Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape.

Phenotypic heterogeneity exists within collectively invading packs of tumor cells, suggesting that cellular subtypes cooperate to drive invasion and metastasis. Here, we take a chemical biology approach to probe cell:cell cooperation within the collective invasion pack. These data reveal metabolic heterogeneity within invasive chains, in which leader cells preferentially utilize mitochondrial respiration and trailing follower cells rely on elevated glucose uptake. We define a pyruvate dehydrogenase (PDH) dependency in leader cells that can be therapeutically exploited with the mitochondria-targeting compound alexidine dihydrochloride. In contrast, follower cells highly express glucose transporter 1 (GLUT1), which sustains an elevated level of glucose uptake required to maintain proliferation. Co-targeting of both leader and follower cells with PDH and GLUT1 inhibitors, respectively, inhibits cell growth and collective invasion. Taken together, our work reveals metabolic heterogeneity within the lung cancer collective invasion pack and provides rationale for co-targeting PDH and GLUT1 to inhibit collective invasion.

Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet remains largely underexplored in invasion and metastasis. Here, we investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and revealed the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and three-dimensional (3D) collective invasion. We further identified JAG1 signaling as a previously unknown upstream activator of MYO10 expression in leader cells. Using live-cell imaging, we found that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion exclusively in leaders. Our data fit a model where epigenetic heterogeneity and JAG1 signaling jointly drive collective cancer invasion through MYO10 up-regulation in epigenetically permissive leader cells, which induces filopodia dynamics necessary for linearized fibronectin micropatterning.

SUMMARYUnlike the adult mammalian retina, Müller glia (MG) in the adult zebrafish retina are able to dedifferentiate into a ‘‘stem cell’’-like state and give rise to multipotent progenitor cells upon retinal damage. We show that miR-216a is downregulated in MG after constant intense light lesioning and that miR-216a suppression is necessary and sufficient for MG dedifferentiation and proliferation during retina regeneration. miR-216a targets the H3K79 methyltransferase Dot1l, which is upregulated in proliferating MG after retinal damage. Loss-of-function experiments show that Dot1l is necessary for MG reprogramming and mediates MG proliferation downstream of miR-216a. We further demonstrate that miR-216a and Dot1l regulate MG-mediated retina regeneration through canonical Wnt signaling. This article reports a regulatory mechanism upstream of Wnt signaling during retina regeneration and provides potential targets for enhancing regeneration in the adult mammalian retina.

Myosin-X (MYO10) is a noncanonical myosin that drives filopodia formation and extension, and is necessary for invasion and metastasis of breast cancer, prostate cancer and melanoma. It is not yet known by what mechanism MYO10 increases cancer cell invasion and metastasis. Two independently published MYO10 knockout mouse models showed severe developmental defects dependent upon collective migration of several cell types, suggesting that MYO10 may also regulate collective invasion of cancer cells. In order to study collective cancer cell invasion, our lab previously developed a technique to isolate and culture purified highly-invasive leader cells and highly-proliferative follower cells from collectively invading lung cancer cell lines. Using this method, we demonstrated distinct gene expression patterns, DNA methylation patterns, and phenotypic properties of leader versus follower cells. MYO10 is highly overexpressed in leader cells but is not expressed in follower cells, and MYO10 overexpression in leaders correlates with highly significant CpG island promoter DNA hypomethylation and gene body DNA hypermethylation. We hypothesize that MYO10 regulates cancer cell collective invasion by driving the formation of long filopodia necessary for leading-edge fibronectin patterning and subsequent leader cell invasion. MYO10 localizes at leader cell filopodia tips but not in follower cell filopodia within both 2D culture and 3D invading spheroids of four non-small cell lung cancer cell lines. MYO10 knockdown in purified leader cells and in these four lung cancer cell lines decreases the number and length of filopodia, 2D cell motility and 3D spheroid invasion; in contrast, MYO10 overexpression in follower cells increases filopodia length, cell motility and spheroid invasion. In addition, proteomic analysis shows that leader cells produce and secrete fibronectin (FN1), unlike follower cells. Immunofluorescence within invading spheroids shows the formation of long FN1 fibrils, i.e. elongated parallel bundles of FN1 that extend far past the leader cell body in multiple cell lines. FN1 fibrils preferentially localized with MYO10+ filopodia. Knockdown of MYO10 disrupts the formation of these fibronectin fibrils. We previously reported that knockdown of fibronectin completely abrogates lung cancer spheroid collective invasion. Therefore, our data suggest that MYO10 regulates cancer cell collective invasion by driving the formation of long filopodia that then regulate fibronectin architecture and subsequent invasion by leader cells. Note: This abstract was not presented at the meeting. Citation Format: Emily R. Summerbell, Jessica Konen, Jeanne Kowalski, Paula Vertino, Adam Marcus. MYO10 aberrant methylation and overexpression in leader cells regulates lung cancer collective cell invasion and fibronectin patterning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 178.