Design and Characterization of Bioengineered Cancer-Like Stem Cells

<div><p>Cancer stem cells (CSCs) are a small subset of cancer cells responsible for maintenance and progression of several types of cancer. Isolation, propagation, and the differentiation of CSCs in the proper stem niches expose the intrinsic difficulties for further studies. Here we show that induced cancer like stem cells (iCLSCs) can be generated by <i>in vitro</i> oncogenic manipulation of mouse embryonic stem cells (mESCs) with well-defined oncogenic elements; SV40 LTg and H<i>ras</i>V12 by using a mouse stem virus long terminal repeat (MSCV-LTR)-based retroviral system. The reprogrammed mESCs using both oncogenes were characterized through their oncogenic gene expression, the enhancement of proliferation, and unhampered maintenance of stem properties <i>in vitro</i> and <i>in vivo</i>. In addition, these transformed cells resulted in the formation of malignant, immature ovarian teratomas <i>in viv</i>o. To successfully further expand these properties to other organs and species, more research needs to be done to fully understand the role of a tumor- favorable microenvironment. Our current study has provided a novel approach to generate induced cancer like stem cells through <i>in vitro</i> oncogenic reprogramming and successfully initiated organ-specific malignant tumor formation in an orthotopic small animal cancer model.</p></div

Sub-cloning of H<i>ras</i>V12 and LTg into pMSCV plasmids.

<p>(A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-H<i>ras</i>V12-GFP; 3: pMSCV-GFP^cut; 4: pMSCV-H<i>ras</i>V12-GFP^cut; 5:pBABE-H<i>ras</i>V12^cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFP^cut 9: pMSCV-SV40 LTg-RFP^cut; 10: pBABE-SV40 LTg^cut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.</p

Histo-Pathological analysis of representative images (hematoxylin-eosin (H&E) tissue stain).

<p>(A) Ovarian Panel: (a). Normal right (non-injected) ovary (100X). Scale bar is 100 μm; (b). Left Ovarian mass (following orthotopic inoculation with mESC) showing signs of a mature teratoma (small window, 100X) characterized with a focus of mature, keratinizing squamous epithelium within the area depicted inside the box (200X). Scale bar is 100 μm; (c). Ovarian mass (following orthotopic inoculation with mESC-H<i>ras</i>V12/SV40-LTg) showing signs of immature teratoma (small window, 100X) characterized with scattered foci of a high-grade malignant neoplasm within the area depicted inside the box (400X). Scale bar is 100 μm. (B) Breast Panel: (a). Normal murine mammary tissue (100X). Scale bar is 100 μm; (b). Breast mass (following orthotopic inoculation of mESC into cleared inguinal mammary fat pad) exhibiting signs of mature teratoma (small window, 100X) characterized with the respiratory-like epithelium having well formed cilia within the area depicted inside the box (200X). Scale bar is 100 μm.</p

Characterization of genetically modified, retrovirally transduced mESCs.

<p>Representative images from immunoblot analysis: (A) H<i>ras</i>V12, (B) SV40 LTg. (C) CCK cell proliferation assay for 7 days of proliferation period of mESCs and transformed mESCs. (D) Comparison of proliferations at day 7. Mean ± S.D. (n = 3), *, **, and #, ## p<0.05. ANOVA test was performed with Tukey’s post-test using the GraphPad Prism software.</p

Establishment and characterization of GP2-293 derivatives for stable generation of retrovirus.

<p>(A) Confirmation of successful GFP expression in GP2-293 cells, and (B) Confirmation of successful RFP expression in GP2-293 cells after introduction of pMSCV plasmids. BF: bright field image; FL: fluorescence image. Scale bare is 400 μm. (C) Immunoblot analysis illustrating stable expression of H<i>ras</i>V12 and SV40 LTg in GP2-293 cell derivatives; 1. BL: GP2-293 blank cell; 2. GFP: GFP containing GP2-293 cell 3. HrasV12-GFP: H<i>ras containing</i> GP2-293 cell; 4. BL: GP2-293 blank cell; 5. RFP: RFP containing GP2-293 cell 6. SV40LTg-RFP: SV40LTg and RFP containing GP2-293 cell; M: Protein ladder. (D) Flow cytometry analysis (FACS) of 1x 10⁵ NIH-3T3 mouse fibroblast after infection with retrovirus produced from GP2-293 derivatives.</p

Characterization of stem cell properties of genetically modified, retrovirally transduced mESCs.

<p>(A) Alkaline phosphatase (AP) staining. Red-colored areas indicate alkaline phosphatase activity. (a). Bright field image of mESCs; AP staining: (b). mESCs; (c). mESC-H<i>ras</i>V12; (d). mESC-SV40 LTg; (e). mESC-H<i>ras</i>V12/SV40 LTg. Scale bare is 10 μm. (B) Immunocytochemistry staining of live-cells expressing SSEA1. Pairs of bright filed and SSEA1 images: (a). mESCs; (b). mESC-H<i>ras</i>V12; (c).mESC-SV40 LTg; (d). mESC-H<i>ras</i>V12/SV40 LTg. The representative images were obtained from fluorescent microscope with TRITC filter. Scale bar is 100 μm.</p

Ovarian mass at 31 days after orthotopic inoculation of mESCs transformed by both retroviral HrasV12 and SV40 LTg (mESC-HrasV12/SV40 LTg) into ovarian bursa of C57BL/6 mice.

<p>(A). Abdominal site with ovarian mass (blue circle); (B). Cervix with Uterus, right normal ovary (blue arrow) and left ovarian mass (white arrow); (C). Left ovary with partially intact capsule and mass breaking through the ovarian surface (blue circle).</p

Clinical and Genomic Crosstalk between Glucocorticoid Receptor and Estrogen Receptor α In Endometrial Cancer

Summary: Steroid hormone receptors are simultaneously active in many tissues and are capable of altering each other’s function. Estrogen receptor α (ER) and glucocorticoid receptor (GR) are expressed in the uterus, and their ligands have opposing effects on uterine growth. In endometrial tumors with high ER expression, we surprisingly found that expression of GR is associated with poor prognosis. Dexamethasone reduced normal uterine growth in vivo; however, this growth inhibition was abolished in estrogen-induced endometrial hyperplasia. We observed low genomic-binding site overlap when ER and GR are induced with their respective ligands; however, upon simultaneous induction they co-occupy more sites. GR binding is altered significantly by estradiol with GR recruited to ER-bound loci that become more accessible upon estradiol induction. Gene expression responses to co-treatment were more similar to estradiol but with additional regulated genes. Our results suggest phenotypic and molecular interplay between ER and GR in endometrial cancer. : Estrogen receptor α (ER) and glucocorticoid receptor (GR) are expressed in the uterus and have differential effects on growth. Vahrenkamp et al. find that expression of both receptors is associated with poor outcome in endometrial cancer and that simultaneous induction of ER and GR leads to molecular interplay between the receptors. Keywords: estrogen receptor, glucocorticoid receptor, endometrial cance