Tang, Cha-Mei

Abstract Circulating tumor cells (CTCs) are cancer cells that detach from tumors, enter the blood and seed distant sites. CTCs spread as single cells, or cluster aggregates, and can be heterogenous with differing biological phenotypes, i.e. epithelial, mesenchymal, progenitor, & stem cell. Three clinically relevant CTC subtypes are often described in patients (pts) 1) CTCs with normal epithelial traits (strong filamentous cytokeratin (CK+ & CD45-/CD14-), 2) Epithelial-mesenchymal CTCs (EMTs), cancer cells with downregulated epithelial & upregulated mesenchymal traits (low diffused CK+, CD45-/CD14-) and 3) tumor-macrophage fusion cells (TMFCs), cancer cells that fuse with macrophages (polynucleated DAPI+, diffuse CK+ and CD14+/CD45+). To better elucidate the biological and clinical importance of the 3 CTC subtypes, we isolated CTCs by a marker agnostic method from the blood of n=49 metastatic cancer pts. We then enumerated and phenotypically subtyped CTCs comparing clinical utility of clustering and CTC heterogeneity over 2 years. Peripheral blood samples were collected from 49 metastatic cancer pts, breast (n=38), pancreatic (n=9), colon (n=3), lung (n=1), and prostate (n=1). Whole blood (7.5mL) was filtered by size exclusion using CellSieve microfilters and fluorescently stained for CD45, CD14, CK, and DAPI to identify CTCs, EMTs and TMFCs. Cells were imaged, enumerated and fluors quenched by the QUAS-R technique; followed by re-stained for CD44, CD24, CD133, CD144, N-Cadherin, EpCAM, and Vimentin. Markers were categorized as negative, low, medium, or high. A heterogeneity biomarker score was developed as high, 2-3 log deviations between marker expression, or low 0-1. Pt progressive free survival (PFS) and overall survival (OS) were analyzed over 2 years. CTCs were found in 40% of pts, EMTs in 51%, & TMFCs in ∼93%. For subtyping, CD44 was in CTCs 88%, EMTs 59%, & TMFCs 91%; CD24 in CTCs 40%, EMTs 41%, & TMFCs 6%; CD133 in CTCs 100%, EMTs 8%, & TMFCs 100%; CD144 in CTCs 100%, EMTs 77%, & TMFCs 82%; N-cadherin in CTCs 100%, EMTs 0%, & TMFCs 45%; Vimentin in CTCs 51%, EMTs 100%, & TMFCs 83%; EpCAM in CTCs 74%, EMTs 12%, & TMFCs 20%. CTCs and EMTs were found to have high heterogeneity in 30% of samples in both individual cells and clusters, with a high biomarker heterogeneity significantly correlated with poorer outcomes (PFS: HR=15.4, p=0.0014, CI=3.4-68.9; OS: HR=12.4, p=0.0056, CI=2.6-59.1). TMFCs were found to have high heterogeneity, 40% of samples, but this was not correlated with outcomes. In this pilot study, it appeared that isolated CTC, EMTs and TMFCs subtypes have differing heterogenous expressions of epithelial, mesenchymal, progenitor, and stem cell markers. Further, high rates biomarker heterogeneity in CTCs and EMTs, but not TMFCs, correlated with faster rates of progression & death. Larger validation experiments with biomarker stratification is ongoing. Citation Format: Alexis B. Duffy, Massimo Cristofanilli, Carolina Reduzzi, Cha-Mei Tang, Saranya Chumsri, Susan Tsai, Mohammed Aldakkak, Daniel L. Adams. Heterogeneity of epithelial, mesenchymal, progenitor, and stem cell phenotypes on CTCs correlates to clinical outcomes in metastatic cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1989.

Abstract Tumor associated macrophages are known to fuse with cancer cells resulting in the formation of a cellular phenomenon called tumor-macrophage fusion cells (TMFCs). TMFCs are a highly aggressive cell subtype that may move into circulation, evade immune surveillance, and possibly aid in seeding of metastatic sites. The formation of TMFCs involves CD47, a ubiquitous surface receptor that acts as a “don’t eat me” signal, interacting with the macrophage protein SIRPa, a down regulator of phagocytosis, and syncytin-1, a mediator of hyperploidy cancer fusion. In this analysis, we isolated circulating TMFCs from the blood of n=39 metastatic cancer patients (pts) to evaluate the expressions of CD47, SIRPa and syncytin-1 as it relates to stable or progressive disease via 30 day imaging by PET scan. We conducted a prospective pilot study of n=39 pts with pathologically confirmed metastatic breast (n=17), prostate (n=7), pancreas (n=7), or colon (n=8) cancer receiving standard of care systemic therapy. We isolated TMFCs from 7.5ml peripheral blood using a CellSieve microfilter, and stained with CD47, SIRPa, & syncytin-1. TMFCs were identified based on large cell size (>30 µm) & multinucleated structure, and were categorized into 4 distinct subtypes: A) >2 distinct non-contacting nuclei within a TMFC, B) 2 distinct non-contacting nuclei, C) 2 contacting nuclei, D) a single multinucleated nuclei. CD47, SIRPa, and syncytin-1 expressions were categorized as negative, low, or high. TMFCs subtypes and marker expressions were compared to pts’ clinical status (i.e. progressive, stable or responding) at first available PET/CT scans after blood draw (∼45 days). TMFCs were identified in 72% of pts (n=28/39), average 8 cells/sample, and ranged 1-77 cells. Of the TMFCs, 12% were subtype A, 11% as B, 11% as C, and 66% as D. CD47 expression was seen in 100% of TMFs, with low expression in 39% of subtype A cells, 58% subtype B, 39% subtype C, & 62% subtype D. Notably, CD47 was significantly lower (p=0.010) in subtype B than other groups. Further, the expression of syncytin-1 in subtype B cells was significantly lower (p=0.02) than in other subtypes, and subtype D cells had 75% higher syncytin-1 localized to the nuclear regions. No correlations were found in the SIRPa expression and subtypes. Interestingly, progressing pts had 200% more TMFCs, more subtype B (83%), and significantly lower (p<0.001) CD47 expressing TMFCs than non-progressing pts. In this pilot study on the expression profiles of circulating TMFCs, it appears that specific subtypes of TMFC with low CD47 expression may be associated with progressive disease. Interestingly, this contrasts existing studies on primary tumors which often correlate higher CD47 with more aggressive cancers. This finding suggests that low CD47 cancer cells circulate blood in some pts with progressing disease and may be pertinent to efforts targeting CD47 in cancer. Citation Format: Sonia Muthuraj, Massimo Cristofanilli, Saranya Chumsri, Susan Tsai, Raymond C. Bergan, Cha-Mei Tang, Daniel L. Adams. Low CD47 expression in specific populations of tumor macrophage fusion cells in blood correlates with progressive disease in metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1979.

Abstract Background: Blood-based testing can be used as a noninvasive method to recover and analyze circulating tumor-derived cells for clinical use. Circulating cancer-associated macrophage-like cells (CAML) are specialized myeloid cells found in peripheral blood and associated with the presence of solid malignancies. We measured CAMLs prospectively in peripheral blood to ascertain their prevalence, specificity, and sensitivity in relation to breast disease status at clinical presentation. Methods: We report on two related but separate studies: 1) CellSieve microfilters were used to isolate CAMLs from blood samples of patients with known malignant disease (n = 41). Prevalence and specificity was compared against healthy volunteers (n = 16). 2) A follow-up double-blind pilot study was conducted on women (n = 41) undergoing core-needle biopsy to diagnose suspicious breast masses. Results: CAMLs were found in 93% of known malignant patients (n = 38/41), averaging 19.4 cells per sample, but none in the healthy controls. In subjects undergoing core biopsy for initial diagnosis, CAMLs were found in 88% of subjects with invasive carcinoma (n = 15/17) and 26% with benign breast conditions (n = 5/19). Conclusion: These preliminary pilot studies suggest that the presence of CAMLs may differentiate patients with malignant disease, benign breast conditions, and healthy individuals. Impact: We supply evidence that this previously unidentified circulating stromal cell may have utility as a screening tool to detect breast cancer in various malignancies, irrespective of disease stage. Cancer Epidemiol Biomarkers Prev; 25(7); 1037–42. ©2016 AACR.