Tumor cells transmigrated to the bottom were measured 24?h after seeding. we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by?200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC. test *test. *test *test **test **stood out with a 60- to 200-fold increase in the lung metastases Mouse monoclonal to KSHV ORF45 (Supplementary Fig.?1aCc). This gene encodes a cell surface protein, intercellular adhesion molecule 1 (ICAM1), also known as CD54, which is involved in vascular adhesion30,31. Strikingly, we found that expression marked a subset cells of lung metastases in the PDXs with concurrent gene expression patterns related to metastasis, tumor initiation, and stem cell functions, such as (Fig.?1b), suggesting may contribute to metastasis initiation of TNBC. Using flow cytometry analyses and IHC staining, we first confirmed the highly enriched ICAM1 expression (17C99%) in the lung metastases of three TNBC PDX models (TN1, TN2, and TN3) compared to that of primary tumor cells (0.5C36%, test ****test *test ***test ****vector, named M1-M5, including M1 with truncated DZ2002 domain name II (112-211), M2 with truncated domain name IV (310-394), M3 with predicted homodimer-disruptive mutations R287D/N296E/Q297A in domain name III, M4 with combined mutations (M1-M2-M3), and M5 with mutated binding sites for LFA1 and MAC1, E34A in domain name I and D290H/Q291L/R292E in domain name III, respectively (Fig.?2l top panels). We found that all of the ICAM1-Myc mutants lost their homophilic interactions with ICAM1-Flag in the co-IP assays (Fig.?2l bottom panels), suggesting that multiple regions of ICAM1 are involved in the self-dimerization. ICAM1-regulated pathways and targets Next, we examined the role of ICAM1-induced molecular alterations of breast cancer cells in metastasis. We employed RNA sequencing and mass spectrometry for transcriptome and proteome analyses of MDA-MB-231 cells upon siICAM1-mediated knockdown (Fig.?3a; Supplementary Fig.?6aCb; and Supplementary Tables?2 and 3). Multiple pathways were down-regulated in siICAM1-transfected cells, including stemness, the cell cycle, hypoxia and HIF-1 targets, microtubule-based processes, plasma membrane cell projection assembly, telomere maintenance, and cell survival (Fig.?3a and Supplementary Fig.?6bCd). Other pathways involved in histone modification, autophagy, and mammary differentiation were up-regulated in siICAM1-transfected cells (Fig.?3b and Supplementary Fig.?6eCh). These data suggest that ICAM1 signaling enhances cancer stemness and cell-cycle progression, and suppresses epithelial differentiation. Using immunoblotting analyses of cell lysates, we confirmed that ICAM1 knockdown not only reduced protein levels of the top targets related to stemness and cell-cycle regulation, such as CDK6, OCT3/4, NOTCH1, MCM3, ZEB1, Sec23a, and HIF1A; but also DZ2002 up-regulated proteins related to epithelial differentiation and stress signaling, such as KRT19, PAI1, and HMGA2 (Fig.?3c and Supplementary Fig.?6c, f). Open in a separate window Fig. 3 Downstream targets of ICAM1 in regulating metastasis.a Down-regulated pathways upon knockdown in MDA-MB-231 cells, analyzed by RNA sequencing (top) and mass spectrometry analysis (bottom). b GSEA of the gene sets for histone deacetylase targets, H3K27ME3, and EZH2 targets enriched among the up-regulated genes in MDA-MB-231 knockdown cells in comparison to siRNA control, identified by RNA sequencing. c Immunoblots of ICAM1 and CDK6 in MDA-MB-231 cells transfected with control siRNAs (siCon) and siICAM1 for gene knockdown. test **test *test *with mRNA levels (Fig.?3k and Supplementary Fig.?7k). We also compared the effects of knocking down ICAM 1 and CDK6 on early metastatic seeding and long-term growth of lung metastasis. At 6C8?h after tail vein injection, siICAM1-transfected cells showed decreased seeding to the lungs, whereas si-CDK6 transfected cells had comparable efficiency of seeding as the control cells (Supplementary Fig.?8a, b), suggesting ICAM1-mediated metastatic seeding is cell-cycle DZ2002 independent. Nevertheless, the long-term.