This work was partly supported by an Extramural Collaborative Research Grant of Cancer Research Institute Kanazawa University, Ono cancer research grant, and Matching Planner Program from Japan Science and Technology Agency JST Grant Number MP27115663029. Footnotes CONFLICTS OF INTEREST The authors declare no conflicts of PD 123319 ditrifluoroacetate interest. REFERENCES 1. feeder cells induces Ad-MCA formation in PDAC cells before the onset of EMT, and Ad-MCA formation converts GM-sensitive CD44v3-10high/CD44slow PDAC cells into GM-resistant quiescent CSC-like cells. Furthermore, our work demonstrates the transcriptomes of PDAC cells are very rapidly and significantly changed by coculture PD 123319 ditrifluoroacetate with HEK293T cells. The quick phenotypic changes of PDAC cells observed in this coculture system appear to mimic those happening at the early phase of metastatic colonization of PDAC cells. This coculture system should be useful for understanding the molecular mechanisms underlying the emergence of intractable PDAC cells and the true nature of collective cell behavior. RESULTS Coculture with HEK293T cells induces Ad-MCA formation and GM resistance in epithelial cell phenotype CD44vhigh/CD44slow PDAC cells Modified expression of CD44 from CD44v to CD44s induces EMT and promotes malignancy progression . This suggests that the classification of splicing isoforms can be used as an indication of the EMT process. Thus, to distinguish whether the PDAC cell lines used in this study exhibited an epithelial cell or mesenchymal cell phenotype, we examined the manifestation patterns of CD44 variant isoform transcripts in the following CD44+ PDAC cell lines: PCI-55, PCI-24, PCI-43, PCI-6, PCI-35, MIA-PaCa-2, and PANC-1 (Number ?(Figure1A).1A). PCI-55, PCI-24, PCI-6, and PCI-35 cells showed an epithelial cell phenotype that exhibits high manifestation of CD44v mRNA and low manifestation of CD44s mRNA (CD44vhigh/CD44slow), of which PCI-55, PCI-24, and PCI-43 showed high manifestation of CD44v3-10 mRNA (CD44v3-10high/CD44slow), and PCI-6 and PCI-35 cells showed high manifestation of CD44v8-10 mRNA (CD44v8-10high/CD44slow). These CD44 variants were confirmed by direct sequencing of PCR products. In contrast, MIA-PaCa-2 and PANC-1 cells showed a mesenchymal cell phenotype, exhibiting low manifestation of CD44v mRNA and high manifestation of CD44s mRNA (CD44vlow/CD44shigh). Next, we evaluated GM level of sensitivity in each PDAC cell collection by measuring the percentage of apoptotic cells induced by treatment with 0.8 M GM for Rabbit Polyclonal to Glucagon 48 h. PCI-55, PCI-24, and PCI-43 were more sensitive to GM (30% and 20% of apoptotic cells) than PCI-6, PCI-36, MIA-PaCa-2, and PANC-1 (less than 6% of apoptotic cells) (Number ?(Figure1B).1B). Interestingly, PDAC cell lines expressing different CD44 isoforms showed different behavior when they were cocultured with HEK293T cells (Number ?(Number1C).1C). CD44v3-10high/CD44slow PDAC cells such as PCI-55 and PCI-24, and CD44v8-10high/CD44slow PDAC cells such as PCI-6 adhered to a monolayer of HEK293T cells and created Ad-MCAs. In contrast, CD44vlow/CD44shigh PDAC cells such as MIA-PaCa-2 and PANC-1 failed to form Ad-MCAs. We then examined whether coculture with HEK293T cells affected level of sensitivity to GM in GM-sensitive PCI-55 and PCI-24 cells. Coculture with HEK293T cells made PCI-55 and PCI-24 cells more resistant to GM (Number ?(Figure1D).1D). Treatment with GM affected Ad-MCA formation by neither PCI-55 (Number ?(Figure1E)1E) nor PCI-24 cells (data not shown). Taken together, these results show that coculture with HEK293T cells induces Ad-MCA formation and GM resistance in CD44v3-10high/CD44slow PDAC cells. Open in a separate window Number 1 Direct coculture with HEK293T cells induces Ad-MCAs in CD44vhigh/CD44slow epithelial PDAC cells(A) RT-PCR analysis of CD44 variant isoform manifestation in seven CD44+ PDAC cell lines. (B) Percentage of apoptotic PDAC cells induced by treatment with GM. PDAC cell lines were cultured in the presence of 0.8 M GM for 48 h. Apoptotic PDAC cells were evaluated from the percentage of sub G0/G1 phase cells by circulation cytometry. (C) Ad-MCA formation by CD44vhigh/CD44slow epithelial PDAC cells. (D) Percentage of apoptotic cells in PCI-55 and PCI-24 cells treated with GM for 48 h. (E) Ad-MCA formation by PCI-55 cells is not affected by treatment with 0.8 M GM (right). The data are offered as the mean ideals of three self-employed experiments. *< 0.05, **< 0.01, ***< 0.001. Bars: 50 m (C), 25 m (E). CD44v3-10high/CD44slow PDAC cells forming Ad-MCAs upregulate CD44v8-10 manifestation Trans-axial images of cocultured cells captured by confocal microscopy exposed that CD44 was indicated specifically by Ad-MCA-forming PCI-55 cells (Number ?(Number2A,2A, remaining panels). Three-dimensional analysis showed strong and clean membranous staining for CD44 on the surface of Ad-MCAs that anchored to a monolayer of HEK293T cells (Number ?(Number2A,2A, right panels). Immunofluorescence staining for CD44 exposed that PD 123319 ditrifluoroacetate filopodia were induced on the surface of some Ad-MCAs (Number ?(Figure2B).2B). Next, we examined the manifestation of CD44 isoforms in sorted Ad-MCA-forming PDAC cells. HEK293T cultured only did not express CD44 transcripts, and NHDFs cultured only expressed only CD44s transcripts (Number ?(Figure2C).2C). When PCI-55 and PCI-24 cells were cocultured with HEK293T cells, they markedly improved expression of CD44v8-10 (Number ?(Figure2D).2D). Consistent with increased manifestation of CD44v8-10 transcripts, strong staining.