Circulating giant macrophages as a potential biomarker of solid tumors.

Tumor-associated macrophages (TAMs) derived from primary tumors are believed to facilitate circulating tumor cell (CTC) seeding of distant metastases, but the mechanisms of these processes are poorly understood. Although many studies have focused on the migration of CTCs, less attention has been given to TAMs that, like CTCs, derive from tumor sites. Using precision microfilters under low-flow conditions, we isolated circulating cancer-associated macrophage-like cells (CAMLs) from the peripheral blood of patients with breast, pancreatic, or prostate cancer. CAMLs, which are not found in healthy individuals, were found to express epithelial, monocytic, and endothelial protein markers and were observed bound to CTCs in circulation. These data support the hypothesis that disseminated TAMs can be used as a biomarker of advanced disease and suggest that they have a participatory role in tumor cell migration.

Mitosis in circulating tumor cells stratifies highly aggressive breast carcinomas.

BACKGROUND: Enumeration of circulating tumor cells (CTCs) isolated from the peripheral blood of breast cancer patients holds promise as a clinically relevant, minimally invasive diagnostic test. However, CTC utility has been limited as a prognostic indicator of survival by the inability to stratify patients beyond general enumeration. In comparison, histological biopsy examinations remain the standard method for confirming malignancy and grading malignant cells, allowing for cancer identification and then assessing patient cohorts for prognostic and predictive value. Typically, CTC identification relies on immunofluorescent staining assessed as absent/present, which is somewhat subjective and limited in its ability to characterize these cells. In contrast, the physical features used in histological cytology comprise the gold standard method used to identify and preliminarily characterize the cancer cells. Here, we superimpose the methods, cytologically subtyping CTCs labeled with immunohistochemical fluorescence stains to improve their prognostic value in relation to survival. METHODS: In this single-blind prospective pilot study, we tracked 36 patients with late-stage breast cancer over 24 months to compare overall survival between simple CTC enumeration and subtyping mitotic CTCs. A power analysis (1-ß = 0. 9, α = 0.05) determined that a pilot size of 30 patients was sufficient to stratify this patient cohort; 36 in total were enrolled. RESULTS: Our results confirmed that CTC number is a prognostic indicator of patient survival, with a hazard ratio 5.2, p = 0.005 (95 % CI 1.6-16.5). However, by simply subtyping the same population based on CTCs in cytological mitosis, the hazard ratio increased dramatically to 11.1, p < 0.001 (95 % CI 3.1-39.7). CONCLUSIONS: Our data suggest that (1) mitotic CTCs are relativity common in aggressive late-stage breast cancer, (2) mitotic CTCs may significantly correlate with shortened overall survival, and (3) larger and more defined patient cohort studies are clearly called for based on this initial pilot study.

Cytometric characterization of circulating tumor cells captured by microfiltration and their correlation to the CellSearch(®) CTC test.

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(®) 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™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ 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 non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley Periodicals, Inc.

Parthenolide and costunolide reduce microtentacles and tumor cell attachment by selectively targeting detyrosinated tubulin independent from NF-κB inhibition.

INTRODUCTION: Detyrosinated tubulin, a post-translational modification of α-tubulin and a hallmark of stable microtubules, has gained recent attention given its association with tumor progression, invasiveness, and chemoresistance. We also recently reported that epithelial-to-mesenchymal transition (EMT) promotes tubulin detyrosination through tubulin tyrosine ligase (TTL) suppression. Furthermore, detyrosinated tubulin-enriched membrane protrusions, termed microtentacles (McTN), facilitate tumor cell reattachment to endothelial layers. Given the induction of EMT associated with inflammation and cancer progression, we tested anti-inflammatory nuclear factor-kappaB (NF-κB) inhibitors on a panel of human breast carcinoma cells to examine their effects on detyrosinated tubulin to identify more specific tubulin-directed anti-cancer treatments. METHODS: Using metastatic human breast carcinoma cells MDA-MB-157, MDA-MB-436, and Bt-549, we measured the impact of NF-κB inhibitors parthenolide, costunolide, and resveratrol on detyrosinated tubulin using protein expression analysis and immunofluorescence. A luciferase reporter assay and a viability screen were performed to determine if the effects were associated with their NF-κB inhibitory properties or were a result of apoptosis. Real-time monitoring of cell-substratum attachment was measured utilizing electrical impedance across microelectronic sensor arrays. We compared the selectivity of the NF-κB inhibitors to specifically target detyrosinated tubulin with traditional tubulin-targeted therapeutics, paclitaxel and colchicine, throughout the study. RESULTS: Sesquiterpene lactones, parthenolide and costunolide, selectively decrease detyrosinated tubulin independent of their inhibition of NF-κB. Live-cell scoring of suspended cells treated with parthenolide and costunolide show reduction in the frequency of microtentacles and inhibition of reattachment. Structural analysis shows that parthenolide and costunolide can decrease detyrosinated microtubules without significantly disrupting the overall microtubule network or cell viability. Paclitaxel and colchicine display indiscriminate disruption of the microtubule network. CONCLUSIONS: Our data demonstrate that selective targeting of detyrosinated tubulin with parthenolide and costunolide can reduce McTN frequency and inhibit tumor cell reattachment. These actions are independent of their effects on NF-κB inhibition presenting a novel anti-cancer property and therapeutic opportunity to selectively target a stable subset of microtubules in circulating tumor cells to reduce metastatic potential with less toxicity in breast cancer patients.

Precision Microfilters as an all in one System for Multiplex Analysis of Circulating Tumor Cells.

Enumeration of circulating tumor cells (CTCs) from cancer patient blood is an established diagnostic assay used to evaluate patient status as a singleplex test. However, in the coming age of personalized medicine, multiplex analysis of patient CTCs, including proteomic and genomic techniques, will have to be integrated with CTC isolation platform technologies. Advancements in microfabrication have demonstrated that CTCs can be isolated and analyzed using microfluidic lab-on-a-chip devices. However, to date, most microfluidic devices are either still in the development phase, not applicable to all clinical tests, or are not commercially available. To overcome these discrepancies, we describe an all-in-one device for the isolation and multiplexing of clinically applicable CTC assays. Microfilters present an ideal lab-on-a-chip platform for analysis of CTCs as non-toxic and inert materials allow for a multitude of tests from cell growth through clinical staining techniques, all without background interference. Lithographically fabricated microfilters, can be made with high porosity, precise pore dimensions, arrayed pore distribution, and optimized for CTC size-based isolation. In this study we describe microfilter use in isolation and in situ analysis of CTCs using multiple sequential techniques including culture, FISH, histopathological analysis, H&E staining, photobleaching and re-staining. Further, as a proof of principle, we then describe the ability to quantitatively release patient derived CTCS from the microfilters for potential use in downstream genomic/proteomic analysis.

ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cells.

The presence of circulating tumor cells (CTCs) in blood predicts poor patient outcome and CTC frequency is correlated with higher risk of metastasis. Recently discovered, novel microtubule-based structures, microtentacles, can enhance reattachment of CTCs to the vasculature. Microtentacles are highly dynamic membrane protrusions formed in detached cells and occur when physical forces generated by the outwardly expanding microtubules overcome the contractile force of the actin cortex. Rho-associated kinase (ROCK) is a major regulator of actomyosin contractility and Rho/ROCK over-activation is implicated in tumor metastasis. ROCK inhibitors are gaining popularity as potential cancer therapeutics based on their success in reducing adherent tumor cell migration and invasion. However, the effect of ROCK inhibition on detached cells in circulation is largely unknown. In this study, we use breast tumor cells in suspension to mimic detached CTCs and show that destabilizing the actin cortex through ROCK inhibition in suspended cells promotes the formation of microtentacles and enhances reattachment of cells from suspension. Conversely, increasing actomyosin contraction by Rho over-activation reduces microtentacle frequency and reattachment. Although ROCK inhibitors may be effective in reducing adherent tumor cell behavior, our results indicate that they could inadvertently increase metastatic potential of non-adherent CTCs by increasing their reattachment efficacy.

α-Tubulin acetylation elevated in metastatic and basal-like breast cancer cells promotes microtentacle formation, adhesion, and invasive migration.

Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies that remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle (McTN) protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this posttranslational modification exerted a significant impact on McTN frequency and the reattachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of more than 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared with primary tumors. Proteomic analysis of an independent cohort of more than 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high-intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in patients with breast cancer.

The combinatorial activation of the PI3K and Ras/MAPK pathways is sufficient for aggressive tumor formation, while individual pathway activation supports cell persistence.

A high proportion of human tumors maintain activation of both the PI3K and Ras/MAPK pathways. In basal-like breast cancer (BBC), PTEN expression is decreased/lost in over 50% of cases, leading to aberrant activation of the PI3K pathway. Additionally, BBC cell lines and tumor models have been shown to exhibit an oncogenic Ras-like gene transcriptional signature, indicating activation of the Ras/MAPK pathway. To directly test how the PI3K and Ras/MAPK pathways contribute to tumorigenesis, we deleted PTEN and activated KRas within non-tumorigenic MCF-10A breast cells. Neither individual mutation was sufficient to promote tumorigenesis, but the combination promoted robust tumor growth in mice. However, in vivo bioluminescence reveals that each mutation has the ability to promote a persistent phenotype. Inherent in the concept of tumor cell dormancy, a stage in which residual disease is present but remains asymptomatic, viable cells with each individual mutation can persist in vivo during a period of latency. The persistent cells were excised from the mice and showed increased levels of the cell cycle arrest proteins p21 and p27 compared to the aggressively growing PTEN-/-KRAS(G12V) cells. Additionally, when these persistent cells were placed into growth-promoting conditions, they were able to re-enter the cell cycle and proliferate. These results highlight the potential for either PTEN loss or KRAS activation to promote cell survival in vivo, and the unique ability of the combined mutations to yield rapid tumor growth. This could have important implications in determining recurrence risk and disease progression in tumor subtypes where these mutations are common.

Curcumin targets breast cancer stem-like cells with microtentacles that persist in mammospheres and promote reattachment.

Cancer stem-like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non-stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell-cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy.

The systematic study of circulating tumor cell isolation using lithographic microfilters.

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.

Interplay of Stem Cell Characteristics, EMT, and Microtentacles in Circulating Breast Tumor Cells.

Metastasis, not the primary tumor, is responsible for the majority of breast cancer-related deaths. Emerging evidence indicates that breast cancer stem cells (CSCs) and the epithelial-to-mesenchymal transition (EMT) cooperate to produce circulating tumor cells (CTCs) that are highly competent for metastasis. CTCs with both CSC and EMT characteristics have recently been identified in the bloodstream of patients with metastatic disease. Breast CSCs have elevated tumorigenicity required for metastatic outgrowth, while EMT may promote CSC character and endows breast cancer cells with enhanced invasive and migratory potential. Both CSCs and EMT are associated with a more flexible cytoskeleton and with anoikis-resistance, which help breast carcinoma cells survive in circulation. Suspended breast carcinoma cells produce tubulin-based extensions of the plasma membrane, termed microtentacles (McTNs), which aid in reattachment. CSC and EMT-associated upregulation of intermediate filament vimentin and increased detyrosination of α-tubulin promote the formation of McTNs. The combined advantages of CSCs and EMT and their associated cytoskeletal alterations increase metastatic efficiency, but understanding the biology of these CTCs also presents new therapeutic targets to reduce metastasis.