Live cell reporters of genetic changes in stiff vs soft surroundings - Causes & Consequences

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Project Summary/Abstract Aneuploidy is defined as the existence of an abnormal number of chromosomes in a cell, and it has long been a defining characteristic of cancers. 90% of all solid tumors, for example, show chromosomal gains/losses. Gaining and/or losing chromosomes generates phenotypic heterogeneity within tumors that can be beneficial to survival by creating subpopulations that may become resistant to therapeutics, providing new mechanisms to evade immune cells, facilitating growth and proliferation in challenging microenvironments, among other advantages. However, we currently struggle to easily identify aneuploid cells—typically we need to kill them for sequencing, meaning that we can only at best infer possible causes, consequences, and possible long-term viability and evolution. This also limits our ability to identify how aneuploid cells interact with other cells and study if and how these interactions differ from other “less” aneuploid cells. These interactions become increasingly important when the engaging cell is from the immune repertoire. In this proposed research, we tackle two aspects of aneuploidy: (1) providing a simple, effective method to easily identify aneuploidy in live cells and (2) understanding how aneuploidy can modulate macrophage- mediated phagocytosis of cancer cells. We already have successfully generated several chromosome reporters, in which we fuse fluorescent proteins to only single alleles of constitutively expressed genes. For these appropriate genes, we find that fluorescence loss equates to chromosomal loss—thoroughly characterized from genomic PCR to deep single-cell sequencing. We further seek to expand this toolkit to make it accessible to more appropriate mouse models. On the immunobiology end, we already find that high levels of aneuploid help promote an initial macrophage-immune response. This suggests that initial stages of aneuploidy (before a cancer can adapt and evolve to tolerate it) are susceptible to macrophage clearance. Although seemingly two distinct ideas, we finally plan to merge our reporter approach in analyzing how macrophages are phenotypically molded by aneuploid cells that we can easily identify. Do macrophages eat/clear these reporter-negative aneuploid cells? Are reporter-negative cells simply more susceptible to macrophages or are profound paracrine signaling pathways involved? We will extend our studies to both in vitro and in vivo models to better characterize macrophage response under these circumstances, and we will also ultimately combine these studies with powerful single-cell sequencing to see how aneuploidy sculpts macrophage behavior and their overall effect on the immune landscape.