Background/Objectives: The lack of canonical biomarkers and strategies to target radioresistance contribute to poor patient outcomes in triple-negative breast cancer (TNBC). Identifying and targeting novel radioresistance genes will benefit in enhancing radiotherapy response and treatment outcomes in TNBC patients. Methods: A genome-wide CRISPR screen was performed to identify radioresistance genes in the TNBC cell line. An in vitro clonogenic assay was used to assess the antiproliferative effects of Artemis knockout or pharmacologic inhibition of Artemis, either alone or in combination with RT. Tumor doubling time and animal survival were assessed using an in vivo xenograft model. RNA-seq analysis was performed to identify genes and pathways deregulated under Artemis knockout conditions, both alone and in combination with RT. Cellular senescence was evaluated using a β-galactosidase assay. Results: Our CRISPR screen identified Artemis as a top hit in RT-treated TNBC cells, whose depletion led to radiosensitization in TNBC. Artemis knockout significantly reduced cell proliferation and enhanced the antiproliferative effects of RT in vitro. Compared to mice-bearing control MDA-MB-231 xenografts, Artemis knockout exhibited prolonged survival that was further enhanced with RT. Bulk RNA-sequencing indicated that the antiproliferative and radiosensitization effects of Artemis depletion were mediated by the activation of cellular senescence which was confirmed with a β-galactosidase assay. Conclusions: Taken together, our results highlight the critical role of Artemis in TNBC cell proliferation and response to radiation. Our findings identify Artemis as a potential biomarker indicative of sensitivity to radiation and a putative target that could be inhibited to enhance the efficacy of RT in TNBC.

e15030 Background: Programmed death-ligand 1 (PD-L1) is a predictive biomarker for immunotherapy in the treatment of non-small cell lung cancer (NSCLC). Assessing PD-L1 expression from the tumor specimen can be challenging because of tissue accessibility, heterogeneity, and dynamic changes in PD-L1 expression that may impact the status of PD-L1 during disease evolution and treatment. Hence, assessing PD-L1 status from archival tumor might not reflect its actual state on the tumor and having a real-time assessment of its expression with the use of non-invasive techniques such circulating tumor cells (CTCs) is useful. Methods: We conducted a single centre prospective study to detect CTCs in the blood of patients with stage III-IV NSCLC treated with immunotherapy using the standard CellSearch technology for CTC enumeration and the Epic Sciences technology for CTCs enumeration and assessment of PD-L1 protein expression on CTCs. CTCs were detected at baseline before treatment initiation and after 2 cycles of treatment. Study endpoints included CTCs detection and concordance between the 2 technologies, PD-L1 expression assessment on CTCs with the Epic Sciences technology, and concordance with tissue-based expression. Comparisons were made using Pearson correlation coefficient (PCC) and intraclass correlation coefficient (ICC) for count data and percentage of perfect agreement. Results: Between 2019 and 2022, 48 patients treated with immunotherapy were enrolled in the study. The mean ± standard deviation (SD) age was 66.8 ± 10.3 years, 63% were females, 42% received combination chemotherapy and immunotherapy, and 44% had adenocarcinoma histology. The tissue PD-L1 expression was high (≥50%), intermediate (1-49%), and low (<1%) in 36%, 31% and 33% of patients respectively. The mean ± SD baseline CTCs count per 7.5ml was 1.97 ± 4 with CellSearch and 1.38 ± 2.72 per ml with Epic Sciences. After 2 treatment cycles, 22% and 41% of patients had an increase in their CTCs count with CellSearch and Epic Sciences, respectively and 30% and 29% had a decrease in their CTCs. The PCC and ICC for baseline CTCs detected by CellSearch and Epic Sciences were 0.72 and 0.61, respectively and for CTCs detected after 2 cycles of treatment by the 2 technologies were -0.16 and 0.45 respectively. One patient had PD-L1 expression on their CTCs with the Epic Sciences technology and there was no association between the PD-L1 expression on CTCs and on matched tissue samples. Conclusions: This study showed a moderate to strong correlation between the 2 technologies for baseline CTCs detection and moderate to poor correlation for CTCs detection after 2 cycles of therapy. No association was found between CTCs and tissue PD-L1 expression. Future work needs to further investigate the role of PD-L1 expression on CTCs.

Patients with triple-negative breast cancer (TNBC) have an increased propensity to develop lung metastasis. Our previous studies demonstrated that stem-like ALDH hi CD44 + breast cancer cells interact with lung-derived soluble factors, resulting in enhanced migration and lung metastasis particularly in TNBC models. We have also observed that the presence of a primary TNBC tumor can ‘prime’ the lung microenvironment in preparation for metastasis. In this study, we hypothesized that soluble lung-derived factors secreted in the presence of a primary TNBC tumor can influence stemness/plasticity of breast cancer cells. Using an ex vivo pulmonary metastasis assay (PuMA), we observed that the lung microenvironment supports colonization and growth of ALDH hi CD44 + TNBC cells, potentially via interactions with lung-derived FGF2. Exposure of TNBC cells to lung-conditioned media (LCM) generated from mice bearing TNBC primary tumors (tbLCM) significantly enhanced the proportion of ALDH hi CD44 + cells compared to control or LCM from tumor-naïve mice (tnLCM). Further analysis using a human cancer stem cell qPCR array revealed that, relative to tnLCM or control, exposure of TNBC cells to tbLCM leads to downregulation of the transcription factor and putative tumor suppressor Dachshund homolog 1 (DACH1), a downstream regulator of FGF2. In addition, inhibition of DACH1 using siRNA or treatment with recombinant FGF2 enhanced the ALDH hi CD44 + phenotype. Taken together, our findings suggest that the FGF2-DACH1 signaling axis supports stemness/plasticity of TNBC cells in the lung microenvironment and lays the foundation for future evaluation of FGF2 as a potential novel therapeutic target for treatment or prevention of breast cancer metastasis to the lung.

Circulating tumor cells (CTCs) present an opportunity to detect/monitor metastasis throughout disease progression. The CellSearch® is currently the only FDA-approved technology for CTC detection in patients. The main limitation of this system is its reliance on epithelial markers for CTC isolation/enumeration, which reduces its ability to detect more aggressive mesenchymal CTCs that are generated during metastasis via epithelial-to-mesenchymal transition (EMT). This Technical Note describes and validates two EMT-independent CTC analysis protocols; one for human samples using Parsortix® and one for mouse samples using VyCap. Parsortix® identifies significantly more mesenchymal human CTCs compared to the clinical CellSearch® test, and VyCap identifies significantly more CTCs compared to our mouse CellSearch® protocol regardless of EMT status. Recovery and downstream molecular characterization of CTCs is highly feasible using both Parsortix® and VyCap. The described CTC protocols can be used by investigators to study CTC generation, EMT and metastasis in both pre-clinical models and clinical samples.

Gene vectors regulated by tumor-specific promoters to express transgenes specifically in cancer cells are an emerging approach for cancer diagnosis and treatment. Minicircles are shortened plasmids stripped of prokaryotic sequences that have potency and safety characteristics beneficial for clinical translation. Previously, we developed minicircles driven by the tumor-specific survivin promoter, which exhibits elevated transcriptional activity in aggressive cancers, to express a secreted reporter for blood-based cancer detection. Here we present the first activatable, cancer theranostic minicircle system featuring a pair of diagnostic and therapeutic minicircles expressing Gaussia luciferase for urine-based cancer detection or cytosine deaminase:uracil phosphoribosyltransferase for gene-directed enzyme prodrug therapy. Diagnostic minicircles revealed urinary reporter output related to cellular survivin levels. Notably, mice with aggressive prostate tumors exhibited significantly higher urine reporter activity than mice with non-aggressive tumors and healthy mice after intratumoral minicircle administration. Therapeutic minicircles displayed specific cytotoxicity in survivin-rich cancer cells and significantly attenuated growth of aggressive orthotopic prostate tumors in mice. Use of these minicircles together creates a theranostic system that can first identify individuals carrying aggressive prostate cancer via a urinary test, followed by stringent control of tumor progression in stratified individuals who carry high-risk prostate lesions.

The lung is one of the deadliest sites of breast cancer metastasis, particularly for triple negative breast cancer (TNBC). We have previously shown that the lung produces several soluble factors that may enhance the metastatic behavior of TNBC, including E-, L-, and P-selectin. In this paper, we hypothesize that lung-derived selectins promote TNBC metastatic behavior and may serve as a potential therapeutic target. Lungs were isolated from mice and used to generate lung-conditioned media (CM). Lung-derived selectins were immunodepleted and TNBC migration and proliferation were assessed in response to native or selectin-depleted lung-CM. A 3D ex vivo pulmonary metastasis assay (PuMA) was used to assess the metastatic progression of TNBC in the lungs of wild-type versus triple-selectin (ELP-/-) knockout mice. We observed that individual lung-derived selectins enhance in vitro migration (p ≤ 0.05), but not the proliferation of TNBC cells, and that ex vivo metastatic progression is reduced in the lungs of ELP-/- mice compared to wild-type mice (p ≤ 0.05). Treatment with the pan-selectin inhibitor bimosiamose reduced in vitro lung-specific TNBC migration and proliferation (p ≤ 0.05). Taken together, these results suggest that lung-derived selectins may present a potential therapeutic target against TNBC metastasis. Future studies are aimed at elucidating the pro-metastatic mechanisms of lung-derived selectins and developing a lung-directed therapeutic approach.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

The lung is one of the deadliest sites of breast cancer metastasis, particularly in patients with triple-negative (TN) disease. We hypothesized that the presence of a TN primary breast tumor induces changes in the extracellular matrix (ECM) and soluble components of the lung microenvironment that support metastatic behavior. SUM159 (TN) and MCF7 (luminal A) breast cancer cells were injected into mice, and primary breast tumors were established prior to assessing metastatic niche changes. We observed increased CD117(+) hematopoietic progenitor cells in the bone marrow of SUM159 mice versus MCF7 or control mice (p < 0.05). Relative to mice bearing MCF7 tumors and non-tumor controls, mice bearing SUM159 tumors demonstrated enhanced expression of ECM proteins in the lung (fibronectin, tenascin-c and periostin), with similar changes observed in lung fibroblasts treated with extracellular vesicles (EVs) from TN breast cancer cells (p < 0.05). Exposure to lung-conditioned media (LCM) from SUM159 tumor-bearing mice resulted in increased migration/proliferation of both SUM159 and MCF7 cells relative to the control (p < 0.05). In contrast, LCM from MCF-7 tumor-bearing mice had no such effect. LCM from SUM159 tumor-bearing mice contained 16 unique proteins relative to other LCM conditions, including the metastasis-associated proteins CCL7, FGFR4, GM-CSF, MMP3, thrombospondin-1 and VEGF. These findings suggest for the first time that the TN breast cancer molecular subtype may be an important determinant of premetastatic changes to both the ECM and soluble components of the lung, potentially mediated via breast cancer-derived EVs.

Spheroidal microparticles versatility as a drug carrier makes it a real workhorse in drug delivery applications. Despite of their long history, few research publications emphasize on how to improve their potential targeting ability, production rate, and dissolution characteristics. The current research presents an example of the combined state of the art of nano- and microparticles development technologies. Here in a novel on-chip, microfluidics approach is developed for encapsulating amphiphilic nanomicelles-in-sodium alginate spheroid. The designed nano-in-micro drug delivery system revealed a superior cytotoxicity against triple-negative human breast cancer cell line (MDA-MB-231), besides, a more sustained release of the drug. Hydrodynamics of the designed microchip was also investigated as a function of different flow rates with an insight on the dimensionless numbers; capillary number and Weber number throughout the microchannels. Our study confirmed the efficient encapsulation of nanomicelles within the alginate shell. The current microfluidics approach can be efficiently applied for uniform production of nano-in-microparticles with potential anticancer capability.

Magnetic nanocarriers are useful in targeted cancer therapy. Dasatinib (DAS)-loaded magnetic micelles were prepared for magnetically guided drug delivery. The magnetic nanoplatform is composed of hydrophobic oleic acid-coated magnetite (Fe3O4) core along with DAS encapsulated in amphiphilic zein-lactoferrin self-assembled polymeric micelles. Transmission electron microscope analysis manifested formation of these magnetic micelles with a mean diameter of about 100 nm. In addition, drug-loaded magnetic micelles displayed a saturation magnetization of about 10.01 emu.g-1 with a superparamagnetic property. They also showed good in vitro serum stability and hemocompatibility accompanied with a sustained release of DAS in acidic pH. More importantly, they exhibited 1.35-fold increase in their in vitro cytotoxicity against triple-negative human breast cancer cell line (MDA-MB-231) using an external magnetic field compared to drug-loaded magnetic micelles in the absence of a magnetic field. Enhanced inhibition of p-c-Src protein expression level and in vitro cellular migration under the effect of magnetic field was noted owing to the dual-targeting strategy offered by the presence of a magnetic sensitive core, as well as the active targeting property of lactoferrin corona. Taken all together, these results suggest that DAS-loaded magnetic micelles possess a great potential for targeted therapy of breast cancer.

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.

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.

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.

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.

Abstract Breast cancer remains a leading cause of mortality among women, with the majority of deaths attributed to metastatic disease. In particular, the lung is one of the most common and deadly sites of breast cancer metastasis, particular in women with more aggressive molecular subtypes such as Her2+ and triple-negative breast cancer. However, it still remains unclear whether the propensity of breast cancer cells to metastasize to the lung reflects properties of cancer cells themselves, properties of the lung microenvironment, or a combination of both. Both cancer initiation and progression can in part be attributed to small subsets of “stem cell-like” tumor cells, which in breast cancer are characterized by high aldehyde dehydrogenase (ALDH) activity and/or expression of the CD44+CD24- phenotype (ALDHhiCD44+). We have previously shown that ALDHhiCD44+ breast cancer cells demonstrate preferential migration and growth in response to lung-derived soluble factors, and an increased propensity to metastasize to the lung in vivo. The objective of this study was to use an innovative 3D ex vivo pulmonary metastasis model (PuMA) that incorporates the native 3D architecture of the lung in order to test the hypothesis that whole population and stem-like ALDHhiCD44+ breast cancer cell metastatic behavior is influenced by the lung microenvironment. Red fluorescent protein (RFP)-expressing MDA-MB-231 and MDA-MD-468 breast cancer cells were seeded (5×105 cells) to the lungs of female nude mice by tail vein injection. Following euthanasia, lungs were excised, sliced into 1 mm transverse sections, and grown in culture under serum-free conditions over 21 days. Sections were imaged at 0, 7, 14 and 21 days to observe growth and progression. Using H&E and Masson's Trichrome staining, we confirmed that lung sections remained healthy with intact pulmonary architecture over 21 days in culture. Over a 14-day period, we observed significant growth of MDA-MB-231 whole breast cancer cell populations in this assay at day 14 relative to day 7 and day 0 (p≤0.05). Sorted populations of MDA-MB-231 ALDHhiCD44+ cells showed significant growth at days 14 (p≤0.001) and 21 (p≤0.0001) compared to their ALDHlowCD44- counterparts. ALDHhiCD44+ cells also progressed from the single cell state (<50 μm) to micrometastatic (200-400 μm) to macrometastatic (>400 μm) colonies during 21 days. Conversely, ALDHlowCD44- cells showed no such progression and remained predominantly as single cells throughout the assay. Together these results demonstrate that the PuMA assay sustains healthy lung architecture over 21 days, and that aggressive breast cancer cells interact with the lung to grow and progress as metastatic colonies. Ultimately, the results of this study will provide a greater understanding of the contribution of the lung microenvironment in mediating breast cancer metastasis. Citation Format: Matthew Piaseczny, David Goodale, Alison Allan. The lung microenvironment influences the metastatic behavior of breast cancer cells in an innovative 3D ex vivo pulmonary metastasis model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2015-2379

Breast cancer preferentially metastasizes to lung, lymph node, liver, bone, and brain. However, it is unclear whether properties of cancer cells, properties of organ microenvironments, or a combination of both is responsible for this observed organ tropism. We hypothesized that breast cancer cells exhibit distinctive migration/growth patterns in organ microenvironments that mirror common clinical sites of breast cancer metastasis and that receptor-ligand interactions between breast cancer cells and soluble organ-derived factors mediate this behavior. Using an ex vivo model system composed of organ-conditioned media (CM), human breast cancer cells (MDA-MB-231, MDA-MB-468, SUM149, and SUM159) displayed cell line-specific and organ-specific patterns of migration/proliferation that corresponded to their in vivo metastatic behavior. Notably, exposure to lung-CM increased migration of all cell lines and increased proliferation in two of four lines (P < .05). Several cluster of differentiation (CD) 44 ligands including osteopontin (OPN) and L-selectin (SELL) were identified in lung-CM by protein arrays. Immunodepletion of SELL decreased migration of MDA-MB-231 cells, whereas depletion of OPN decreased both migration and proliferation. Pretreatment of cells with a CD44-blocking antibody abrogated migration effects (P < .05). "Stemlike" breast cancer cells with high aldehyde dehydrogenase and CD44 (ALDH(hi)CD44(+)) responded in a distinct chemotactic manner toward organ-CM, preferentially migrating toward lung-CM through CD44 receptor-ligand interactions (P < .05). In contrast, organ-specific changes in migration were not observed for ALDH(low)CD44(-) cells. Our data suggest that interactions between CD44(+) breast cancer cells and soluble factors present in the lung microenvironment may play an important role in determining organotropic metastatic behavior.

Abstract 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<0.05). These observations closely mirror previously observed patterns of in vivo metastatic behavior, providing validation for our ex vivo model system. ALDHhiCD44+ cells responded in a chemotactic manner towards organ-conditioned media in a similar pattern to whole population results. Notably, ALDHhiCD44+ cells showed preferential migration towards lung-conditioned media relative to all other organ-conditioned media tested (p<0.05). Several CD44 ligands including osteopontin (OPN) and E-, L-, and P-selectin were identified by protein array analysis of lung-conditioned media. Immunodepletion of L-selectin or OPN from lung-conditioned media resulted in decreased migration of MDA-MB-231 cells, however only depletion of OPN also resulted in decreased growth relative to non-depleted media. These effects could be abrogated by pre-treatment with a functional CD44 receptor blocking antibody (p<0.05). 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.

BACKGROUND:Osteopontin (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.METHODS:To 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.RESULTS:All 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.CONCLUSIONS:The 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.

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 (R) 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.