Poster S4 - Unlocking the molecular code of cancer dormancy through 3D engineered models

Introduction: Cancer recurrence involves the activation of dormant cancer cells, which remain quiescent until they initiate secondary tumors. Understanding the molecular mechanisms governing dormancy is crucial for preventing recurrence and metastasis. In this study, we investigate the regulatory role of GAS6 in triple-negative breast cancer (TNBC) dormancy using spheroids and microtissues.

Materials and

Methods: To identify dormant cancer cells we are using a construct called mVenus-p27 that fluoresces when cells are in the G0 phase of the cell cycle. All experiments were performed using spheroids or/and microtissues (cancer cells embedded in collagen that are fabricated via microfluidic devices). Cell cycle was performed using FxCycle™ PI/RNase Staining Solution.

Results, Conclusions, and Discussions: Our initial investigation revealed an increase in GAS6 expression in Hs578T cells subjected to serum starvation, coinciding with a greater presence of dormant cells identified through the mVenus-p27 construct, indicating G0 phase arrest (Figure 1). This prompted us to explore whether this upregulation of GAS6 was merely a consequence of cells entering a quiescent state or if GAS6 played a regulatory role in this process. To address this question, we supplemented Hs578T spheroids with exogenous GAS6 and observed a corresponding increase in p27+ cells, suggesting that GAS6 might indeed induce dormancy in these cells in vitro (Figure 2). Notably, we also found that blocking the GAS6 receptor AXL reduced the number of p27-positive cells, indicating a potential regulatory role for AXL in this axis (Figure 2). To further confirm GAS6's involvement in inducing cancer dormancy, we performed cell cycle assays, which revealed an accumulation of cells in the G0/G1 phases following the addition of recombinant GAS6 protein (Figure 3). Considering the metastatic tendencies of breast cancer, particularly in the bone marrow, we investigated the interaction between HS-5 bone marrow stromal cells (known for their high levels of GAS6 expression) and Hs578T cells. Co-culture experiments demonstrated an increase in dormant cells (mVenus-p27+) which was significantly reduced upon prior blockade of AXL or MERTK receptors (Figure 4). Additionally, conditioned media from HS-5 cells modified to not express GAS6 failed to induce cancer dormancy in Hs578T cells (Figure 5), underscoring the crucial role of GAS6 secretion by HS-5 cells in inducing dormancy. Currently, we are also investigating the role of another cellular player in the bone marrow capable of producing large quantities of GAS6: plasma cells (Figure 6). Through single-cell RNA-seq data and analyses from the GEO database, we hypothesize that these activated B cells may be responsible not only for antibody production but possibly also for inducing cancer dormancy in the bone marrow. Similarly, we will co-culture these cells with Hs578T tumor cells, as well as use conditioned media from them to test our hypothesis. We are also transitioning several of these experiments with spheroids to microtissues (Figure 7).

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