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Antiangiogenic therapies have failed to confer survival benefits in patients with metastatic breast cancer (mBC). However, to date, there has not been an inquiry into the roles for acquired versus innate drug resistance in this setting. In this study, we report roles for these distinct phenotypes in determining therapeutic response in a murine model of mBC resistance to the antiangiogenic tyrosine kinase inhibitor sunitinib. Using tumor measurement and vascular patterning approaches, we differentiated tumors displaying innate versus acquired resistance. Bioluminescent imaging of tumor metastases to the liver, lungs, and spleen revealed that sunitinib administration enhances metastasis, but only in tumors displaying innate resistance to therapy. Transcriptomic analysis of tumors displaying acquired versus innate resistance allowed the identification of specific biomarkers, many of which have a role in angiogenesis. In particular, aquaporin-1 upregulation occurred in acquired resistance, mTOR in innate resistance, and pleiotrophin in both settings, suggesting their utility as candidate diagnostics to predict drug response or to design tactics to circumvent resistance. Our results unravel specific features of antiangiogenic resistance, with potential therapeutic implications. Cancer Res; 77(4); 1008-20. ©2016 AACR ., (©2016 American Association for Cancer Research.)

The structure and molecular signature of tumor-associated vasculature are distinct from those of the host tissue, offering an opportunity to selectively target the tumor blood vessels. To identify tumor-specific endothelial markers, we performed a microarray on tumor-associated and nonmalignant endothelium collected from patients with renal cell carcinoma (RCC), colorectal carcinoma, or colorectal liver metastasis. We identified a panel of genes consistently upregulated by tumor blood vessels, of which melanoma cell adhesion molecule (MCAM) and its extracellular matrix interaction partner laminin alpha 4 (LAMA4) emerged as the most consistently expressed genes. This result was subsequently confirmed by immunohistochemical analysis of MCAM and LAMA4 expression in RCC and colorectal carcinoma blood vessels. Strong MCAM and LAMA4 expression was also shown to predict poor survival in RCC, but not in colorectal carcinoma. Notably, MCAM and LAMA4 were enhanced in locally advanced tumors as well as both the primary tumor and secondary metastases. Expression analysis in 18 different cancers and matched healthy tissues revealed vascular MCAM as highly specific in RCC, where it was induced strongly by VEGF, which is highly abundant in this disease. Lastly, MCAM monoclonal antibodies specifically localized to vessels in a murine model of RCC, offering an opportunity for endothelial-specific targeting of anticancer agents. Overall, our findings highlight MCAM and LAMA4 as prime candidates for RCC prognosis and therapeutic targeting. Cancer Res; 76(8); 2314-26. ©2016 AACR., (©2016 American Association for Cancer Research.)

The development of anticancer therapies that target the angiogenic process is an area of major growth in oncology. A method of noninvasively measuring tumor vascular endothelial growth factor (VEGF) in vivo could provide important efficacy information for VEGF-dependent antiangiogenic agents and the role of VEGF in cancer biology. We have developed a novel radiotracer for use with positron emission tomography (PET) that enables noninvasive imaging of VEGF. This radiotracer comprises an IgG1 monoclonal antibody, known as VG76e, that binds to human VEGF, labeled with a positron-emitting radionuclide, iodine-124 ([(124)I]-SHPP-VG76e). Three radiolabeling strategies were evaluated to synthesize the radiotracer with optimal radiochemical yield, purity, and immunoreactivity. To evaluate the pharmacokinetics and VEGF-specific localization of [(124)I]-SHPP-VG76e, two subclones of the HT1080 human fibrosarcoma selected on the basis of differing VEGF production (26.6 and 1/3C, the former producing 2-4-fold more in vitro) were established in culture and grown as solid tumor xenografts in immune-deficient mice. A single i.v. injection of the radiotracer into tumor-bearing mice revealed a time dependent and specific localization of [(125)I]-SHPP-VG76e to the tumor tissue. Three validation studies established the VEGF specificity and potential for use of [(124)I]-SHPP-VG76e in vivo: (a) uptake of [(125)I]-SHPP-VG76e was 1.8-fold higher in HT1080-26.6 compared with HT1080-1/3C tumors (P < 0.05); (b) uptake of [(125)I]-SHPP-VG76e in HT1080-26.6 tumors was specifically blocked by prior administration of excess unlabeled VG76e (P < 0.05); and (c) tumor uptake of the IgG1, [(125)I]-SHPP-CIP5, which has a similar molecular weight as [(125)I]-SHPP-VG76e but does not recognize VEGF, was the same for both HT1080-26.6 and HT1080-1/3C (P > 0.05). Other than tumor localization, [(125)I]-SHPP-VG76e was present in urine and blood and to a lesser extent in heart, lungs, liver, kidney, and spleen. Whole-animal PET imaging studies revealed a high tumor-to-background contrast and also revealed [(124)I]-SHPP-VG76e distributions in the major organs. These studies support further development of [(124)I]-SHPP-VG76e as a radiotracer for measuring tumor levels of VEGF in humans.

Tumor-associated macrophages (TAMs) produce angiogenic factors and in breast cancer are associated with high vascular grade and poor survival. TAMs preferentially migrate to hypoxic areas within tumors and strongly express hypoxia-inducible factor (HIF)-2 alpha. This study examined whether HIF-2 alpha was involved in TAM angiogenic activation by correlating its expression with tumor microvessel density as a marker of angiogenesis, and other tumor variables, in a series of human primary invasive breast carcinomas. A correlation was found between high TAM HIF-2 alpha and high tumor vascularity (P < 0.0001), as well as high tumor grade (P = 0.007). The relation of HIF-2 alpha expression to a recently described oxygen-dependent pathway of angiogenesis was also studied, and an inverse relationship was found between TAM HIF-2 alpha and tumor thymidine phosphorylase expression (P = 0.02). These results suggest that TAM HIF-2 signaling may be a useful target for future antiangiogenic strategies but show that tumors use both oxygen-dependent and oxygen deficiency-regulated pathways for angiogenesis. Thus, combined blockade of pathways and careful assessment of these pathways in trials are necessary.

Thymidine phosphorylase (TP) (E.C. 2.4.2.4), also known as platelet-derived endothelial cell growth factor, is a potent angiogenic factor. The expression of TP correlates with poor prognosis in a range of tumor types. 2-Deoxy-D-ribose-1-phosphate, a product of thymidine catabolism by TP, is a strongly reducing sugar that generates oxygen radical species during the early stages of protein glycation. We show that thymidine induces oxidative stress in TP-overexpressing carcinoma cells, promoting secretion of the stress-induced angiogenic factors vascular endothelial growth factor and interleukin-8, and inducing matrix metalloproteinase-1. Our findings outline a putative mechanism for TP-induced angiogenesis and identify novel targets for intervention.

The assessment of angiogenesis in breast cancer is of importance as a key indicator of survival and response to therapy. Circulating vascular endothelial growth factor (VEGF) measurements may provide a less subjective analysis than microvessel density (MVD) or immunohistochemical analysis of VEGF expression; however, most studies have used serum, which is now known to largely reflect platelet-derived VEGF concentrations. This study examined for the first time both plasma (VEGFp) and serum (VEGFs) VEGF concentrations in 201 blood samples from pre- and postmenopausal healthy controls and from patients with benign breast disease, localized breast cancer, breast cancer in remission, or metastatic breast cancer and related these to other clinicopathological markers. VEGFp but not VEGFs concentrations of patients with localized disease were significantly elevated compared with normal controls (P = 0.016). Patients with metastatic disease had higher VEGFp and VEGFs levels than normal controls (P < 0.001, P = 0.044 respectively), and higher VEGFp, but not VEGFs, than patients with benign disease (P = 0.009) and patients with localized disease (P = 0.004). However, the highest VEGFp and VEGFs concentrations were seen in patients in remission compared with normal controls (P < 0.001 and P = 0.008, respectively). VEGFp concentrations in patients in remission were also higher than in patients with benign disease (P = 0.01) or patients with localized disease (P = 0.005). Tamoxifen treatment was significantly associated with higher circulating and platelet-derived VEGF levels. Circulating VEGF did not correlate with any clinicopathological factor, including MVD or VEGF expression. VEGF expression was significantly correlated with estrogen receptor status and inversely correlated with tumor grade. MVD correlated with tumor size. Tamoxifen-induced increases in VEGF may be important in clinical prognosis or associated pathologies.

Tumor development is angiogenesis dependent, and vascular endothelial growth factor (VEGF) is a key growth factor in this process. We demonstrate that high expression of VEGF mRNA in 55 superficial bladder cancers was associated with earlier recurrence (P = 0.001; hazard ratio, 3.09) and progression to a more invasive phenotype (P = 0.02; hazard ratio, 5.33). VEGF mRNA expression correlated with protein levels in superficial tumors (r = 0.59, P = 0.003) and normal bladder (r = 0.65, P < 0.05), although the ratio of VEGF protein to mRNA was elevated in tumors compared to normal bladder (P = 0.004), suggesting posttranscriptional regulation. In this study, VEGF is implicated as a major downstream mediator of the effects of the p53 tumor suppressor gene by the association between high p53 protein (determined immunochemically) and high VEGF protein and mRNA expression (P < 0.02), although in cases without high p53 protein expression, high VEGF mRNA also predicts a poor prognosis. The relationship between VEGF and early tumor recurrence suggests that seeding via angiogenesis may be a major mechanism in the pathogenesis of recurrence. These studies indicate that VEGF can predict the behavior of superficial bladder tumors and is a therapeutic target for intravesical therapy.

Angiogenesis is a significant prognostic factor in breast cancer, but the factors that control angiogenesis in vivo are not well defined. Multiple angiogenic polypeptides are known, and we have determined the expression of seven of these in primary human breast cancers; the relationship of expression to estrogen receptor and vascular density was also examined. Vascular endothelial growth factor (VEGF) and its four isoforms (121, 165, 189, and 206 amino acids), transforming growth factor (TGF)-beta1, pleiotrophin, acidic and basic fibroblast growth factor (FGF), placental growth factor, and thymidine phosphorylase (platelet-derived endothelial cell growth factor) were quantitated by RNase protection analysis. beta-FGF was also measured by ELISA. The estrogen receptor (ER), epidermal growth factor receptor, and vascular density were analyzed in 64 primary breast cancers. All tumors expressed at least six different vascular growth factors. VEGF was most abundant, and the transcript for the 121-amino acid form predominated. Other angiogenic factors expressed at high levels were thymidine phosphorylase and TGF-beta1. Expression of most of the angiogenic factors did not correlate with that of ER or vascular density. However, thymidine phosphorylase did, with a correlation coefficient of 0.3 (P = 0.03). There were significant associations of pleiotrophin with acidic FGF expression (P = 0.001) and TGF-beta with platelet-derived endothelial cell growth factor expression (P = 0.001). Thus, angiogenesis may involve a coordinate regulation of some vascular growth factors. High VEGF expression correlated with poor prognosis in univariate analysis (P = 0.03), as did ER and epidermal growth factor receptor expression. Basic FGF was also assessed by ELISA and was more highly expressed in tumors than normal breast tissues (median, 346 microg/ml cytosol; range, 54-1323 versus median, 149; range, 32-509; P = 0.01). Implications for therapy are that broad spectrum agents that block features common to these factors may be useful (e.g., antagonism of heparin-binding activity agents), because so many angiogenic factors are expressed. Inhibiting endothelial migration or agents directly toxic to endothelium would be of value in a combined approach to therapy.

We report that hypoxia regulates and influences the level of the angiogenic enzyme platelet-derived endothelial cell growth factor (PD-ECGF), also called thymidine phosphorylase, in vitro and in vivo. Levels of PD-ECGF protein increased 6-fold in the breast cancer cell line MDA 231 after 16 h of growth in 0.3% oxygen. A simultaneous increase in enzyme activity was observed. Immunohistochemical staining of MDA 231 tumors grown in nu/nu mice showed increased expression of PD-ECGF in those parts of the tumor that are proximal to the areas of necrosis. In addition, increased and widespread staining for PD-ECGF protein was obtained when the tumor vascular supply was occluded for 2 h by clamping. Lowering the media pH to 6.3-6.7 in vitro also resulted in an increase in PD-ECGF protein levels. This study demonstrates that tumor microenvironmental factors can result in the specific up-regulation of an angiogenic enzyme that can also activate 5-fluorouracil prodrugs and hence is exploitable therapeutically.

In the urine of patients with bladder cancer, levels of the angiogenio peptide basic fibroblast growth factor (bFGF) may be elevated 100-fold. To date, levels of expression of bFGF in bladder tumor tissue have not been determined, nor has the cellular source of the urinary bFGF been identified. bFGF mRNA expression was quantified using RNase protection analysis in 32 primary bladder tumors and 8 normal bladder specimens. In addition, bFGF protein expression in the tumor cytosol was determined using a Quantikine ELISA, and bFGF protein expression was localized with immunohistochemistry. bFGF mRNA expression was absent in 28 of 32 (87%) bladder cancers despite detectable expression in 7 of 8 (87%) normal bladder specimens (P = 0.0001). In only one tumor was bFGF mRNA expression higher than in normal bladder tissue. Median bFGF protein expression was also higher in the normal bladder specimens than in the superficial tumors (3800 pg/g protein versus 1140 pg/g protein; P < 0.02), but there was no statistically significant difference between protein expression in normal bladder and invasive cancers (3800 pg/g versus 3600 pg/g). Median bFGF protein expression was higher in invasive cancers than in superficial tumors (P < 0.05). Intense bFGF immunoreactivity was seen in the basal lamina of normal transitional epithelium, in normal human detrusor muscle, and in vessels within tumors. Tumor cell immunoreactivity was rare and was usually weak. Only in the tumor which strongly overexpressed bFGF mRNA and protein was cytoplasmic staining detectable in the neoplastic cells. There are two mechanisms of bFGF-induced angiogenesis in bladder cancer. Rarely, neoplastic cells synthesize bFGF but more commonly bFGF is released by degradation of epithelial basement membranes and detrusor muscle, from where it can diffuse into the tumor microenvironment and bind to blood vessels. Mechanisms of extracellular matrix degradation may be important in bladder cancer angiogenesis and progression and as such are potential therapeutic targets.

Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor, has been implicated in bladder cancer angiogenesis. To examine its role more clearly, we have quantified and localized its expression using Western analysis and immunohistochemistry in a series of 105 bladder cancers. We have also assessed the relationship between TP expression and other tumor parameters including quantitative angiogenesis, p53 status, ploidy, and survival. By Western analysis, TP expression was 5-fold higher in tumors than in normal bladder samples (P < 0.02). Expression was 15-fold higher in invasive tumors than in normal bladder (P < 0.001) and 8-fold higher than in superficial tumors (P < 0.005). Immunohistochemistry of the tumors showed TP was present in the neoplastic epithelium in 27% of the tumors, in the inflammatory cells in 72% of the tumors, in stromal cells in 30% of the tumors, and in tumor-associated endothelium in 11% of the tumors. Expression by Western blotting and immunohistochemistry was significantly up-regulated in tumors compared with normal bladder (P < 0.05). Tumor cell TP expression correlated with tumor grade (P < 0.02), but there was no correlation between tumor cell TP expression and tumor stage (P = 0.46), ploidy (P = 0.52), p53 expression (P = 0.9), tumor vascularity (P = 0.8), relapse-free survival (P = 0.57), or overall survival (P = 0.94). TP protein is expressed in bladder cancers, and expression is associated with an aggressive phenotype. Because TP can activate a number of cytotoxic agents, it provides a potential therapeutic target in bladder cancer.

Midkine (MK) is a member of a family of heparin-binding growth factors, which are reported to be angiogenic. We have investigated by RNase protection analysis the expression of MK in 47 primary bladder tumors and 7 normal bladder samples. MK mRNA transcripts were detectable in 46 (98%) of 47 of the tumors and in 5 (70%) of 7 of the normal bladder samples. However, median MK expression was 4-fold higher in tumors than in the normal bladder (P < 0.004). In eight tumors (17%), MK expression was elevated more than 10-fold compared with the median value of the normal bladder specimens. There was no statistically significant difference in expression between superficial and invasive tumors (P < 0.50). Seven (32 %) of 22 patients with invasive cancers are alive at 1 year with no evidence of recurrence; in 5 (70%) of these patients, MK expression in the tumor was within the normal range at the time of presentation. By contrast, in only 2 (13%) of 15 patients who died or whose tumors recurred or progressed was MK expression in the normal range (P < 0.01). Overall, median MK expression in invasive tumors that caused death, progressed, or recurred within 12 months was 3-fold higher than that found in the tumors of those patients who were clear of disease at 12 months (P < 0.04). Thus, overexpression of MK is associated with the development of bladder cancer and in invasive cancers predicts a poor clinical outcome in the short term.

We have investigated by RNase protection analysis the expression of 2 angiogenic factors in 45 primary bladder tumors and 8 normal bladders. Expression of vascular endothelial growth factor (VEGF) was 3-fold higher and that of platelet-derived endothelial cell growth factor was 40-fold higher in tumors compared to normal bladder. However, the factors were differentially expressed in different stages of the cancer. Expression of VEGF in superficial tumors was 4-fold higher than in invasive tumors and 10-fold higher than in normal bladder (superficial versus invasive, P < 0.0006; superficial versus normal, P < 0.0002). Expression of platelet-derived endothelial cell growth factor in invasive tumors was 33-fold higher than in superficial tumors and 260-fold higher than in normal bladder (invasive versus superficial, P < 0.0001; invasive versus normal, P < 0.0003). In well differentiated or moderately differentiated superficial tumors which had invaded the lamina propria (pT1G1/2) VEGF expression was 4-fold higher in tumors which subsequently recurred at 3 months compared to those which did not recur (P < 0.002). Thus there are two distinct angiogenic pathways involved in different stages of bladder cancer, which is in keeping with the evidence for two different genetic pathways. Elevated VEGF expression predicts early recurrence of pT1G1/2 tumors.

Wnt gene expression was investigated by ribonuclease protection analysis in human breast cancer, nontumorous breast tissue, and a variety of human breast cell lines. We report the expression of Wnt3, Wnt4, and Wnt7b in human breast cell lines and Wnt2, Wnt3, Wnt4, and Wnt7b in human breast tissues. Wnt3a and Wnt7a were absent in the cell lines and tissues tested. The level of expression of Wnt2 and Wnt4 was 10- to 20-fold higher in fibroadenomas than it was in normal or malignant breast tissue, and in 10% of tumors Wnt7b expression was 30-fold higher than in normal or benign breast tissues. In contrast to the mouse, in which Wnt1 and Wnt3 are involved in tumorigenesis, our results suggest that Wnt2, Wnt4, and Wnt7b may be associated with abnormal proliferation in human breast tissue.

Current studies of tumor angiogenesis rely on the concept that endothelium proliferates 30-40 times faster in tumors than in normal tissues. This evidence is based on histological autoradiographic data largely from animal studies. To assess endothelial cell proliferation in human cancer we used the more sensitive and specific technique of immunohistochemistry. We measured the frequency and distribution of endothelial cell proliferation and examined their relationship to tumor cell proliferation. For the first time, we also correlated endothelial and tumor cell proliferation with tumor vascularity. Twenty breast carcinomas from patients exposed to bromodeoxyuridine 3-8 h prior to surgery were double immunostained using antibodies to CD31 (as a marker of endothelium) and bromodeoxyuridine (as a marker of proliferation). The labeling index (LI) for both tumor and endothelial cells was determined and tumor vascularity was assessed by counting the number of CD31 positive vessels. Endothelial cell proliferation was predominantly at the tumor periphery while tumor cell proliferation occurred throughout the lesion. The mean LIs for endothelium and tumor were 2.2% (range, 0.8-5.3) and 7.3% (range, 1.3-17.1), respectively. There was no correlation between tumor and endothelial cell LI (P = 0.414) or between the tumor LI or endothelial cell LI and tumor vascularity (P = 0.08 and P = 0.39, respectively). These findings suggest that previous studies in animal tumors have significantly overestimated endothelial cell proliferation and that its importance in tumor angiogenesis may be related more to continual remodeling and migration of vessels than to proliferation alone.

Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose-dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB-468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth.