Unknown,Transcriptomics,Genomics,Proteomics

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Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures

ABSTRACT: The study of breast cancer pathogenesis relies heavily on the use of established cell lines often derived from metastatic lesions, which while having significantly contributed to the knowledge of breast cancer biology may inadvertently limit the understanding of the mechanisms governing the metastatic process. Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. These cellular models differ from recently developed patient derived xenograft models (PDX) in that they can be used for both in vitro and in vivo studies. Here we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as “dissociated tumor (DT) cells”. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, and a group of cancer-associated fibroblasts (CAFs). In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein and gene expression profiling, including PAM50 predictor analysis. The latter showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes, offering novel cellular models of these ER-negative breast cancer subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. DT cultures comprised of CAFs were isolated from luminal-A, Her2-enriched and basal primary tumors, providing subtype-specific components of the tumor microenvironment. Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis and tumor microenvironment. reference x sample

ORGANISM(S): Homo sapiens

SUBMITTER: Charles Perou

PROVIDER: E-GEOD-55070 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures.

Drews-Elger Katherine K Brinkman Joeli A JA Miller Philip P Shah Sanket H SH Harrell J Chuck JC da Silva Thiago G TG Ao Zheng Z Schlater Amy A Azzam Diana J DJ Diehl Kathleen K Thomas Dafydd D Slingerland Joyce M JM Perou Charles M CM Lippman Marc E ME El-Ashry Dorraya D

Breast cancer research and treatment 20140225 3

Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as "dissociated tumor (DT) cells." In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, in ...[more]

PMID: 24567196

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Project description:Abstract Maspin is a tumor and metastasis suppressor playing an essential role as gatekeeper of tumor progression. It is highly expressed in epithelial cells but is silenced in the onset of metastatic disease by epigenetic mechanisms. Reprogramming of Maspin epigenetic silencing offers a therapeutic potential to lock metastatic progression. Herein we have investigated the ability of the Artificial Transcription Factor 126 (ATF-126) designed to upregulate the Maspin promoter to inhibit tumor progression in pre-established breast tumors in immunodeficient mice. ATF-126 was transduced in the aggressive, mesenchymal-like and triple negative breast cancer line, MDA-MB-231. Induction of ATF expression in vivo by Doxycycline resulted in 50% reduction in tumor growth and totally abolished tumor cell colonization. Genome-wide transcriptional profiles of ATF-induced cells revealed a gene signature that was found over-represented in estrogen receptor positive (ER+) “Normal-like” intrinsic subtype of breast cancer and in poorly aggressive, ER+ luminal A breast cancer cell lines. The comparison transcriptional profiles of ATF-126 and Maspin cDNA defined an overlapping 19-gene signature, comprising novel targets downstream the Maspin signaling cascade. Our data suggest that Maspin up-regulates downstream tumor and metastasis suppressor genes that are silenced in breast cancers, and are normally expressed in the neural system, including CARNS1, SLC8A2 and DACT3. In addition, ATF-126 and Maspin cDNA induction led to the re-activation of tumor suppressive miRNAs also expressed in neural cells, such as miR-1 and miR-34, and to the down-regulation of potential oncogenic miRNAs, such as miR-10b, miR-124, and miR-363. As expected from its over-representation in ER+ tumors, the ATF-126-gene signature predicted favorable prognosis for breast cancer patients. Our results describe for the first time an ATF able to reduce tumor growth and metastatic colonization by epigenetic reactivation of a dormant, normal-like, and more differentiated gene program. A total of six cell lines were used for gene expression analyses: CONTROL –DOX, CONTROL +DOX, ATF-126 –DOX, ATF-126 +DOX (all with 3 technical replicates), p-RetoX-Tight-Maspin –DOX, and p-RetoX-Tight-Maspin +DOX (with 2 technical replicates). For each cell line, total RNA was purified, amplified, labeled, and hybridized [46] using Agilent Agilent 4X44K oligo microarrays (Agilent Technologies, United States). The probes/genes were filtered by requiring the lowest normalized intensity values in both –DOX and +DOX samples to be >10. The normalized log2 ratios (Cy5 sample/Cy3 control) of probes mapping to the same gene were averaged to generate independent expression estimates. We also used available microarrays from the breast cancer cell lines [21], the UNC337-patient [20], the MERGE 550-patient dataset [47] and the NKI (295 patients [48,49]). All microarray cluster analyses were displayed using Java Treeview version 1.1.3. Average-linkage hierarchical clustering was performed using Cluster v2.12 [50]. ANOVA tests for gene expression data were performed using R (http://cran.r-project.org).

Project description:Background: Human breast cancer is a heterogeneous disease consisting of multiple molecular subtypes. Genetically engineered mouse models (GEMMs) are useful resources for studying breast cancers in vivo under genetically controlled and immune competent conditions. Identifying murine models with conserved human tumor features will facilitate etiology determinations, highlight the effects of mutations on pathway activation, and improve preclinical drug validation. Results: Transcriptomic profiles of 27 murine models of mammary carcinoma and normal mammary tissue were determined using gene expression microarrays. Hierarchical clustering analysis identified 17 distinct murine subtypes (classes). Across species analyses using three independent human breast cancer datasets identified eight murine classes that represent specific human breast cancer subtypes. Multiple models were associated with human basal-like tumors including TgC3(1)-Tag, TgWap-Myc, and Trp53-/-. Interestingly, the TgWAPCre-Etv6 model mimicked the HER2-enriched subtype, a group of human tumors without a murine counterpart in previous comparative studies. Gene signature analysis identified hundreds of commonly expressed pathways between linked mouse and human subtypes, highlighting potentially common genetic drivers of tumorigenesis and candidate pathways for therapeutic intervention. Conclusion: This study consolidates murine models of breast carcinoma into the largest comprehensive transcriptomic dataset to date to identify human-mouse disease subtype counterparts. This approach illustrates the value of comparisons between species to identify murine models that faithfully mimic the human condition and indicates that multiple GEMMs are needed to represent the diversity of human breast cancers. These trans-species associations should guide model selection during preclinical study design to ensure appropriate representatives of the human disease subtypes are used. Keywords: breast cancer, comparative genomics, genetically engineered mouse models, and molecular pathway signatures reference x sample

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Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures

Publications

Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures.

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