Live Cell Lineage Tracing of Dormant Cancer Cells

Abstract

Breast cancer is a leading cause of global cancer-related deaths, and metastasis is the overwhelming culprit of poor patient prognosis. The most nefarious aspect of metastasis is dormancy, a prolonged period between primary tumor resection and relapse. Current therapies are insufficient at killing dormant cells; thus, they can remain quiescent in the body for decades until eventually undergoing a phenotypic switch, resulting in metastases that are more adaptable and drug resistant. Unfortunately, dormancy has few in vitro models, largely because lab-derived cell lines are highly proliferative. Existing models address tumor dormancy, not cellular dormancy, because tracking individual cells is technically challenging. To combat this problem, a live cell lineage approach to find and track individual dormant cells, distinguishing them from proliferative and dying cells over multiple days, is adapted. This approach is applied across a range of different in vitro microenvironments. This approach reveals that the proportion of cells that exhibit long-term quiescence is regulated by both cell intrinsic and extrinsic factors, with the most dormant cells found in 3D collagen gels. This paper envisions that this approach will prove useful to biologists and bioengineers in the dormancy community to identify, quantify, and study dormant tumor cells.

Keywords: Matrigel; biomaterials; breast cancer; collagen; poly(ethylene glycol) hydrogels.

Fig. 1.. Experimental setups and timelines for 2D environments.

(a). Shows a cell lineage tree of HCC1954 cells on TCPS obtained by randomly tracking 30 individual cell proliferation across 50 hours. Each time a cell splits, it is denoted in the graph by splitting into two lines. Increasing on the y-axis shows least proliferating to most. If a cell doesn’t split, it will have a straight line across the entire graph. When encountering a dying cell, the time of death is marked with a red ‘x’. (b). Bar graph distribution of cell division and death from the lineage analysis. (c). This schematic consists of the seeding procedure and experimental timeline of two cell lines (MCF7 and HCC1954) in different media conditions on top of 2D Matrigel in a 96-well plate. (d). Schematic of a 24-well plate containing protein treated coverslip conditions with an included timeline. (e-g). Cell lineage analyses of HCC1954 in serum containing media (RPMI) across the coverslip conditions: (e). PBS-treated coverslip, (f). Collagen I protein treated coverslip, and (g). Collagen I peptide (GFOGER) treated coverslip. (h). Stacked bar graph for coverslip conditions from figures e-g.

Fig. 2.. Distribution of cell proliferation in 2D and 3D gel environments.

(a) The stacked bar graph showing proportion of cells with 0, 1, 2+ cell divisions or with cell death, which data was taken from the cell lineage analysis conducted on HCC1954 cells seeded on 2D collagen gel in regular RPMI. (b-c). Bar graph showing proportion of cells with 0, 1, 2+ cell divisions or with cell death for (b) HCC1954 and (c) MCF7 cells on 2D Matrigel. (d). Bar graph showing proportion of cells with 0, 1, 2+ cell divisions or with cell death for HCC1954 cells in 3D collagen gel. (e). Bar graph showing proportion of cells with 0, 1, 2+ cell divisions or with cell death for HCC1954 in 3D Matrigel. (f). Stacked bar graphs showing proportion of cell populations with 0, 1, 2+ cell divisions or with cell death for MCF7 in 3D Matrigel. (g). Stacked bar graphs showing proportion of cell populations with 0, 1, 2+ cell divisions or with cell death for MCF7 in 3D collagen gel.

Fig. 3.. Mouse breast cancer cells that have proliferative (D2A1) or dormant (D2.0R) phenotype in 3D Matrigel.

(a). Cell lineage trees of D2A1 (proliferative) and D2.0R (dormant) cells in serum containing and serum free conditions. Top row is showing D2A1 lineages, and bottom row is showing D2.0R lineages. Left column is the lineage trees of cells under serum containing condition, and right column is the lineage trees of cells under serum free condition. (b). A stacked bar graph showing proportion of cells with 0, 1, 2+ cell divisions or with cell death of D2 series cells, and the number of each group was calculated from the cell lineage trees shown in (a). (c). IF staining images of D2 series cells in serum-containing and serum-free conditions. Images on the far-left column are brightfield images, and the images from 2nd to 5th columns are IF staining images. Cells were stained with DAPI (blue), Ki-67 (red), ERK (yellow), and p38 (green). Scale bar = 50μm. (d). Quantification of fluorescent levels of Ki-67, ERK, and p38 in each condition. (e-f). Scattered plot of ERK and p38 expression levels in individual cells of (e) D2A1 and (f) D2.0R. Each dot indicates data from a single cell.

Fig. 4.. Triple negative breast cancer lines in PEG-RGD gels.

(a-b). Stacked bar graphs from the cell lineage analysis conducted on HCC1143 cells in (a) 3D Collagen and (b) 3D Matrigel. (c). Stacked bar graphs from cell lineage analysis of MDA-MB-231 on TCPS and in various 3D environments. Four columns on the left are with FBS, and three columns on the right are without FBS. (d) A stacked bar graph from the cell lineage analysis conducted on HCC1143 cells in a 3D PEG-RGD system with different serum and EGF conditions (e). Bar graph of the MDA-MB-231 cell line with its subclones 2-6E and 2-11F in 3D PEG-RGD gels. (f). IF-stained images of MDA-MB-231 parental and subclones in 3D PEG-RGD gels with FBS. DAPI is shown in blue, Ki-67 in red, ERK in yellow, p38 in green. Scale bar = 100μm.

Fig. 5.. Cell lineage tracing of D2 series cells in 3D collagen and Matrigel during serum recovery.

(a-d). Cell lineage analysis for (a) D2A1 in 3D collagen, (b) D2A1 in 3D Matrigel, (c) D2.0R in 3D collagen, and (d) D2.0R in 3D Matrigel. Imaging started from Time=0hr in the graphs, which was 6 hours after serum deprivation started. 48 hours after imaging cells under serum deprivation, we placed full serum media (10% FBS) and continued imaging for additional 48 hours. Blue horizontal lines indicate the cells showed proliferation during the 48-hour serum recovery window. (e). Stacked bar graphs showing the distribution of each cell population, such as dormant/reactivation, senescent, cell death, 1 cell division, and 2 or more cell divisions during 96-hour tracing.