Abstract PR004: In vivo tracking of clonal dynamics during UV-induced skin carcinogenesis

Abstract

Abstract Background: The impact of chronic UV exposure on clonal dynamics and genomic diversity remains unclear. Our central hypothesis is that skin cancer is induced not by solely accumulation of somatic mutations, but rather a combination of mutations and disruption of the spatial and temporal constraints imposed by the skin’s 3-D architecture. Here we characterize clonal dynamics and transcriptional signatures during skin carcinogenesis using multicolor lineage tracing. Methods: We generated a K14Cre-ERT2 Confetti mice with inducible fluorophore (flr) expression. Mice were UV-irradiated for 3 months. Clones were 3-D digitized using confocal microscopy (z-stacks) and clone volumes estimated computationally. scRNAseq was used to compare UV-exposed (EXP) vs. non-exposed (NON) epidermis vs. skin tumors. Results: We generated 914 serial images of the EXP/NON skin over the course of 6 months following initiation of UV. We analyzed 16,135 clones from the EXP and 21,506 clones from the NON skin. We classified clone sizes into 3 classes represented by the small (<50,000 μm3), medium (50,000-500,000 μm3), and large “goliath” (> 500,000 μm3). The median size of clones does not differ between UV treatments and does not change with time. However, clones from EXP samples have significantly greater mean size than NON ones. Their mean sizes differed by some 1.5-fold, with an over 6-fold increase in variance, resulting in the sizes distribution to be highly skewed towards large clones with a long, narrow tail. Goliath clones are rarely present in the NON skin; however, they increase in number dramatically by months 3-4, plateauing between months 5-6. Using 3 ecological metrics (clone size, clone numbers, and coefficient of variation) we see phase shifts, which primarily distinguish months 1 & 2 from months 3 & 4. scRNAseq of EXP/NON epidermis and tumors revealed differential representation of 16 clusters, the majority of which could be mapped to previously defined keratinocyte populations. We observe dynamic changes to these clusters when progressing from normal skin to chronically exposed skin, and then to tumors. EXP clusters were associated with expression of cystatins (Scfa 3, BC100530), and alarmins/proliferative keratins (Krt16, Krt6a), which have been associated with skin injury. Clusters expressing cystatins and alarmins also increased in tumors. Flr-expressing keratinocytes harvested from large clones in EXP epidermis exhibited altered keratinocyte differentiation (downregulation of Krt77, Loricrin and Nfkbia, upregulation of cystatin), inflammation (downregulation of Nfkbia), and upregulation of metabolic regulators (carbonic anhydrase II and retinol transport (Rbp1)). Genes differentially expressed in exposed skin and retained in tumors may be required for carcinogenesis, while those expressed only in exposed skin likely required for adaptive responses to UV. Our findings have important implications for understanding cancer through an eco-evolutionary framework and designing novel approaches to cancer prevention. Citation Format: Stanislav Avdieiev, Leticia Tordesillas, Omar Chavez Chiang, Zhihua Chen, Luiza Silva Simoes, Y. Ann Chen, Noemi Andor, Robert Gatenby, Elsa R. Flores, Joel S. Brown, Kenneth Y. Tsai. In vivo tracking of clonal dynamics during UV-induced skin carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR004.