Adhesion Strength of Stromal-Resident Tumor Cells Predicts Metastatic Disease in vivo

Introduction: It is difficult to determine metastatic potential within breast cancer due to a lack of a universal molecular prognostic marker. Our prior evidence strongly supports a biophysical marker, i.e., the weakly adherent fraction of a cell population–when selected by a parallel-plate flow chamber (PPFC), as a more accurate means of stratifying tumor metastatic potential. We now demonstrate that cells with weak adhesion strength in vivo have increased metastatic potential compared to strongly adherent cells. Additionally, utilizing a divergent parallel plate flow chamber (dPPFC) we have assayed the adhesion strength of cells from human patient breast cancers or reduction mammoplasties and found that cells from cancerous tissue have a lower adhesion strength than cells from reduction tissue

Materials and

Methods: GFP+Luc+ MDAMB231 cells were seeded (~18,000 cells/cm^2) overnight onto fibronectin-coated glass slides. Using the PPFC, the slides were exposed to 30 dynes/cm^2 shear stress for 2 minutes to collect weakly adherent cells, then exposed to 60 dynes/cm^2 shear stress for 2 minutes. Any cells that remained on the slide were removed via 0.25% trypsin and collected as strongly adherent cells. 10^6 cells from the strongly adherent fraction, weakly adherent fraction, or the unsorted control were suspended in 40 μL of Matrigel-PBS (1:1) mixture and were injected into the inguinal mammary fat pad of 11-week-old female NOD/SCID mice. Human samples were collected from patients that underwent lumpectomies/mastectomies or reduction mammoplasties. Samples were digested into a single cell suspension and using the dPPFC, slides were exposed to a gradient of shear stresses. Τ50, or the shear stress at which 50% of seeded cells are sheared off, was found.

Results, Conclusions, and Discussions: We sorted GFP+Luc+ MDAMB231 population into strongly and weakly adherent cells and, along with unselected control, injected cells into mammary fat pads of mice (Fig 1A). The number of lung metastases was significantly higher for mice receiving weakly adherent cells (Fig. 1B-C). dPPFC analyses provide label-free measurements of adhesion strength and cell abundance, which when used for admixed cancer lines enables separation and when applied to metastatic disease can retrospectively predict metastatic potential (Fig. 1D). dPPFC analysis of human samples show that cells from breast cancers have a weaker adhesion strength than cells from reduction mammoplasties (Fig. 1E). Results suggest that weakly adherent cells migrate out of the primary tumor microenvironment and can be isolated to prognostically assess future patient outcomes. Patient derived breast tumors are composed of a larger population of weakly adherent cells than patient derived reduction mammoplasties, confirming results from in vitro and in vivo assays.

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