Abstract
Simple SummaryPredicting metastatic bone tropism in breast cancer by simply analyzing cell morphology would be highly desirable, in order to identify breast cancer patients at high risk for bone metastasis or to develop innovative bone-targeting drugs. In the current in vitro study, we show that MDA-MB-231 breast cancer cells are morphologically heterogeneous and exhibit a wide range of stable morphologies on a single cell level. Using the highly osteotropic derivative cell lines of MDA-MB-231 (MDA-MET, MDA-BONE), we demonstrate that single cell morphology reflects the molecular, migratory and biophysical nature of a given breast cancer cell and is specifically altered upon adoption of a bone-tropic phenotype. Thus, we propose that cell morphology could be an informative readout for understanding breast cancer heterogeneity and for predicting bone-metastases in breast cancer patients.Breast cancer is a heterogeneous disease and the mechanistic framework for differential osteotropism among intrinsic breast cancer subtypes is unknown. Hypothesizing that cell morphology could be an integrated readout for the functional state of a cancer cell, we established a catalogue of the migratory, molecular and biophysical traits of MDA-MB-231 breast cancer cells, compared it with two enhanced bone-seeking derivative cell lines and integrated these findings with single cell morphology profiles. Such knowledge could be essential for predicting metastatic capacities in breast cancer. High-resolution microscopy revealed a heterogeneous and specific spectrum of single cell morphologies in bone-seeking cells, which correlated with differential migration and stiffness. While parental MDA-MB-231 cells showed long and dynamic membrane protrusions and were enriched in motile cells with continuous and mesenchymal cell migration, bone-seeking cells appeared with discontinuous mesenchymal or amoeboid-like migration. Although non-responsive to CXCL12, bone-seeking cells responded to epidermal growth factor with a morphotype shift and differential expression of genes controlling cell shape and directional migration. Hence, single cell morphology encodes the molecular, migratory and biophysical architecture of breast cancer cells and is specifically altered among osteotropic phenotypes. Quantitative morpho-profiling could aid in dissecting breast cancer heterogeneity and in refining clinically relevant intrinsic breast cancer subtypes.
Highlights
Breast cancer is the most common malignancy in females worldwide [1]
To study the morphological difference between parental triple-negative MB-231 and bone-colonizing variants MET and BONE at the single cell level, cells were cultured on fibronectin-coated substrates in a subconfluent state, and labeled with fluorochromeconjugated Wheat Germ Agglutinin (WGA) to stain the cell surface and phalloidin to stain the actin filaments (Figure 1A)
A sheet-like lamellipodium emerged from its terminal point, while part of its structure was not fully in direct contact with the substrate
Summary
Breast cancer is the most common malignancy in females worldwide [1]. Due to early detection and innovative treatment, the prognosis of breast cancer patients is generally favorable. The occurrence of bone metastasis is a severe complication in breast cancer and osteolytic bone metastases often have a detrimental impact on quality of life and survival, e.g., by inducing pathological fractures, nerve compression syndromes or severe pain [4,5]. Loco-regional treatment of bone metastasis and current bone targeting agents, such as anti-resorptive drugs or bone-seeking radiopharmaceuticals, are often ineffective, so that bone-metastatic breast cancer is essentially incurable [6]. The metastatic cascade of breast cancer cells comprises five key steps: (1) acquisition of an invasive phenotype; (2) entrance of tumor cells into the blood vessels (intravasation);
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