Spatial domain analysis predicts risk of colorectal cancer recurrence and infers associated tumor microenvironment networks.

An unmet clinical need in solid tumor cancers is the ability to harness the intrinsic spatial information in primary tumors that can be exploited to optimize prognostics, diagnostics and therapeutic strategies for precision medicine. Here, we develop a transformational spatial analytics computational and systems biology platform (SpAn) that predicts clinical outcomes and captures emergent spatial biology that can potentially inform therapeutic strategies. We apply SpAn to primary tumor tissue samples from a cohort of 432 chemo-naïve colorectal cancer (CRC) patients iteratively labeled with a highly multiplexed (hyperplexed) panel of 55 fluorescently tagged antibodies. We show that SpAn predicts the 5-year risk of CRC recurrence with a mean AUROC of 88.5% (SE of 0.1%), significantly better than current state-of-the-art methods. Additionally, SpAn infers the emergent network biology of tumor microenvironment spatial domains revealing a spatially-mediated role of CRC consensus molecular subtype features with the potential to inform precision medicine.

NDRG1 regulates neutral lipid metabolism in breast cancer cells.

BACKGROUND: Altered lipid metabolism is an emerging hallmark of aggressive breast cancers. The N-myc downstream regulated gene (NDRG1) gene plays a critical role in peripheral nervous system myelination, as inactivating mutations cause severe demyelinating neuropathy. In breast cancer, elevated NDRG1 expression has been linked to clinical outcomes, but its functional role in breast cancer physiology has remained unclear. METHODS: A meta-analysis of NDRG1 expression in multiple large publicly available genomic databases was conducted. Genome-wide expression correlation and Cox proportional hazards and Kaplan-Meier modeling of clinical outcomes associated with elevated expression were assessed. To study NDRG1 function, gene silencing and overexpression phenotypic studies were carried out in a panel of cell lines representing all major breast cancer molecular subtypes. Changes in cell proliferation, morphology, and neutral lipid accumulation due to altered NDRG1 expression were assessed by high throughput, quantitative microscopy. Comprehensive lipidomics mass spectrometry was applied to characterize global changes in lipid species due to NDRG1 silencing. Labeled fatty acids were used to monitor cellular fatty acid uptake and subcellular distribution under nutrient replete and starvation culture conditions. RESULTS: NDRG1 overexpression correlated with glycolytic and hypoxia-associated gene expression, and was associated with elevated rates of metastasis and patient mortality. Silencing NDRG1 reduced cell proliferation rates, causing lipid metabolism dysfunction including increased fatty acid incorporation into neutral lipids and lipid droplets. Conversely, NDRG1 expression minimized lipid droplet formation under nutrient replete and starvation conditions. CONCLUSIONS: Here we report that NDRG1 contributes to breast cancer aggressiveness by regulating the fate of lipids in cells that exhibit an altered lipid metabolic phenotype. In line with its role in promoting myelination and its association with altered metabolism in cancer, our findings show that NDRG1 is a critical regulator of lipid fate in breast cancer cells. The association between NDRG1 and poor prognosis in breast cancer suggests it should play a more prominent role in patient risk assessment. The function of NDRG1 in breast cancer lipid metabolism may represent a promising therapeutic approach in the future.

Single-Cell Mass Spectrometry of Subpopulations Selected by Fluorescence Microscopy.

Specific subpopulations in a heterogeneous collection of cells, for example, cancer stem cells in a tumor, are often associated with biological or medical conditions. Fluorescence microscopy, based on biomarkers labeled with fluorescent probes, is a widely used technique for the visualization and selection of such cells. Phenotypic differences for these subpopulations at the molecular level can be identified by their untargeted analysis by single-cell mass spectrometry (MS). Here, we combine capillary microsampling MS with fluorescence microscopy for the analysis of metabolite and lipid levels in single cells to discern the heterogeneity of subpopulations corresponding to mitotic stages. The distributions of ATP, reduced glutathione (GSH), and UDP- N-acetylhexosamine (UDP-HexNAc) levels in mitosis reveal the presence of 2-3 underlying subpopulations. Cellular energy is found to be higher in metaphase compared to prometaphase and slightly declines in anaphase, telophase, and cytokinesis. The [GTP]/[GDP] ratio in cytokinesis is significantly higher than in prometaphase and anaphase. Pairwise correlations between metabolite levels show that some molecules within a group, including certain amino acids and nucleotide sugars, are strongly correlated throughout mitosis, but this is not related to their pathway distances. Correlations are observed between monophosphates (AMP and GMP), diphosphates (ADP and GDP), and triphosphates (ATP and GTP) of different nucleosides. In contrast, there is low correlation between diphosphates and triphosphates of the same nucleoside (ADP and ATP).

Bruton's Tyrosine Kinase Inhibitors Prevent Therapeutic Escape in Breast Cancer Cells.

We have reported that a novel isoform of BTK (BTK-C) expressed in breast cancer protects these cells from apoptosis. In this study, we show that recently developed inhibitors of BTK, such as ibrutinib (PCI-32765), AVL-292, and CGI-1746, reduce breast cancer cell survival and prevent drug-resistant clones from arising. Ibrutinib treatment impacts HER2(+) breast cancer cell viability at lower concentrations than the established breast cancer therapeutic lapatinib. In addition to inhibiting BTK, ibrutinib, but not AVL-292 and CGI-1746, efficiently blocks the activation of EGFR, HER2, ErbB3, and ErbB4. Consequently, the activation of AKT and ERK signaling pathways are also blocked leading to a G1-S cell-cycle delay and increased apoptosis. Importantly, inhibition of BTK prevents activation of the AKT signaling pathway by NRG or EGF that has been shown to promote growth factor-driven lapatinib resistance in HER2(+) breast cancer cells. HER2(+) breast cancer cell proliferation is blocked by ibrutinib even in the presence of these factors. AVL-292, which has no effect on EGFR family activation, prevents NRG- and EGF-dependent growth factor-driven resistance to lapatinib in HER2(+) breast cancer cells. In vivo, ibrutinib inhibits HER2(+) xenograft tumor growth. Consistent with this, immunofluorescence analysis of xenograft tumors shows that ibrutinib reduces the phosphorylation of HER2, BTK, Akt, and Erk and histone H3 and increases cleaved caspase-3 signals. As BTK-C and HER2 are often coexpressed in human breast cancers, these observations indicate that BTK-C is a potential therapeutic target and that ibrutinib could be an effective drug especially for HER2(+) breast cancer. Mol Cancer Ther; 15(9); 2198-208. ©2016 AACR.

Stromal-Based Signatures for the Classification of Gastric Cancer.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

ARID1A expression in early stage colorectal adenocarcinoma: an exploration of its prognostic significance.

ARID1A is a chromatin remodeling gene that is mutated in a number of cancers including colorectal carcinoma (CRC). Loss of ARID1A has been associated with an adverse outcome in some types of cancer. However, literature data have not been consistent. Major limitations of some outcome studies include small sample size and heterogeneous patient population. In this study, we evaluated the prognostic value of ARID1A in a homogeneous group of early stage CRC patients, a population where prognostic markers are particularly relevant. We collected a retrospective series of 578 stage I or II CRCs. All patients underwent surgery with curative intent and without neoadjuvant or adjuvant therapy. ARID1A expression was analyzed by immunohistochemistry using tissue microarray. We found ARID1A loss in 49 of 552 analyzable tumors (8.9%). Compared with the ARID1A-retained group, cases with ARID1A loss were associated with female sex (P<.001), mismatch-repair protein deficiency (P<.001), poor differentiation (P<.001), lymphovascular invasion (P=.001), and higher pT stage (P=.047). However, at a median follow-up of 49months, ARID1A loss did not correlate with overall, disease-specific, or recurrence-free survival. This is the first systematic analysis to evaluate the prognostic significance of ARID1A in stage I/II CRCs, and our data indicate that ARID1A loss lacks prognostic significance in this population despite its association with other adverse features. Such data are clinically relevant, as efforts are ongoing in identifying markers that can detect the small but significant subset of early stage CRCs that will have a poor outcome.

Bruton's tyrosine kinase is a potential therapeutic target in prostate cancer.

Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that has mainly been studied in haematopoietic cells. We have investigated whether BTK is a potential therapeutic target in prostate cancer. We find that BTK is expressed in prostate cells, with the alternate BTK-C isoform predominantly expressed in prostate cancer cells and tumors. This isoform is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. Prostate cancer cell lines and prostate tumors express more BTK-C transcript than the malignant NAMALWA B-cell line or human lymphomas. BTK protein expression is also observed in tumor tissue from prostate cancer patients. Down regulation of this protein with RNAi or inhibition with BTK-specific inhibitors, Ibrutinib, AVL-292 or CGI-1746 decrease cell survival and induce apoptosis in prostate cancer cells. Microarray results show that inhibiting BTK under these conditions increases expression of apoptosis related genes, while overexpression of BTK-C is associated with elevated expression of genes with functions related to cell adhesion, cytoskeletal structure and the extracellular matrix. These results are consistent with studies that show that BTK signaling is important for adhesion and migration of B cells and suggest that BTK-C may confer similar properties to prostate cancer cells. Since BTK-C is a survival factor for these cells, it represents both a potential biomarker and novel therapeutic target for prostate cancer.

Emerging understanding of multiscale tumor heterogeneity.

Cancer is a multifaceted disease characterized by heterogeneous genetic alterations and cellular metabolism, at the organ, tissue, and cellular level. Key features of cancer heterogeneity are summarized by 10 acquired capabilities, which govern malignant transformation and progression of invasive tumors. The relative contribution of these hallmark features to the disease process varies between cancers. At the DNA and cellular level, germ-line and somatic gene mutations are found across all cancer types, causing abnormal protein production, cell behavior, and growth. The tumor microenvironment and its individual components (immune cells, fibroblasts, collagen, and blood vessels) can also facilitate or restrict tumor growth and metastasis. Oncology research is currently in the midst of a tremendous surge of comprehension of these disease mechanisms. This will lead not only to novel drug targets but also to new challenges in drug discovery. Integrated, multi-omic, multiplexed technologies are essential tools in the quest to understand all of the various cellular changes involved in tumorigenesis. This review examines features of cancer heterogeneity and discusses how multiplexed technologies can facilitate a more comprehensive understanding of these features.

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Autofluorescence removal using a customized filter set.

Quantitative fluorescence microscopy is severely hindered by intrinsic autofluorescence (AF). Endogenous fluorescent molecules in tissue and cell samples emit fluorescence that often dominates signals from specific dyes. This makes AF removal critical to the development and practice of quantitative fluorescence microscopy. In this study, we showed that AF signal could be separated from specific signal using a customized filter set. The filter set used the same excitation and beam splitter as the standard filter set, but the emission filter was red-shifted 40-60 nm from the peak of the specific dye. This filter set configuration collected mostly AF with minimum contribution from the specific dye. A linear transformation of AF images was required to correct for the difference in exposure and filter configuration. The constants (slope and intercept) in linear transformation were obtained through a pixel to pixel comparison between AF images (no staining) obtained by the standard filter set and the customized AF filter set. After staining of specific dye, the standard filter collecting target dye spectra was used to capture both target signal and AF, whereas customized filter was used to capture only AF. AF removal was accomplished by subtracting the linear transformed AF image from the image obtained from the standard filter. To validate our approach, we examined weak staining of androgen receptor in an AF abundant prostate tissue sample. Our method revealed a similar but cleaner nuclear staining of androgen receptor in a specimen, when compared to a traditional autofluorescence removal method.

Lipid biology of breast cancer.

Alterations in lipid metabolism have been reported in many types of cancer. Lipids have been implicated in the regulation of proliferation, differentiation, apoptosis, inflammation, autophagy, motility and membrane homeostasis. It is required that their biosynthesis is tightly regulated to ensure homeostasis and to prevent unnecessary energy expenditure. This review focuses on the emerging understanding of the role of lipids and lipogenic pathway regulation in breast cancer, including parallels drawn from the study of metabolic disease models, and suggestions on how these findings can potentially be exploited to promote gains in HER2/neu-positive breast cancer research. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Multiplexed analysis of proteins in tissue using multispectral fluorescence imaging.

We present a new application of multispectral analysis for subcellular measurement of multiple proteins in formalin-fixed paraffin embedded tissue and cells. Typically, the targets of interest are present in the same or spatially overlapping cellular compartments. Such co-localization can complicate analysis and interpretation of the images obtained using traditional fluorescence, especially when spectrally overlapping labels are present. The spectral properties of currently available fluorescent dyes set an upper limit to the number of molecules that can be detected simultaneously with traditional fluorescence. By exciting a set of fluorophores at the same wavelength and unmixing their emission signals from background autofluorescence, we were able to image three targets in a single channel. This parallel imaging approach provides significant advantages for multiplexed analysis of tissues and cells.