Implementation of Systematic Bioanalysis of Antibody-Drug Conjugates for Preclinical Pharmacokinetic Study of Ado-Trastuzumab Emtansine (T-DM1) in Rats.

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies covalently bound to cytotoxic drugs by a linker. They are designed to selectively bind target antigens and present a promising cancer treatment without the debilitating side effects of conventional chemotherapies. Ado-trastuzumab emtansine (T-DM1) is an ADC that received US FDA approval for the treatment of HER2-positive breast cancer. The purpose of this study was to optimize methods for the quantification of T-DM1 in rats. We optimized four analytical methods: (1) an enzyme-linked immunosorbent assay (ELISA) to quantify the total trastuzumab levels in all drug-to-antibody ratios (DARs), including DAR 0; (2) an ELISA to quantify the conjugated trastuzumab levels in all DARs except DAR 0; (3) an LC-MS/MS analysis to quantify the levels of released DM1; and (4) a bridging ELISA to quantify the level of anti-drug antibodies (ADAs) of T-DM1. We analyzed serum and plasma samples from rats injected intravenously with T-DM1 (20 mg/kg, single dose) using these optimized methods. Based on these applied analytical methods, we evaluated the quantification, pharmacokinetics, and immunogenicity of T-DM1. This study establishes the systematic bioanalysis of ADCs with validated assays, including drug stability in matrix and ADA assay, for future investigation on the efficacy and safety of ADC development.

Analysis of Medication Errors Reported by Community Pharmacists in the Republic of Korea: A Cross-Sectional Study.

Background and objectives: We aimed to describe medication-related incidents or medication errors (MEs) reported by community pharmacists and analyze the prevalent medications involved. Materials and Methods: We extracted ME reports from databases comprising patient safety incidents reported to the Korean Pharmaceutical Association between January 2013 and June 2021. Medications were analyzed according to the second (therapeutic subgroup) and fifth (chemical substance) levels of the Anatomical Therapeutic Chemical classification. Results: A total of 9046 MEs were identified, most of which were near miss reports (88.3%). Among the errors that reached the patients (521 cases), harmful incidents accounted for 76.8%. Most MEs occurred during prescription (89.5%), while harmful MEs occurred mainly during dispensing (73.3%). In the prescription step, wrong drugs (44.8%), dosing errors (27.0%), and wrong durations (14.0%) were common. Anti-inflammatory and anti-rheumatic products (M01), drugs for acid-related disorders (A02), and antihistamines for systemic use (R06) were the most frequently reported medication classes involved. Harmful incidents were most common for dosing errors (31.0%) and wrong drugs (26.8%) and were common with warfarin, levothyroxine, and glimepiride. Conclusions: The MEs reported by community pharmacists were mainly prescribing errors, most of which were rectified before reaching patients. The prevalent medications involved in harmful errors include anti-diabetic, anti-thrombotic, and anti-inflammatory agents.

Treatment of Actinic Keratosis: The Best Choice through an Observational Study.

Actinic keratosis (AK) is a precancerous lesion that can progress to invasive squamous cell carcinoma if untreated. However, no gold standard treatment has been established. We aimed to investigate the management of AK by comparing the effectiveness and treatment duration of treatment modalities, including cryotherapy, imiquimod (IMQ), and photodynamic therapy (PDT). We reviewed the medical records of 316 patients diagnosed with AK at Seoul St. Mary's Hospital from February 2015 to May 2020, and a total of 195 patients were included. The clearance rate was the highest in PDT, followed by cryotherapy and IMQ (82.4%, 71.2%, and 68.0%, respectively). The recurrence rate was the lowest in cryotherapy, followed by PDT and IMQ (3.5%, 6.7%, and 10.5%, respectively, p < 0.05). The average treatment duration was shortest with PDT, followed by IMQ and cryotherapy (5.5 weeks, 6.8 weeks, and 9.1 weeks, respectively, p < 0.05). The number of hospital visits was lowest for PDT, followed by cryotherapy and IMQ (1.8, 2.8, and 3.6, respectively, p < 0.05). PDT showed the highest clearance rate, a moderate recurrence rate, the shortest treatment duration, and the least number of visits, suggesting that PDT could be the first choice for treatment of AK. Considering the advantages as a topical agent, IMQ could also be a treatment option.

Value Frameworks: Adaptation of Korean Versions of Value Frameworks for Oncology.

This study sought to adapt the existing value framework (VF) to produce a reliable and valid Korean oncology VF. Two VFs developed by The American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) were selected for examination in the present study. Forward and backward translations were conducted for six high-priced drugs indicated for non-small-cell lung cancer and multiple myeloma. Inter-rater reliability was measured based on the intraclass correlation coefficient (ICC) and variation was described using the coefficient of variation. The relative weights of factors critically considered by Korean oncologists were derived following the analytic hierarchy process (AHP), and focus group interviews (FGIs) were used to obtain qualitative data regarding the applications of these two VFs in the Korean setting. The ICCs of the Korean VFs were 0.895 (0.654-0.983) for ASCO and 0.726 (0-0.982) for ESMO translations, suggesting excellent reliability for ASCO and good reliability for ESMO. AHP demonstrated that clinical benefit has the highest priority, which is consistent with the ASCO VF. The FGIs suggested that the result for AHP is acceptable and that both ESMO and ASCO VFs should be used complementarily. Although further evaluation with a larger sample size is needed, the Korean versions of ESMO/ASCO VFs are valid and reliable tools and are acceptable to Korean stakeholders, yet they should be applied with caution.

A Meta-Regression Analysis of Utility Weights for Breast Cancer: The Power of Patients' Experience.

To summarize utility estimates of breast cancer and to assess the relative impacts of study characteristics on predicting breast cancer utilities. We searched Medline, Embase, RISS, and KoreaMed from January 1996 to April 2019 to find literature reporting utilities for breast cancer. Thirty-five articles were identified, reporting 224 utilities. A hierarchical linear model was used to conduct a meta-regression that included disease stages, assessment methods, respondent type, age of the respondents, and scale bounds as explanatory variables. The utility for early and late-stage breast cancer, as estimated by using the time-tradeoff with the scales anchored by death to perfect health with non-patients, were 0.742 and 0.525, respectively. The severity of breast cancer, assessment method, and respondent type were significant predictors of utilities, but the age of the respondents and bounds of the scale were not. Patients who experienced the health states valued 0.142 higher than did non-patients (P <0.001). Besides the disease stage, the respondent type had the highest impact on breast cancer utility.

ß-Lactoglobulin Peptide Fragments Conjugated with Caffeic Acid Displaying Dual Activities for Tyrosinase Inhibition and Antioxidant Effect.

The regulation of tyrosinase activity and reactive oxygen species is of great importance for the prevention of dermatological disorders in the fields of medicine and cosmetics. Herein, we report a strategy based on solid-phase peptide chemistry for the synthesis of ß-lactoglobulin peptide fragment/caffeic acid (CA) conjugates (CA-Peps) with dual activities of tyrosinase inhibition and antioxidation. The purity of the prepared conjugates, CA-MHIR, CA-HIRL, and CA-HIR, significantly increased to 99%, as acetonide-protected CA was employed in solid-phase coupling reactions on Rink amide resins. The tyrosinase inhibitory activities of all CA-Pep derivatives were higher than the activity of kojic acid, and CA-MHIR exhibited the highest tyrosinase inhibition activity (IC50 = 47.9 µM). Moreover, CA-Pep derivatives displayed significantly enhanced antioxidant activities in the peroxidation of linoleic acid as compared to the pristine peptide fragments. All CA-Pep derivatives showed no cytotoxicity against B16-F1 melanoma cells.

Prescription Trends of Psychotropics in Children and Adolescents with Autism Based on Nationwide Health Insurance Data.

Children with autism are often medicated to manage emotional and behavioral symptoms; yet, data on such pharmacotherapy is insufficient. In this study, we investigated the Korean National Health Insurance Claims Database (NHICD) information related to autism incidence and psychotropic medication use. From the 2010-2012 NHICD, we selected a total of 31,919,732 subjects under 19 years old. To examine the diagnostic incidence, we selected patients who had at least one medical claim containing an 10th revision of International Statistical Classification of Diseases and Related Health Problems (ICD-10) code for pervasive developmental disorder, F84, not diagnosed in the previous 360 days. Psychotropics were categorized into seven classes. Then, we analyzed the data to determine the mean annual diagnostic incidence and psychotropic prescription trends. Diagnostic incidence was 17,606 for the 3 years, with a mean annual incidence per 10,000 population of 5.52. Among them, 5,348 patients were prescribed psychotropics. Atypical antipsychotics were the most commonly used, followed by antidepressants. An older age, male sex, and the availability of medical aid were associated with a higher rate of prescription than observed for a younger age, female sex, and the availability of health insurance. Psychotropic drugs were used for less than one-third of patients newly diagnosed with autism, and prescription differed by sex and age. Increased diagnostic incidence is associated with an increased prescription of psychotropic drugs. Therefore, medication-related safety data and policies for psychotropic drugs in autism should be prepared.

Decreased expression of Toll-like receptor 4 and 5 during progression of prostate transformation in transgenic adenocarcinoma of mouse prostate mice.

Chronic inflammation has been considered an important risk factor for development of prostate cancer. Toll-like receptors (TLRs) recognize microbial moieties or endogenous molecules and play an important role in the triggering and promotion of inflammation. In this study, we examined whether expression of TLR4 and TLR5 was associated with progression of prostate transformation in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The expression of TLR4 and TLR5 was evaluated by immunohistochemisty in formalin-fixed paraffin-embedded prostate tissue from wild-type (WT) and TRAMP mice. Normal prostate tissue from WT mice showed strong expression of TLR4 and TLR5. However, TLR4 expression in the prostate tissue from TRAMP mice gradually decreased as pathologic grade became more aggressive. TLR5 expression in the prostate tissue from TRAMP mice also decreased in low-grade prostate intraepithelial neoplasia (PIN), high-grade PIN and poorly differentiated adenocarcinoma. Overall, our results suggest that decreased expression of TLR4 and TLR5 may contribute to prostate tumorigenesis.

The first case of Capillaria hepatica infection in a nutria (Myocastor coypus) in Korea.

This study reports the first case of Capillaria hepatica infection in a nutria in Korea. Ten nutrias, captured near the Nakdong River, were submitted to our laboratory for necropsy. White-yellowish nodules were found in the liver of 1 of the nutrias at necropsy. Histologically, the lesions were granulomatous, and infiltrations of lipid-laden macrophages, eosinophils, and several multinucleated giant cells were observed. The lesions consisted of numerous eggs and necrotic hepatocytes. The eggs were lemon-shaped and had polar plugs at the ends of both long sides. The eggs were morphologically identified as those of C. hepatica. Worldwide, C. hepatica infection in nutrias is very rare. Nutrias are a kind of livestock, as well as wildlife; therefore, an epidemiological study for parasitic infections needs to be conducted.

High animal fat intake enhances prostate cancer progression and reduces glutathione peroxidase 3 expression in early stages of TRAMP mice.

BACKGROUND: Prostate cancer is the most frequently diagnosed cancer in Western men, and more men have been diagnosed at younger ages in recent years. A high-fat Western-style diet is a known risk factor for prostate cancer and increases oxidative stress. METHODS: We evaluated the association between dietary animal fat and expression of antioxidant enzymes, particularly glutathione peroxidase 3 (GPx3), in the early stages of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Six-week-old male nontransgenic and TRAMP mice were placed on high animal fat (45% Kcal fat) or control (10% Kcal fat) diets and sacrificed after 5 or 10 weeks. RESULTS: The histopathological score increased with age and high-fat diet consumption. The histopathological scores in dorsal and lateral lobes increased in the 10-week high-fat diet group (6.2±0.2 and 6.2±0.4, respectively) versus the 10-week control diet group (5.3±0.3 and 5.2±0.2, respectively). GPx3 decreased both at the mRNA and protein levels in mouse prostate. GPx3 mRNA expression decreased (∼36.27% and ∼23.91%, respectively) in the anterior and dorsolateral prostate of TRAMP mice fed a high-fat diet compared to TRAMP mice fed a control diet. Cholesterol treatment increased PC-3 human prostate cancer cell proliferation, decreased GPx3 mRNA and protein levels, and increased H2 O2 levels in culture medium. Moreover, increasing GPx3 mRNA expression by troglitazone in PC-3 cells decreased cell proliferation and lowered H2 O2 levels. CONCLUSIONS: Dietary fat enhances prostate cancer progression, possibly by suppressing GPx3 expression and increasing proliferation of prostate intraepithelial neoplasia (PIN) epithelial cells.

TLR7 expression is decreased during tumour progression in transgenic adenocarcinoma of mouse prostate mice and its activation inhibits growth of prostate cancer cells.

PROBLEM: Although various Toll-like receptors (TLRs) have been associated with immune response and tumorigenesis in the prostate cells, little is known about the role of TLR7. Accordingly, we examined the expression of TLR7 during tumour progression of TRMAP (transgenic mouse model for prostate cancer) mice and its role on cell growth. METHOD OF STUDY: Toll-like receptor7 expression was examined by RT-polymerase chain reaction (PCR), Western blot, and immunohistochemistry. Cell growth was examined by MTT assay. Colony formation was investigated by crystal violet staining. RESULTS: Strong expression of TLR7 was detected in the normal prostate epithelia of Wild-type (WT) mice, but not in TLR7-deficient mice. In contrast, TLR7 expression was weak in transgenic adenocarcinoma of mouse prostate (TRAMP)-C2 cells, as compared with murine bone marrow-derived macrophages (BMDMs). Moreover, TLR7 mRNA was markedly expressed in RWPE-1 cells (non-cancerous prostate epithelial cells), but not in PC3 and DU145 (prostate cancer cells). Immunohistochemically, TLR7 expression gradually decreased in TRAMP mice depending on the pathologic grade of the prostate cells. TLR7 agonists increased both the gene and protein expression of TLR7 and promoted production of proinflammatory cytokines/chemokines and IFN-ß gene expression in prostate cancer cell lines. Moreover, loxoribine inhibited the growth and colony formation of TRAMP-C2 cells dependent of TLR7. CONCLUSION: These findings suggest that TLR7 may participate in tumour suppression in the prostate cells.

In vitro and in vivo growth inhibition of prostate cancer by the small molecule imiquimod.

Prostate cancer is the second leading cause of cancer death in men worldwide. In the present study, we examined in vitro and in vivo antitumor effect of the small molecule imiquimod, also known as a TLR7 agonist, against prostate cancer. Imiquimod inhibited the growth of mouse (TRAMP­C2) and human (PC-3) prostate cancer cells. Treatment with imiquimod induced cell cycle arrest at the G2/M phase in TRMPA-C2 cells, confirmed by the changes of G2/M checkpoint regulators such as reduction of cyclin B1 expression and increase of phospho-CDC2 and p21 in TRAMP-C2 cells treated with imiquimod. Flow cytometry and western blot analysis revealed that imiquimod induced direct apoptosis in TRAMP-C2 cells via a mitochondrial­dependent pathway. Intratumoral injection with imiquimod reduced significantly tumor growth and increased apoptotic cells in mice subcutaneously implanted with TRAMP-C2 cells. Our results indicate that imiquimod can be an alternative therapeutic for locally generated prostate cancer.

Nanog reverses the effects of organismal aging on mesenchymal stem cell proliferation and myogenic differentiation potential.

Although the therapeutic potential of mesenchymal stem cells (MSCs) is widely accepted, loss of cell function due to donor aging or culture senescence are major limiting factors hampering their clinical application. Our laboratory recently showed that MSCs originating from older donors suffer from limited proliferative capacity and significantly reduced myogenic differentiation potential. This is a major concern, as the patients most likely to suffer from cardiovascular disease are elderly. Here we tested the hypothesis that a single pluripotency-associated transcription factor, namely Nanog, may reverse the proliferation and differentiation potential of bone marrow-derived MSC (BM-MSC) from adult donors. Microarray analysis showed that adult (a)BM-MSC expressing Nanog clustered close to Nanog-expressing neonatal cells. Nanog markedly upregulated genes involved in cell cycle, DNA replication, and DNA damage repair and enhanced the proliferation rate and clonogenic capacity of aBM-MSC. Notably, Nanog reversed the myogenic differentiation potential and restored the contractile function of aBM-MSC to a similar level as that of neonatal (n)BM-MSC. The effect of Nanog on contractility was mediated--at least in part--through activation of the TGF-ß pathway by diffusible factors secreted in the conditioned medium of Nanog-expressing BM-MSC. Overall, our results suggest that Nanog may be used to overcome the effects of organismal aging on aBM-MSC, thereby increasing the potential of MSC from aged donors for cellular therapy and tissue regeneration.

Activation of Nod1 and Nod2 induces innate immune responses of prostate epithelial cells.

BACKGROUND: Nod1 and Nod2 are cytosolic receptors which are responsible for sensing bacterial peptidoglycan derivatives. In this study, we determined whether Nod1 and Nod2 are involved in the innate immune responses of prostate epithelial cells. METHODS: The expression of Nod1 and Nod2 was examined by RT-PCR and immunohistochemistry. ELISA was performed to determine the production of cytokines/chemokines. Activation of NF-κB and MAPK was examined using western blot analysis. RESULTS: The Nod1 gene was distinctly expressed in all tested cells including DU145, PC3, and TRAMP-C2 cells, whereas Nod2 expression was weak. Both Nod1 and Nod2 proteins were expressed in normal mouse prostate epithelia with difference of expression levels. Tri-DAP (Nod1 agonist), but not MDP (Nod2), increased the production of IL-8 (or KC) and IL-6 in prostate epithelial cells. Tri-DAP and MDP could upregulate the gene expression of COX-2 and activate NF-κB and MAPK. In addition, Tri-DAP and MDP synergized with TLR agonists to induce the production of IL-8/KC or IL-6 in PC3 and TRAMP-C2 cells. We finally showed that Nod1 and Nod2 were also expressed in a wide range of prostate lesions including prostate intraepithelial neoplasm (PIN), phyllodes-like tumor, and adenocarcinoma in TRAMP (transgenic adenocarcinoma of the mouse prostate) mice, even though the expression level of Nod1 and Nod2 was different. CONCLUSION: These results indicate that Nod1 and Nod2 may play important roles in the innate immune response of prostate epithelial cells and the development and progression of prostate cancer.

Detection of sarcocystic infection in a wild rodent (Apodemus agrarius chejuensis) captured on Jeju island.

Sarcocystis spp is a causative agent of sarcocystosis. They have a characteristic life cycle infecting both prey and predator. Sarcocystis can cause myositis, atrophy of the adjacent cells and abortion in cattle. In mice, sarcocystosis causes mild cellular reactions without clinical disease. Severe haemorrhage and abortion were also reported. For monitoring the disease in wild rodents of the Korean peninsula, we captured Apodemus agrarius chejuensis on Jeju island and examined the specimen histopathologically. Intramuscular cysts were found and diagnosed as Sarcocystis. Sarcocystic infection has been reported in worldwide. There have been many reported infections in cattle and pigs in Korea. To our knowledge, this is the first report of Sarcocystis in Apodemus agrarius chejuensis captured in Korea.

Molecular and functional effects of organismal ageing on smooth muscle cells derived from bone marrow mesenchymal stem cells.

AIMS: Bone marrow-derived smooth muscle cells (BM-SMCs) have high potential as an autologous cell source of vascular progenitors but normal cell function and turnover frequency may decline with age. In this study we set out to study the effects of organismal ageing on the molecular and functional properties of BM-SMCs. METHODS AND RESULTS: To address this issue, we employed a smooth muscle alpha-actin promoter (alphaSMA) driving expression of enhanced green fluorescence protein (EGFP) to isolate SMCs from bone marrow of neonatal (nBM-SMCs) or adult (aBM-SMCs) sheep and examined their proliferation potential and contractility. Compared with nBM-SMCs, aBM-SMCs exhibited lower clonogenicity and proliferation potential that could be improved significantly by addition of basic fibroblast growth factor. Vascular constructs from aBM-SMCs showed reduced ability to generate force and contract fibrin hydrogels and this function could be partially restored by addition of transforming growth factor-beta1. They also exhibited lower receptor- and non-receptor-mediated vascular contractility and mechanical strength, which was comparable to that of tissue constructs prepared with vascular SMCs from neonatal umbilical veins. In agreement with the contractile properties and mechanical strength of vascular constructs, aBM-SMCs displayed significantly lower expression of alphaSMA, smoothelin, desmin, type I collagen, and tropoelastin transcripts compared with nBM-SMCs. CONCLUSION: Understanding the effects of organismal ageing on BM-SMCs and the properties of the resulting vascular constructs may lead to innovative ways to facilitate application of these cells in the treatment of cardiovascular disease which is especially prevalent in the elderly.

Cre/loxP-regulated transgenic zebrafish model for neural progenitor-specific oncogenic Kras expression.

Ras proteins regulate signaling pathways that control many cellular responses, such as proliferation, survival, and differentiation. However, there are intriguing questions about the relationship between the developmental timing of specific mutations and the resultant phenotypes in individual cells. In this study, we used the Cre/loxP system for maintaining transgenic zebrafish lines harboring oncogenic Kras(V12) under the nestin promoter, and investigated the developmental effects of Ras activation in neural progenitor cells. Activated human Kras(V12) was induced within pDSNesLCherryLEGFPKRas(V12) transgenic fish by Cre mRNA injection. Cre-mediated gene excision was confirmed by polymerase chain reaction, and the injected embryos were investigated for Kras(V12) effects using the hemotoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and in situ hybridization. pDSNesLCherryLEGFPKRas(V12) transgenic embryos normally expressed mCherry in their central nervous system throughout the developmental stage. However, Cre mRNA injection efficiently excised the flanking stop sequence, and the injected embryos expressed enhanced green fluorescent protein in their brain with severe edema. Brain histology showed that neuronal cell differentiation could occur in spite of oncogenic Kras(V12) overexpression, but massive apoptosis and brain edema caused early embryonal death. In summary, the overexpression of Kras(V12) induces extensive apoptosis of neural progenitor cells followed by severe edema of the brain. However, some neural progenitor cells are resistant to Kras(V12) and can retain their ability to differentiate into neurons. Finally, our transgenic model demonstrates the inability of Kras(V12) alone to induce brain tumors at the early stage of development.

Bone morphogenetic protein 7 induces mesenchymal-to-epithelial transition in melanoma cells, leading to inhibition of metastasis.

Bone morphogenetic protein (BMP) 7 counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity in developmental stages. Because epithelial-to-mesenchymal transition and its reversed process mesenchymal-to-epithelial transition (MET) are also involved in cancer progression, we investigated whether BMP7 plays a role in WM-266-4 melanoma cell growth and metastasis. An MTT assay was conducted in WM-266-4 and HEK293T cell lines to show the cell growth inhibition ability of BMP7 and cisplatin. Semiquantitative RT-PCR was used to determine MET in morphologically changed BMP7-treated melanoma cells. MET-induced cells expressed less a basic helix-loop-helix transcription factor (TWIST) in western blot analysis, and we confirm that BMP receptor (Alk2) siRNA transduction could restore TWIST protein expression via blocking of Smad 1, 5 and 8 signaling. Matrigel invasion and cell migration assays were done to investigate the BMP7-induced metastasis inhibition ability. BMP7 treatment only slightly reduced cell growth rate, but induced apparent MET. BMP7 also reduced the invasion and migration ability. Furthermore, BMP7 reduced the resistance of WM-266-4 cells to cisplatin. Collectively, our findings indicate that the metastatis inhibition ability of BMP7 is involved in MET, and that BMP7 could be used as a potential metastasis inhibitor in human melanoma cells.

Zebrafish embryo extracts promote sphere-forming abilities of human melanoma cell line.

Sphere-forming abilities in culture condition are considered a hallmark of cancer stem-like cells, which represents tumor cell invasiveness and stem-like characteristics. We aimed to show that the sphere-forming subpopulation of human malignant melanoma cell line WM-266-4 acts differently to zebrafish embryo extracts compared with their bulk counterpart. Spheres were maintained in neural stem cell culture conditions. The embryos of zebrafish at specific developmental stages were collected and the extracts were purified under 100 kDa. Spheres were treated with embyo extracts and proliferation assay and immunocytochemistry were conducted. Spheroid cells expressed nestin and epidermal growth factor receptor (EGFR) but not melanoma antigen recognized by T-cells (MART)1, indicating their stem-like character. Zebrafish embryo extracts at 50% epiboly stage inhibited melanoma bulk cell proliferation in a dose-dependent manner. However, sphere-forming abilities were significantly enhanced under 1 microg/mL concentration of 50% epiboly stage embryo extract treatment. Our findings implicate that we should consider cell subsets of a different character from the tumor origin that can respond differently to exogenous substances or tumor microenvironments. We suggest that cancer research should consider both minor stem-like subpopulations and the other major bulk tumor cells.