Circulating Breast Cancer Cells Research Articles

Abstract IA06: Postpartum breast involution informs breast cancer metastasis to the liver

Abstract Young women with breast cancer have disproportionally higher rates of metastasis, and effective treatment strategies are lacking. Our lab has focused on investigating how normal reproductive changes in tissue biology interface with breast cancer metastasis in young patients. These studies identify weaning-induced involution of the breast as a tissue remodeling process similar to wound healing that promotes metastasis in rodent models. Relevance to women is suggested by the fact that a breast cancer diagnosis within 5 years of childbirth is a strong and independent risk factor for metastasis. Further, these studies find that up to 50% of all young onset breast cancer is diagnosed within this high risk postpartum window. This subset of common, young onset breast cancer is referred to as postpartum breast cancer (PPBC). We find that PPBC appears durably altered by involution, as PPBC tumors display involution, metastatic, and treatment resistant molecular signatures. PPBC also specifically associates with increased liver metastases, a metastatic site associated with very poor prognosis. In rodents, we identified a functional link between the mammary gland and the liver, including weaning-induced liver involution. Liver involution is characterized by hepatocyte cell death, fibroblast activation, ECM deposition, and an immune regulatory infiltrate similar to that found in the postpartum involuting breast. In rodent models of breast cancer liver metastasis (BCLM), the involuting liver supports outgrowth of circulating breast cancer cells more than the nulliparous host liver. Evidence for similar liver biology occurring in women is suggested by studies in healthy women, where we find liver volume increase during pregnancy and loss post wean, data consistent with liver involution. In our mouse models of BCLM, the involution metastatic advantage coincided with weaning-induced immune tolerance in the liver. We next targeted the pro-tumor, immune suppressed state of involution with the immune modulatory agent vitamin D. These studies revealed dramatic down regulation of liver metabolism with weaning, including liver cytochrome P450 enzymes, which effectively nullified the protective effect of vitamin D in involution hosts. Combined, these novel findings are anticipated to have implications for sex-specific liver diseases broadly, efficacy of pro-drugs in the postpartum window, and for breast cancer outcomes in young patients. Citation Format: Pepper Schedin. Postpartum breast involution informs breast cancer metastasis to the liver [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA06.

Ganoderma lucidum spore oil synergistically enhances the function of cyclophosphamide in the prevention of breast cancer metastasis.

Ganoderma lucidum ( G . lucidum ) is a traditional Chinese herbal medicine that has shown potential as an alternative adjuvant therapy for cancer patients. However, the mechanisms and adjuvant therapeutic effects of G . lucidum in cancer treatment remain unclear. In this work, G . lucidum spore oil (GanoOil), a newly developed oily G . lucidum spore extract was used to investigate the mechanisms and adjuvant therapeutic effects of GanoOil in conjunction with the chemotherapeutic drug cyclophosphamide (CTX) for preventing breast cancer metastasis. In the model of lung metastasis, orally administered GanoOil increased the population of CD8 + T cells and interleukin (IL)-6 cytokine levels in mouse blood, whereas also enhancing the activity of natural killer cells in the spleen. Furthermore, the combination of GanoOil and CTX effectively suppressed the lung metastasis of circulating breast cancer cells, alleviated CTX-induced weight loss, and reduced the ratio of lung and spleen weight to body weight in mice. Moreover, high concentrations of GanoOil exhibited no significant toxicity or side effects in both in vitro and in vivo experiments. In conclusion, GanoOil is a safe drug that can enhance immune activity in mice to achieve therapeutic effects on cancer, and can also synergistically inhibit tumor metastasis with CTX.

EMT status of circulating breast cancer cells and impact of fluidic shear stress

Circulating tumor cells (CTCs) play a vital role in the metastasis and recurrence of breast cancer. CTCs are highly heterogeneous at the stage of Epithelial-to-Mesenchymal Transition (EMT), but the phenotypic and biological characteristics in different EMT stages remain poorly defined. We conducted an orthotopic mouse (4T1) model of breast cancer to isolate CTCs and identified two phenotypes of CTCs: intermediate E/M and mesenchymal CTCs. MTT, Colony formation, Transwell migration and invasion assays were utilized to examined cell proliferation, colony forming, migration and invasion ability. Both the intermediate E/M and mesenchymal CTCs exhibited lower rates of proliferation, colony formation and invasion, as compared to primary tumor cells. The mesenchymal CTCs had a higher rate of invasion but lower rates of proliferation and colony formation than the intermediate E/M CTCs. They also exhibited lower rates of growth and metastasis than the primary tumor cells in vivo, but the mesenchymal CTCs had a higher rate of metastasis than the intermediate E/M CTCs. Fluid shear stress induced the EMT transition of CTCs. The comprehensive analysis of CTCs proteomics discovered proteins that differentially expressed in the two types of CTCs and their primary tumor cells.

Simultaneous Detection and HER2 Profiling of Circulating Breast Cancer Cells in Clinical Patients Using a Rare Cell Sorter.

This study describes a rare cell sorter (RCS) method to detect circulating tumor cells (CTCs) and CTC clusters in whole blood without pretreatment. We collected samples from breast cancer patients at the University of Tsukuba Hospital. A total of 15 whole-blood specimens from patients with breast cancer were collected and analyzed via a microfluidics chip, fluorescence-conjugated antibody staining, and fluorescence microscopy. Of 15 total cases, eight were analyzed by RCS ver3 and seven were analyzed by RCS ver3.5 to reveal potential clinical differences in scanning methods. We then examined the HER2 status on 4 of the 15 patients using our RCS system. RCS efficiently detected all subtypes of CTCs and CTC clusters from the peripheral blood of cancer patients. The concordance rate of HER2 status between tissue and CTCs in 4 tested clinical samples was 100%. RCS is a non-invasive method that allows for simultaneous detection of CTCs, cluster presence, and surface marker (e.g., HER2) status. Frequent sampling is, thus, possible and the large amount of data obtained will be clinically useful to predict response to therapy as well as plan adjunct support therapies in cancer patients.

PO-25: Elevated plasma von Willebrand Factor in patients with breast cancer may contribute to the early metastatic niche, promoting endothelial adhesion, invasion and angiogenic properties of circulating breast cancer cells

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Cell specific variation in viability in suspension in in vitro Poiseuille flow conditions

The influence of Poiseuille flow on cell viability has applications in the areas of cancer metastasis, lab-on-a-chip devices and flow cytometry. Indeed, retaining cell viability is important in the emerging field of adoptive cell therapy, as cells need to be returned to patients’ bodies, while the viability of other cells, which are perhaps less accustomed to suspension in a fluidic environment, is important to retain in flow cytometers and other such devices. Despite this, it is unclear how Poiseuille flow affects cell viability. Following on from previous studies which investigated the viability and inertial positions of circulating breast cancer cells in identical flow conditions, this study investigated the influence that varying flow rate, and the corresponding Reynolds number has on the viability of a range of different circulating cells in laminar pipe flow including primary T-cells, primary fibroblasts and neuroblastoma cells. It was found that Reynolds numbers as high as 9.13 had no effect on T-cells while the viabilities of neuroblastoma cells and intestinal fibroblasts were significantly reduced in comparison. This indicates that in vitro flow devices need to be tailored to cell-specific flow regimes.

Alteration of the F1Fo ATP Synthase Causes Metabolic Remodeling in Breast Cancer Cells

Abstract Objectives The F1Fo ATP synthase is a multienzyme complex that produces mitochondrial ATP. Aberrant expression or assembly of F1Fo ATP synthase subunits leads to alterations in energy metabolism. We recently found that breast cancer cells exposed to fluid shear stress (FSS) have significantly enhanced metastatic behavior including chemoresistance and cell proliferation. Chemoresistance depends upon active transport systems, and cell division and growth require ATP. Therefore, we hypothesized that circulating breast cancer cells undergo altered energy metabolism via FSS-induced changes in F1Fo ATP synthase subunits and subsequent mitochondrial remodeling. Methods Non-metastatic MCF7 and metastatic MDA-MB-231 human breast cancer cells were treated with or without FSS and cultured. Cellular proliferation was assayed by measuring cell number and gap distance. Metabolic profile including intracellular ATP and oxygen consumption rate were analyzed. We also quantified abundance of F1Fo ATP synthase subunits using immunoblotting. Results Treatment with FSS significantly increased proliferation of both MCF7 and MDA-MB-231 human breast cancer cells. FSS significantly increased intracellular ATP in MDA-MB-231 breast cancer cells while ATP levels in MCF7 were not significantly changed. MDA-MB-231 cells retained increased ATP after treatment with the uncoupler FCCP, indicating remodeling and decreased reliance on mitochondrial energy metabolism. Interestingly, oxygen consumption rate was significantly increased in both MCF7 and MDA-MB-231 by FSS. We further quantified the abundance of F1Fo ATP synthase subunits in both cell lines. The β- and c-subunits of the F1Fo ATP synthase were significantly depleted in both lines of FSS-treated breast cancer cells. Conclusions Our data show that FSS alters abundance of the F1Fo ATP synthase subunits leading to metabolic remodeling. We suggest that FSS may influence non-metastatic (MCF7) and metastatic cancer cells (MDA-MB-231) differently. Underlying changes in mitochondrial and cytoplasmic ATP production in these cells is still under investigation. However, it is possible that reactive oxygen species generated during FSS may signal a switch to cytoplasmic intracellular energy metabolism. Funding Sources Alabama Life Research Institute Pilot Project (University of Alabama)

Immune Milieu Established by Postpartum Liver Involution Promotes Breast Cancer Liver Metastasis.

Simple SummaryCancer becomes lethal when it metastasizes to secondary sites, and for breast cancer metastasis to the liver is a serious clinical problem. Liver metastasis is promoted, in part, by changes to the liver environment, resulting in the formation of a metastatic niche that supports circulating tumor cells. Understanding how the liver niche support breast cancer cells may lead to development of treatments for patients with metastatic breast cancer. Here, we report that the developmentally regulated process of weaning-induced liver involution increases liver metastasis in cancer cells with otherwise low metastatic potential. Increased metastasis associates with unique immunological properties in the involuting liver, including reduced ability to activate T cells required for tumor cell clearance. These data establish physiologic liver involution as a model to understand the liver metastatic niche and suggest future research into whether the immune milieu identified in the involuting liver could be targeted to treat metastases more generally.In rodents, we identified a physiologic process within the normal liver that creates a pre-metastatic niche. This physiology is weaning-induced liver involution, characterized by hepatocyte cell death, immune influx, and extracellular matrix remodeling. Here, using weaning-induced liver involution as a model of a physiologically regulated pro-metastatic niche, we investigate how liver involution supports breast cancer metastasis. Liver metastases were induced in BALB/c immune competent hosts by portal vein injection of D2OR (low metastatic) or D2A1 (high metastatic) mouse mammary tumor cells. Tumor incidence and multiplicity increased in involution hosts with no evidence of a proliferation advantage. D2OR tumor cell extravasation, seeding, and early survival were not enhanced in the involuting group compared to the nulliparous group. Rather, the involution metastatic advantage was observed at 14 days post tumor cell injection. This metastatic advantage associated with induction of immune tolerance in the involution host liver, reproductive state dependent intra-tumoral immune composition, and CD8-dependent suppression of metastases in nulliparous hosts. Our findings suggest that the normal postpartum liver is in an immune suppressed state, which can provide a pro-metastatic advantage to circulating breast cancer cells. Potential relevance to women is suggested as a postpartum diagnosis of breast cancer is an independent predictor of liver metastasis.

Abstract LT004: Immune suppression established by postpartum liver involution promotes liver metastasis

Abstract A postpartum diagnosis of breast cancer in women ≤45 yoa is an independent predictor of liver metastasis, suggesting a liver metastatic advantage (PMID: 27974414). In rodents, we identified a postpartum liver biology that supports liver metastasis in mouse models. This biology is weaning-induced liver involution and is characterized by a transient period of hepatocyte cell death, catabolic metabolism, immune influx, and stromal remodeling (PMID: 27974414). Here, we sought to investigate the mechanism by which post-wean liver involution supports breast cancer metastasis. Liver metastases were induced by portal vein injection of mouse mammary tumor cells into nulliparous or post-wean, involution hosts (PMID: 28060292). Compared to nulliparous mice, involution mice had increased incidence of liver metastasis, as well as more liver lesions per mouse. However, there was no group differences in tumor size or proliferation index. These data are consistent with the involution liver providing an environment that promotes tumor cell establishment, not proliferation. To further investigate, we intraportally injected GFP-tagged tumor cells into GFP-tolerant mice, and assessed tumor cell abundance in the liver at 90 minutes, and 1, 3, and 14 days post injection. Contrary to our expectations, the nulliparous group had more tumor cells present at 90 minutes, 1 day, and 3 days post tumor cell injection. We saw the metastatic advantage in the involution group emerge by 14 days after tumor cell injection. We also observed increased abundance of PD1+ CD4 T cells in addition to heightened IL-10 signaling in the involution group at baseline (i.e. no tumor). Based on these observations, we hypothesized that the involution metastatic niche may be characterized by immune suppression, permitting tumor cell escape from immune surveillance. To address if the involution host liver is immune suppressed, we used an in vivo T cell activation assay where we adoptively transferred antigen-specific T cells systemically, and subsequently injected the cognate antigen into the liver of tumor free mice. Five days later, we assessed antigen-specific T cell number as a measure of T cell priming and activation. We found significantly impaired naïve T cell priming in the normal involution host liver compared to the nulliparous host. These data raise the possibility that impaired adaptive immunity could be responsible for increased liver metastasis in the involution group. In support, when we depleted CD8 T cells in our liver metastasis model, we found that metastasis in the anti-CD8 treated nulliparous group was similar to the untreated involution group, showing that depletion of cytotoxic immune cells in nulliparous hosts can recapitulate the involution host metastatic advantage. Taken together, our findings suggest the normal postpartum liver is in an immune suppressed state, which can provide a pro-metastatic advantage to circulating breast cancer cells. Future work will investigate therapeutic targeting of the postpartum liver niche. Citation Format: Alexandra Quackenbush, Nathan Penncok, Pepper Schedin. Immune suppression established by postpartum liver involution promotes liver metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT004.

Tumor-derived exosomes can specifically prevent cancer metastatic organotropism

Each type of cancer has its own specific metastatic route developed by disseminating circulating tumor cells (CTCs) and related extracellular vesicles to the target organ, i.e., metastasis organotropism. Tumor-derived small extracellular vesicles (herein exosomes, EXO) play an important role in determining cancer organotropic metastases to pre-metastasis niches. We therefore hypothesized that drug-loaded EXO may mix well with their companion small extracellular vesicles to specifically target the aimed metastatic organ via organotropism. Here, we demonstrate that the circulating breast-cancer-derived EXO loaded with doxorubicin (EXO-DOX) can mingled with their original companion EXO and inhibit breast cancer metastasis to lungs. The CD47 on the EXO-DOX prevented EXO-DOX from immune attack and prolonged their circulation in blood. The tissue distribution ratio of EXO-DOX is identical to the ratio of their companion EXO due to the specific affinity of EXO to integrins in targeted tissues. Quantitative accumulation of EXO-DOX in the mouse lungs is proportional to the organotropism of the circulating breast cancer cells that disseminate from subcutaneously-implanted human breast cancer cells in mice. EXO-DOX inhibited angiogenesis and cancer cell proliferation, resulting in prevention of breast cancer metastasis to the lungs. This study opens a novel path to use Trojan small extracellular vesicles for specifically controlled release of active components by small extracellular vesicles organotropism mechanism to the targeted organ for disease chemoprevention.

Identification and Characterization of Cancer Cells That Initiate Metastases to the Brain and Other Organs.

Specific biological properties of those circulating cancer cells that are the origin of brain metastases (BM) are not well understood. Here, single circulating breast cancer cells were fate-tracked during all steps of the brain metastatic cascade in mice after intracardial injection over weeks. A novel in vivo two-photon microscopy methodology was developed that allowed to determine the specific cellular and molecular features of breast cancer cells that homed in the brain, extravasated, and successfully established a brain macrometastasis. Those BM-initiating breast cancer cells (BMIC) were mainly originating from a slow-cycling subpopulation that included only 16% to 20% of all circulating cancer cells. BMICs showed enrichment of various markers of cellular stemness. As a proof of principle for the principal usefulness of this approach, expression profiling of BMICs versus non-BMICs was performed, which revealed upregulation of NDRG1 in the slow-cycling BMIC subpopulation in one BM model. Here, BM development was completely suppressed when NDRG1 expression was downregulated. In accordance, in primary human breast cancer, NDRG1 expression was heterogeneous, and high NDRG1 expression was associated with shorter metastasis-free survival. In conclusion, our data identify temporary slow-cycling breast cancer cells as the dominant source of brain and other metastases and demonstrates that this can lead to better understanding of BMIC-relevant pathways, including potential new approaches to prevent BM in patients. IMPLICATIONS: Cancer cells responsible for successful brain metastasis outgrowth are slow cycling and harbor stemness features. The molecular characteristics of these metastasis-initiating cells can be studied using intravital microscopy technology.

Pharmacological Modulation of Blood-Brain Barrier Permeability by Kinin Analogs in Normal and Pathologic Conditions.

The blood–brain barrier (BBB) is a major obstacle to the development of effective diagnostics and therapeutics for brain cancers and other central nervous system diseases. Peptide agonist analogs of kinin B1 and B2 receptors, acting as BBB permeabilizers, have been utilized to overcome this barrier. The purpose of the study was to provide new insights for the potential utility of kinin analogs as brain drug delivery adjuvants. In vivo imaging studies were conducted in various animal models (primary/secondary brain cancers, late radiation-induced brain injury) to quantify BBB permeability in response to kinin agonist administrations. Results showed that kinin B1 (B1R) and B2 receptors (B2R) agonists increase the BBB penetration of chemotherapeutic doxorubicin to glioma sites, with additive effects when applied in combination. B2R agonist also enabled extravasation of high-molecular-weight fluorescent dextrans (155 kDa and 2 MDa) in brains of normal mice. Moreover, a systemic single dose of B2R agonist did not increase the incidence of metastatic brain tumors originating from circulating breast cancer cells. Lastly, B2R agonist promoted the selective delivery of co-injected diagnostic MRI agent Magnevist in irradiated brain areas, depicting increased vascular B2R expression. Altogether, our findings suggest additional evidence for using kinin analogs to facilitate specific access of drugs to the brain.

Response to growth hormone in patients with RNPC 3 mutations

Corrigendum7 September 2020Open Access Response to growth hormone in patients with RNPC3 mutations Gabriel Á Martos-Moreno Gabriel Á Martos-Moreno Search for more papers by this author Lourdes Travieso-Suárez Lourdes Travieso-Suárez Search for more papers by this author Jesús Pozo-Román Jesús Pozo-Román Search for more papers by this author María T Muñoz-Calvo María T Muñoz-Calvo Search for more papers by this author Julie A Chowen Julie A Chowen Search for more papers by this author Mikko J Frilander Mikko J Frilander Search for more papers by this author Luis A Pérez-Jurado Luis A Pérez-Jurado Search for more papers by this author Federico G Hawkins Federico G Hawkins Search for more papers by this author Jesús Argente Jesús Argente orcid.org/0000-0001-5826-0276 Search for more papers by this author Gabriel Á Martos-Moreno Gabriel Á Martos-Moreno Search for more papers by this author Lourdes Travieso-Suárez Lourdes Travieso-Suárez Search for more papers by this author Jesús Pozo-Román Jesús Pozo-Román Search for more papers by this author María T Muñoz-Calvo María T Muñoz-Calvo Search for more papers by this author Julie A Chowen Julie A Chowen Search for more papers by this author Mikko J Frilander Mikko J Frilander Search for more papers by this author Luis A Pérez-Jurado Luis A Pérez-Jurado Search for more papers by this author Federico G Hawkins Federico G Hawkins Search for more papers by this author Jesús Argente Jesús Argente orcid.org/0000-0001-5826-0276 Search for more papers by this author Author Information Gabriel Á Martos-Moreno, Lourdes Travieso-Suárez, Jesús Pozo-Román, María T Muñoz-Calvo, Julie A Chowen, Mikko J Frilander, Luis A Pérez-Jurado, Federico G Hawkins and Jesús Argente EMBO Mol Med (2020)12:e13132https://doi.org/10.15252/emmm.202013132 This article corrects the following: Response to growth hormone in patients with RNPC3 mutations06 June 2018 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The authors recently noticed that one of the reported mutations, chr1:104093621C>A/p.P474T, was incorrectly listed as c.1320C>A at the cDNA level. The correct mutation is c.1420C>A; the nomenclature at the protein level was correct. This genetic variant is correctly listed in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/587367/), has an allele frequency of 0.00003185 in gnomAD (https://gnomad.broadinstitute.org/variant/1-104093621-C-A?dataset=gnomad_r2_1) and has been reported in a second family with growth hormone deficiency due RNPC3 dysfunction (Verberne et al, 2020). References Verberne EA, Faries S, Mannens MMAM, Postma AV, van Haelst MM (2020) Expanding the phenotype of biallelic RNPC3 variants associated with growth hormone deficiency. Am J Med Genet A https://doi.org/10.1002/ajmg.a.61632Wiley Online LibraryWeb of Science®Google Scholar Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 12,Issue 9,07 September 2020This month's cover highlights the article Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity by Claudia Koch, Andra Kuske, Klaus Pantel and colleagues. New estrogen receptor‐positive breast cancer cell line CTC‐ITB‐01 enables functional studies of circulating tumor cells. Cytoplasmatic keratin (orange) and nuclear estrogen receptor (green) protein expression in CTC‐ITB‐01 cells. DAPI nuclear staining (blue). (Scientific image by Claudia Koch et al., Institute of Tumor Biology, University Medical Center Hamburg, Eppendorf, Hamburg, Germany) Volume 12Issue 97 September 2020In this issue ReferencesRelatedDetailsLoading ...

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC‐derived breast cancer cell line, designated CTC‐ITB‐01, established from a patient with metastatic estrogen receptor‐positive (ER+) breast cancer, resistant to endocrine therapy. CTC‐ITB‐01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC‐ITB‐01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA‐sequencing data indicate that CTC‐ITB‐01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC‐ITB‐01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC‐ITB‐01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

Clinical value and feasibility of ISET in detecting circulating tumor cells in early breast cancer.

BackgroundPatients with operable breast cancer have a better prognosis for recovery. However, once distant organ metastasis occurs, the chance of a long-term survival or a cure is limited. The collection and counting of circulating tumor cells (CTCs) by reliable detection techniques are of increasing importance in the diagnosis of early metastasis and prognosis of disease progression. Isolation by size of epithelial tumor cells (ISET) has the advantage of simplicity of operation and high homogeneity. It is practical for large-scale clinical detection showing cell abundance. The value of ISET in the detection of circulating breast cancers in the blood has not been determined. The purpose of this study is to explore the feasibility of applying ISET to detect CTCs by determining the detection rate of ISET in operable breast cancer and by evaluating the correlation between detection rate, cell count and clinical factors such as molecular typing and pathological staging.MethodsThe experiment included 193 breast cancer patients who were diagnosed by core needle biopsy before the operation. 10 mL of venous blood was collected from the patients preoperatively, and CTCs in their blood samples were counted and analyzed by ISET.ResultsPatients were divided into groups according to pathology and immunohistochemistry. The overall detection rate of CTCs by ISET was 41.24%. The detection rate, the number of overall CTCs and the average number of CTCs in each group were analyzed individually. No significant differences were observed between the different groups.ConclusionsAlthough ISET has a relatively good detection rate for circulating breast cancer cells, it fails to provide more information on pathological staging, molecular classification and so forth.

RANKL/RANK System-Based Mechanism for Breast Cancer Bone Metastasis and Related Therapeutic Strategies.

Breast cancer remains one of the most life-threatening tumors affecting women. Most patients with advanced breast cancer eventually develop metastatic diseases, which cause significant morbidity and mortality. Approximately two-thirds of patients with advanced breast cancer exhibit osteolytic-type bone metastasis, which seriously reduce the quality of life. Therefore, development of novel therapeutic strategies for treating breast cancer patients with bone metastasis is urgently required. The “seed and soil” theory, which describes the interaction between the circulating breast cancer cells (seeds) and bone microenvironment (soil), is widely accepted as the mechanism underlying metastasis. Disruption of any step in this cycle might have promising anti-metastasis implications. The interaction of receptor activator of nuclear factor-κB ligand (RANKL) and its receptor RANK is fundamental in this vicious cycle and has been shown to be a novel effective therapeutic target. A series of therapeutic strategies have been developed to intervene in this cross-talk. Therefore, in this review, we have systematically introduced the functions of the RANKL/RANK signaling system in breast cancer and discussed related therapeutic strategies.

Rapid Detection of Circulating Breast Cancer Cells Using a Multiresonant Optical Fiber Aptasensor with Plasmonic Amplification.

The detection of circulating tumor cells (CTCs), which are responsible for metastasis in several forms of cancer, represents an important goal in oncological diagnosis and treatment. These cells remain extremely challenging to detect, despite numerous previous studies, due to their low concentration (1-10 cells/mL of blood). In this work, an all-fiber plasmonic aptasensor featuring multiple narrowband resonances in the near-infrared wavelength range was developed to detect metastatic breast cancer cells. To this aim, specific aptamers against mammaglobin-A were selected and immobilized as receptors on the sensor surface. In vitro assays confirm that the label-free and real-time detection of cancer cells [limit of detection (LOD) of 49 cells/mL] occurs within 5 min, while the additional use of functionalized gold nanoparticles allows a 2-fold amplification of the biosensor response. Differential measurements on selected optical resonances were used to process the sensor response, and results were confirmed by microscopy. The detection of only 10 cancer cells/mL was achieved with relevant specificity against control cells and with quick response time.

β2ARs stimulation in osteoblasts promotes breast cancer cell adhesion to bone marrow endothelial cells in an IL-1β and selectin-dependent manner

Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to impact the hypothalamic-pituitary axis and the autonomic nervous system. Preclinical and clinical evidence support the involvement of the sympathetic nervous system in the control of bone remodeling and in pathologies of the skeleton, including bone metastasis. In experimental mouse models of skeletal metastasis, administration of the βAR agonist isoproterenol (ISO), used as a surrogate of norepinephrine, the main neurotransmitter of sympathetic neurons, was shown to favor bone colonization of metastatic breast cancer cells via an increase bone marrow vascularity. However, successful extravasation of cancer cells into a distant organ is known to be favored by an activated endothelium, itself stimulated by inflammatory signals. Based on the known association between high sympathetic outflow, the expression of inflammatory cytokines and bone metastasis, we thus asked whether βAR stimulation in osteoblasts may alter the vascular endothelium to favor cancer cell engraftment within the skeleton. To address this question, we used conditioned medium (CM) from PBS or ISO-treated bone marrow stromal cells (BMSCs) in adhesion assays with bone marrow endothelial cells (BMECs) or the endothelial cell line C166. We found that ISO treatment in differentiated BMSCs led to a robust induction of the pro-inflammatory cytokines interleukin-1 beta (IL-1β) and interleukin-6 (IL-6). The CM from ISO-treated BMSCs increased the expression of E- and P-selectin in BMECs and the adhesion of human MDA-MB-231 breast cancer cells to these cells in short-term static and dynamic adhesion assays, and a blocking antibody against IL-1β, but not IL-6, reduced this effect. Direct IL-1β treatment of BMECs had a similar effect, whereas the impact of IL-6 treatment on the expression of adhesion molecules by BMECs and on the adhesion of cancer cells to BMECs was negligible. Collectively, these in vitro results suggest that in the context of the multicellular and dynamic bone marrow environment, sympathetic activation and subsequent βAR stimulation in osteoblasts may profoundly remodel the density but also the activation status of bone marrow vessels to favor the skeletal engraftment of circulating breast cancer cells.

Abstract 4589: Expression of PD-L1 on circulating breast cancer cells: Correlation with clinicopathologic data and impact on prognosis

Abstract Purpose Immune therapies, including anti-PD-L1 therapies, are widely evaluated in the treatment of solid tumors, including metastatic breast cancer (MBC). While tumor PD-L1 expression has been largely reported, with conflicting results regarding its theranostic value, few data are available regarding the prognostic value of the determination of PD-L1 expression on circulating tumor cells (CTCs), allowing a minimally invasive determination of this biomarker of interest. We previously published a validated and robust method for PD-L1(+)-CTC detection. In this new report, we focused on the clinicopathological correlations and prognostic value of the presence of PD-L1(+)-CTCs in an expanded MBC cohort. Methods We investigated PD-L1(+)-CTCs as previously reported (Mazel et al., Molecular Oncology, 9 (2015); 1773-1782) using the CellSearch® system in 72 MBC patients from our previous report and a prospective dedicated biomarker study (NCT02866149). Results Median age was 65 years (range 35-87). 69.4% of tumors were classified ER(+)/HER2(-), 18.1% HER2(+) and 12.5% triple negative (TN). 40 patients (55.6%) were treated in ≥ fourth metastatic line. 58.3% of the patients presented with more than 2 metastatic sites. Archival tissue was available for 56 patients (77.8%) and 13 samples (23.2%) showed positive (≥1%) PD-L1 expression. Baseline CTCs were detected in 57 patients (79.2%), with 43 (59.7%) and 36 (50%) patients with ≥4 and ≥10 CTCs, respectively. PD-L1(+)-CTCs were detected in 26 (36.1%) cases. No statistically significant correlation was found between PD-L1 tumor tissue expression and the presence of PD-L1(+)-CTCs (p=0.694). At the time of analysis, with a median follow-up of 13 months, 55 patients (76.4%) had progressed and 33 (45.8%) died. Median progression-free survival (PFS) was 5 months (95%CI 3.5-8.0). In univariate analysis, TN phenotype (p=0.020), more than 3 previous metastatic treatment lines (p<0.001) and detection of PD-L1(+)-CTCs (p=0.047) were significantly associated with PFS, while it was not the case for the CTC count (≥4 CTCs threshold) or the PD-L1 expression in the biopsy (p=0.100 and p=0.280). Median overall survival (OS) was 20 months (95%CI 11.1-38.3). In univariate analysis, TN phenotype (p=0.007) and the CTC count, either at the ≥4 (p=0.024) or ≥10 (p=0.027) threshold were significantly associated with OS, while there was a trend for association with OS of PD-L1(+)-CTCs detection (p=0.078). Conclusion In this heterogeneous MBC cohort, PD-L1 expression on CTCs appears independent of PD-L1 expressed in the primary tumor site. MBC patients harboring PD-L1(+)-CTCs seems to have a worse PFS. Evaluation of the respective impact of PD-L1 expression in the tumor and by CTCs is needed in a population treated by anti-PD-1/PD-L1 antibodies in order to evaluate their respective predictive values. An update of the OS data is warranted. Citation Format: William Jacot, Martine Mazel, Caroline Mollevi, Stéphane Pouderoux, Véronique D'Hondt, Laure Cayrefourcq, Angélique Ducteil, Marie Viala, Thierry Maudelonde, Séverine Guiu, Catherine Alix-Panabières. Expression of PD-L1 on circulating breast cancer cells: Correlation with clinicopathologic data and impact on prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4589.

Abstract 35: Triple-negative breast cancer cells alter the pulmonary landscape to favor metastasis via exosome-mediated release of NM23

Abstract There is strong evidence implicating exosomes, cell-derived nanoscale vesicles, and their protein cargo in facilitating dialogue between primary tumor cells and the premetastatic niche. Identified by our lab as one such protein, secreted nucleoside diphosphate kinase (NM23/NDPK) has been shown to activate endothelial remodeling in support of proangiogenic and prometastatic events. We have previously demonstrated that triple-negative breast cancer (MDA-MB-231) cells elaborate exosomes enriched with NDPK transphosphorylase activity and target the lung as a future site of metastasis. Using in vitro and in vivo approaches, we interrogate the functional role of NDPK in exosome-mediated lung remodeling. In cell-based assays, we demonstrate that MDA-MB-231 exosomes stimulate migration of pulmonary vascular endothelial cells and enhance monolayer permeability. These effects are reversed following treatment with an inhibitor of NDPK activity or an antagonist to the purinergic P2Y1 receptor. Further, we show that immune-compromised mice receiving intravenous injections of MDA-MB-231 exosomes exhibit enhanced pulmonary vascular leakage, whereas treatment with an inhibitor of NDPK attenuates this effect. Using tandem mass tag labeling and mass spectrometry, we profiled proteomic changes to the lung and identified the purinergic signaling pathway as among those significantly affected by exosome treatment. These results elucidate the functional role of exosomal NDPK in compromising vascular integrity and transforming the pulmonary landscape to support colonization by circulating breast cancer cells. Citation Format: Suzann Duan, Senny Nordmeier, Aidan E. Byrnes, Miles A. Brown, Iain L. Buxton. Triple-negative breast cancer cells alter the pulmonary landscape to favor metastasis via exosome-mediated release of NM23 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 35.