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Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids are therefore a promising model system to identify and characterize novel drivers of breast cancer that would be unlikely to be identified using 2D cell lines. Antisense oligonucleotides can be used to efficiently and specifically knockdown target genes in the cell (Bennett et al., 2017). They can be taken up freely by organoids without the need for a transfection agent, making them a convenient tool for routine lab studies and screens.
Breast cancer is the most frequent malignancy in women worldwide and the second leading cause of cancer mortality in women (Siegel et al., 2017). To improve existing treatment regimens, it is critical to identify and investigate new molecular targets that have the potential to prevent breast cancer progression. We applied RNA-seq to generate a comprehensive catalog of long non-coding RNAs (lncRNAs) that are dysregulated in primary mammary tumors compared to normal mammary epithelial cells and prioritized 30 previously uncharacterized lncRNAs as Mammary Tumor Associated RNAs (MaTARs). In order to functionally assess MaTARs as key drivers of tumor progression, we performed antisense oligonucleotide (ASO)-mediated knockdown assays of all 30 MaTARs in 3D mammary tumor organoids (Diermeier et al., 2016).
ASOs are short (20-mers), single stranded DNA molecules containing phosphorothioate-modified nucleotides as well as modifications of the 2’-ribose (5-10-5 2’-MOE gapmer) (Geary et al., 2015). Upon binding of the ASO to its complementary target, the RNA-DNA duplex stimulates degradation of the lncRNA by RNase H and thereby reduces the level of the respective transcript (Wu et al., 2004). Importantly, we found that ASO uptake in primary mammary tumor cells and organoids is efficient without the use of transfection agents, a mechanism that has been studied in detail in hepatocytes (Koller et al., 2011). ASO-mediated knockdown is particularly efficient for nuclear retained lncRNAs (Lennox and Behlke, 2016).
Organoids represent an ex vivo model of mammary gland development and model branching morphogenesis in 3D culture (Ewald, 2013; Fata et al., 2007), which is driven by two physiological processes: collective cell migration and cell proliferation. As the same processes also drive tumor invasion, the mammary organoid system can be utilized to model invasive breast cancer in vitro. Loss of branching was observed upon ASO-mediated knockdown of 20 MaTARs in organoids (Diermeier et al., 2016) as well as the lncRNA Malat1 (Arun et al., 2016), indicating that these RNAs are involved in mammary tumor cell proliferation and/or collective cell migration. Hence, we suggest that antisense-mediated knockdown in mammary tumor organoids can be used to identify and characterize novel drivers of tumor progression.
|10x TaqMan RT buffer||5 μl|
|Random hexamers||2.5 μl|
|MgCl2 (25 mM)||11 μl|
|RNase Inhibitor||1 μl|
|Nuclease-free H2O||8.25 μl|
|Total volume||39 μl|
|Use immediately after preparation|
|PowerUp SYBR Green Master mix||10 μl|
|Nuclease-free H2O||7 μl|
|Primer mix (for + rev, 10 μM)||1/well (conc.: 0.5 μM/well)|
|Use immediately after preparation|
1Organoids can also be grown in 48-well and 96-well plates for assays requiring higher throughput. The size for the Matrigel domes has to be scaled down accordingly, e.g., 40 μl/well in 48-well plates and 20 μl/well in 96-well plates.
2If live-cell imaging is to be performed to analyze organoids, use glass-bottom plates (e.g., MatTek)
3If using new, untested ASOs, it is recommended to test several concentrations (e.g., 0.1, 0.5, 1 and 5 μM) and compare the knockdown efficiency.
4According to our RNA-seq data, culturing of organoids in an artificial extracellular matrix (ECM) for up to seven days does not significantly alter the expression patterns of lncRNAs (Diermeier et al., 2016).
5For optimal results, primers for qPCR should not overlap with the ASO binding site, and the ASO should not bind within the qPCR amplified region.