Sayedmohammad Hasheminasab, MD

Tumor Microenvironment - Genomic stability of tumor endothelium

Being a dynamic cellular system which traffics oxygen, nutrients, and immune modulators to cancer cells, vascular endothelium plays an integral role in cancer pathophysiology. Tumor endothelial cells (TECs) are long known to be deregulated, possibly as a result of hypoxia and constant growth factor stimulation. Irregularities such as abnormal vessel diameters, lack of structural stability, abnormal cellular sprouts and intercellular gaps are observed in TECs in various cancers.

These abnormalities lead to tumor progression and facilitate metastasis. Leaky vasculature with increased gaps and holes, has been shown to be associated with increased metastasis in both mouse and human cancers (Xian et al., 2006; Yonenaga et al., 2005). This leakiness may also negatively affect the distribution of antineoplastic agents. Bevacizumab (an anti-VEGF antibody) caused a prompt and stable decrease in vessel permeability; therefore resulting in tumor growth inhibition (Dickson et al., 2007).

In the recent decades, anti-angiogenic therapy has swiftly taken an essential part in standard anti-cancer treatment. Many of the primarily successful therapies eventually fail in the course of treatment because tumors achieve resistance to drugs by means of plasticity and evolution. Compared to genetically unstable tumor cells TECs are genetically more stable and therefore less susceptible to the development of acquired drug resistance.

It has long been considered that the TECs are genetically normal. However recently strong evidence provided by Michael Klagsbrun’s laboratory, displayed the genomic instability in forms of centrosome and cytogenetic abnormalities (Hida et al., 2004). They observed a gain in chromosome 17 in 30% to 50% of the TECS; however they did not observed the same aberrations in the tumor cells and also these were aberration were not clonal. Supporting the former findings, it has been shown that the vascular endothelial growth factor induces centrosome duplications (Taylor et al., 2010). On the other hand, Streubel has found identical genomic alterations between TECs and follicular B cell lymphoma (Streubel et al., 2004). Their finding highlights the role of horizontal transfer of genetic material through apoptotic vesicles or tunneling nano-tubes.

The controversy mentioned above has motivated us to tackle this field with state of the art technologies, going even down to the single cell resolution. Firstly we aimed to screen the TECs in different types of cancers. We have dissociated the tumor samples with the means of mechanical and enzymatic disaggregation methods. Using image-based single cell sorting methods we isolated the CD31 and VE-cadherin positive TECs. We will then prepare sequencing libraries utilizing the ultra-low input libraries preparation methods to achieve unbiased sequencing data.

Additionally, we aim to investigate effects of hypoxia on TECs’ genome and transcriptome. We have injected mice with the hypoxia-stain pimonidazole and Hoechst dye (which stains proliferating tissue usually found in normoxic tissues) just before taking the tumor tissue. This combination is used to distinguish areas of hypoxia (pimo-high/Hoechst-low) from normoxic tissue. The TECs can then be isolated from pathology slides and genetically investigated.

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References:

Dickson, P. V., Hamner, J. B., Sims, T. L., Fraga, C. H., Ng, C. Y., Rajasekeran, S., Davidoff, A. M. (2007). Bevacizumab-induced transient remodeling of the vasculature in neuroblastoma xenografts results in improved delivery and efficacy of systemically administered chemotherapy. Clin Cancer Res, 13(13), 3942-3950. doi:10.1158/1078-0432.CCR-07-0278

Hida, K., Hida, Y., Amin, D. N., Flint, A. F., Panigrahy, D., Morton, C. C., & Klagsbrun, M. (2004). Tumor-associated endothelial cells with cytogenetic abnormalities. Cancer Res, 64(22), 8249-8255. doi:10.1158/0008-5472.CAN-04-1567

Streubel, B., Chott, A., Huber, D., Exner, M., Jager, U., Wagner, O., & Schwarzinger, I. (2004). Lymphoma-specific genetic aberrations in microvascular endothelial cells in B-cell lymphomas. N Engl J Med, 351(3), 250-259. doi:10.1056/NEJMoa033153

Taylor, S. M., Nevis, K. R., Park, H. L., Rogers, G. C., Rogers, S. L., Cook, J. G., & Bautch, V. L. (2010). Angiogenic factor signaling regulates centrosome duplication in endothelial cells of developing blood vessels. Blood, 116(16), 3108-3117. doi:10.1182/blood-2010-01-266197

Xian, X., Hakansson, J., Stahlberg, A., Lindblom, P., Betsholtz, C., Gerhardt, H., & Semb, H. (2006). Pericytes limit tumor cell metastasis. J Clin Invest, 116(3), 642-651. doi:10.1172/JCI25705

Yonenaga, Y., Mori, A., Onodera, H., Yasuda, S., Oe, H., Fujimoto, A., . . . Imamura, M. (2005). Absence of smooth muscle actin-positive pericyte coverage of tumor vessels correlates with hematogenous metastasis and prognosis of colorectal cancer patients. Oncology, 69(2), 159-166. doi:10.1159/000087840

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