The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.

The colony formation assay (CFA) is the gold standard for measuring cytotoxic effects on cells. To increase efficiency, the CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation (IR) on the FaDu and A549 cancer cell lines. Its ability to evaluate combination therapies was investigated using cisplatin and IR. The 96-well CFA was transferred to a robotic platform for evaluation as a high-throughput screen (HTS) readout for the discovery of novel anti-cancer compounds, and radiosensitizers. Screening yielded eight putative anti-cancer hits, and five putative radiosensitizing hits. Secondary screening confirmed 6/8 anti-cancer compounds, and 0/5 radiosensitizing compounds. Thus, the 96-well CFA can be adopted as an alternative assay to the 6-well CFA in the evaluation of cytotoxicity in vitro, providing a possible readout to be utilized in HTS for discovering anti-cancer compounds, but with limited applicability in discovering radiosensitizers.

No description available.

Colorectal cancer (CRC) is the third most widespread and fourth most fatal malignancy disease. The CRC from a primary site can spread to other tissues, forming secondary tumours. CRC can metastasise to the liver through the effect of K-Ras and Pten mutation (Mt.) (Abbas et al. 2020). This study aimed to assess the hypothesis that the Ran inhibitor mebendazole MBZ reduces cell invasion and metastasis of CRC. I have investigated MBZ effect on the CRC isogenic human cell lines with specific mutations (HCT-116 K-Ras, DLD-1 K-Ras and Pten deletion and wild type HCT-116 and DKO-3. I used qRT-PCR and western blotting to identify expression levels of various genes and signalling molecules after treatment with 0.5 mM MBZ. In addition, several assays were performed to investigate MBZ effect on biological properties of the cells such as proliferation, migration, invasion, and colony formation. MBZ downregulated Ran and induced apoptosis through inhibition of Bcl-2 expression as well as inducing caspase -3, -7, -9 and PARP cleavage. Moreover, MBZ showed an effect on immune response by down regulating C5a, IL-1ß and IL-1α analysed at mRNA level. When treated with MBZ, the migration, invasion and colony formation abilities of HCT-116 K-Ras Mt., DLD-1 K-Ras Mt. and HCT-116 Pten-/- were significantly reduced compared to a control treated cell line. This was also the case with wild type cell lines such as HCT-116 and DKO-3. Furthermore, signalling molecules such as p- Erk 1/2 and p- Akt were upregulated after MBZ treatment and exert inhibition on Akt 1/2/3 and VEGFR1/2 mRNA levels. In conclusion; MBZ which is a Ran inhibitor, has significantly reduced proliferation, colony formation, and migration in colorectal cell lines with K-Ras and Pten gene deletion compared to wild type cells in a dose-dependent manner. This work paves the way to clinical validation of MBZ as a combination therapy for reducing the invasion of CRC cells.

Background: Pembrolizumab and cetuximab are antibodies under investigation in head and neck squamous cell carcinoma (HNSCC) either as single agents or combined with cisplatin and other chemotherapeutic drugs, e.g., 5-fluorouracil and/or docetaxel. However, also the combination of both antibodies may have potential in recurrent/ metastatic (R/M) HNSCC, in particular in cisplatin-resistant or -refractory cases or patients with comorbid disease, e.g. patients with impaired renal function. Methods: To clarify potential benefit that may result from such combination, we used the FLAVINO assay, a short-time ex vivo assay to compare responsiveness of HNSCC to pembrolizumab, cetuximab and both combined regarding colony formation of epithelial cells of biopsy-derived tumor samples and their cytokine production within three days either without or with stimulation with 10 ng/mL interferon gamma (IFN-g). Vascular endothelial growth factor A (VEGF), monocyte chemoattractant protein 1 (MCP-1 or CCL2), interleukin 6 (IL-6), IL-8, IFN-g, and interferon gamma-induced protein 10 (IP-10 or CXCL10) in supernatants were measured by ELISA. Results: We detected huge heterogeneity in response to cetuximab, pembrolizumab and both combined with and without IFN-g stimulation. Moreover, we detected a link between IFN-g induced IP-10 release and improved outcome in those HNSCC patients who were capable to respond to IFN-g and pembrolizumab, cetuximab and both combined with a further increase in IP-10 production. We derived an “IP-10 score” that independent from clinical characteristics of HNSCC patients and therapy regimens applied was able to predict their outcome. Conclusions: The heterogeneity in the ex vivo response of cetuximab, pembrolizumab and both combined with and without IFN-g stimulation identifies subgroups of HNSCC patients with deviating OS.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23817号 / 医科博第138号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 川口 義弥, 教授 藤田 恭之, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM