Circulating microrna-214 and -126 as potential biomarkers for canine neoplastic disease

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1 Supplementary Materials Circulating microrna-214 and -126 as potential biomarkers for canine neoplastic disease Kazuki Heishima, 1,* Yukie Ichikawa, 1, Kyoko Yoshida, 1 Ryota Iwasaki, 1 Hiroki Sakai, 2 Takayuki Nakagawa, 3 Yuiko Tanaka, 3 Yuki Hoshino, 4 Yasuhiko Okamura, 5 Mami Murakami, 1 Kohji Maruo, 1,6 Yukihiro Akao, 7 and Takashi Mori 1 1 Department of Veterinary Clinical Oncology, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu, Gifu , Japan 2 Laboratory of Veterinary Pathology, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu, Gifu , Japan 3 Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo , Japan 4 Veterinary Teaching Hospital, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo , Japan. 5 Cooperative Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, , Ueda, Morioka , Japan 6 Comparative Cancer Laboratory, Department of Animal Nursing, Faculty of Animal Nursing, Yamazaki Gakuen University, 4-7-2, Minami-osawa, Hachioji, Tokyo , Japan 7 United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1, Yanagido, Gifu, Gifu , Japan * Corresponding author. These authors equally contributed to this work.

2 a Case counts Mix Chihuahua Maltese Pomeranian Yorkshire Terrier Papillon Toy poodle Miniature dachshund Norfolk Terrier Jack Russell Terrier Miniature Pinscher Miniature schnauzer Cairn Terrier Shih Tzu Bichon Frise Cavalier King Charles Spaniel Miniature Poodle Pug Welsh Corgi Shetland Sheepdog Beagle French Bulldog Shiba Staffordshire Bull Terrier Border Collie Kishu Siberian Husky English Setter American Pit Bull Terrier Belgian Tervuren Saluki Irish setter Standard Poodle Golden Retriever Labrador Retriever Flat-coated Retriever Great Pyrenees Bernese Mountain Dog Larger sized breeds b Larger sized breeds Control Epithelial Non-epithelial Miscellaneous Mix Chihuahua 4 2 Maltese 1 Pomeranian 2 1 Yorkshire Terrier 3 Papillon 2 1 Toy poodle Miniature dachshund Norfolk Terrier 1 Jack Russell Terrier 2 1 Miniature Pinscher 1 Miniature schnauzer Cairn Terrier 2 1 Shih Tzu 2 1 Bichon Frisé 1 Cavalier King Charles Spaniel 1 Poodle 2 Pug 1 Welsh Corgi Shetland Sheepdog 1 2 Beagle 1 3 French Bulldog Shiba Staffordshire Bull Terrier 1 Border Collie 3 Kishu 1 Siberian Husky 1 English Setter 1 American Pit Bull Terrier 1 Belgian Tervuren 1 Saluki 1 Irish setter 1 Poodle (Standard) 1 Golden Retriever 4 8 Labrador Retriever Flat-coated Retriever 2 1 Great Pyrenees 1 1 Bernese Mountain Dog Larger sized breeds Control Squamous cell carcinoma Adenocarcinoma Transitional cell carcinoma Mammary carcinoma Hepatocellular carcinoma Throid carcinoma Hemangiosarcoma Osteosarcoma Chondrosarcoma Histiocytic sarcoma Melanoma Soft tissue sarcoma Mast cell tumor Lymohoma Other endocrine tumors Genital tumor Odontogenic tumor Mix Chihuahua Maltese 1 Pomeranian Yorkshire Terrier Papillon Toy poodle Miniature dachshund Norfolk Terrier 1 Jack Russell Terrier Miniature Pinscher 1 Miniature schnauzer Cairn Terrier 2 1 Shih Tzu Bichon Frisé 1 Cavalier King Charles Spaniel 1 Poodle 1 1 Pug 1 Welsh Corgi Shetland Sheepdog Beagle French Bulldog Shiba Staffordshire Bull Terrier 1 Border Collie 1 2 Kishu 1 Siberian Husky 1 English Setter 1 American Pit Bull Terrier 1 Belgian Tervuren 1 Saluki 1 Irish setter 1 Poodle (Standard) 1 Golden Retriever Labrador Retriever Flat-coated Retriever Great Pyrenees 1 1 Bernese Mountain Dog 1 1 Supplemental figur Supplemental Figure 1. Breeds of dogs enrolled in this study Various breeds were included in this study. The top 6 major breeds were Miniature Dachshund, Mixed-breed, Welsh Corgi, Shiba, Labrador, and Golden Retriever. The breeds were arranged in order of ascending average body weight. (a) Total case number of each breed for each tumour type is given. (b) Distribution and case number of breeds in each category. (c) Distribution and case number of breeds in each subcategory. c

3 10-2 n=2 n=4 n=7 n= n=2 n=4 n=7 n=7 mir-214 Relative expression to mir I II III IV Stages of melanoma mir-126 Relative expression to mir I II III IV Stages of melanoma Supplemental Figure 2. Levels of circulating mir-214 and -126 in each clinical stage of melanoma Statistical analysis was not available due to the small number of cases included in stage 1. The median levels of circulating mir-126 calculated in this study increased stage-dependently but the levels of circulating mir-214 did not. The red centre bars and error bars indicate the median and interquartile range, respectively. The staging was based on the World Health Organization staging scheme for dogs.

4 a (Homo sapiens) 2588 mirnas 453 mirnas Common mirnas with identical sequences b mir-214 stem-loop (Homo sapiens) ggccu acaga u aca aacau 5' ggcugg guugucaugug cugccugucu cuugcugugcag c c 3' ccgacc caacaguacac gacggacaga ggacgacauguc g ----u u cac cacuc Mature sequence (from the 3' arm) 5 -acagcaggcacagacaggcagu-3 ggccu acgga u aca aacau 5' ggcugg guugucaugug cugccugucu cuugcugugcag c c 3' ccgacc caacaguacac gacggacaga ggacgacauguc g ----u u cac cacuc 5 -acagcaggcacagacaggcagu-3 c mir-126 stem-loop (Homo sapiens) c gc - a u c cug c 5' gcug gacgg g cauuauuacuu ugguacg g uga a 3' cggc cugcc c guaauaaugag gccaugc c acu c a ac g - u u -aa u Mature sequence (from the 3' arm) 5 -ucguaccgugaguaauaaugcg-3 -- u c cug c 5' cauuauuacuu ugguacg g uga a 3' guaauaaugag gccaugc c acu c gc u u -aa u 5 -ucguaccgugaguaauaaugcg-3 d mir-16 stem-loop ag c - a c u gauu 5' gucagc ugc uuagcagcac gu aauauugg g uaa c (Homo sapiens) 3' caguug aug agucgucgug ca uuaugacc c auu u ga a u a u u aaaa Mature sequence (from the 5' arm) 5 -uagcagcacguaaauauuggcg a c u gauu 5' uagcagcac gu aauauugg g uaa c 3' gucgucgug ca uuaugacc c auu u aa u a u u aaaa 5 -uagcagcacguaaauauuggcg-3 Supplemental Figure 3. Homology between human and canine mirna sequences Mature mirna sequences were conserved well between human and canine. mature mir-214, -126, and -16 showed perfectly identical sequences to human ones, whereas the stem-loop sequences of these mirnas were slightly diff erent between human and canine. (a) Venn diagram comparing human 2588 and canine 453 mature mirna sequences registered in the database of mirbase version 21. mirnas showed a perfect match with human ones in 172 of the total 453 mirnas (38.0%). The blue and red circle represent the groups of human and canine mirnas, respectively. (b) and canine mir-214 stem-loops and the mature sequences from 3' arm. (c) and canine mir-126 stem-loops and the mature sequences from 3' arm. (d) and canine mir-16 stem-loops and the mature sequences from 5' arm. The red letters in the stem-loops indicate the mature sequences in each mirna.

5 50 40 Ct (mir-16) (97.5%) 22.5 (75%) 21.7 (median) 20.8 (25%) 19.0 (2.5%) Relative frequency (%) Supplemental Figure 4. Ct value distribution of circulating mir-16 Histogram and Boxplot showing a Ct value variability of circulating mir-16, the internal control in the present study. Circulating mir-16 was stably detected in the plasma samples from the dogs bearing cancers. The median Ct value was 21.7, and the Ct values ranged (Difference: 1.7) in half of the cases. The percentile was (Diff erence: 6.5).

6 Categories (n) Subcategories (n) Diagnosis (n) Primary site (n) Control (22) Epithelial (78) Squamous cell carcinoma (18) Squamous cell carcinoma (18) Oral cavity (5) Nasal cavity (4) Skin (3) Tonsil (3) Anus (2) Tongue (1) Adenocarcinoma (27) Adenocarcinoma (27) Anal sac (7) Nasal cavity (5) Rectum (5) Gastric (3) Pancreas (2) Salivary gland (2) Lung (1) Prostate gland (1) Skin (1) Transitional cell carcinoma (8) Transitional cell carcinoma (8) Bladder (5) Prostate gland (2) Kidney (1) Mammary carcinoma (7) Mammary carcinoma (7) Mammary gland (7) Hepatocellular carcinoma (12) Hepatocellular carcinoma (12) Liver (12) Throid carcinoma (6) Thyroid follicular cell carcinoma (6) Thyroid gland (6) Non-epithelial (77) Hemangiosarcoma (13) Hemangiosarcoma (13) Spleen (10) Abdominal cavity (1) Kidney (1) Subcutis (1) Osteosarcoma (9) Osteosarcoma (9) Extremities (5) Cranial bone (2) Nasal cavity (2) Chondrosarcroma (5) Chondrosarcroma (5) Nasal cavity (2) Oral cavity (2) Extremities (1) Histiocytic sarcoma (5) Histiocytic sarcoma (5) Bone (2) Joint (2) Thoracic cavity (1) Melanoma (20) Malignant melanoma (17) Oral cavity (16) Amelanotic malignant melanoma (3) Tongue (2) Lip (1) Nailbed (1) Soft tissue sarcoma (6) Hemangiopericytoma (5) Subcutis (5) Fibrosarcoma (1) Spleen (1) Mast cell tumor (7) Mast cell tumor (7) Skin (6) Lip (1) Lymphoma (12) B-cell lymphoma (5) Lymph node (9) T-cell lymphoma (7) Colon (1) Skin (1) Uterus (1) Miscellaneous (14) Genital tumor (5) Glanulosa cell tumor (2) Ovary (4) Dysgerminoma (1) Testis (1) Adenocarcinoma (1) Seminoma (1) Other endcrine tumor (4) Pituitary tumor (2) Pituitary gland (2) Pheochromocytoma (2) Adrenal gland (2) Odontogenic tumor (5) Peripheral odontogenic fibroma (2) Oral cavity (5) (Fibromatous epulis) Periferal odontogenic fibroma (2) (Ossifying epulis) Acanthomatous ameloblastoma (1) (Acanthomatous epulis) Total (191) Supplemental Table 1. Histopathological diagnosis and primary sites of tumours The case numbers of each diagnosis and primary site are indicated in parentheses.

7 Statistical methods Figures Softwares Kruskal-Wallis test Figure 1b, c, d, e GraphPad Prism 7 Chi-square Test Figure 1f, Figure 4 GraphPad Prism 7 Mann-Whitney U test Figure 2a, Figure 3a GraphPad Prism 7 Steel Test Figure 2b, c, Figure 3b, c JMP Version 12, 64-bit ROC curve analysis Figure 5ab GraphPad Prism 7 Cluster analysis, dendrogram, and heatmap JMP Version 12, 64-bit illustration Figure 2e, Figure 3e, Figure 4 Multivariate correlation analysis Figure 7 JMP Version 12, 64-bit Linear regression analysis Figure 7 GraphPad Prism 7 Supplemental table 2 Supplemental Table 2. Summary of statistical methods used in this study Nine statistical methods were used in this study. Disease Sample type Masured mirna fraction Separation method Quantification Evaluated clinical significance Levels in Tumors References mir-214 Increased Breast cancecr Serum Total circulating mirnas - qrt-pcr Diagnosis, LN metastasis Increased Schwarzenbach, H. et al. Gastric cancer Plasma Total circulating mirnas - qrt-pcr Diagnosis, Distant metastasis - Zhang, K.-C. C. et al. Myeloma Serum Total circulating mirnas - qrt-pcr and Array Diagnosis, Prognosis - Hao, M. et al. Osteosarcoma Plasma Total circulating mirnas - qrt-pcr Diagnosis, Prognosis Increased Allen-Rhoades, W. et al. Ovarian cancer Serum Exosome MACS with anti-epcam Array Diagnosis - Taylor, D. D. et al. Decreased Coronary artery disease Plasma Total circulating mirnas - qrt-pcr Signiture only - Lu, H. Q. et al. mir-126 Increased Allergic rhinitis and asthma Plasma Total circulating mirnas - qrt-pcr Signiture only - Panganiban, R. P. et al. Metastatic sporadic melanoma Plasma Exosome ExoQuick solution qrt-pcr and Array Signiture only Increased Pfeffer, S. R. et al. Moyamoya disease Serum Total circulating mirnas - qrt-pcr and Array Signiture only - Dai, D. et al. Non-small cell lung carcinoma Serum Total circulating mirnas - qrt-pcr Diagnosis, Early diagnosis - Zhu, W. et al. Decreased Atherosclerosis Whole blood Total blood mirnas - qrt-pcr Diagnosis - Jiang, Y. et al. Atrial fibrillation and heart failure Serum Total circulating mirnas - qrt-pcr Signiture only - Wei, X. J. et al. Type-2 diabetes mellitus Serum Total circulating mirnas - qrt-pcr Diagnosis, Treatment response - Liu, Y. et al. and Olivieri, F. et al. Supplemental table Supplemental Table 3. Summary of previously reported human diseases with dysregulated levels of circulating mir-214 and -126 myeloma, osteosarcoma, breast, gastric, and ovarian cancers reportedly showed increased levels of circulating mir-214. The levels of circulating mir-126, in contrast, was increased in human diseases, such as allergic rhinitis and asthma, metastatic sporadic melanoma, Moyamoya disease, and non-small cell lung carcinoma, but was decreased in atherosclerosis, atrial fibrillation, heart failure, and type-2 diabetes mellitus. The term Total blood mirnas refers to cellular mirnas in white blood cells and cell-free circulating mirnas in the plasma. The term Total circulating mirnas from plasma or serum included protein-binding mirnas and mirnas contained in extracellular vesicles such as exosomes and microvesicles. MACS, magnetic-activated cell sorting; qrt-pcr, quantitative reverse transcription polymerase chain reaction.