Abstract B01: Autocrine fibronectin supports metastatic latency in the bone marrow extracellular matrix
Authors: Barney Lauren , Hall Christopher , Schwartz Alyssa , Peyton Shelly 
Published: March 2017

Introduction: Dormancy is most commonly observed in the bone marrow, which is a protective niche providing an extracellular matrix (ECM) and soluble factors to support disseminated tumor cell survival. We hypothesize that the ability of metastatic cells to survive dormancy, exit quiescence, and colonize a specific tissue depends upon the ability of the soil to first sustain survival, and subsequently trigger outgrowth. To this end, we created a biomaterial platform to investigate how the bone marrow matrix regulates entrance into dormancy. When inducing dormancy in vitro via mitogen withdrawal, we have found that both a capable seed and a hospitable soil are required for sustained quiescence, and that the tumor cells remodel the microenvironment to support their indefinite survival.

Results: A systematic screen of matrix protein type, density, and stiffness identified matrix composition as the most critical determinant of survival across several breast cancer cell lines. We expanded our screen to 23 breast cancer cell lines and three hospitable ECMs (TCPS, collagen I, and a bone marrow mimicking matrix) and identified 11 dormancy-competent cell lines that remain viable for at least 8 weeks on at least one ECM, and then grow when re-stimulated with serum. Survival most strongly depends on β1 integrin, and cell lines with a more varied integrin profile, even at lower expression levels, were capable of surviving on many matrices (e.g. HCC 1954). The surviving latent cells are ~90% Ki67-negative, upregulate p21, and surprisingly, a small population is still actively cycling. Dormant cells are thought to have high stress signaling, but interestingly, we only observe activation of JNK and p38 when cells are serum deprived on TCPS, and stress signaling is suppressed in latent cells on a biomaterial matrix. Surprisingly, basal p-ERK1/2 is unchanged, suggesting that sustained ERK signaling supports survival. Accordingly, within seven days, ERK inhibition decreases the number of surviving cells by greater than 70%.

We then hypothesized that this sustained ERK signaling is mediated by the matrix. More than half of the integrin genes are upregulated in our latent cells, and we also observe that cells secrete collagen I, fibronectin, laminin and vitronectin as a result of serum deprivation. Fibronectin was the most significantly upregulated matrix protein, and surviving cells secrete a more dense and organized matrix when cultured on a collagen or a bone marrow matrix. Even on a hospitable collagen I matrix, the fibronectin binding α5 integrin subunit was transcriptionally upregulated in latent cells and is expressed in focal adhesions, suggesting that long-term survival is mediated by α5β1 integrin binding to cell-secreted fibronectin. Finally, we characterized expression of 58 soluble factors, and identified a latency-induced secretome that includes factors that are known to induce dormancy (e.g. TGFβ2) and factors that may recruit stromal cells (e.g. SDFα). We have found that fibroblasts chemotax toward BT549 secreted factors, while mesenchymal stem cells chemotax toward HCC 1954 secreted factors, suggesting that the latent cells are actively recruiting their stroma.

Conclusions: We suggest that the matrix metastatic cells initially encounter determines short-term survival, but that long-term latency requires organized matrix production initiated by the tumor cells. When serum is reintroduced, cells return to basal levels of integrin expression and proliferation within seven days, and the recovered populations are not more invasive or drug resistant, indicating that this latent phenotype is reversible. This suggests that both a capable seed and a hospitable soil are required for metastatic latency, and emphasizes the phenotypic plasticity and dynamic changes in the microenvironment that can promote survival of disseminated tumor cells in the bone marrow.

Citation Format: Lauren Barney, Christopher Hall, Alyssa Schwartz, Shelly Peyton. Autocrine fibronectin supports metastatic latency in the bone marrow extracellular matrix. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B01.