Poster S19 - AXL as a driver of breast cancer dormancy

Introduction: Metastatic breast cancer remains one of the leading causes of cancer-related deaths. Despite removal of the primary tumor, many patients experience a relapse event after a latency period – a phenomenon known as dormancy. This poses a challenging issue as current therapeutics are unable to target and eliminate these dormant cells. AXL is such a potential future target. AXL is overexpressed in many cancer types, and promotes drug resistance and cancer progression, however its role in dormancy is not yet known. In prior work, we developed a live cell lineage tracing method to study how the tumor microenvironment, namely the surrounding extracellular matrix composition, drives cellular dormancy in 3D. In this presentation I will use this live cell lineage approach to specifically study the role of AXL in driving breast cancer cell dormancy in engineered 3D tunable environments.

Materials and

Methods: Our modified induced-dormancy lab protocol subjects MDA-MB-231, MCF7, and HCC1954 cells to serum deprivation prior to imaging and mimicked ‘dormancy awakening’. Utilizing several 2D and 3D environments including protein-treated glass coverslips, Matrigel and collagen gel, we can quantify different subpopulations—including potentially dormant and senescent cells in dormancy-capable lines. Additionally, as an added validation we will be conducting serum-deprivation as part of our model to confidently quantify senescence; a necessary when quantifying cell divisions after serum-awakening. This improved model is used as a platform to study the effects of an AXL inhibitor, RXDX-106 on triple negative MDA-MB-231 breast cancer wild-type (WT) and AXL knockout (KO) cells. This is done by dosing the cells at 0, 50, 100, and 500nM on 2D gel environments, Matrigel and collagen. Afterwards, we proceed to in vivo tissue validations using a BALB/C nude mouse model and 3D gel analysis. Quantifying AXL expression in different subpopulations of this cell line will be done through multiplexed tissue staining.

Results, Conclusions, and Discussions: Preliminary 2D data suggested that inhibiting AXL resulted in an increase of dormant behavior in the AXL WT cells. When introducing a ‘dormancy awakening step’, the trend remained the same with dormant and senescent cell distributions increasing with greater drug concentration. More promisingly, the cell distribution data for the wild-type cells mimicked the trends of the AXL KO data when subjected to AXL inhibition through the media changes. This suggests that AXL inhibition and knockout negatively affect cell proliferation and growth (ERK/MAPK) even with the aid of a 2D microenvironment. To compare this with in vivo results, we intracardially injected the WT and AXL KO cells tagged with luciferase into BALB/C nude mice before imaging on the IVIS at time points 7, 14, 21, and 28. With the full use of and availability of AXL, the WT cells actively metastasized to the hindlegs and jaw by day 28; whereas the AXL KO cells showed no visible fluorescence. Preliminary optimization staining showed small pockets of AXL expression in metastatic bone. Further optimization over the summer is needed for the other immunofluorescent stains as well as an added senescent step to distinguish the dormant/senescent population which will be included with this work. Here, we propose that AXL is a driving force for senescent and dormant behavior and provides a novel dormancy target.

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