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BACKGROUND: Pancreatic cancer is one of the most lethal tumors. The aim of this study is to provide an effective therapeutic discovery platform for pancreatic cancer by establishing and characterizing patient-derived organoids (PDOs). METHODS: PDOs were established from pancreatic tumor surgical specimens, and the mutations were examined using a panel sequence. Expression of markers was assessed by PCR, immunoblotting, and immunohistochemistry; tumorigenicity was examined using immunodeficient mice, and drug responses were examined in vitro and in vivo. RESULTS: PDOs were established from eight primary and metastatic tumors, and the characteristic mutations and expression of cancer stem cell markers and CA19-9 were confirmed. Tumorigenicity of the PDOs was confirmed in subcutaneous transplantation and in the peritoneal cavity in the case of PDOs derived from disseminated nodules. Gemcitabine-sensitive/resistant PDOs showed consistent responses in vivo. High throughput screening in PDOs identified a compound effective for inhibiting tumor growth of a gemcitabine-resistant PDO xenograft model. CONCLUSIONS: This PDO-based platform captures important aspects of treatment-resistant pancreatic cancer and its metastatic features, suggesting that this study may serve as a tool for the discovery of personalized therapies.

Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell–germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.

mDia1/3 generate cortical F-actin meshwork in Sertoli cells that is continuous with contractile F-actin bundles and indispensable for spermatogenesis and male fertility. Sakamoto S, Thumkeo D, Ohta H, Zhang Z, Huang S, et al. PLOS Biology. 2018. 16(9) : e2004874. doi:10.1371/journal.pbio.2004874, Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell–germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.

The small GTPase Rho and mDia2, a Rho-regulated actin nucleator, function as critical regulators of cytokinesis in cultured cells. However, their involvement in cytokinesis during mammalian development remains unknown. Here, we generated mice deficient in mDia2 and examined the role of Rho signaling in cytokinesis during development. mDia2-deficient mice survive until embryonic day 11.5 (E11.5), exhibit severe anemia with multinucleate erythroblasts, and die in utero by E12.5. mDia2-deficient erythroid cells differentiate normally, though in a delayed manner, but exhibit cytokinesis failure with decreased accumulation of F-actin in the cleavage furrow during late differentiation from proerythroblasts. On the other hand, inactivation of Rho induces cytokinesis failure from the earlier progenitor stage. mDia2-deficient erythroblasts, however, are able to enucleate their nuclei. Our findings have thus revealed that mDia2 functions critically in cytokinesis in vivo during erythropoiesis and further suggest that the cytokinesis mechanism in development diverges downstream of Rho. They also demonstrate that cytokinesis and enucleation utilize different mechanisms.

Diaphanous-related formin, mDia, is an actin nucleation/polymerization factor functioning downstream of the small GTPase Rho. Although Rho is critically involved in cytokinesis, it remains elusive how Rho effectors and other regulators of cytoskeletons work together to accomplish this process. Here we focused on mDia2, an mDia isoform involved in cytokinesis of NIH 3T3 cells, and analyzed mechanisms of its localization in cytokinesis. We found that targeting of mDia2 to the cleavage furrow requires not only its binding to RhoA but also its diaphanous-inhibitory domain (DID). We then performed pulldown assays using a fragment containing the latter domain as a bait and identified anillin as a novel mDia2 interaction partner. The anillin-binding is competitive with the diaphanous autoregulatory domain (DAD) of mDia2 in its autoinhibitory interaction. A series of RNA interference and functional rescue experiments has revealed that, in addition to the Rho GTPase-mediated activation, the interaction between mDia2 and anillin is required for the localization and function of mDia2 in cytokinesis.

mDia1/3 generate cortical F-actin meshwork in Sertoli cells that is continuous with contractile F-actin bundles and indispensable for spermatogenesis and male fertility. Sakamoto S, Thumkeo D, Ohta H, Zhang Z, Huang S, et al. PLOS Biology. 2018. 16(9) : e2004874. doi:10.1371/journal.pbio.2004874, Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell–germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.

mDia1/3 generate cortical F-actin meshwork in Sertoli cells that is continuous with contractile F-actin bundles and indispensable for spermatogenesis and male fertility. Sakamoto S, Thumkeo D, Ohta H, Zhang Z, Huang S, et al. PLOS Biology. 2018. 16(9) : e2004874. doi:10.1371/journal.pbio.2004874, Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell–germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.