Gabi W. van Pelt

Presence of a high amount of stroma and downregulation of SMAD4 predict for worse survival for stage I-II colon cancer patients.

Background: For stage I–II colon cancer a significant number (5–25%) of patients has recurrent disease within 5 years. There is need to identify these high-risk patients as they might benefit from additional treatment. Stroma-tissue surrounding the cancer cells plays an important role in the tumor behavior. The proportion of intra-tumor stroma was evaluated for the identification of high-risk patients. In addition, protein expression of markers involved in pathways related to stroma production and epithelial-to-mesenchymal transition (EMT) was analyzed: β-catenin, TGF-β-R2 and SMAD4. Methods: In a retrospective study of 135 patients with stage I–II colon cancer, the amount of stroma was estimated on routine haematoxylin–eosin stained histological sections. Sections were also immunohistochemically stained for β-catenin, TGF-β-R2 and SMAD4. Results: Of 135 analyzed patients 34 (25.2%) showed a high proportion of stroma (stroma-high) and 101 (74.8%) a low proportion (stroma-low). Significant differences in overall-survival and disease-free-survival were observed between the two groups, with stroma-high patients showing poor survival (OS p < 0.001, HZ 2.73, CI 1.73–4.30; DFS p < 0.001, HZ 2.43, CI 1.55–3.82). A high-risk group was identified with stroma-high and SMAD4 loss (OS p = 0.008, HZ 7.98, CI 4.12–15.44, DFS p = 0.005, HZ 6.57, CI 3.43–12.56); 12 of 14 (85.7%) patients died within 3 years. In a logistic-regression analysis a high proportion of stroma and SMAD4 loss were strongly related (HZ 5.42, CI 2.13–13.82, p < 0.001). Conclusion: Conventional haematoxylin–eosin stained tumor slides contain more prognostic information than previously fathomed. This can be unleashed by assessing the tumor–stroma ratio. The combination of analyzing the tumor–stroma ratio and staining for SMAD4 results in an independent parameter for confident prediction of clinical outcome.

Linkage-Specific in Situ Sialic Acid Derivatization for N-Glycan Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded Tissues.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.

Clinical application of circulating tumor cells in breast cancer

Circulating tumor cells (CTCs) play a major role in the metastatic spread of breast cancer. CTC detection has proven to be an important parameter for predicting progression free and overall survival. Collection of CTCs is minimally invasive and can be performed more often than disseminated tumor cell (DTC) collection from bone marrow, thus providing a real-time “liquid biopsy”. In this review, the most important techniques for enrichment and detection of CTCs are discussed for clinical application in low and higher staged breast cancer, as well as the genetic and molecular characterization of CTCs. For CTCs, the use of immunology-based enrichment techniques with multiple antibodies is recommended in a clinical setting, as well as the use of cytometric detection techniques, combined with RT-PCR for confirmation. Special attention is given to the value of cancer stem cell (CSC) activity, which may be the main cause of ineffectiveness of the control over metastatic lesions due to intratumor heterogeneity. Accumulating information on CSCs offers new paradigms to generate effective targets for the treatment of metastatic disease. Genetic and molecular characterization of CTCs has potential to stratify patients for optimal personalized treatment regimens. CTCs can be used for monitoring patients during treatment schedules.

c-Myb enhances breast cancer invasion and metastasis through the Wnt/β-catenin/Axin2 pathway

The molecular underpinnings of aggressive breast cancers remain mainly obscure. Here we demonstrate that activation of the transcription factor c-Myb is required for the prometastatic character of basal breast cancers. An analysis of breast cancer patients led us to identify c-Myb as an activator of Wnt/β-catenin signaling. c-Myb interacted with the intracellular Wnt effector β-catenin and coactivated the Wnt/β-catenin target genes Cyclin D1 and Axin2 Moreover, c-Myb controlled metastasis in an Axin2-dependent manner. Expression microarray analyses revealed a positive association between Axin2 and c-Myb, a target of the proinflammatory cytokine IL1β that was found to be required for IL1β-induced breast cancer cell invasion. Overall, our results identified c-Myb as a promoter of breast cancer invasion and metastasis through its ability to activate Wnt/β-catenin/Axin2 signaling. Cancer Res; 76(11); 3364-75. ©2016 AACR.

N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.

Multimodal Mass Spectrometry Imaging of N-Glycans and Proteins from the Same Tissue Section.

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.

Stroma-High Lymph Node Involvement Predicts Poor Survival More Accurately for Patients with Stage III Colon Cancer

Objective: The tumor microenvironment has ample impact on the behavior of the malignant process in colon cancer (CC). Patients with a high percentage of stroma within the primary tumor, determined by the tumor-stroma ratio (TSR), have a poor prognosis. In metastatic lymph nodes from patients with stage III CC, the TSR is heterogeneous, but the impact on patients’ prognosis is unknown. Methods: Haematoxylin and eosin stained tissue slides of primary tumor (PT) and associated lymph nodes (LNs) metastases from 102 patients with stage III CC were analyzed for the TSR. Stroma-high (>50% stroma) and stromalow (≤ 50% stroma) groups were evaluated with respect to disease free survival (DFS). Results: Of 102 analyzed primary tumors, 47 (46.1%) scored as stroma-high and 55 (53.9%) as stroma-low. In total, 33 patients had at least one stroma-high LN and 69 patients had one or more stroma-low LNs. Interestingly, 28 patients (27.5%) had both stroma-high and stroma-low LNs, but in another 44 cases the TSR between PT and LNs differed: 29 patients had a stroma-high PT with stroma-low LNs, while 15 patients displayed the opposite. As a result of the combination of the TSR analysis of the PT and the involved metastatic LNs, 62 patients (60.8%) were classified as stroma-high and 40 (39.2%) as stroma-low, restaging 14.7% of the patients to stroma-high with a significantly worse 5-year DFS compared to stroma-low patients (59% vs. 82%, HR=2.83 (95%CI 1.34–5.97), P=0.006). In multivariate analysis, the TSR retained its independent prognostic impact (HR=2.85 (95%CI 1.33-6.10), P=0.007). Conclusion: The presence of abundant stroma in metastatic LNs from patients with stage III CC adds to the prognostic information learned from the primary tumor independently, and supports selective patient treatment.

High-Throughput and High-Sensitivity Mass Spectrometry-Based N -Glycomics of Mammalian Cells

The current protocols for glycomic analysis of cells often require a large quantity of material (5-20 million cells). In order to analyze the N-glycosylation from small amounts of cells (≤1 million) as obtained from, for example, primary cell lines or cell sorting, and in a higher throughput approach, we set up a robust 96-well format PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid stabilization, cleanup, and MALDI-TOF-MS analysis. We further evaluated the influence of PNGase F incubation time on the N-glycan profile.

Endoglin expression on cancer-associated fibroblasts regulates invasion and stimulates colorectal cancer metastasis

Purpose: Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis. Experimental Design: CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer–derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model. Results: CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9–induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver. Conclusions: Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations. See related commentary by Becker and LeBleu, p. 6110.

Molecular profiling of colorectal tumors stratified by the histological tumor-stroma ratio-increased expression of galectin-1 in tumors with high stromal content

The tumor microenvironment is a dominant determinant of cancer cell behavior. Reactive tumor stroma is associated with poor outcome perspective. The tumor-stroma ratio (TSR) is a strong independent prognostic factor in colorectal cancer and is easily assessed using conventional hematoxylin and eosin (H&E) stained paraffin sections at the invasive margin of the tumor. We aim to understand the biology of the tumor stroma in colorectal cancer by investigating the transcriptomic profiles of tumors classified by the TSR method. The TSR was assessed in a cohort of 71 colorectal cancer patients undergoing surgery without (neo)adjuvant therapy. In the cohort, stroma-high tumors were distinguished from stroma-low tumors at gene expression level in the upregulation of biological pathways related to extracellular matrix (ECM) remodeling and myogenesis. The activated microenvironment in stroma-high tumors overexpressed different types of collagen genes, THBS2 and 4 as well as INHBA, COX71A and LGALS1/galectin-1. The upregulation of THBS2, COX7A1 and LGALS1/galectin-1. The upregulation of THBS2, COX7A1 and LGALS1/galectin-1 in stroma-high tumors was validated in The Cancer Genome Atlas. In conclusion, the gene expression data reflects the high stromal content of tumors assessed based on the histological method, the TSR. The composition of the microenvironment suggests an altered proteolysis resulting in ECM remodeling and invasive capacity of tumor cells.