P. Parmar1, B. Traub2, S. Shukla2, S. Kelly1, J. Condeelis2, M. Oktay2, D. Entenberg2 1Albert Einstein College Of Medicine, Department Of Surgery, Bronx, NY, USA 2Albert Einstein College Of Medicine, Department Of Pathology, Bronx, NY, USA
Introduction: While localized breast cancer has a 99% 5-year survival rate, the rate for distant metastasis plummets to 29%. This highlights limitations in current therapies and a critical gap in our understanding of the metastatic process. Cancer dissemination is complex, and while intravasation is well-studied, extravasation is less understood. Prior studies have identified key time points of tumor cell extravasation in the lung. In this work, we aimed to determine the gene expression profile of tumor cells as they undergo extravasation.
Methods: Experimental metastases were generated by intravascularly injecting a fluorescently labeled mouse breast cancer cell line, E0771-GFP, into C57BL/6 mice. Time points post-injection previously shown to correspond to pre-extravasation, peri-extravasation, and post-extravasation were used to harvest lungs, which were then digested into single-cell suspensions and flow-sorted for GFP+ tumor cells and sent for bulk RNA sequencing. Differential gene expression analysis between extravasation time points was performed in Python using PyDESeq2. Pathway and GO term enrichment were carried out in R with clusterProfiler, focusing on genes with an FDR >0.01 and adjusted p-value <0.05.
Results: We identified 923 genes differentially expressed between pre-extravasation and peri-extravasation, and 1,221 genes between pre-extravasation and post-extravasation. Pathway analysis showed upregulation of pathways related to cell migration, cytoskeleton reorganization, adhesion, and chemotaxis during peri-extravasation. During post-extravasation, the same pathways were upregulated, along with additional pathways related to monocyte, macrophage, and endothelial cell interactions (Figure 1a, b). Downregulated genes during both peri-extravasation and post-extravasation were predominantly associated with cell cycle progression. Upregulated genes during peri-extravasation included Csf1, Slpi, Tgm2, Adm, Phlda1, Amoti1, Bcl3, Itprip, and Nav2. Upregulated genes during post-extravasation included Csf1, Slpi, Wdr92, Serpine1, 1500012F01Rik, Pvt, 1110038B12Rik, Socs3, Snhg12, Gfpt2, Cxcl2, Chac1, and Tnfaip3 (Figure 1c, d).
Conclusion: This study provides the initial identification of new additional players driving extravasation of tumor cells. Our data indicate that, upon extravasation, disseminated tumor cells reprogram for adhesion, migration, and survival while cell cycle genes are repressed. After extravasation, DTCs begin to interact with macrophages and endothelial cells in the microenvironment. Future work to validate and test the impact of altering these genes on extravasation is necessary to develop targeted therapies to inhibit metastasis and improve patient outcomes.