Reproduction of the Antitumor Effect of Cisplatin and Cetuximab Using a Three-dimensional Spheroid Model in Oral Cancer

Background/Aim: Cancer research has been conducted using cultured cells as part of drug discovery testing, but conventional two-dimensional culture methods are unable to reflect the complex tumor microenvironment. On the other hand, three-dimensional cultures have recently been attracting attention as in vitro models that more closely resemble the in vivo physiological environment. The purpose of this study was to establish a 3D culture method for oral cancer and to verify its practicality. Materials and Methods: Three-dimensional cultures were performed using several oral cancer cell lines. Western blotting was used for protein expression analysis of the collected cell masses (spheroids), and H-E staining was used for structural observation. The cultures were exposed to cisplatin and cetuximab and the morphological changes of spheroids over time and the expression changes of target proteins were compared. Results: Each cell line formed spheroidal cell aggregates and showed enhancement of cell adhesion molecules over time. H-E staining showed tumor tissue-like structures specific to each cell line. Cisplatin showed concentration-dependent antitumor effects due to loss of cell adhesion and spheroid disruption in each cell line, while cetuximab exhibited antitumor effects that correlated with EGFR expression in each cell line. Conclusion: Spheroids made from oral cancer cell lines appeared to have tumor-like characteristics that may reflect their clinical significance. In the future, it may become possible to produce tumor spheroids from tissue samples of oral cancer patients, and then apply them to drug screening and to develop individualized diagnostic and treatment methods

乳牛における簡易型検査装置を用いた血乳の発生に関する現況調査

Reddish or pinkish milk contaminated with red blood cells due to diapedesis of the mammary gland is a rare occurrence at dairy farms. There is not much information about the frequency and causal factors of haemolactia (i.e., the presence of blood in milk). In this study, haemolactia in dairy cows was investigated by using a device for quantitative determination of blood in milk, which is being developed by the Tokachi Foundation. Milk samples (colostrum) from 4 cows and pinkish milk samples from 2 cows with mastitis or an external wound to the udder were obtained at two dairy farms in eastern Hokkaido between July and November 2019. The average concentrations of blood in the colostrum on the 3rd day after calving was 0.0028±0.0027%,which is less than the 0.01% detection limit of visual inspection. The concentration of blood in the milk from the cow with mastitis ranged from 0.25% to 0.0012% over the 4 days, which gave the milk a pinkish tinge. The concentration of blood in the milk from the cow with an external wound to the udder ranged from 0.24% to 0.011% over 4 days; this milk was not noticeably pink on the 4th day by visual inspection.酪農現場では、乳房内出血による血乳が搾乳されることがある。一般的に血乳は前搾り時に発⾒できるため、生乳出荷事故につながることは少ないが、その場で廃棄されるため、原因や血液混⼊割合などの詳細が明らかではない。本研究では、とかち財団が開発している簡易型血乳検査装置を用いて、現場での現況調査を実施した。調査対象は釧路地方の２酪農場において、2019年6月に分娩した４個体から搾乳した「①初乳」ならびに、同年10～11 月に、前搾り時に血乳と判断された個体乳（「②乳房炎」、「③乳房の打撲」が原因と推定される血乳が突発的に生じたそれぞれ１個体）とした。これらの血液濃度を同装置で推定した上で、体細胞数をブリード法により測定した。分娩後３日目における「①初乳」中の推定血液濃度は0.0028±0.0027％で、目視（色沢）で確認できる限界濃度（約0.01％）より低かった。「②乳房炎」が原因と推定される血乳の推定血液濃度は0.25～0.012％で、明らかなピンク色を呈し、出血前の体細胞数は2,660[千個/ml]と高い値であった。「③乳房の打撲」が原因と推定される血乳の体細胞数は51±39[千個/ml]と比較的低いことから、炎症性の可能性は低いと推察された。その後４日間の推定血液濃度は0.24～0.011％で、日にちの経過とともに低下した

TLC screening of thraustochytrid strains for squalene production

Screenings of thraustochytrids (Labyrinthulomycetes) have been conducted for 176 strains isolated from various sites in the Asian region to investigate what type of species and strains accumulate high levels of squalene. Thin layer chromatography (TLC) screening for squalene production revealed that 38 strains were rated as "+" (high), 29 as "±" (medium), and 109 as "-" (low). Further, high performance liquid chromatography analysis strongly supported the TLC screening results. Besides the 18W-13a strain of Aurantiochytrium sp., which was previously recognized as a squalene-rich strain, several strains produced squalene at approximately 1 g L-1 of culture volume. Squalene production was strongly related to locality, colony color, and phylogenetic clade. Most strains with "+" squalene spots were isolated from Okinawa, a subtropical region of Japan, while the strains with "±" and "-" squalene spots were isolated from wide geographical regions from tropical to subarctic. Approximately half the strains with orange colonies on GTY medium plates produced a high amount of squalene, whereas the other strains with different colors showed less or no squalene spots on TLC. All the squalene-rich strains were assigned to the Aurantiochytrium clade. Overall, our results suggest that (1) the thraustochytrids show tendentious locality in terms of squalene production, (2) a relationship exists between the metabolic synthesis of carotenoid pigments and squalene production, and (3) the Aurantiochytrium clade may have evolved to accumulate squalene