FGF2 and EGF Are Required for Self-Renewal and Organoid Formation of Canine Normal and Tumor Breast Stem Cells

Abstract

Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self-renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570-584, 2017. © 2016 Wiley Periodicals, Inc.

Epidermal growth factor promotes proliferation and maintains multipotency of continuous cultured adipose stem cells via activating STAT signal pathway in vitro

“…Previous studies also indicated that growth factors not only promoted the proliferation of stem cells, but also maintained the differentiation potency of stem cells. [ 16 , 17 ] Similar findings were also obtained in this study: EGF not only accelerates the proliferation of ASCs and delays their senescence, but also maintains the differentiation potency of ASCs.…”

Establishment and characterization of mammary organoids from non-traditional model organisms

“…Since our initial attempts to establish equine mammary organoids (EqMaOs) from mammospheres and mammosphere-derived epithelial cells (MDECs) resulted in stellate colonies incapable of recapitulating mammary tissue polarity (data not shown) and attempts outside of our group using unsorted cells from canine mammospheres resulted in mammary organoids (MaOs) appearing stellate and lacking complex branching structures as well (Cocola et al 2017), we assessed whether we could grow EqMaOs from mammary tissue fragments (MTFs) instead, using a modified protocol for growing MaOs from mice (Ngyuen-Ngoc et al 2015). A key difference between the latter and our protocol was that the equine mammary tissues were collected and pre-digested using collagenase type II and cryopreserved for long-term storage ( Fig.…”

Identification and characterization of cancer stem cells in canine mammary tumors

“…In a 3D assay in vitro, the canine CSC cultured on collagen gels generated branching structures reminiscent of primitive tubes of the mammary gland, and when implanted into the fat pads of NOD/SCID mice, induced tumor growth. The regeneration potential as well as the number of spheroids varied in subsequent studies [25–33]. Cells isolated from the REM134 canine mammary carcinoma cell line formed spheres 16 times in long-term serial passaging.…”

“…Differences in sphere culturing media however have also been shown to influence the regeneration potential of CSC from CMT. The results of these experiments revealed that in contrast to stem cells isolated from normal mammary gland tissue, self-renewal of CSC was repressed by increased concentrations of EGF and FGF2 rather than promoted [33]. In another study, cells obtained from four canine mammary gland adenoma cell lines (1 × 10 4 ) formed between 45 and 273 spheres demonstrating the existence of different percentages of progenitor cells among samples [27].…”