC2 was highly expressed in gastric cancer tissues, and the expression levels were positively correlated with clinicopathological features, but negatively with the patients' outcome. Cox regression analysis identified PLXDC2 as an independent prognostic indicator for the patients. Knockdown of PLXDC2 markedly suppressed the in vitro invasion and in vivo metastasis of gastric cancer cells, while overexpression of PLXDC2 resulted in opposite effects. Mechanistically, PLXDC2 enhanced the level of phosphorylated Cortactin (p-Cortactin) by physically interacting with protein tyrosine phosphatase 1B (PTP1B), an important dephosphorylase, to prevent its dephosphorylating of p-Cortactin, thereby promoting the formation of invadopodia. Collectively, our results indicate that PLXDC2 contributes to the invasion and metastasis of gastric cancer by inhibiting PTP1B to facilitate the invadopodium formation, and may serve as a potential prognostic biomarker and a therapeutic target for this disease. [ABSTRACT FROM AUTHOR]

Abstract Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods. The quality and function of purified antibody preparations were checked by electrophoresis and western blot analysis. The secreted rabbit mAb produced by a combination of culturing at 32 °C, purification by ammonium sulfate fractionation, and diethylaminoethyl resin (DEAE) ion exchange chromatography was of high quality. In contrast, the antibody produced by the cells grown at 37 °C for 6 days after transfection and purified by Protein A/G affinity method was low quality. Hypothermic conditions during production reduced protein heterogeneity probably by favorably affecting the levels of glycosylation and aggregation. In particular, according to western blotting data, CIMmultus DEAE chromatography that utilizes monolithic columns not only excluded inferior charge variants resulting from nonspecific reactions but also yielded rabbit mAb that was of better quality than commercially available rabbit polyclonal antibodies. The combination of techniques suggested by us may be a general approach to enhance product quality of rabbit mAbs produced by transient expression systems. Highlights • Optimized method of rabbit monoclonal antibodies by HEK293-6E cells is proposed. • HEK293-6E cells grown hypothermically at 32 °C produced best quality antibodies. • CIMmultus DEAE chromatography was the optimal method of antibody purification. [ABSTRACT FROM AUTHOR]

Abstract: Plexin-domain containing 2 (Plxdc2) is a relatively uncharacterised transmembrane protein with an area of nidogen homology and a plexin repeat (PSI domain) in its extracellular region. Here, we describe Plxdc2 expression in the embryonic mouse, with particular emphasis on the developing central nervous system. Using light microscopy and optical projection tomography (OPT), we analyse RNA in situ hybridization patterns and expression of two reporter genes, β-geo (a fusion of β-galactosidase to neomycin phosphotransferase) and placental alkaline phosphatase (PLAP) in a Plxdc2 gene trap mouse line (KST37; [Leighton, P.A., Mitchell, K.J., Goodrich, L.V., Lu, X., Pinson, K., Scherz, P., Skarnes, W.C., Tessier-Lavigne, M., 2001. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410, 174–179]). At mid-embryonic stages (E9.5–E11.5) Plxdc2-βgeo expression is prominent in a number of patterning centres of the brain, including the cortical hem, midbrain–hindbrain boundary and the midbrain floorplate. Plxdc2 is expressed in other tissues, most notably the limbs, lung buds and developing heart, as well as the spinal cord and dorsal root ganglia. At E15.5, expression is apparent in a large number of discrete nuclei and structures throughout the brain, including the glial wedge and derivatives of the cortical hem. Plxdc2-βgeo expression is particularly strong in the developing Purkinje cell layer, especially in the posterior half of the cerebellum. The PLAP marker is expressed in a number of axonal tracts, including the posterior commissure, mammillotegmental tract and cerebellar peduncle. We compare Plxdc2-βgeo expression in the embryonic brain with the much more restricted expression of the related gene Plxdc1 and with members of the Wnt family (Wnt3a, Wnt5a and Wnt8b) that show a striking overlap with Plxdc2 expression in certain areas. [Copyright &y& Elsevier]

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-( N- morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.