Chemosensitivity of Patient-Derived Cancer Stem Cells Identifies Colorectal Cancer Patients with Potential Benefit from FGFR Inhibitor Therapy

Some colorectal cancer patients harboring FGFR (fibroblast growth factor receptor) genetic alterations, such as copy number gain, mutation, and/or mRNA overexpression, were selected for enrollment in several recent clinical trials of FGFR inhibitor, because these genetic alterations were preclinically reported to be associated with FGFR inhibitor sensitivity as well as poor prognosis, invasiveness, and/or metastatic potential. However, few enrolled patients were responsive to FGFR inhibitors. Thus, practical strategies are eagerly awaited that can stratify patients for the subset that potentially responds to FGFR inhibitor chemotherapy. In the present study, we evaluated the sensitivity to FGFR inhibitor erdafitinib on 25 patient-derived tumor-initiating cell (TIC) spheroid lines carrying wild-type RAS and RAF genes, both in vitro and in vivo. Then, we assessed possible correlations between the sensitivity and the genetic/genomic data of the spheroid lines tested. Upon their exposure to erdafitinib, seven lines (7/25, 28%) responded significantly. Normal colonic epithelial stem cells were unaffected by the inhibitors. Moreover, the combination of erdafitinib with EGFR inhibitor erlotinib showed stronger growth inhibition than either drug alone, as efficacy was observed in 21 lines (84%) including 14 (56%) that were insensitive to erdafitinib alone. The in vitro erdafitinib response was accurately reflected on mouse xenografts of TIC spheroid lines. However, we found little correlation between their genetic/genomic alterations of TIC spheroids and the sensitivity to the FGFR inhibitor. Accordingly, we propose that direct testing of the patient-derived spheroids in vitro is one of the most reliable personalized methods in FGFR-inhibitor therapy of colorectal cancer patients

An improved method for culturing patient-derived colorectal cancer spheroids

患者由来大腸がん幹細胞培養を用いた薬剤感受性試験を開発 --個別化医療の実現へ期待--. 京都大学プレスリリース. 2018-09-03.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

患者由来の大腸がん幹細胞から得た高品質DNAによるミスマッチ修復欠損に対する正確な診断検査法

京都大学0048新制・課程博士博士(医学)甲第21686号医博第4492号新制||医||1036(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 妹尾 浩, 教授 小川 誠司, 教授 武田 俊一学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Accurate diagnosis of mismatch repair deficiency in colorectal cancer using high-quality DNA samples from cultured stem cells

Mismatch repair (MMR)-deficient or microsatellite instability (MSI) colorectal cancer includes two subtypes; Lynch syndrome and sporadic MSI cancer, both of which generate multiple neoantigens due to unrepaired mutations. Although such patients respond very well to immune checkpoint therapy, their diagnosis can be confused by low quality DNA samples owing to formalin fixation and/or low cancer cell content. Here we prepared high-quality DNA samples from in vitro-cultured cancer spheroids that consisted of the pure cell population. We evaluated their diagnostic power by on-chip electrophoresis, mutational burden assessment, and direct sequencing. Because formalin-fixed paraffin-embedded (FFPE) tissues are widely used as the DNA source, we compared such samples with spheroid DNA. Additionally, we performed immunohistochemistry (IHC) for MMR proteins on spheroids as well as primary tumor sections. Of 111 cases of colorectal cancer patients, we found seven MSI-high cases in which all diagnostic results agreed on spheroid-based assays, whereas the results with the FFPE DNA were less reliable though analyzable. Importantly, there was an MSS case that appeared as MSI by IHC on primary tumor sections. Based on these results, we propose to employ cultured cancer spheroids as the source of both DNA and IHC specimens for more reliable clinical diagnosis