Skbr3

Manufactured by American Type Culture Collection

Sourced in United States, Germany, United Kingdom, China, Sweden, France, Italy, Japan

SKBR3 is a cell line derived from a breast adenocarcinoma. It is a commonly used model for breast cancer research.

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1 329 protocols using skbr3

Effect of PIP2 on ERBB2 Expression

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Human fibroblast BJ from neonatal foreskin and SKBR3 (breast adenocarcinoma, human) cell lines were purchased from ATCC. Fibroblast cells were grown in DMEM supplemented with 10% HyClone FBS, non essential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, at 37°C in 5% CO₂. SKBR3 cells were grown in ATCC-formulated McCoy's 5a modified medium complemented with 10% FBS and were maintained under an atmosphere of 5% CO₂ at 37°C.
The effect of alkylating PIP 2 on the expression of ERBB2 mRNA was determined in both fibroblast and SKBR3 cell lines using real-time PCR. BJ skin fibroblast cells were seeded at a density of 5 × 10⁴ cells/well of a 6-well plate and SKBR3 cells were plated into the 6-well plate at 4 × 10⁵ cells/well. The cells were then treated with 50 and 100 nM of alkylating PIP 2 for 48 h with DMSO as a corresponding control sample. After 48 h, total RNA was isolated using RNEasy Kit (Qiagen) and cDNA was synthesized by ReverTra Ace qPCR RT Master mix with genomic DNA remover (Toyobo, Japan) following the manufacturer's instructions. The expression level of ERBB2 was normalized using β-actin, as an internal control.
The primers used in this study includes, β-actin sense, 5′-CAATGTGGCCGAGGACTTTG-3′ and antisense, 5′-CATTC TCCTTAGAGAGAAGTGG-3′. The sense primer of ERBB2 is 5′-AGCCGCGAGCA CCCAAGT-3′ and antisense, 5′-TTGGTGGGCAGGTAGGTGAGTT-3′.

Chandran A., Syed J., Taylor R.D., Kashiwazaki G., Sato S., Hashiya K., Bando T, & Sugiyama H. (2016). Deciphering the genomic targets of alkylating polyamide conjugates using high-throughput sequencing. Nucleic Acids Research, 44(9), 4014-4024.

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Transfection of miR-133a-1 in Breast Cancer Cells

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Human miR-133a-1 and miR-negative control (NC) were purchased from Vigene Biosciences (Jinan, China; Cat. #CR207927). The human breast cancer cell line SKBR3 was purchased from ATCC (American Type Culture Collection; Manassas, VA, USA). The SKBR3 cells were seeded onto 6-well plates and cultured in ATCC-formulated McCoy’s 5a Medium Modified (Catalog No. 30-2007; Manassas, VA, USA) plus 10% fetal bovine serum (Catalog No. 30-2020; Manassas, VA, USA) overnight at 37 °C. Cells were then transfected with miR-133a-1 or mi-NC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 hours, the cells were harvested for further experiments.

Meng Y., Tang H., Luo Z., Tan W., Chen L., Du Y., Tao Z., Huang M., Li W., Cao J., Wang L., Li T., Liu X., Lv F., Liu X., Zhang J., Zheng L, & Hu X. (2021). An integrated bioinformatics analysis to investigate the targets of miR-133a-1 in breast cancer. Translational Cancer Research, 10(3), 1238-1248.

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Breast cancer cell culture protocol

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Breast cancer cell lines MCF7, MDA-MB-231 and SK-BR-3 were purchased from ATCC and cultured at 37 °C in a humidified atmosphere with 5% CO₂ using high-glucose GlutaMAX™ DMEM (MCF-7, MDA-MB-231) and McCoy’s 5A (Modified) Medium (SK-BR-3) supplemented with 10% (v/v) FBS (fetal bovine serum), 1% (v/v) penicillin–streptomycin. Cell lines were characterized by flow cytometry and immunofluorescence for HER2 and HER3 expression (Figure S1). For microchip experiments, 10 k cells of each cell line were injected inside Ephesia.

Tulukcuoglu Guneri E., Lakis E., Hajji I., Martin E., Champ J., Rampanou A., Pierga J.Y., Viovy J.L., Proudhon C., Bidard F.C, & Descroix S. (2022). Deciphering HER2-HER3 Dimerization at the Single CTC Level: A Microfluidic Approach. Cancers, 14(8), 1890.

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Breast Cancer Cell Line Culture

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Human breast cancers SK-BR-3 (high HER-expressing cells) and MDA-MB-231 (basal HER-expressing cells) cell lines were purchased from ATCC. The ZR-75-1 cell line (which overexpresses HER2 [27 (link)] without gene amplification [28 (link)]) was a generous gift from Dr Elisa Caiola, Mario Negri Institute, Milan. SK-BR-3 cell line (ATCC HTB-30) was cultured in McCoy’s 5a medium (Euroclone) supplemented with 10% Fetal Bovine Serum (FBS). ZR-75-1 cells (ATCC CRL-1500) were cultivated in RPMI-1640 medium (Euroclone) supplemented with 10% FBS. MDA-MB-231 cell line (ATCC HTB-26) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Euroclone) supplemented with 10% FBS.
Cells were cultured in 100 mm dishes at 37 °C in a humidified atmosphere containing 5% CO₂, and when they reached confluence, cells were passaged using a Trypsin-EDTA solution.

Damiani I., Castiglioni S., Sochaj-Gregorczyk A., Bonacina F., Colombo I., Rusconi V., Otlewski J., Corsini A, & Bellosta S. (2021). Purification and In Vitro Evaluation of an Anti-HER2 Affibody-Monomethyl Auristatin E Conjugate in HER2-Positive Cancer Cells. Biology, 10(8), 758.

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Cancer Cell Culturing and Xenograft Processing

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MCF7, MDA-MB-453, A-431, H1781, H1819, Calu-3, BT-474, SKBR3, and NCI-N87 cell lines were purchased from the ATCC. MCF7, MDA-MB-453, and A-431 were cultured in DMEM; H1781, H1819, Calu-3, and NCI-N87 in RPMI-1640; BT-474 in ATCC HybriCare Medium with 1.5 g/L sodium bicarbonate; SKBR3 in the ATCC-formulated McCoy’s 5a Medium Modified. To make the complete medium, all media were supplemented with 10% FBS and 1% streptomycin/penicillin. All cells used in this study were periodically tested negative for Mycoplasma using the Mycoplasma Plus PCR Primer Kit (Agilent). All patient-derived cancer cells or tumor spheroids in this study were collected and studied according to the Declaration of Helsinki and DFCI IRBs. DFCI 429 was established from a pleural effusion sample. Next-Generation Sequencing confirmed de novo HER2 amplification. DFCI315 and DFCI359 were characterized to harbor HER2 exon20 V777_G778insGSP and HER2 exon19 755_757LREdelinsRP, respectively (23 (link)). Xenograft tumors were processed for short-term ex vivo as previously described (23 (link)). The spheroids in a range of 40 to 70 μmol/L were resuspended in type I collagen (Corning) and loaded into the DAX-1 3-D cell culture chip (AIM Biotech). After solidification, the media were applied to the outer channels with indicated drugs.

Inoue M., Kobayashi M., Kozaki S., Zimmer A, & Ueda H. (1998). Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice. Proceedings of the National Academy of Sciences of the United States of America, 95(18), 10949-10953.

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Comparative Analysis of Prostate and Breast Cancer Cell Lines

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Four prostate cancer cell lines (VCaP, LNCaP, PC-3 and PC-3M) and two breast cancer cell lines (SKBR3 and MDAMB-231) were used in our analysis. VCaP, SKBR3, and MDAMB231 were obtained from American Type Culture Collection (ATCC). VCaP cells were cultured in Dulbecco’s Modified Eagles’s Medium (DMEM, ATCC). SKBR3 cells were cultured in McCoy’s 5a Medium Modified (ATCC) and MDA-MB-231 cells were cultured in Leibovitz’s L-15 medium (ATCC). LNCaP, PC-3 and PC-3M cell lines were obtained from Dr. Alison Allan’s research group at London Health Sciences, London, ON, Canada. LNCaP and PC-3M cells were cultured in RPMI-1640. PC-3 cells were maintained in F12K media (ATCC). All media were supplemented with 10% fetal bovine serum (FBS). Cells were cultured at 37 °C and 5% CO₂.

Kermanshah L., Poudineh M., Ahmed S., Nguyen L.N., Srikant S., Makonnen R., Cantu F.P., Corrigan M, & Kelley S.O. (2018). Dynamic CTC Phenotypes in Prostate Cancer Metastatic Models Visualized Using Magnetic Ranking Cytometry. Lab on a chip, 18(14), 2055-2064.

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Breast Cancer Cell Line Culture

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Human breast cancer cell lines; luminal-type (MDA-MB-361 and MCF7), HER2-type (SK-BR-3) and TNBC-type (HCC1937, MDA-MB-231) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). According to manufacturer’s instructions, MDA-MB-361 cells were cultured in in Leibovitz’s L-15 medium (ATCC) containing 20% FBS (without heat inactivation) without CO₂ aeration. MCF7 cells were cultured in Eagle’s Minimum Essential Medium (ATCC) containing 10% FBS (without heat inactivation) and 0.01 mg/ml of human recombinant insulin under the 5% CO₂ aeration. SK-BR-3 cells were cultured in McCoy’s 5a Medium (ATCC) containing 10% FBS (without heat inactivation) under the 5% CO₂ aeration. HCC1937 cells were cultured in RPMI-1640 medium (ATCC) containing 10% FBS (without heat inactivation) under the 5% CO₂ aeration. MDA-MB-231 cells were cultured in Leibovitz’s L-15 medium (ATCC) containing 10% FBS (without heat inactivation) without CO₂ aeration.

Murayama M.A., Takada E., Takai K., Arimitsu N., Shimizu J., Suzuki T, & Suzuki N. (2022). Nicotine treatment regulates PD-L1 and PD-L2 expression via inhibition of Akt pathway in HER2-type breast cancer cells. PLoS ONE, 17(1), e0260838.

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Lung and Breast Cancer Cell Lines

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Eight lung cancer cell lines (A549, Calu‐3, HCC827, NCI‐H1299, NCI‐H1781, NCI‐H1975, NCI‐H1993, and NCI‐H2170), two breast cancer cell lines (SK‐BR‐3 and BT‐474), and one normal human bronchial epithelial cell line (BEAS‐2B) were used in this study. These cell lines, except A549, SK‐BR‐3, BT‐474 and BEAS‐2B, were kindly provided by Dr. Adi F. Gazdar (The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA), who established these lines with Dr. John D. Minna.19, 20 A549, SK‐BR‐3, and BT‐474 were purchased from ATCC (Manassas, VA, USA). BEAS‐2B was purchased from European Collection of Cell Cultures (Porton Down, UK). All lung cancer cells were cultured in RPMI‐1640 media supplemented with 10% FBS and the other cells were maintained in DMEM with 10% FBS. They were grown in a humidified incubator with 5% CO₂ at 37°C. Afatinib and gefitinib were purchased from Sykkinase (San Diego, CA, USA) and InvivoGen (San Diego, CA, USA), respectively.

Suzawa K., Toyooka S., Sakaguchi M., Morita M., Yamamoto H., Tomida S., Ohtsuka T., Watanabe M., Hashida S., Maki Y., Soh J., Asano H., Tsukuda K, & Miyoshi S. (2015). Antitumor effect of afatinib, as a human epidermal growth factor receptor 2‐targeted therapy, in lung cancers harboring HER2 oncogene alterations. Cancer Science, 107(1), 45-52.

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HER2+ Breast Cancer Cell Lines Characterization

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Two HER2+BC cell lines, SK-BR-3 and AU565, were purchased from American Type Culture Collection (ATCC; Manassas, VA), with Cat. Nos. of ATCC^®HTB-30™ and ATCC^®CRL-2351™, respectively. All the cells underwent short tandem repeat (STR) authentication, and mycoplasma detection was performed before each cell experiment. The medium of SK-BR-3 cells was McCoy’s 5a (ATCC, 30–2007)+10% fetal bovine serum (ATCC, 30–2020), while that of AU565 cells was RPMI-1640 (ATCC, 30–2001). Both medium contained 1% L-glutamine, 1% sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cell culture conditions were 37.0°C, 95% air+5% CO₂.

Zhang H., Zhang X.Y., Kang X.N., Jin L.J, & Wang Z.Y. (2020). LncRNA-SNHG7 Enhances Chemotherapy Resistance and Cell Viability of Breast Cancer Cells by Regulating miR-186. Cancer Management and Research, 12, 10163-10172.

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Culturing and Characterizing Circulating Tumor Cells

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Viable CTCs were enriched from a cryo-conserved DLA product by using the Parsortix system and were cultured as previously reported [25 (link)]. Briefly, CTCs were cultured in low attachment plates (Corning, Corning, NY, USA) with RPMI 1640 medium supplemented with 1 × B27 (Thermo Fisher Scientific), 20 ng/mL human epidermal growth factor (Merck), 20 ng/mL fibroblast growth factor (Merck), and 1% penicillin-streptomycin (Thermo Fisher Scientific) in a humidified atmosphere with 5% CO2 and 4% O2.
For drug testing 100 CTCs were seeded per well of a 96-well plate after ten days of pre-culture. The cells were treated with capivasertib (MedChem Express, Monmouth Junction, NJ, USA), everolimus (Merck), epirubicin (Merck), and paclitaxel (Merck). Each drug concentration was tested in triplicates. After incubation for 6 days, cells were spun on glass slides, stained for cytokeratin, and numbers were determined by counting. As references, cell lines MDA-MB-231, SK-BR-3, T-47D, and MCF7 were used (ATCC, Manassas, VA, USA; catalog numbers: MDA-MB 231: HTB-26, SK-BR-3: HTB-30, T-47D: HTB133, and MCF7: HTB-22). Cells were authenticated via short tandem repeat analysis and regularly tested negative for Mycoplasma.

Franken A., Behrens B., Reinhardt F., Yang L., Rivandi M., Marass F., Jaeger B., Krawczyk N., Cieslik J.P., Honisch E., Asperger H., Jeannot E., Proudhon C., Beerenwinkel N., Schölermann N., Esposito I., Dietzel F., Stoecklein N.H., Niederacher D., Fehm T, & Neubauer H. (2021). Multiparametric Circulating Tumor Cell Analysis to Select Targeted Therapies for Breast Cancer Patients. Cancers, 13(23), 6004.

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