Significance Using microfiltration as a liquid biopsy for the recovery of circulating tumor cells (CTCs) has revealed an accompanying macrophage subset that we use as a highly sensitive biomarker for solid tumors. We supply evidence that this circulating giant cell is a subset of disseminated tumor-associated macrophages capable of binding CTCs in peripheral blood of cancer patients. The presence of this cell expands the concept of using a liquid biopsy not only to indicate cancer presence but also to track cancer treatment effects sequentially using other circulating blood cells. Further, we supply observational evidence hypothesizing a metastasis pathway model in which CTCs migrate with pro-angiogenic macrophages, linking cancer cell intravasation, migration, and extravasation and the formation of metastatic microenvironments.

Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch V R CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve TM ) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)1/CD452/DAPI1 cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK1/CD452 cells than CellSearch. Furthermore, analysis showed low correlation between the total CK1/CD452 cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK1/CD452/DAPI1 cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using nonEpCAM dependent isolation.

Circulating tumor cells (CTCs) disseminated into peripheral blood from a primary, or metastatic, tumor can be used for early detection, diagnosis and monitoring of solid malignancies. CTC isolation by size exclusion techniques have long interested researchers as a simple broad based approach, which is methodologically diverse for use in both genomic and protein detection platforms. Though a variety of these microfiltration systems are employed academically and commercially, the limited ability to easily alter microfilter designs has hindered the optimization for CTC capture. To overcome this problem, we studied a unique photo-definable material with a scalable and mass producible photolithographic fabrication method. We use this fabrication method to systematically study and optimize the parameters necessary for CTC isolation using a microfiltration approach, followed by a comparison to a “standard” filtration membrane. We demonstrate that properly designed microfilters can capture MCF-7 cancer cells at rate of 98 ± 2% if they consist of uniform patterned distributions, ≥160 000 pores, and 7 μm pore diameters.

Background Blood based testing can be used as a non-invasive 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/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.

Purpose: Evidence suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Monitoring this response is limited, as repetitive biopsies during therapy are impractical, dangerous, and miss tumor stromal cells. Monitoring PD-L1 expression in both circulating tumor cells (CTCs) and circulating stromal cells (CStCs) in blood-based biopsies might be a practical alternative for sequential, noninvasive assessment of changes in tumor and stromal cells.Experimental Design: Peripheral blood was collected before and after radiotherapy from 41 patients with lung cancer, as were primary biopsies. We evaluated the expression of PD-L1 and formation of RAD50 foci in CTCs and a CStC subtype, cancer-associated macrophage-like cells (CAMLs), in response to DNA damage caused by radiotherapy at the tumor site.Results: Only 24% of primary biopsies had sufficient tissue for PD-L1 testing, tested with IHC clones 22c3 and 28-8. A CTC or CAML was detectable in 93% and 100% of samples, prior to and after radiotherapy, respectively. RAD50 foci significantly increased in CTCs (>7Â, P < 0.001) and CAMLs (>10Â, P ¼ 0.001) after radiotherapy, confirming their origin from the radiated site. PD-L1 expression increased overall, 1.6Â in CTCs (P ¼ 0.021) and 1.8Â in CAMLs (P ¼ 0.004): however, individual patient PD-L1 expression varied, consistently low/negative (51%), consistently high (17%), or induced (31%).Conclusions: These data suggest that RAD50 foci formation in CTCs and CAMLs may be used to track cells subjected to radiation occurring at primary tumors, and following PD-L1 expression in circulating cells may be used as a surrogate for tracking adaptive changes in immunotherapeutic targets.