Tumor heterogeneity, in cell behavior, cell microenvironment and gene expression, has become a hallmark of
tumor progression, and provides an important challenge in understanding cancer. For example, the adoption of
an invasive phenotype by some tumor cells is a key step as carcinomas advance from the benign tumor state.
Migration of tumor cells from the epithelial layers of the epidermis to the dermis provides tumor cell access to
the vasculature, offering a direct path for distal metastasis. Understanding how and why some cells are capable
of invading surrounding tissues is key to understanding tumor invasion, but current in vivo models of invasion do
not readily allow specific invasive cells to be recovered and studied to elucidate drivers of tumor invasion and
Our goal is to create a new pipeline for following tumorigenesis, permitting direct studies of tumor cell
diversification and invasion. This experimental pipeline is designed to offer capabilities to:
(i) track tumorigenesis longitudinally over time from the first origins of a tumor to key events such as invasion;
(ii) label and selectively purify tumor cell subsets identified in these time-course studies;
(iii) characterize individual tumor cells in isolated populations in unbiased way.
This approach would allow identification of the cellular events and developmental trajectories that accompany
tumor growth and invasiveness. Molecular correlates characterized in the key sub-fractions of the tumors can
be directly related to the event history of the cell sub-fractions, and the importance of these cellular and molecular
events can be tested through perturbation, ablation and function blocking experiments. Such a functional and
longitudinal experimental pipeline is key to creating more targeted and informative drug screening systems.
We have developed a set of transgenic zebrafish lines and imaging tools that allow repeated, non-invasive
visualization of endogenous tumor development in adult animals. This permits tumors to be tracked from their
origins, over long periods, after oncogene expression in skin epithelial cells. Our preliminary results show that
heterogeneity is present from the earliest stages of tumor formation, as only a small fraction of cells induced to
express oncogenes go on to form invasive tumors; such results provide the motivation for and the foundation for
our analyses of the emergence and elaboration of tumor heterogeneities.
Here, we propose to employ imaging and labeling of selected cell populations, in combination with single-cell
RNA-sequencing analyses, to study the cell behaviors and differentiation routes key to invasive tumor
development. We will establish an experimental pipeline by investigating: the cell-types specific to invasive
tumors (Aim 1); the key differentiation pathways leading to an invasive phenotype (Aim 2); and leveraging these
insights by functional assays (Aim 3). This pipeline will offer new insights into invasive tumor development,
supporting larger studies and the design of anti-tumor therapeutics targeting key cell subsets.