Stromal targets for fluorescent-guided oncologic surgery

Pre-operative imaging techniques are essential for tumor detection and diagnosis, but offer limited help during surgery. Recently, the applicability of imaging during oncologic surgery has been recognized, using near-infrared fluorescent dyes conjugated to targeting antibodies, peptides, or other vehicles. Image-guided oncologic surgery (IGOS) assists the surgeFon to distinguish tumor from normal tissue during operation, and can aid in recognizing vital structures. IGOS relies on an optimized combination of a dedicated fluorescent camera system and specific probes for targeting. IGOS probes for clinical use are not widely available yet, but numerous pre-clinical studies have been published and clinical trials are being established or prepared. Most of the investigated probes are based on antibodies or peptides against proteins on the membranes of malignant cells, whereas others are directed against stromal cells. Targeting stroma cells for IGOS has several advantages. Besides the high stromal content in more aggressive tumor types, the stroma is often primarily located at the periphery/invasive front of the tumor, which makes stromal targets particularly suited for imaging purposes. Moreover, because stroma up-regulation is a physiological reaction, most proteins to be targeted on these cells are “universal” and not derived from a specific genetic variation, as is the case with many upregulated proteins on malignant cancer cells

Tissue levels of matrix metalloproteinases MMP-2 and MMP-9 are related to the overall survival of patients with gastric carcinoma.

Proteinases are involved in tumour invasion and metastasis. Several matrix metalloproteinases (MMPs) have been shown to be increased in various human carcinomas. We assessed the levels of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in 50 gastric carcinomas and corresponding mucosa using quantitative gelatin zymography. Both MMP levels were significantly enhanced in gastric carcinomas compared with adjacent mucosal tissue, showed a relatively poor intercorrelation and no relation was found with histopathological carcinoma classifications according to Laurén, WHO and tumour-node-metastasis (TNM). Cox's multivariate proportional hazards analyses revealed that high carcinomatous MMP values are of prognostic significance for a poor overall survival of the patients, independent of the major clinicopathological parameters

Contribution of plasminogen activators and their inhibitors to the survival prognosis of patients with Dukes' stage B and C colorectal cancer.

Despite the advances in pre-, peri- and post-operative medical care of colorectal carcinoma patients, the prognosis has improved only marginally over recent decades. Thus, additional prognostic indicators would be of great clinical value to select patients for adjuvant therapy. In previous studies we found that colorectal carcinomas have a marked increase of the urokinase-type of plasminogen activator (u-PA), and the inhibitors PAI-1 and PAI-2, whereas the tissue-type plasminogen activator (t-PA) is found to be decreased in comparison with adjacent normal mucosa. In the present study we evaluated the prognostic value of several plasminogen activation parameters, determined in both normal and carcinomatous tissue from colorectal resection specimens, for overall survival of 136 Dukes' stage B and C colorectal cancer patients, in relation to major clinicopathological parameters. Uni- and multivariate analyses indicated that a high PAI-2 antigen level in carcinoma, a low t-PA activity and antigen level and a high u-PA/t-PA antigen ratio in adjacent normal mucosa are significantly associated with a poor overall survival. A high ratio of u-PA antigen in the carcinomas and t-PA antigen in normal mucosa, i.e. u-PA(C)/t-PA(N), was found to be predictive of a poor overall survival as well. All these parameters were found to be prognostically independent of the clinicopathological parameters. Multivariate analysis of combinations of these prognostically significant plasminogen activation parameters revealed that they are important independent prognostic indicators and have in fact a better prognostic value than their separate components. Based on these combined parameters, subgroups of patients with Dukes' stage B and C colorectal cancer could be identified as having either a high or a low risk regarding overall survival. In conclusion, these findings emphasize the relevance of the intestinal plasminogen activation system for survival prognosis of patients with colorectal cancer and, in the future, might constitute a patient selection criterion for adjuvant therapy

Tissue level, activation and cellular localisation of TGF-β1 and association with survival in gastric cancer patients

Transforming growth factor-β1 (TGF-β1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-β1 activation and localisation of TGF-β1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-β1 staining by immunohistochemistry. Active TGF-β1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-β1. Active TGF-β1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-β1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-β1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-β1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-β1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-β1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-β1 activity levels in gastric cancer

Cell-based tracers as Trojan horses for image-guided surgery

Surgeons rely almost completely on their own vision and palpation to recognize affected tissues during surgery. Consequently, they are often unable to distinguish between different cells and tissue types. This makes accurate and complete resection cumbersome. Targeted image-guided surgery (IGS) provides a solution by enabling real-time tissue recognition. Most current targeting agents (tracers) consist of antibodies or peptides equipped with a radiolabel for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), magnetic resonance imaging (MRI) labels, or a near-infrared fluorescent (NIRF) dye. These tracers are preoperatively administered to patients, home in on targeted cells or tissues, and are visualized in the operating room via dedicated imaging systems. Instead of using these ‘passive’ tracers, there are other, more ‘active’ approaches of probe delivery conceivable by using living cells (macrophages/monocytes, neutrophils, T cells, mesenchymal stromal cells), cell(-derived) fragments (platelets, extracellular vesicles (exosomes)), and microorganisms (bacteria, viruses) or, alternatively, ‘humanized’ nanoparticles. Compared with current tracers, these active contrast agents might be more efficient for the specific targeting of tumors or other pathological tissues (e.g., atherosclerotic plaques). This review provides an overview of the arsenal of possibilities applicable for the concept of cell-based tracers for IGS

Identifying Biomarkers in Lymph Node Metastases of Esophageal Adenocarcinoma for Tumor-Targeted Imaging

INTRODUCTION: Tumor-targeted imaging is a promising technique for the detection of lymph node metastases (LNM) and primary tumors. It remains unclear which biomarker is the most suitable target to distinguish malignant from healthy tissue in esophageal adenocarcinoma (EAC). OBJECTIVE: We performed an immunohistochemistry study to identify viable tumor markers for tumor-targeted imaging of EAC. METHODS: We used samples from 72 patients with EAC to determine the immunohistochemical expression of ten potential tumor biomarkers for EAC (carbonic anhydrase IX [CA-IX], carcinoembryonic antigen [CEA], hepatic growth factor receptor, epidermal growth factor receptor, epithelial membrane antigen [EMA], epithelial cell adhesion molecule [EpCAM], human epidermal growth factor receptor 2 [HER-2], urokinase plasminogen activator receptor, vascular endothelial growth factor-A [VEGF-A], and VEGF receptor 2). Immunohistochemistry was performed on tissue microarrays of LNM (n = 48), primary EACs (n = 62), fibrotic tissues (n = 11), nonmalignant lymph nodes (n = 24), and normal esophageal and gastric tissues (n = 40). Tumor marker staining was scored on intensity and percentage of positive cells. RESULTS: EMA and EpCAM showed strong expression in LNM (> 95%) and primary EACs (> 95%). Significant expression was also observed for LNM and EAC using VEGF-A (85 and 92%), CEA (68 and 54%), and CA-IX (4 and 34%). The other tumor biomarkers showed expression of 0-15% for LNM and primary EAC. Except for VEGF-A, nonmalignant lymph node staining was scored as slight or absent. CONCLUSIONS: High expression rates and correlation between LNM in EAC combined with low expression rates in healthy lymph nodes and esophagus tissues were observed for EpCAM and CEA, meaning these are promising targets for tumor-targeted imaging approaches for lymph nodes in patients with EAC

Matrix metalloproteinase-2 is a consistent prognostic factor in gastric cancer

In a pioneer study, we showed 10 years ago that enhanced tissue levels of the matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in gastric cancers, as determined by zymography, were related with worse overall survival of the patients. To corroborate these observations, we now assessed MMP-2 and MMP-9 with new techniques in an expanded group of gastric cancer patients (n=81) and included for comparison MMP-7, MMP-8 and the tissue inhibitors of MMPs, TIMP-1 and -2. All MMPs and TIMP-1 were significantly increased in tumour tissue compared to normal gastric mucosa. Matrix metalloproteinase-7, -8 and -9, and the TIMPs showed some correlations with the clinicopathologic parameters TNM, WHO and Laurén classification, but their levels were not related with survival. Regardless of the determination method used, that is, enzyme-linked immunosorbent assay or bioactivity assay, an enhanced tumour MMP-2 level did not show a significant correlation with any of the clinicopathological parameters, but was confirmed to be an independent prognostic factor in gastric cancer

Endoglin targeting inhibits tumor angiogenesis and metastatic spread in breast cancer

Endoglin, a transforming growth factor-beta co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical trials for anti-angiogenic therapy. In this project, the redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis and the effects of targeting both pathways on breast cancer metastasis were explored. In patient samples, increased endoglin signaling after VEGF inhibition was observed. In vitro TRC105, an endoglin-neutralizing antibody, increased VEGF signaling in endothelial cells. Moreover, combined targeting of the endoglin and VEGF pathway, with the VEGF receptor kinase inhibitor SU5416, increased antiangiogenic effects in vitro and in a zebrafish angiogenesis model. Next, in a mouse model for invasive lobular breast cancer, the effects of TRC105 and SU5416 on tumor growth and metastasis were explored. Although TRC105 and SU5416 decreased tumor vascular density, tumor volume was unaffected. Strikingly, in mice treated with TRC105, or TRC105 and SU5416 combined, a strong inhibition in the number of metastases was seen. Moreover, upon resection of the primary tumor, strong inhibition of metastatic spread by TRC105 was observed in an adjuvant setting. To confirm these data, we assessed the effects of endoglin-Fc (an endoglin ligand trap) on metastasis formation. Similar to treatment with TRC105 in the resection model, endoglin-Fc-expressing tumors showed strong inhibition of distant metastases. These results show, for the first time, that targeting endoglin, either with neutralizing antibodies or a ligand trap, strongly inhibits metastatic spread of breast cancer in vivo.Surgical oncolog