Circulating Tumor Cell Analysis: Technical and Statistical Considerations for Application to the Clinic

Solid cancers are a leading cause of death worldwide, primarily due to the failure of effective clinical detection and treatment of metastatic disease in distant sites. There is growing evidence that the presence of circulating tumor cells (CTCs) in the blood of cancer patients may be an important indicator of the potential for metastatic disease and poor prognosis. Technological advances have now facilitated the enumeration and characterization of CTCs using methods such as PCR, flow cytometry, image-based immunologic approaches, immunomagnetic techniques, and microchip technology. However, the rare nature of these cells requires that very sensitive and robust detection/enumeration methods be developed and validated in order to implement CTC analysis for widespread use in the clinic. This review will focus on the important technical and statistical considerations that must be taken into account when designing and implementing CTC assays, as well as the subsequent interpretation of these results for the purposes of clinical decision making

Recent Advances in the Molecular Characterization of Circulating Tumor Cells

Although circulating tumor cells (CTCs) were first observed over a century ago, lack of sensitive methodology precluded detailed study of these cells until recently. However, technological advances have now facilitated the identification, enumeration, and characterization of CTCs using a variety of methods. The majority of evidence supporting the use of CTCs in clinical decision-making has been related to enumeration using the CellSearch((R)) system and correlation with prognosis. Growing evidence also suggests that CTC monitoring can provide an early indication of patient treatment response based on comparison of CTC levels before and after therapy. However, perhaps the greatest potential that CTCs hold for oncology lies at the level of molecular characterization. Clinical treatment decisions may be more effective if they are based on molecular characteristics of metastatic cells rather than on those of the primary tumor alone. Molecular characterization of CTCs (which can be repeatedly isolated in a minimally invasive fashion) provides the opportunity for a. real-time liquid biopsy that allows assessment of genetic drift, investigation of molecular disease evolution, and identification of actionable genomic characteristics. This review focuses on recent advances in this area, including approaches involving immunophenotyping, fluorescence in situ hybridization (FISH), multiplex RT-PCR, microarray, and genomic sequencing

The Role of Cancer Stem Cells in the Organ Tropism of Breast Cancer Metastasis: A Mechanistic Balance between the “Seed” and the “Soil”?

Breast cancer is a prevalent disease worldwide, and the majority of deaths occur due to metastatic disease. Clinical studies have identified a specific pattern for the metastatic spread of breast cancer, termed organ tropism; where preferential secondary sites include lymph node, bone, brain, lung, and liver. A rare subpopulation of tumor cells, the cancer stem cells (CSCs), has been hypothesized to be responsible for metastatic disease and therapy resistance. Current treatments are highly ineffective against metastatic breast cancer, likely due to the innate therapy resistance of CSCs and the complex interactions that occur between cancer cells and their metastatic microenvironments. A better understanding of these interactions is essential for the development of novel therapeutic targets for metastatic disease. This paper summarizes the characteristics of breast CSCs and their potential metastatic microenvironments. Furthermore, it raises the question of the existence of a CSC niche and highlights areas for future investigation

Circulating Tumor Cell Analysis in Preclinical Mouse Models of Metastasis

The majority of cancer deaths occur because of metastasis since current therapies are largely non-curative in the metastatic setting. The use of in vivo preclinical mouse models for assessing metastasis is, therefore, critical for developing effective new cancer biomarkers and therapies. Although a number of quantitative tools have been previously developed to study in vivo metastasis, the detection and quantification of rare metastatic events has remained challenging. This review will discuss the use of circulating tumor cell (CTC) analysis as an effective means of tracking and characterizing metastatic disease progression in preclinical mouse models of breast and prostate cancer and the resulting lessons learned about CTC and metastasis biology. We will also discuss how the use of clinically-relevant CTC technologies such as the CellSearch((R)) and Parsortix platforms for preclinical CTC studies can serve to enhance the study of cancer biology, new biomarkers, and novel therapies from the bench to the bedside

Circulating Tumor Cell Analysis: Technical and Statistical Considerations for Application to the Clinic

Solid cancers are a leading cause of death worldwide, primarily due to the failure of effective clinical detection and treatment of metastatic disease in distant sites. There is growing evidence that the presence of circulating tumor cells (CTCs) in the blood of cancer patients may be an important indicator of the potential for metastatic disease and poor prognosis. Technological advances have now facilitated the enumeration and characterization of CTCs using methods such as PCR, flow cytometry, image-based immunologic approaches, immunomagnetic techniques, and microchip technology. However, the rare nature of these cells requires that very sensitive and robust detection/enumeration methods be developed and validated in order to implement CTC analysis for widespread use in the clinic. This review will focus on the important technical and statistical considerations that must be taken into account when designing and implementing CTC assays, as well as the subsequent interpretation of these results for the purposes of clinical decision making

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs

What do self-efficacy items measure? : Examining the discriminant content validity of self-efficacy items

© 2018 The British Psychological Society.Peer reviewedPostprin

Soluble bone-derived osteopontin promotes migration and stem-like behavior of breast cancer cells

Breast cancer is a leading cause of cancer death in women, with the majority of these deaths caused by metastasis to distant organs. The most common site of breast cancer metastasis is the bone, which has been shown to provide a rich microenvironment that supports the migration and growth of breast cancer cells. Additionally, growing evidence suggests that breast cancer cells that do successfully metastasize have a stem-like phenotype including high activity of aldehyde dehydrogenase (ALDH) and/or a CD44(+)CD24(-)phenotype. In the current study, we tested the hypothesis that these ALDH (hi) CD44 (+) CD24(-)breast cancer cells interact with factors in the bone secondary organ microenvironment to facilitate metastasis. Specifically, we focused on bone-derived osteopontin and its ability to promote the migration and stem-like phenotype of breast cancer cells. Our results indicate that bone-derived osteopontin promotes the migration, tumorsphere-forming ability and colony-forming ability of whole population and ALDH hi CD44(+)CD24-breast cancer cells in bone marrow-conditioned media (an ex vivo representation of the bone microenvironment) (p \u3c= 0.05). We also demonstrate that CD44 and RGD-dependent cell surface integrins facilitate this functional response to bone-derived osteopontin (p \u3c= 0.05), potentially through activation of WNK-1 and PRAS40-related pathways. Our findings suggest that soluble bone-derived osteopontin enhances the ability of breast cancer cells to migrate to the bone and maintain a stem-like phenotype within the bone microenvironment, and this may contribute to the establishment and growth of bone metastases

Epithelial-to-mesenchymal transition leads to disease-stage differences in circulating tumor cell detection and metastasis in pre-clinical models of prostate cancer

Metastasis is the cause of most prostate cancer (PCa) deaths and has been associated with circulating tumor cells (CTCs). The presence of \u3e= 5 CTCs/7.5mL of blood is a poor prognosis indicator in metastatic PCa when assessed by the CellSearch (R) system, the gold standard clinical platform. However, similar to 35% of metastatic PCa patients assessed by CellSearch (R) have undetectable CTCs. We hypothesize that this is due to epithelial-to-mesenchymal transition (EMT) and subsequent loss of necessary CTC detection markers, with important implications for PCa metastasis. Two pre-clinical assays were developed to assess human CTCs in xenograft models; one comparable to CellSearch (R) (EpCAM-based) and one detecting CTCs semi-independent of EMT status via combined staining with EpCAM/HLA (human leukocyte antigen). In vivo differences in CTC generation, kinetics, metastasis and EMT status were determined using 4 PCa models with progressive epithelial (LNCaP, LNCaP-C42B) to mesenchymal (PC-3, PC-3M) phenotypes. Assay validation demonstrated that the CellSearch (R)-based assay failed to detect a significant number (similar to 40-50%) of mesenchymal CTCs. In vivo, PCa with an increasingly mesenchymal phenotype shed greater numbers of CTCs more quickly and with greater metastatic capacity than PCa with an epithelial phenotype. Notably, the CellSearch (R)-based assay captured the majority of CTCs shed during early-stage disease in vivo, and only after establishment of metastases were a significant number of undetectable CTCs present. This study provides important insight into the influence of EMT on CTC generation and subsequent metastasis, and highlights that novel technologies aimed at capturing mesenchymal CTCs may only be useful in the setting of advanced metastatic disease

High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis, and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435, MDA-MB-231, MDA-MB-468, MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase (ALDH), an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation, colony-forming ability, adhesion, migration, invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDHlowCD44low/- cells, ALDHhiCD44+CD24- (MDA-MB-231) and ALDHhiCD44+CD133+ (MDA-MB-468) cells demonstrated increased growth (P \u3c 0.05), colony formation (P \u3c 0.05), adhesion (P \u3c 0.001), migration (P \u3c 0.001) and invasion (P \u3c 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2 gamma receptor null mice, ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells showed enhanced tumorigenicity and metastasis relative to ALDHlowCD44low/- cells (P \u3c 0.05). These novel results suggest that stem-like ALDHhiCD44+CD24- and ALDHhiCD44+CD133+ cells may be important mediators of breast cancer metastasis