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

Introduction:: Although breast cancer has high rates of early-stage detection, metastasis of the primary tumor can reduce the patient’s 5-year survival rate by 73%; it is difficult to determine metastatic potential due to a lack of a universal molecular prognostic marker, and thus calibrating patient care can be challenging. Our prior evidence^1-3 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 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 and a transcriptome consistent with reduced overall survival, suggesting PPFC as a novel device.


Materials and Methods:: GFP⁺Luc⁺ MDAMB231 cells were seeded (~5000 cells/cm²) overnight onto fibronectin-coated glass slides. Using the PPFC, the slides were exposed to 25 dynes/cm² shear stress for 2 minutes to collect weakly adherent cells, then exposed to 90 dyn/cm² shear stress for 2 minutes. Any cells that remained on the slide were removed via 0.25% trypsin and collected as strongly adherent cells. 10⁶ 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/SCIDg mice.


Results, Conclusions, and Discussions:: We resected GFP⁺Luc⁺ MDAMB231 cells from mammary fat pads 6-weeks post-injection and found that GFP⁺ cells that invaded the fat pad had a significantly lower average adhesion strength compared to cells that remained in the tumor bolus (Fig. 1A-B). To enrich for a particular phenotype, we sorted an isogenic GFP⁺Luc⁺ MDAMB231 population into strongly and weakly adherent cells and, along with unselected control cells, injected cells again into mammary fat pads. While there was no difference in the primary tumor weight, the number of lung metastases were significantly higher for mice receiving weakly adherent cells (Fig. 1C-D). Transcriptional analyses of primary tumors stratify metastatic outcomes as did their pre-injection counterparts, and their gene signature predicts disease outcome from analyses of triple negative breast cancer patients in The Cancer Genome Atlas. Finally, PPFC analyses provide label-free measurements of adhesion strength and cell abundance relative to the analyzed population (determined from deconvolving host and tumor samples in a logistic regression model), which when used for admixed cancer lines enables separation and when applied to metastatic disease can retrospectively predict metastatic potential (Fig. 1E).These results suggest that weakly adherent cells migrate out of the primary tumor microenvironment and can be isolated to prognostically assess future patient outcomes. Continuing work is applying this device to tumor samples from TNBC and Ductal Carcinoma In Situ patients.


Acknowledgements (Optional): :

References (Optional): : ¹Fuhrmann, et al Biophys J 2017; ²Beri, et al Cancer Res 2020; ³Yeoman, et al Cell Reports 2021