IntroductionReal-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.MethodsCTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.ResultsIn total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.ConclusionThis study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0508-5) contains supplementary material, which is available to authorized users.

Human epidermal growth factor receptor-2-overexpressing (HER2+) breast cancer is an aggressive tumor. Despite the clinical success of anti-HER2 drugs such as lapatinib (L) and trastuzumab (T), intrinsic and acquired drug resistance occurs in many patients. Identification of novel drug targets in HER2+ breast cancer is an unmet clinical need. In this context, G-protein coupled receptors (GPCRs) may be excellent drug targets because they cross-talk with the HER family members. However, the expression and function of the majority of GPCRs are unknown in HER2+ breast cancer. In a preliminary study, we examined the differential gene expression of GPCRs in anti-HER2 treatment-resistant derivatives as well as in the tumorigenic cell population, suggested to be involved in resistance, of a BT474 cell line model of HER2+ breast cancer. Anti-HER2 resistant derivatives of BT474 cells were established by long-term exposure of parental cells to increasing concentrations of L, T, or their combination (L+T). Tumorigenic cells were identified as aldehyde dehydrogenase-positive (ALDH+) cells using the Aldefluor assay. RNA was profiled using TaqMan real time RT-PCR GPCR 384-well microarray to quantify the expression of mRNA encoding 343 GPCRs. The publically available TCGA dataset was interrogated to determine differential mRNA expression of selected GPCRs in HER2+ and other subtypes of breast cancer. To determine the functional role of GPR110, BT474 cells were infected with lentiviral GPR110 construct (GPR110-OE) or empty vector (EV), and stable pools were obtained. Anchorage-dependent cell growth was evaluated using MTT cell proliferation assay over 8 days. Tumorigenic potential was determined by calculating the% of ALDH+ cells using Aldefluor assay and by evaluating the anchorage-independent cell growth using soft agar assay over 14 days. The influence of GPR110 overexpression on HER signaling pathway was investigated by measuring the levels of phosphorylated (active) and total protein levels of HER1 and HER2 using immunoblotting. GPR110 was the only GPCR overexpressed in resistant derivatives versus parental cells as well as in ALDH+ versus ALDH- cells of BT474 cells. In TCGA dataset, GPR110 expression was significantly higher in HER2+ and basal subtypes of breast cancer compared to ER+ luminal A and B subtypes. Overexpression of GPR110 in BT474 cells (9-fold in GPR110-OE vs. EV cells) resulted in a marked 5-fold increase in the number of colonies when grown in soft agar compared to EV cells even though anchorage-dependent cell growth was not significantly different between EV and GPR110-OE cells. In addition, GPR110-OE cells had a significantly higher% of ALDH+ population compared to EV cells. Phosphorylated (but not total) HER1 and HER2 protein levels were significantly higher in GPR110-OE cells compared to EV cells, suggesting hyperactive HER signaling with GPR110 overexpression. Experiments with L and T treatment will reveal the role of GPR110 in drug efficacy and acquired resistance. In summary, we show for the first time a pro-tumorigenic role of GPR110 in HER2+ breast cancer. Therefore, GPR110 may be a novel pharmacological target in HER2+ breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-05.

Background: Recent reports suggest that circulating tumor cells (CTCs) mimic cancer progenitor cells, which are commonly resistant to various treatment modalities and are capable of invasion and tumor initiation at a single cell level. We hypothesized that CTCs isolated from metastatic breast cancer (MBC) patients can be propagated in 3-D in vitro systems, representing a clinically relevant model to study MBC biology and to test efficacy of multiple anti-cancer drugs. Methods: CTCs were isolated from MBC patient peripheral blood by depleting leucocytes and red blood cells. Enriched CTCs were immunostained for detection as CD45-negative (-) and nuclear counter stain-positive (+) cells, and for expression of estrogen receptor (ER) and HER-2. In order to obtain a large and continuous supply of CTCs for our culture studies, we screened 19 primary transplantable xenograft lines, established by directly transplanting human breast tumors into epithelium-free mammary fat pads of SCID/Beige mice, for their production of CTCs. The CTCs were isolated together with mouse mononuclear blood cells by either gradient centrifugation or red blood cell lysis, and identified by human pan-cytokeratin immunostaining. Viable CTCs isolated from both patients and xenograft mice were plated in mammosphere conditions either alone or in co-culture with mammary fibroblasts tagged with green fluorescent protein (GFP). the propagated CTCs were identified as GFP-, cytokeratin 19+ cells. Results: To date, we have collected and processed blood samples from 14 MBC patients. CTCs were detected in 5 out of the 8 samples (62%) already analyzed. Interestingly, in 2 out of 4 patients (50%) with positive CTC counts and ER+ primary tumor, all the isolated CTCs were ER-. Moreover, we found HER-2+ CTCs in 1 out of 4 patients (25%) with HER-2 negative disease. CTC count and characterization of the additional collected samples is ongoing and will be presented at the conference. In the mice bearing primary xenografts, we detected CTCs in 13 out of 19 lines (68%) and in 26 out of 54 mice (48%). When CTCs isolated from either human patients or primary xenografts were cultured alone, no mammospheres were observed. In contrast, when CTCs were cultured in direct contact with fibroblasts we observed the formation of mixed CTC/fibroblast spheres that may reproduce the tumor cell/stroma interaction observed in vivo. Moreover, in the presence of GFP+ fibroblasts, the numbers of GFP- cells and cell aggregates were clearly higher at all time points compared to the single culture. Conclusions: Our study suggests that viable CTCs isolated from human patients and primary xenograft bearing mice can be propagated in vitro in presence of mammary fibroblasts. The successful development of a CTC-based cell culture platform will provide a unique model to better understand the biological mechanisms of the metastatic process and to guide treatment decisions in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-305. doi:1538-7445.AM2012-LB-305

Introduction: Real-time monitoring of biological changes in tumors that metastasize may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in patients with localized disease pose major hurdles. The goal of this study was to determine if breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes. Methods: CTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counter staining of RBC-lysed blood and bone marrow fractions, respectively. The lung metastasis (LM) rate was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed using the gene expression arrays of primary tumors from different PDX lines and were subsequently overlapped to identify common genes. Results: A total of 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present either as single cells or as clusters, were detected in 83% (15/18) and 62.5% (10/16) of the lines, respectively. There was a positive association between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 out of 18 (50%) lines, of which all 9 had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the 2 genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified 4 genes (HLA-DP1A, GJA1, PEG3, and XIST). This 4-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients. Conclusion: This study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases. Citation Format: Mario Giuliano, Sabrina Herrera, Pavel Christiny, Chad Shaw, Chad J Creighton, Tamika Mitchell, Raksha Bhat, Xiaomei Zhang, Sufeng Mao, Lacey Dobrolecki, Ahmed Al-rawi, Fengju Chen, Bianca M Veneziani, Xiang H Zhang, Susan G Hilsenbeck, Alejandro Contreras, Carolina Gutierrez, Rinath Jeselsohn, Mothaffar F Rimawi, C Kent Osborne, Michael T Lewis, Rachel Schiff, Meghana V Trivedi. Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-06.

Background: G protein-coupled receptors (GPCRs) are excellent drug targets. However, not many GPCRs have been identified as potential drug targets in breast cancer (BC). This may be due to two factors: (1) The primary mode of GPCR regulation is at the level of signaling rather than gene expression, which is the common method for large-scale target identification. (2) GPCR expression in BC stem cells or drug-resistant cells could have been overlooked because these cells exist in small fractions. In this study, we aimed to identify novel GPCR targets based on large panel functional screening and differential gene expression in HER2+ BC stem cells and drug resistant cells. Methods: (I) For the functional screening, each of the 370 GPCR genes was knocked down using targeted Silencer® Select siRNA library comprising of 3 individual siRNAs per target in BT474 and SKBR3 cell lines (N = 3). Candidate GPCRs were selected based on at least 20% alteration in cell growth by at least 2 out of 3 siRNAs. (II) For the expression screening, gene expression of 343 GPCRs was measured in ALDH+ BC stem cells vs. ALDH- cells as well as in anti-HER2 drug-resistant derivatives vs. parental cells of BT474 cells using Taqman RT-PCR arrays (N = 2-4). Melatonin MT1 receptor (MTNR1A) gene knockdown and pharmacological modulation were employed to functionally validate its role in anchorage-dependent (MTT assay) and -independent (soft agar colony formation assay) cell growth. Results: Gene knockdown of 11 GPCRs (ADORA, ADRA1D, CCR8, GNRHR, GPR26, GPR113, GPR120, LPAR3, MC5R, MTNR1A, NTSR2) altered cell growth of HER2+ BC cells by at least 20%. The MT1 receptor was selected as a candidate GPCR for this study because (1) its knockdown resulted in at least 20% inhibition of cell growth in both BT474 and SKBR3 cells, and (2) its expression was increased in ALDH+ vs. ALDH- as well as in drug-resistant cells vs. parental BT474 cells. MTNR1A knockdown resulted in 25-35% inhibition in the anchorage-dependent cell growth of both BT474 and SKBR3 cells. Furthermore, MTNR1A knockdown caused a marked (3-fold) decrease in the number of colonies in soft agar assay. MT1 receptor antagonist luzindole reduced the anchorage-dependent cell growth of BT474 cells in a concentration-dependent manner (IC50 = 20-55 nM). However, the efficacy of luzindole was only ∼50% of lapatinib efficacy in reducing cell growth. Luzindole (1μM) also significantly reduced (by 2-fold) anchorage-independent cell growth of BT474 cells. Neither MTNR1A knockdown nor luzindole significantly improved the efficacy of lapatinib (1nM) in inhibiting cell growth of BT474 cells. Conclusion: In summary, functional knockdown screening and differential gene expression of GPCRs is a powerful tool for identifying GPCRs as candidate targets in BC. Using the combination of these two approaches, we have discovered that melatonin MT1 receptor as a potential drug target in HER2+ BC cells. The place in therapy for MT1 receptor antagonists, however, still remains to be determined. Citation Format: Raksha Bhat, Puja Yadav, Pavel Christiny, Rachel Schiff, Meghana V. Trivedi. Novel G protein-coupled receptor targets in HER2+ breast cancer. [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 3573. doi:10.1158/1538-7445.AM2015-3573