An <em>In Vitro</em> Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

Abstract

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of>30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

Therapeutically targeting tumor microenvironment–mediated drug resistance in estrogen receptor–positive breast cancer

“…Applying this filter highlighted FGF2 as the top validated cytokine that conferred resistance to both anti-estrogens and PI3K–mTOR pathway inhibitors and is highly expressed in ER+ breast cancer–relevant tissues and TME cell types. Indeed, several lines of evidence indicate that FGF2 is expressed in the ER+ breast TME: (a) FGF2 was observed in stroma in 34/54 (63%) breast tumors ( Linder et al, 1998 ); (b) a broad range of FGF2 levels were detected in stroma, but not breast epithelial cells, in 149/149 (100%) breast tumors ( Smith et al, 1999 ); and (c) FGF2 is robustly expressed by bone marrow stromal cells ( Tivari et al, 2015 ), and bone is the most common site of metastasis for ER+ breast cancer. FGF2 IHC of tumors from four breast cancer patients confirmed FGF2 expression in stromal cells, including adipocytes, endothelial cells, and fibroblasts, but not cancer cells (Fig.…”

Reawakening of dormant estrogen-dependent human breast cancer cells by bone marrow stroma secretory senescence

“…MCF-7 cells were incubated at clonogenic density with 1000 or 1500 cells/well in quadruplicate on human fibronectin coated 24 well plates in 1 ml DMEM/10% FCS and allowed to adhere overnight [ 34 , 46 ]. The next day, wells were supplemented with 100 μl of 100 ng/ml of FGF-2 for a final concentration of 10 ng/ml with or without 10 ng/ml recombinant human IL-6, IL-8 or TGFβ1.…”

In vitro Models of Breast Cancer Metastatic Dormancy

“…BCCs that are plated onto fibronectin-coated plates undergo quiescence in presence of FGF2 and activation of integrin α5β1, PI3K and ERK pathways. These cells express partial EMT markers and can re-enter proliferation upon treatment with IL6/8 and TGFβ) (Korah et al, 2004;Najmi et al, 2005;Barrios and Wieder, 2009;Tivari et al, 2015).…”

“…To distinguish between quiescence and senescence (or even apoptosis), cells must re-enter the proliferative state upon withdrawal of the factors used to trigger dormancy or upon treatment with signals able to drive exit from dormancy. Examples of such signals for BCCs are inflammation (LPS, smoke) (Cock et al, 2016;Albrengues et al, 2018), POSTN , TGFβ1 (Ghajar et al, 2013), RTKs (Tivari et al, 2015;, IL6, Collagen I (Barkan et al, 2010), Src (Barkan et al, 2010;, SFRP2 , IKKβ (Lamiaa et al, 2017), integrins activation (as discussed below); while examples of inhibitors are: TSP1 (Ghajar et al, 2013), p38 (Marlow et al, 2013), Alk5 (Marlow et al, 2013), BMP2 (Gao et al, 2012), TGFβ2 (Bragado et al, 2013), MSK1 (Gawrzak et al, 2018), IFN-β (Lan et al, 2019).…”