Abstract
Recent advances allowed culturing and examination of patient-derived colorectal cancer (PD-CRC) cells as organoids or spheroids. To be applied to practical personalized medicine, however, current methods still need to be strengthened for higher efficiency. Here we report an improved method to propagate PD-CRC tumor initiating cells (TICs) in spheroid culture. We established > 100 cancer spheroid lines derived from independent colorectal cancer patients employing a serum-containing medium with additional inhibitors, Y27632 and SB431542. Because colorectal cancer spheroids showed wide-range growth rates depending on the patient tumors, we searched for supplementary factors that accelerated proliferation of slow-growing CRC-TIC spheroids. To this end, we introduced a convenient growth-monitoring method using a luciferase reporter. We found that epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) were critical for steady propagation of a subset of CRC-TIC spheroids carrying the wild-type RAS and RAF genes. We also identified 5′-(N-ethyl-carboxamido)-adenosine (NECA), an adenosine receptor agonist, as an essential supplement for another subset of spheroids. Based on these results, we propose to optimize culture conditions for CRC-TIC spheroids by adjusting to the respective tumor samples. Our method provides a versatile tool that can be applied to personalized chemotherapy evaluation in prospective clinical studies.
Highlights
Colorectal cancer is one of the commonest cancers worldwide [1]
We found that epidermal growth factor (EGF) and/or basic fibroblast growth factor were critical for steady propagation of a subset of CRC-tumor initiating cells (TICs) spheroids carrying the wild-type RAS and RAF genes
The increments of growth effect index (GEI) were critical for their propagation because they barely grew in the cancer medium alone (Supplementary Figure 3E). These results suggest that activation of the cyclic AMP (cAMP)-protein kinase A (PKA) and mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) signaling enhances proliferation of CRC-TIC spheroids in an additive manner though not synergistic
Summary
Colorectal cancer is one of the commonest cancers worldwide [1]. the mortality of colorectal cancer is decreasing thanks to early detection efforts and treatment improvements, the disease is often lethal once it reaches the metastatic stage, even with molecular-targeted therapeutics such as antibodies against epidermal growth factor receptor (EGFR) and/or against vascular endothelial growth factor (VEGF) [2]. It has become a practical alternative to culture patient-derived tumor initiating cells of the colorectal cancer (PD-CRC-TICs) as organoids or spheroids [9,10,11,12] Their clinical applications have been hampered by some technical difficulties, including the requirement for supplements such as growth factors, minerals, vitamins, and hormones. To propagate tissue-derived intestinal epithelial cells rapidly, a recent method employed a serum-containing medium conditioned by L-WRN fibroblasts that secrete three components (Wnt3a, R-spondin 3, and Noggin) essential for tissue stem cells [13] Such media can support the proliferation of various types of epithelial stem cells from humans and mice when supplemented with a Rho-associated coiled-coil protein kinase (ROCK) inhibitor, Y27632, and a transforming growth factor β (TGF-β) type I receptor inhibitor, SB431542 [14]
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