Although the bulk of research on the development of biomarkers and therapeutic targets has focused on the tumor cell, the role of host cell populations in tumor growth has become quite apparent, and the investigation of such populations has the potential to identify additional biomarkers and therapeutic targets. Isolating and studying the small population of tumor vascular cells has been quite challenging. However, we have been able to characterize the gene expression signature of these cells in vivo with the use of immunohistochemistry-guided laser capture microdissection coupled with transcriptional profiling. We analyzed all genes we have identified to date from the ovarian cancer vasculature and applied a number of filters to identify tumor-specific candidates for diagnosis or therapy. First, we selected TVMs with known or putative transmembrane or secretory protein products. Then, we used publicly available Affymetrix expression data for over 1,000 normal and cancer tissues to select genes with low or no expression in normal tissues. Through this approach we identified 50 candidate genes, which were then validated by qRT-PCR. We chose 13 genes for detailed validation based on affinity reagents that could reliably identify the protein products. Most of these genes play important roles in processes that are in fact vital for tumor growth, such as regulation of endothelial permeability, leukocyte extravasation,26
extracellular matrix degradation29–31
Nine of the genes (ADAM12, CDCP1, CSPG2, EGFL6, ESM1, FLJ46072, LGALS3BP, ST14 and TSG6) exhibited higher expression in ovarian cancer relative to normal tissues. It should be noted that these expression data are based on whole tissue analysis, but we normalized against endothelial gene CD31 to eliminate the influence of vascular density. The difference in expression between cancer and normal was quite pronounced when we compared purified endothelial cells. Thus, it is plausible that targeted imaging or therapy may reveal a higher tumor to normal ratio than analysis of whole tissue would suggest. After confirmation of our results at the protein level, we conclude that select transmembrane (ADAM12, CDCP1) as well as secreted (ESM1, EGFL6) proteins represent promising tumor vascular targets for imaging or therapy of EOC. These add to TVM genes identified in our previous work1
(adlican, COL11A1, F2RL1, FZD10 and OLFML2B), which also provide good candidates for therapeutic or imaging strategies. ESM1 showed low expression in freshly isolated TECs relative to HUVECs at the RNA level, but this may be due to sampling bias, as we show that ESM1 is expressed only in a subset of tumors, or to mRNA instability. The expression of ESM1 specifically in tumor endothelial cells (TECs) was established at the protein level in tumor tissue by immunohistochemistry. In addition, BLAME was not expressed at the RNA level in immunopiurified tumor endothelial cells, but was expressed by tumor-purified vascular related leukocytes (VLCs). These cells of monocyte-macrophage lineage associate structurally with tumor vasculature and perivascular stroma, and may play an important role in tumor vascular development.33
Pericyte-like expression of BLAME was confirmed by immunostaining. Because BLAME is not expressed by PBMC, it could be an interesting therapeutic target.
Tumor vascularization is a dynamic biological process regulated by complex microenvironment conditions that cannot be precisely recapitulated in vitro. Nevertheless, we attempted to subject HUVEC to known environmental/paracrine factors that are likely to play an important role in the tumor vasculature milieu: normoxic or hypoxic tumor cell supernatants; VEGF; and inflammatory mediators activating endothelial NFκB (TNFα). Under these controlled laboratory conditions that only in part reproduce the tumor microenvironment, 10 of 16 candidate genes were upregulated. For example, BLAME, EGFL6 and LGALS3BP showed strong expression after exposure to TNFα, while the expression was undetectable at baseline. CDCP1 (long) showed over 10-fold increase under tumor conditioned media as well as TNFα; TSG6 expression increased over 100-fold by exposure to tumor cell supernatants and almost 1,000-fold by TNFα. Other genes showed a more moderate or no increase. Interestingly, none of the gene candidates were upregulated by VEGF alone, suggesting that the signature we have uncovered does not pertain to physiologic angiogenesis, where VEGF is upregulated but inflammation or other tumor-derived paracrine factors are absent.
Our discovery rules required that vascular endothelial gene candidates be upregulated in many, not all
, tumor vascular endothelial cell samples relative to normal endothelial cells. Thus, some of the identified genes were expressed at low levels in some tumors. This does not diminish their potential importance as tumor vascular markers or therapeutic targets in a subset of patients. Importantly, this is the case for tumor vascular markers that have already been validated and are currently being developed as therapeutic targets, e.g., TEM1,34
for which therapeutic antibodies are already under early phase clinical testing. Ideally, for the purpose of tumor detection, development of molecular imaging tools should focus on genes that are highly expressed in most EOC tumors. For example, as shown in , genes such as EGFL6, CDCP1 and FLJ46072 seem to be consistently elevated in most ovarian cancer samples. Thus, especially cell surface proteins may be suitable targets for tumor imaging. Of note, some markers were found to have high expression in some normal tissue. These could still serve as useful imaging targets, as long as one is aware of their expression in the normal adult. For example, BLAME shows its highest levels of expression in tonsil and lung tissue and therefore could be suitable for abdominal imaging. Furthermore, EGFL6 was only highly expressed in mammary gland, which again makes it a suitable target for abdominal imaging. Cell surface vascular proteins that are highly expressed in most tumors could be universal targets for therapy. In particular, the short CDCP1 isoform and EGFL6 appear promising candidates for therapy as normal tissues exhibit mostly undetectable expression (except for placenta in the case of EGFL6). On the other hand, ADAM12 is very highly expressed in some EOC samples compared with normal ovaries but a significant number of cancer samples exhibited low levels of ADAM12. Thus, ADAM12 may hold predictive value, and it could be used for therapy, especially the long isoform, for patients whose tumors express it. It is important to note that all of these molecules exhibit high levels of expression in many other solid tumors as well (), which underlines the possible application of these targeted approaches in cancers other than ovarian.
Cellular and molecular changes characterizing the angiogenic switch are essential for tumor growth beyond few millimeters.7
Interestingly, specialization of the vasculature may even precede tumor establishment at metastatic sites.35
We tested the notion that secreted or cleaved vascular proteins can function as serum biomarkers. We were restricted by the availability of antibodies for Luminex to the validation of three biomarkers: DR6, ESM1 and CSPG2. The discovery of DR6 as a tumor vascular marker was previous described.1
In the present manuscript we show the development of a new assay for detecting DR6 in serum using Luminex technology, followed by screening and evaluation of serum samples from patients and healthy individuals, and evaluation of DR6-based screening as well as a composite screening with both DR6 and CA-125 for increasing EOC detection specificity and sensitivity. DR6, ESM1 and CSPG2 were predicted to differentiate cancer from control cases but with suboptimal specificity, based on expression in normal tissues and suboptimal sensitivity based on lack of expression in many tumors. Screening sera from 61 EOC and 19 normal controls confirmed the predicted performance of these markers in cancer discrimination. All three markers could differentiate between normal controls and cancer patients. Furthermore, the bivariable logistic regression models that used two biomarkers, either CA125 and TNFRSF21/DR6 or CA125 and ESM1/endocan, fit the data better than the univariable model with CA125 alone. Classification using both CA125 and DR6 in patients with CA125 less than 89 IU/ml improved specificity with no change in sensitivity. Thus, DR6 appears promising for future biomarker validation in larger patient cohorts. It should be noted that these three markers were chosen as Luminex substrates merely based on feasibility, as proof of principle. It is possible that other TVM, predicted by our tissue-based analyses to have better discriminatory value, might in fact perform better as serum biomarkers and development of affinity reagents and assays then becomes a high priority.
In summary, our work emphasizes that the field of tumor vascular biology may provide applications in cancer therapeutics and diagnostics and has provided novel targets for further clinical development.