Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis

Abstract

Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.

Novel therapeutic targets for cancer metastasis

“…Recent advances in the development of human cancer-animal models and improved intravital visualization techniques have led to an explosion of interest toward these previously hard-tovisualize, late-metastasis steps [7,8,10,31,[51][52][53][54]. Conventional imaging technologies used for detection, diagnosis, triaging and prognosis of organ cancers and tumor metastasis include ultrasound (US) and computed tomography (CT) [55,56].…”

“…Intravital imaging allows for visualization of the primary location, proliferation and volume of a tumor, and measurement of the level of cancer cell motility in vivo and in real-time in live animal models [60]. This is essential to study the physics and mechanisms of tumor growth, screen for metastasis-blocking targets, detect and diagnose the cancer early and treat metastatic disease with metastasis-blocking therapeutic agents [3,7,21]. The shell-less avian embryo chorioallantoic membrane (CAM) assay technique is a very useful nanoscale tissue model for noninvasive, real-time, intravital imaging of tumor growth.…”

“…These fully vascularized and leaky tumors proliferate within 7 days on the membrane laterally and shallowly, to an average depth of 200 nm [56]. Tumor cell motility can be visualized via high-resolution confocal microscopy images and videos using differential macromolecular stains that also label the tumor vasculature from within and outside of the tumor [7,8,31,56].…”

“…The identification of genes required for in vivo cell motility has been impeded by the inherent difficulty in visualizing the formation of metastatic lesions in vivo. Recent advances in cellular and nanomolecular imaging and modification technologies, the identity and functionality of host and cancer cell-derived genes, proteins and regulatory species paramount to the metastatic cascade have begun to be uncovered [6][7][8][9][10][11][12].…”

Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen

“…Genome-wide screens employing activating or repressive dCas9 constructs and sgRNA libraries were deployed to identify toxin or drug resistance and vulnerability-conferring genes [9][10][11][12][13][14], genes that mediate cell immune and inflammatory responses [15][16][17][18], and genes required for cell survival, proliferation, and metastasis [10,[19][20][21][22]. The majority of screens performed to date phenotyped cell enrichment or depletion, though some studies measured the rates of differentiation [23], cell motility [24], and the abundance of fluorescent protein [25]. In all cases, the quantified phenotypes were individual-cell attribute.…”