David Goodale

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 < 0.05), colony formation (P < 0.05), adhesion (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2γ receptor null mice, ALDHhiCD44+CD24− and ALDHhiCD44+CD133+ cells showed enhanced tumorigenicity and metastasis relative to ALDHlowCD44low/– cells (P < 0.05). These novel results suggest that stem‐like ALDHhiCD44+CD24− and ALDHhiCD44+CD133+ cells may be important mediators of breast cancer metastasis.

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

The inability to sensitively detect metastatic cells in preclinical models of cancer has created challenges for studying metastasis in experimental systems. We previously developed a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs) in mouse models of breast cancer. We have adapted this methodology for analysis of tumor dissemination to bone marrow (BM) and lymph node (LN), and for analysis of these samples by laser scanning cytometry (LSC). Our objective was to implement these methodologies for characterization of tumor cell dissemination in preclinical models of cancer metastasis. Human cancer cells were injected into mice via mammary fat pad (MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targets bone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificed and blood, LNs, and BM were collected. Samples were immunomagnetically enriched and labeled with human leukocytic antigen–fluorescein isothiocyanate and CD45‐PE antibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis. Following MFP injection, CTCs increased over time, as did disseminated cells to the LN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2 weeks postinjection. Following TV injection, CTCs were initially high but decreased rapidly by 1 week before increasing to peak at endpoint. Combined with an observed concurrent increase in disseminated cells to LN and BM, this suggests that tumor cells may shed into the circulation from lung metastases that establish following initial cell delivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumor cells in the BM (most prevalent site of metastasis after IC injection) remained at moderate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitive quantification of disseminated tumor cells in preclinical models of metastasis. These methods will be valuable for future studies aimed at testing new therapeutics in the metastatic setting.

Flow cytometric assessment of monocyte activation markers and circulating endothelial cells in patients with localized or metastatic breast cancer

Monocyte activation in cancer patients may be reflective of anticancer activity. However, studies indicate that recruitment of macrophages can actually promote tumor growth and angiogenesis. Assessment of other microenvironmental cells such as circulating endothelial cells (CECs) may provide additional information regarding disease progression. The objective of this study was to assess monocyte activation and CECs in breast cancer patients and determine the potential clinical relevance during disease progression.

Image cytometry analysis of circulating tumor cells.

The majority of cancer-related deaths are as a result of metastatic disease, which has been correlated with the presence of circulating tumor cells (CTCs) in the bloodstream. Therefore the ability to reliably enumerate and characterize these cells could provide useful information about the biology of the metastatic cascade; facilitate patient prognosis; act as a marker of therapeutic response; and/or aid in novel anticancer drug development. Several different techniques have been utilized for the enrichment and detection of these rare CTCs, each having their own unique advantages and disadvantages. In this chapter we will briefly discuss each of these techniques as well as the pros and cons of each approach. In particular, we will provide a comprehensive examination of two image cytometry approaches for CTC analysis that are in routine use in our laboratory; the iCys Laser Scanning Cytometer (Compucyte, Cambridge, MA), and the CellSearch® system (Veridex, North Raritan, NJ). The ability to detect, enumerate, and characterize CTCs is an important tool for the study of the metastatic cascade and the improved clinical management of cancer patients. These rare cells could shed light on the basic biology behind this highly lethal process and ultimately change current patient treatment guidelines.

Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

BackgroundOsteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other.MethodsTo study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior.ResultsAll three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells.ConclusionsThe results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer.BACKGROUND The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. METHODS A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. RESULTS Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. CONCLUSION We demonstrated that FFPE specimens retained important prognostic information that could be identified using a recent gene profiling technology. Our study supports the use of FFPE specimens for the development and refinement of prognostic gene signatures for breast cancer. Clinical applications of such prognostic gene profiles await future large-scale validation studies.

Self-assembled amphiphilic zein-lactoferrin micelles for tumor targeted co-delivery of rapamycin and wogonin to breast cancer

Graphical abstract Figure. No caption available. Abstract Protein‐based micelles have shown significant potential for tumor‐targeted delivery of anti‐cancer drugs. In this light, self‐assembled nanocarriers based on GRAS (Generally recognized as safe) amphiphilic protein co‐polymers were synthesized via carbodiimide coupling reaction. The new nano‐platform is composed of the following key components: (i) hydrophobic zein core to encapsulate the hydrophobic drugs rapamycin (RAP) and wogonin (WOG) with high encapsulation efficiency, (ii) hydrophilic lactoferrin (Lf) corona to enhance the tumor targeting, and prolong systemic circulation of the nanocarriers, and (iii) glutaraldehyde (GLA)‐crosslinking to reduce the particle size and improve micellar stability. Zein‐Lf micelles showed relatively rapid release of WOG followed by slower diffusion of RAP from zein core. This sequential release may aid in efflux pump inhibition by WOG thus sensitizing tumor cells to RAP action. Interestingly, these micelles showed good hemocompatibility as well as enhanced serum stability owing to the brush‐like architecture of Lf shell. Moreover, this combined nano‐delivery system maximized synergistic cytotoxicity of RAP and WOG in terms of tumor inhibition in MCF‐7 breast cancer cells and Ehrlich ascites tumor animal model as a result of enhanced active targeting. Collectively, GLA‐crosslinked zein‐Lf micelles hold great promise for combined RAP/WOG delivery to breast cancer with reduced drug dose, minimized side effects and maximized anti‐tumor efficacy.

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 ≥5 CTCs/7.5mL of blood is a poor prognosis indicator in metastatic PCa when assessed by the CellSearch® system, the “gold standard” clinical platform. However, ~35% of metastatic PCa patients assessed by CellSearch® 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® (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®-based assay failed to detect a significant number (~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®-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.

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® 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.

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

Abstract B10: Soluble lung-derived factors mediate breast cancer cell migration and growth via CD44 receptor-ligand interactions

Background: The majority of breast cancer deaths occur due to metastatic disease. Clinical studies have identified a specific pattern for the metastatic spread of breast cancer, where preferential secondary sites include the lymph node, lung, liver, bone, and brain. However, it is not clear whether properties of the cancer cells (the “seeds”); properties of the organ microenvironment (the “soil”), or a combination of both are responsible for this observed organ tropism of breast cancer. It has been proposed that small subsets of stem-like tumor cells are responsible for tumor initiation and progression. In breast cancer, these cells are identified by their high aldehyde dehydrogenase (ALDH) activity and/or CD44+CD24- phenotype. We have previously shown that ALDHhiCD44+ breast cancer cells exhibit key metastatic behaviors in vitro and in vivo. However, the mechanisms underlying the organ-specific metastatic capacity of these cells requires further elucidation. Hypothesis: Breast cancer cells exhibit differential growth and migration patterns in specific organ microenvironments; and ALDHhiCD44+ breast cancer cells exhibit increased organ-specific migration and growth (1) preferentially in the lung microenvironment and (2) compared to their ALDHlowCD44- counterparts. Methods: A novel ex vivo model system comprised of organ-conditioned media generated from athymic nude mice was used to test organotropic migration/growth of whole populations of MDA-MB-231, MDA-MB-468, SUM149, and SUM159 human breast cancer cell lines, and ALDHhiCD44+ and ALDHlowCD44- populations isolated from the MDA-MB-231 cell line. Protein arrays were used to interrogate lung-conditioned media and identify soluble factors of interest. Bead-based immunodepletion of target proteins was performed on lung-conditioned media. The migration/growth response of MDA-MB-231 breast cancer cells was then assessed following exposure to depleted vs. non-depleted lung-conditioned media. Results: Whole populations of all 4 human breast cancer cell lines examined responded via cell line specific patterns of migration towards organ-conditioned media. Notably, all lines demonstrated increased migration towards lung-conditioned media (p Conclusions: Our data suggest an important role for CD44 receptor-ligand interactions in mediating lung-specific breast cancer cell migration and growth. To the best of our knowledge, this is the first study to undertake ex vivo investigation of the role of the microenvironment in metastatic organ tropism in the context of the cancer stem cell hypothesis. Citation Format: Ying Xia, Jenny E. Chu, Benjamin Chin-Yee, David Goodale, Alysha K. Croker, Alison L. Allan. Soluble lung-derived factors mediate breast cancer cell migration and growth via CD44 receptor-ligand interactions. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B10.