Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy

The tumor microenvironment consists of stromal cells and extracellular factors that evolve in parallel with carcinoma cells. To gain insights into the activities of stromal cell populations, we developed and applied multicolor imaging techniques to analyze the behavior of these cells within different tumor microenvironments in the same live mouse. We found that regulatory T-lymphocytes (Tregs) migrated in proximity to blood vessels. Dendriticlike cells, myeloid cells and carcinoma-associated fibroblasts all exhibited higher motility in the microenvironment at the tumor periphery than within the tumor mass. Since oxygen levels differ between tumor microenvironments, we tested if acute hypoxia could account for the differences in cell migration. Direct visualization revealed that Tregs ceased migration under acute systemic hypoxia, whereas myeloid cells continued migrating. In the same mouse and microenvironment, we experimentally subdivided the myeloid cell population and revealed that uptake of fluorescent dextran defined a low-motility subpopulation expressing markers of tumor-promoting, alternatively activated macrophages. In contrast, fluorescent anti-Gr1 antibodies marked myeloid cells patrolling inside tumor vessels and in the stroma. Our techniques allow real-time combinatorial analysis of cell populations based on spatial location, gene expression, behavior and cell surface molecules within intact tumors. The techniques are not limited to investigations in cancer, but could give new insights into cell behavior more broadly in development and disease

High Intratumoral Stromal Content Defines Reactive Breast Cancer as a Low-risk Breast Cancer Subtype

The current study evaluated associative effects of breast cancer cells with the tumor microenvironment and its influence on tumor behavior

Epigenetic silencing of TGFBI confers resistance to trastuzumab in human breast cancer

Background: acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients. Methods: we performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing. Results: epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients. Conclusions: our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients

Comprehensive characterization of 536 patient-derived xenograft models prioritizes candidatesfor targeted treatment

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors

NEFL mRNA Expression Level Is a Prognostic Factor for Early-Stage Breast Cancer Patients

Neurofilament, light polypeptide (NEFL) was demonstrated to be ectopically expressed in breast cancer tissues and decreased in lymph node metastases compared to the paired primary breast cancers in our previous study. Moreover, in several studies, NEFL was regarded as a tumor suppressor gene, and its loss of heterozygosity (LOH) was related to carcinogenesis and metastasis in several types of cancer. To explore the role of NEFL in the progression of breast cancer and to evaluate its clinical significance, we detected the NEFL mRNA level in normal breast tissues, primary breast cancer samples and lymph node metastases, and then analyzed the association between the NEFL expression level and several clinicopathological parameters and disease-free survival (DFS). NEFL mRNA was found to be expressed in 92.3% of breast malignancies and down-regulated in lymph node metastases compared to the paired primary tumors. NEFL mRNA level was lower in primary breast cancers with positive lymph nodes than in cancers with negative lymph nodes. Moreover, a low expression level of NEFL mRNA indicated a poor five-year DFS for early-stage breast cancer patients. Thus, NEFL mRNA is ectopically expressed in breast malignancies and could be a potential prognostic factor for early-stage breast cancer patients

Surface TRAIL decoy receptor-4 expression is correlated with TRAIL resistance in MCF7 breast cancer cells

BACKGROUND: Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. Despite this promising feature, TRAIL resistance observed in cancer cells seriously challenged the use of TRAIL as a death ligand in gene therapy. The current dispute concerns whether or not TRAIL receptor expression pattern is the primary determinant of TRAIL sensitivity in cancer cells. This study investigates TRAIL receptor expression pattern and its connection to TRAIL resistance in breast cancer cells. In addition, a DcR2 siRNA approach and a complementary gene therapy modality involving IKK inhibition (AdIKKβKA) were also tested to verify if these approaches could sensitize MCF7 breast cancer cells to adenovirus delivery of TRAIL (Ad5hTRAIL). METHODS: TRAIL sensitivity assays were conducted using Molecular Probe's Live/Dead Cellular Viability/Cytotoxicity Kit following the infection of breast cancer cells with Ad5hTRAIL. The molecular mechanism of TRAIL induced cell death under the setting of IKK inhibition was revealed by Annexin V binding. Novel quantitative Real Time RT-PCR and flow cytometry analysis were performed to disclose TRAIL receptor composition in breast cancer cells. RESULTS: MCF7 but not MDA-MB-231 breast cancer cells displayed strong resistance to adenovirus delivery of TRAIL. Only the combinatorial use of Ad5hTRAIL and AdIKKβKA infection sensitized MCF7 breast cancer cells to TRAIL induced cell death. Moreover, novel quantitative Real Time RT-PCR assays suggested that while the level of TRAIL Decoy Receptor-4 (TRAIL-R4) expression was the highest in MCF7 cells, it was the lowest TRAIL receptor expressed in MDA-MB-231 cells. In addition, conventional flow cytometry analysis demonstrated that TRAIL resistant MCF7 cells exhibited substantial levels of TRAIL-R4 expression but not TRAIL decoy receptor-3 (TRAIL-R3) on surface. On the contrary, TRAIL sensitive MDA-MB-231 cells displayed very low levels of surface TRAIL-R4 expression. Furthermore, a DcR2 siRNA approach lowered TRAIL-R4 expression on surface and this sensitized MCF7 cells to TRAIL. CONCLUSION: The expression of TRAIL-R4 decoy receptor appeared to be well correlated with TRAIL resistance encountered in breast cancer cells. Both adenovirus mediated IKKβKA expression and a DcR2 siRNA approach sensitized MCF7 breast cancer cells to TRAIL

Maspin is a tumour suppressor that inhibits breast cancer tumour metastasis in vivo

Maspin is a member of the serpin family of serine proteases and functions as a tumour suppressor. A study using a new syngeneic mouse model for breast cancer suggests that maspin can inhibit metastasis in vivo

MMTV-Wnt1 and -ΔN89β-Catenin Induce Canonical Signaling in Distinct Progenitors and Differentially Activate Hedgehog Signaling within Mammary Tumors

Canonical Wnt/β-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-ΔN89β-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin activate canonical Wnt signaling within distinct cell-types. ΔN89β-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18+ER−PR−CD24highCD49flow profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14+/p63+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-ΔN89β-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand