Lipid tethering of breast tumor cells reduces cell aggregation during mammosphere formation

Abstract

Mammosphere assays are widely used in vitro to identify prospective cancer-initiating stem cells that can propagate clonally to form spheres in free-floating conditions. However, the traditional mammosphere assay inevitably introduces cell aggregation that interferes with the measurement of true mammosphere forming efficiency. We developed a method to reduce tumor cell aggregation and increase the probability that the observed mammospheres formed are clonal in origin. Tethering individual tumor cells to lipid anchors prevents cell drift while maintaining free-floating characteristics. This enables real-time monitoring of single tumor cells as they divide to form mammospheres. Monitoring tethered breast cancer cells provided detailed size information that correlates directly to previously published single cell tracking data. We observed that 71% of the Day 7 spheres in lipid-coated wells were between 50 and 150 μm compared to only 37% in traditional low attachment plates. When an equal mixture of MCF7-GFP and MCF7-mCherry cells were seeded, 65% of the mammospheres in lipid-coated wells demonstrated single color expression whereas only 32% were single-colored in low attachment wells. These results indicate that using lipid tethering for mammosphere growth assays can reduce the confounding factor of cell aggregation and increase the formation of clonal mammospheres.

Targeting Mitochondria with ClpP Agonists as a Novel Therapeutic Opportunity in Breast Cancer

“…ADEPs: ClpP has emerged as an anti-bacterial target during the mechanism of action studies on acyldepsipeptide antibiotics (ADEPs) in last two decades. ADEPs were first reported as antibiotics [ 273 ]. Identification of the resistance-mediating mutation within an ADEP-resistant Escherichia coli mutant and affinity chromatography with an immobilized ADEP analog led to identification of ClpP as the direct target of ADEPs [ 274 ].…”

Therapy-resistant breast cancer in focus: Clinically relevant mitigation by flavonoids targeting cancer stem cells

“…BCSCs tend to form spheroids, non-adherent spherical clusters of cells, also known as mammospheres ( Liu et al, 2016 ). Mammospheres are considered a pre-cancerous state and an indicator of stem-ness ( Bhandary et al, 2021 ; Patel et al, 2021 ), specifically self-renewal abilities and increased tumorigenicity and metastatic potential ( Truong et al, 2021 ). Cells from mammospheres can be serially passaged and keep their multipotency ( Ercan et al, 2011 ).…”

The Intricate Interplay between Cancer Stem Cells and Oncogenic miRNAs in Breast Cancer Progression and Metastasis

“…To date, mammosphere assays are widely used to identify the number of cells with self-renewal capability and the stemness potency of BCCs in vitro (mammosphere formation efficiency, MFE ) in combination with other methodologies, such as the CD44 + /CD24 − or ALDH + cell ratio, gene expression profiling, the proliferation and migration rate, etc. [ 10 , 12 , 13 , 14 ].…”