by Will Doss, Northwestern University
Fig. 1: Single-cell RNA sequencing profiles comparing breast cancer cells from primary and metastatic tumor sites. a A schematic showing the single-cell RNA sequencing of the sorted cells from L2T- or L2G-labeled TNBC PDX (mice 1 and 2) or MDA-MB-231 tumor (mouse 3)-bearing mice, both primary breast tumors and lung metastases (early micrometastases). b Heatmap denoting expression magnitude estimates in log scale (red color) for ICAM1 and co-expressed stemness signature genes in primary tumor cells and lung metastases (N = 3 mice, 2 with TN PDXs and 1 with MDA-MB-231 tumor). Genes are sorted according to their correlation with ICAM1 across all cells, as denoted by the gray bars on the right (top highest to bottom lowest). The bottom list of CD44, GAPDH, ACTB, eGFP, and tdTomato serve as control genes without significant changes between primary tumor cells and lung metastases. c Representative ICAM1 expression in L2T+ or L2G+ primary tumor cells and lung metastases determined by flow cytometry from different breast cancer PDX models (TN1, TN2, and TN3). Flow profile gates are shown in Supplementary Fig. 1d. d Quantitative data of the differential ICAM1 expression in PDX primary tumors versus lung metastasis from c. n = 3 biological replicates (mice) for each model. One-sided t test *P = 0.04; **P = 0.01; *P = 0.02. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). e Representative IHC staining images of ICAM1 expression (brown color) in primary tumors and lung metastases from PDX TN1 and TN2 models validating c and d. f Distribution of ICAM1 expression across PAM50 subtypes in the TCGA BRCA cohort (N = 1037). Basal-like and HER2-enriched subtypes are the top two exhibiting significantly higher ICAM1 expression as compared to normal breast tissue (percentage of cases above the blue line value are shown for each subtype). Statistical significance was assessed using a two-sided Student’s t test. *P < 0.05, **P < 0.01, and ****P ≤ 0.0001. g Schematic and flow histogram analyses of the orthotopically implanted TN1-PDX tumors with or without ICAM1 overexpression (OE) at the 4th mammary fat pads. h. PDX TN1 tumor weights 2 months after orthotopic injections of TN1 cells with ICAM1 OE and control vector (Con) (2.5e5 cells into one mammary fat pad/mouse). n = 3 mice per cell group. Two-sided t test *P = 0.04. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). i, j Representative lung images and normalized BLI signals (total flux) of spontaneous lung metastases in orthotopically implanted ICAM1 OE and control TN1 tumor-bearing mice as in g, h. n = 3 mice per group. Two-sided t test **P = 0.003. The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). k Schematic and flow histogram analyses of tail vein injected MDA-MB-231 tumor cells transfectd with siRNA control (siCon) and siICAM1. l, m. Representative images and quantitative data of BLI signal of mice injected with siCon and siICAM1-transfected MDA-MB-231 cells via tail vein (n = 4 mice per cell group. Two-sided t test **P = 0.01 for time point comparisons. N = 6 independent experiments). The boxes range from the first to third quartile with x in a box indicating mean value and whisker lines extending to outliers (minimum and maximum). Credit: DOI: 10.1038/s41467-021-25189-z
Circulating tumor cells (CTCs) with stem cell features use the adhesive protein ICAM1 to facilitate formation of CTC clusters, which can travel from primary tumors to other organs in the body, according to a Northwestern Medicine study published in Nature Communications.
These CTC clusters are a major source of metastases in breast cancer, so inhibiting tumor cell ICAM1 has the potential to stop tumor cell seeding and block cancer progression, according to Huiping Liu, MD, Ph.D., associate professor of Pharmacology and senior author of the study.
“An antibody to neutralize the function of ICAM1 could reduce social networking of tumor cells during circulation and migration, and improve outcomes for patients with breast cancer,” said Liu, who is also a professor of Medicine in the Division of Hematology and Oncology and a member of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.
Compared to cancers with single CTCs, cancers with CTC clusters are associated with decreased survival in breast cancer and are 20 to 100 times more efficient at developing metastases, or additional tumors outside the breast. Breast cancer metastases are present in 90 percent of breast cancer deaths, according to Liu.
“When the disease starts to spread into distant organs, it is almost always incurable,” Liu said.
The seeds of these metastases are multicellular CTC clusters. These clusters, numbering two or three cells, draw strength from numbers while traveling to other organs in the body.
In the current study, Liu and her collaborators used patient-derived cancer cells to study cluster formation, and found they require the protein ICAM1—not only for the initial clustering but also for moving through blood vessel walls as they travel along the bloodstream.
“They present ICAM1 on the cell surface to recognize each other and ‘hold hands,'” Liu said. “It also helps them penetrate the endothelial layer outside of blood vessels.”
Furthermore, cancer cells that are clustered together act like stem cells, more effectively creating offspring that can eventually form a tumor. Depleting ICAM1 from CTCs reduced migration, inhibited formation of distant tumors and interfered with their ability to get in and out of blood vessels.
In the future, an antibody that binds to ICAM1 and reduces its function could be used in breast cancer patients with high levels of ICAM1 or CTC clusters, slowing metastases and hopefully prolonging survival.
“Using ICAM1 as a biomarker, I think these patients would be excellent targets for an antibody treatment,” Liu said. “This could be especially valuable in immunocompromised patients.”
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