Number of injected cells Resetting the Pancreatic Adenocarcinoma(PDAC) cells into non tumorigenic cells via epigenetic reprogramming technology Reyhaneh Khoshchehreh 1,2*,Alexandra Aicher 3,4,Mehdi Totonchi 1,Hossein Baharvand 1,2,Marzieh Ebrahimi 1*,Christopher Heeschen 3,4* 1. Department of Stem Cells and Developmental Biology ,Cell Science Research Centre ,Royan Institute for Stem Cell Biology and Technology, ACECR ,Tehran ,Iran 2. Department of Developmental Biology , ,University of Science and Culture ,Tehran ,Iran 3. Stem Cells & Cancer Group ,Molecular Pathology Programme ,Spanish National Cancer Research Centre (CNIO) ,Madrid ,Spain 4. School of Medical Sciences, University of New South Wales, 2052 Sydney, Australia Objectives Methods Results Immunostaining ,Alkaline phosphates staining and realtime PCR showed that induction with the episomal vectors is the most efficient method to reprogram our fibroblast and PDAC PDX cells and reprogramming of PDAC-PDX cells significantly altered their tumorigenic potential in vitro via differentiation-promoting effect of epigenetic reprogramming.In vivo results in nude mice clearly showed that direct reprogramming decreases the aggressiveness of PDAC-PDX 247 cancer cells as compared with its parental counterpart (2,3). Although cancer is generally believed to develop through accumulation of multiple genetic mutations, there is increasing evidence that cancer cells also acquire epigenetic abnormalities during development, maintenance, and progression. By utilizing the reprogramming technology as a tool to introduce the ‘pressure’ to alter epigenetic regulations, we might be able to clarify the epigenetic behavior that is unique to cancer cells (1). We hypothesized that using cell reprogramming technology would allow the Pancreatic adenocarcinoma cells to exit from tumorigenicity. We therefore sought to reprogram PDX(patient derived xenograft) from human PDAC, by introducing (1) lentiviral mediated induction of Yamanaka Factors (OSKM), (2) the pluripotency associated gene OCT4 and the microRNA mir-302 and (3) Episomal vectors (OCT4, SOX2, KLF4, LMYC and LIN28A combined with P53 knock-down (shP53)) as a safe method to reprogram cells without genome integration. We compare these three different methods to find most efficient and safe method to reprogram PDAC PDX cells into less or non-tumorigenic cells. Result 2: Generation and Characterization of reprogrammed PDAC-247 cells generated by the episomal vectors method. (A) Cells from PDAC-247 were reprogrammed and different passages of the iPS-like clones are shown. (B) Immunofluorescence staining of pluripotency markers in the 247-parental and reprogrammed cells. Both parental and reprogrammed cells were negative for SOX2, OCT4 and TRA-1-60. DAPI was used for nuclear counterstaining; scale bar: 100 μm. (C) Detection of NANOG mRNA expression using SmartFlare mRNA probe for NANOG. Inside a single colony. Dotted area shows cells with higher ratio of nuclei to cytoplasm also expressed NANOG more clearly; scale bar: 100 μm.(D) Expression of pluripotency markers and epigenetic modifier genes in parental and reprogrammed 247 cells by real time PCR. Gene expression levels were normalized to bACTIN; *, p≤ 0.0001; mean ± SD; n=3. (E) ALP activity was only observed in a few of the screened colonies from 247- reprogrammed cell; scale bar: 50 μm. C Number of injected cells 0.5×106 1×106 2.5×106 5×106 7×106 10×106 100% of efficiency 247-PARENTAL 6/6 100 247-REP 0/6 E Result 4: PDAC-PDX 247 cell differentiation following reprogramming and Tumorigenicity in vivo following reprogramming. (A) Immunofluorescence staining of the epithelial differentiation markers cytokeratin 19 and E-cadherin in 247-parental and reprogrammed cells; scale bar: 100 µm. (B) Expression of CD133 in parental and reprogrammed 247 cells by real time PCR. Gene expression levels were normalized to bACTIN; *, p≤ 0.0001, mean ± SD; n=3 (C) Subcutaneous injections of 247 -parental and the reprogrammed cells in different dilutions (0/5×106, 1×106, 2/5×106, 5×106, 7×106 and 10×106 into both flanks). 247 -parental cells formed tumors after 8–10-weeks and all mice were killed three months after injection. (D) Hematoxylin and eosin staining and histological staining for the differentiation marker ECAD and PanCK shows positive cells for epithelial cytokeratins (PanCK) and E-cadherin in tumors derived from 247 -parental cells. Result 1: Induction of PDAC-PDX Cells by three different methods and Characterization of reprogrammed fibroblasts and PDAC-PDX cells as compared with parental cells. (A) Schematic and timeline of cell reprogramming in human pancreatic cancer cells. (B) PDAC-PDX primary cancer cultures (PDAC-215, PDAC-253, PDAC-354, and PDAC-247), the established human pancreatic cancer cell line PANC-1, and human fibroblast cells (HDF and HFF-5) as a control were treated with three reprogramming methods. Referential morphologies are exhibited by HDF and HFF-5; scale bar: 100 μm. Inserts show the red fluorescence of OCT4-mir302-mCherry+ cells, red asterisks refer to the corresponding colonies at brightfield microscopy. (C) Expression of pluripotency markers in parental and reprogrammed cells by real time PCR. (D) Immunofluorescence staining of pluripotency markers NANOG and OCT4 in the parental and reprogrammed HDF cells. DAPI was used for nuclear counterstaining; scale bar: 50 μm. (E) Alkaline phosphatase-positive colonies from reprogrammed HDF cells generated by the episomal vectors method. *P < 0.0001 * Pluripotency genes Epigenetic modifier genes EMT genes Result 5:Correlation of epigenetic modifiers, stemness and EMT genes following reprogramming in PDAC-PDX 247 cells. Schematic representation of gene network analysis for stemness, epigenetic modifiers and EMT genes showing the relative relationship of cancer reprogramming and subsequent differentiation. Result 3 : Tumorigenicity and Invasive capacity of reprogrammed PDAC- 247 cells in vitro. (A) Sphere formation efficiency and (B) 3D tumor sphere formation efficiency of parental and reprogrammed PDAC-247 cells. Quantification (upper) and representative pictures (lower); *, P ≤ 0.05; n=3. (C) Cells were seeded onto the filters of 0.8 µm pore transwells coated with matrigel and invaded cells were assessed after 16h. Photos are representative of one of three performed experiments; scale bar: 100 µm. Quantification of cell invasion; P<0.05, n=3, mean ± SD. (D) Expression of EMT markers in parental and reprogrammed 247 cells by real time PCR. Gene expression levels were normalized to bACTIN; *, p≤ 0.0001, mean ± SD; n=3. (E) Colony formation capacity of single 247- parental and reprogrammed cells after 14 days (left). 247-reprogrammed cells generated colonies with more differentiated morphology compared with their parental counterparts; *, P≤0.05; n=3; mean + SD, (right). (F) Representative flow cytometry for Ki-67 staining in 247-parental versus reprogrammed cell cultures. (G) Cell cycle analysis performed by flow cytometry; n=3; mean + SE. Conclusions This study demonstrated that the Reprogrammed PDAC PDX cancer cells were distinct from natural PDAC cells with regard to their loss of tumorigenicity in vitro and in vivo. References: 1. Yamada, Haga et al. 2014, Stem Cells Translational Medicine. 2. Roe, Hwang et al. 2017, Cell. 3. .Zhou, Zhang et al. 2018, J Cell Physiology.