Intratumoral Endothelial Cells Research Articles

VISTA expression on cancer-associated endothelium selectively prevents T-cell extravasation.

Cancers evade T-cell immunity by several mechanisms such as secretion of anti-inflammatory cytokines, downregulation of antigen presentation machinery, upregulation of immune checkpoint molecules, and exclusion of T cells from tumor tissues. The distribution and function of immune checkpoint molecules on tumor cells and tumor-infiltrating leukocytes is well established, but less is known about their impact on intratumoral endothelial cells. Here, we demonstrated that V-domain Ig suppressor of T-cell activation (VISTA), a PD-L1 homologue, was highly expressed on endothelial cells in synovial sarcoma, subsets of different carcinomas, and immune-privileged tissues. We created an ex vivo model of the human vasculature and demonstrated that expression of VISTA on endothelial cells selectively prevented T-cell transmigration over endothelial layers, under physiological flow conditions, whereas it does not affect migration of other immune cell types. Furthermore, endothelial VISTA correlated with reduced infiltration of T cells and poor prognosis in metastatic synovial sarcoma. In endothelial cells, we detected VISTA on the plasma membrane and in recycling endosomes, and its expression was upregulated by cancer cell-secreted factors in a VEGF-A-dependent manner. Our study reveals that endothelial VISTA is upregulated by cancer-secreted factors and that it regulates T-cell accessibility to cancer and healthy tissues. This newly identified mechanism should be considered when using immunotherapeutic approaches aimed at unleashing T cell-mediated cancer immunity.

Everolimus combined with PD-1 blockade inhibits progression of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Due to rapid progression and a lack of targetable receptors, TNBC is exceptionally difficult to treat. Available treatment options are nonspecific cytotoxic agents, which have had modest success; thus, there is a need for novel therapies for TNBC. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is aberrantly activated in TNBC, and this pathway has been shown to promote cancer cell survival and chemoresistance. As such, mTOR inhibition has been considered a potential therapeutic strategy for TNBC. The mTOR inhibitor everolimus (EVE) has been approved for the treatment of estrogen positive breast cancer; however, its efficacy in TNBC is still undetermined. In this study, we evaluated the effects of EVE monotherapy and the mechanism of EVE resistance in the 4T1 model of TNBC. Whereas EVE monotherapy inhibited mTOR signaling activity, it did not attenuate tumor progression. Additionally, tumors from EVE-treated mice had abnormal vasculature characterized by disorganized architecture and hyperpermeability. We also found that treatment with EVE increased PD-L1 expression in intratumoral vascular endothelial cells, and this increase in endothelial cell-associated PD-L1 corresponded to reduced CD8 + T cell tumor infiltration. Importantly, combination treatment with anti-PD-1 antibody and EVE normalized the tumor vasculature, rescued CD8 + T cell tumor infiltration, and reduced tumor growth. Taken together, our findings improve our current understanding of mechanisms underlying mTOR inhibition resistance in TNBC and identify a novel combination treatment strategy in the treatment of mTOR resistant tumors.

Immunohistochemical Expression Pattern of Theragnostic Targets SSTR2 and PSMA in Endolymphatic Sac Tumors: A Single Institution Case Series

BackgroundEndolymphatic sac tumors are rare neoplasia characterized by slow growth. However, their clinical impact should not be underestimated, considering their potential for local aggressive behavior and strong association with von Hippel–Lindau syndrome. Therefore, early detection with emerging theragnostic examinations such as 68Ga-DOTATATE-PET/CT might improve patient management and reduce morbidity.MethodsWe report the clinicopathological features of seven endolymphatic sac tumors. In this cohort, we performed immunohistochemical analysis of somatostatin receptor 2A (SSTR2A) and prostate specific membrane antigen (PSMA) protein expression patterns; two targets providing rationale for novel imaging modalities such as PSMA- or SSTR-targeted PET.ResultsThe tumor cells of all cases were negative for prostate specific membrane antigen and somatostatin receptor 2A, however immunolabeling was consistently detected in intratumoral endothelial cells of endolymphatic sac tumors for PSMA (7/7 cases, 100%), and for SSTR2A (5/7 cases, 71%).ConclusionsOur results show a high rate of PSMA and SSTR2A expression in the tumor vasculature of endolymphatic sac tumors. PSMA and SSTR2A can be targeted with appropriate radioligands for diagnostic and therapeutic purposes. This finding provides a rationale for prospective clinical studies to test this approach as a sensitive screening tool for patients with suspected endolymphatic sac tumors including an improved management of von Hippel–Lindau syndrome.

Evaluating Established Roles, Future Perspectives and Methodological Heterogeneity for Wilms' Tumor 1 (WT1) Antigen Detection in Adult Renal Cell Carcinoma, Using a Novel N-Terminus Targeted Antibody (Clone WT49).

Renal cell carcinoma (RCC) is arguably the deadliest form of genitourinary malignancy and is nowadays viewed as a heterogeneous series of cancers, with the same origin but fundamentally different metabolisms and clinical behaviors. Immunohistochemistry (IHC) is increasingly necessary for RCC subtyping and definitive diagnosis. WT1 is a complex gene involved in carcinogenesis. To address reporting heterogeneity and WT1 IHC standardization, we used a recent N-terminus targeted monoclonal antibody (clone WT49) to evaluate WT1 protein expression in 56 adult RCC (aRCC) cases. This is the largest WT1 IHC investigation focusing exclusively on aRCCs and the first report on clone WT49 staining in aRCCs. We found seven (12.5%) positive cases, all clear cell RCCs, showing exclusively nuclear staining for WT1. We did not disregard cytoplasmic staining in any of the negative cases. Extratumoral fibroblasts, connecting tubules and intratumoral endothelial cells showed the same exclusively nuclear WT1 staining pattern. We reviewed WT1 expression patterns in aRCCs and the possible explanatory underlying metabolomics. For now, WT1 protein expression in aRCCs is insufficiently investigated, with significant discrepancies in the little data reported. Emerging WT1-targeted RCC immunotherapy will require adequate case selection and sustained efforts to standardize the quantification of tumor-associated antigens for aRCC and its many subtypes.

Abstract P5-06-03: Intratumoral lymphatic endothelial cell infiltration reflects lymphangiogenesis and lymph node metastasis, but is counterbalanced by immune response and better cancer biology in breast cancer tumor microenvironment

Abstract Background: Unfavorable vasculogenesis, such as lymphangiogenesis and hemoangiogenesis are known to be pivotal of cancer progression. Our group reported that S1P pathway link chronic inflammation, such as obesity, and cancer progression by inducing lymphangiogenesis and hemoangiogenesis in breast cancer. During the past decade, complex cytokine network interactions between cancer cells and stromal cells have been found to mediate tumor-associated lymphangiogenesis and hemoangiogenesis. Thus, it is crucial to understand cancer biology as a tumor microenvironment, rather than focusing solely on cancer cells. To this end, we hypothesized that amount of intratumoral lymphatic endothelial cells (iLEC) reflect lymphangiogenesis and associate with poor clinical characteristics and outcomes. Method: We analyzed a total of 4145 breast cancer patient’s clinical and transcriptome data from The Cancer Genome Atlas (TCGA), and GSE96058 datasets. Intratumoral lymphatic endothelial cells (iLEC) and all the other immune cells were determined by xCell algorithm, and a median cutoff was adopted to divide high and low groups. Result: First we performed gene set enrichment assay using Hallmark and PID collections from MSigDB and found that high iLEC breast cancer enriched Lymphangiogenesis-related gene sets (lymph angiogenesis, S1P meta, S1P1, and S1P3 pathway) and hemoangiogenesis-related gene sets (Angiogenesis, VEGFR1 pathway, and VEGFR2 pathway) (all FDR<0.25). Microvascular endothelial cells were also predominant in high iLEC tumors (p < 0.001). Even though iLEC was significantly higher in patients with lymph node metastasis consistently in both TCGA and GSE96058 cohorts, there was no survival difference by the amount of iLEC in neither of the cohorts. High iLEC breast cancer also enriched cancer stem cell-related gene sets (notch signaling, hedgehog signaling, epithelial mesenchymal transition, and Wnt β catenin signaling) and metabolic pathways (bile acid metabolism, fatty acid metabolism), and S1P2 pathway (all FDR<0.25). Stromal cells such as Adipocytes, Preadipocytes and Fibroblasts, were also more prominent in the high iLEC group (all p < 0.001). The immune-related gene sets (allograft rejection, IL2 STAT5 pathway, IL6 Jak STAT3 signaling, inflammatory response, TNFα signaling via NFkB, and complement) and cytolytic activity, reflecting anti-tumor immunity, were elevated in high iLEC group of both cohorts (all FDR<0.25, p < 0.001), but anti-cancer immune cells were not significantly infiltrated. Interestingly, cell proliferation-related gene set (E2F target, G2M checkpoint, MYC target v1) and mTORc1 signaling gene set was strongly enriched in low iLEC breast cancer (all FDR<0.25). Furthermore, Ki67 expression and histological grade as well as aggressive cancer subtype were negatively correlated with iLEC fraction (all p<0.002, r <-0.29). Finally, we found that in the TCGA cohort, low iLEC breast cancer was significantly associated with intratumor heterogeneity, silent and nonsilent mutation rate, fraction altered, and homologous recombination deficiency, based on scoring by Thorrson et al (all p < 0.001). Despite the association with high TIL regional fraction and IFN-γ Response, low iLEC breast cancer was infiltrated by pro-cancer immune cells, such as Th2, Treg, and B-cells (all p < 0.001). Conclusion: High iLEC breast cancer reflected lymphangiogenesis and were associated with cancer stem cell-related genes and lymph node metastasis, while low iLEC breast cancers were associated with accelerated cell proliferation and pro-cancer immune cell infiltrations. Unfavorable cancer characteristics on both high and low iLEC groups may explain the lack of difference in patient survival. Citation Format: Rongrong Wu, Masanori Oshi, Mariko Asaoka, Akimitsu Yamada, Yamato Takabe, Li Yan, Itaru Endo, Takashi Ishikawa, Kazuaki Takabe. Intratumoral lymphatic endothelial cell infiltration reflects lymphangiogenesis and lymph node metastasis, but is counterbalanced by immune response and better cancer biology in breast cancer tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-03.

Abundance of Microvascular Endothelial Cells Is Associated with Response to Chemotherapy and Prognosis in Colorectal Cancer

Simple SummaryThe generation of pathologic, immature, and dysfunctional vessels by angiogenesis is a well-known mechanism of metastasis and has been a therapeutic target for colorectal cancer (CRC). In this study, we investigated the clinical relevance of microvascular endothelial (mvE) cells in CRC by analyzing tumor gene expression profiles of large patient cohorts. We found that the abundance of mvE cells does not mirror angiogenesis, but rather is associated with the number of mature blood vessels in the tumor microenvironment and predicts the response to chemotherapy as well as patient survival in CRC. This is the first study suggesting the clinical relevance of mvE cells in CRC.The generation of pathologic, immature, and dysfunctional vessels by angiogenesis is a mechanism of metastasis that has been a therapeutic target for colorectal cancer (CRC). In this study, we investigated the clinical relevance of intra-tumoral microvascular endothelial (mvE) cells in CRC using the xCell algorithm on transcriptome. A total of 1244 CRC patients in discovery and validation cohorts were analyzed. We found that an abundance of mvE cells did not mirror angiogenesis but reflected mature blood vessels because it was significantly associated with a high expression of vascular stability-related genes, including sphingosine-1-phosphate receptor genes and pericytes. Epithelial–mesenchymal transition and myogenesis gene sets were enriched in mvE cell abundant CRC, while mvE cell-less CRC enriched cell proliferation, oxidative phosphorylation, and protein secretion gene sets. mvE cell abundant CRC was associated with infiltration of M2 macrophages, dendritic cells, and less gamma-delta T cells (all p < 0.001), but not with the interferon-γ response. mvE cell abundant CRC was significantly associated with worse patient survival in CRC. Interestingly, mvE cell abundant CRC was significantly associated with a high response rate to chemotherapy (p = 0.012) and worse patient survival for those that did not receive chemotherapy. However, there was no survival difference in patients who underwent chemotherapy. In conclusion, we estimated the abundance of mvE cells using the xCell algorithm on tumor transcriptome finding its association with the number of mature blood vessels in a tumor microenvironment and its ability to predict response to chemotherapy, thereby patient survival in CRC.

Abstract 4183: Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator

Abstract Background and objectives: Despite the outstanding antitumor activity of halichondrins in mice, the limited supply from the natural sources has prevented drug development using intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) under good manufacturing practice (GMP) conditions. E7130 is not only a novel microtubule dynamics inhibitor, but also a novel tumor-microenvironment ameliorator. E7130 can increase intratumoral CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts (CAFs) at pharmacologically relevant compound concentrations. Here, we analyzed the molecular mechanisms of the α-SMA-positive CAF reduction with E7130. Methods: The expression levels of α-SMA, ECM proteins, ER-TR7 (pan-fibroblast marker), and Ki67 were examined using E7130-treated tumors. In vitro co-culture experiments, normal human fibroblasts were co-cultured with human cancer cells. To examine the effects of E7130 on the TGF-β-induced CAF activation, normal human fibroblasts were treated with TGF-β (final concentration of 1 ng/mL) and E7130 at the several concentrations, and gene expression analysis and immunocytochemical analysis were conducted. Results: E7130 reduced α-SMA-positive CAF and malignant ECM proteins without changing total fibroblast number in tumors. The in vitro co-culture system revealed that TGF-β from cancer cells activate normal human fibroblast and increased α-SMA expression. In addition, treatment with E7130 interfered with α-SMA induction by TGF-β in fibroblasts without growth inhibitory activity. We further found that E7130 inhibited TGF-β-induced PI3K/AKT/mTOR pathway, which resulted in the reduction of α-SMA expression in fibroblasts. Moreover, TGF-β treatment enhanced β-tubulin expression and focal adhesions formation, which were diminished by co-treatment with E7130 in fibroblasts. Many signaling complexes are assembled in focal adhesion sites, and those complexes dispatch several downstream signals, including those involved in the PI3K/AKT/mTOR pathway. Conclusions: Drug discovery using halichondrins was based on their outstanding in vivo antitumor (inhibitory) activity in mice and bone metastasis in mouse melanoma model first reported in 1996. From these results, we hypothesized that halichondrins are not simple microtubule-targeted compounds in tumor cell, and have developed E7130, which harbors unique tumor microenvironment ameliorating effects. Here we proved that E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process without killing the fibroblast by disrupting microtubule network formation, which is important for focal adhesion assembly and thereby the downstream activation of the PI3K/AKT/mTOR pathway. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4183.

Expression of V-domain immunoglobulin suppressor of T cell activation is associated with the advanced stage and presence of lymph node metastasis in ovarian cancer.

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a novel negative immune checkpoint that belongs to the B7 family. VISTA is primarily expressed on hematopoietic cells and inhibits T cell proliferation and cytokine production. The blockade of VISTA has demonstrated promising results in certain murine tumor models. In the present study, an immunohistochemical analysis of VISTA expression on tumor cells, intratumoral immune cells and vascular endothelial cells was performed in a cohort of 65 patients with ovarian cancer (OC). The associations between VISTA expression and different clinicopathological characteristics were evaluated using Fisher's exact test, and the analysis of overall survival in different groups was performed by the construction of Kaplan-Meier curves. The results indicated that high expression of VISTA on tumor cells or ICs was significantly associated with advanced tumor stage and the presence of lymph node metastasis (LNM). However, the percentage of cases with high expression of VISTA on tumor cells (24.6%) was decreased compared with those with high expression on ICs (44.6%). There was no association between VISTA expression and the 5-year overall survival rate, and advanced-stage disease was the only independent predictor of poor prognosis based on multivariate Cox regression analysis. In general, VISTA expression increased with advanced disease stage and LNM, indicating that VISTA expression is involved in the progression of OC. More importantly, these data implicate VISTA as a candidate immunotherapeutic target in OC.

Prognostic role of myoferlin expression in patients with clear cell renal cell carcinoma.

ObjectivesIn patients with cancer, myoferlin protein hyperexpression has been correlated with poor patient prognosis. Here, we evaluated myoferlin expression in patients with clear cell renal cell carcinoma (ccRCC) and investigated the prognostic significance of myoferlin expression in these patients.Materials and MethodsOne hundred and fifty-two patients with ccRCC who underwent treatment at Gyeongsang National University Hospital, Korea, between January 2000 and December 2009 were enrolled. Immunohistochemical analysis was performed on tissue microarray blocks produced from surgical specimens. Surgical specimen cancerous cells were graded as showing myoferlin hyperexpression or hypoexpression by comparison with intratumoral endothelial cells. Disease-free survival was evaluated using Kaplan-Meier analysis. Cox regression analysis was used to determine the relationships between myoferlin expression levels, risk factors, and prognosis.ResultsSeventy-one of 304 cores exhibited myoferlin hyperexpression. T stage was not associated with myoferlin hyperexpression, whereas a high Fuhrman nuclear grade was significantly associated with myoferlin hyperexpression. Kaplan-Meier analysis revealed that patients with T stage >2, Fuhrman nuclear grade >2, and those with myoferlin hyperexpression had poorer disease-free survival compared to those with lower T stage, lower Fuhrman nuclear grade, and myoferlin hypoexpression (all p <0.001). Furthermore, myoferlin hyperexpression was significantly associated with disease-free survival on Cox regression analysis (hazard ratio, 4.604; 95% confidence interval, 1.893–11.199; p = 0.001).ConclusionMyoferlin expression could be a potential prognosticator in patients with ccRCC, and might be a useful marker for oncologic surveillance in such patients.

In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability.

Vascular hyperpermeability is a pathological hallmark of cancer. Previous in vitro studies have elucidated roles of various signaling molecules in vascular hyperpermeability; however, the activities of such signaling molecules have not been examined in live tumor tissues for technical reasons. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we examined the activity of protein kinase A (PKA), which maintains endothelial barrier function. The level of PKA activity was significantly lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analogue alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. Because the vascular endothelial growth factor (VEGF) receptor is a canonical inducer of vascular hyperpermeability and a molecular target of anticancer drugs, we examined the causality between VEGF receptor activity and the PKA activity. Motesanib, a kinase inhibitor for VEGF receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Conversely, subcutaneous injection of VEGF decreased endothelial PKA activity and induced hyperpermeability of subcutaneous blood vessels. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo These data suggested that the VEGF receptor signaling pathway increases vascular permeability, at least in part, by reducing endothelial PKA activity in the live tumor tissue. Cancer Res; 76(18); 5266-76. ©2016 AACR.

Elderly patients at higher risk of laryngeal carcinoma recurrence could be identified by a panel of two biomarkers (nm23-H1 and CD105) and pN+ status.

Laryngeal squamous cell carcinoma (LSCC) recurrences are very difficult to manage in elderly patients (age ≥65 years), because treatment carries significant morbidity and mortality. The aim of this study was to develop a panel of parameters (clinicopathological variables or biomarkers) to improve our ability to detect elderly patients at higher risk of LSCC recurrence. Maspin, nm23-H1, and CD105 were investigated using immunohistochemistry on surgical specimens from 46 elderly patients treated for LSCC. After univariate analysis identified parameters associated with LSCC recurrence, a multivariate prognostic model was constructed. At univariate analysis, a higher recurrence rate was significantly associated with nm23-H1 nuclear expression in carcinoma cells ≤2.0% (p = 0.01), CD105 expression in intratumoral vascular endothelial cells ≥5.28% (p = 0.04), and pN+ status (p = 0.04). Multivariate modeling confirmed that nuclear nm23-H1 ≤2.0% (p = 0.009) and CD105 ≥5.28% (p = 0.013) had a negative prognostic significance in terms of disease recurrence, while pN+ status showed a trend toward significance (p = 0.05). We thus obtained a panel comprising two biomarkers and neck lymph node status that revealed an excellent discriminatory power [AUC (ROC) of 0.81] in terms of the risk of LSCC recurrence. The panel achieved a specificity of 96% and a positive predictive value of 93%. We identified a panel with an excellent discriminatory power in identifying elderly patients at higher risk of recurrence after treatment for LSCC. These patients would benefit from a more aggressive primary treatment.

Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis.

Glioblastomas are the most frequent and most aggressive primary brain tumors in adults. The median overall survival is limited to a few months despite surgery, radiotherapy, and chemotherapy. It is now clearly established that hyperactivity of cyclin-dependent kinases (CDKs) is one of the processes underlying hyperproliferation and tumoral growth. The marine natural products meridianins and variolins, characterized as CDK inhibitors, display a kinase-inhibitory activity associated with cytotoxic effects. In order to improve selectivity and efficiency of these CDK inhibitors, a series of hybrid compounds called meriolins have been synthesized. The potential antitumoral activity of meriolins was investigated in vitro on glioma cell lines (SW1088 and U87), native neural cells, and a human endothelial cell line (HUV-EC-C). The impact of intraperitoneal or intratumoral administrations of meriolin 15 was evaluated in vivo on 2 different nude mice-xenografted glioma models. Meriolins 3, 5, and 15 exhibited antiproliferative properties with nanomolar IC50 and induced cell-cycle arrest and CDK inhibition associated with apoptotic events in human glioma cell lines. These meriolins blocked the proliferation rate of HUV-EC-C through cell cycle arrest and apoptosis. In vivo, meriolin 15 provoked a robust reduction in tumor volume in spite of toxicity for highest doses, associated with inhibition of cell division, activation of caspase 3, reduction of CD133 cells, and modifications of the vascular architecture. Meriolins, and meriolin 15 in particular, exhibit antiproliferative and proapoptotic activities on both glioma and intratumoral endothelial cells, constituting key promising therapeutic lead compounds for the treatment of glioblastoma.

Endothelial cells do not arise from tumor-initiating cells in human hepatocellular carcinoma

BackgroundConventional models of carcinogenesis suggest that tumors recruit blood vessel formation from normal host tissues. This concept has recently been challenged by prominent studies of glioblastoma, which suggest that intratumoral endothelial cells (ECs) may arise from cancer stem cells/tumor-initiating cells (TICs). Hepatocellular carcinoma (HCC) is a common, highly vascularized tumor with few effective therapies, against which anti-angiogenic strategies are being actively explored. TICs are felt to play a role in HCC pathobiology, but their contributions to tumor vasculature have not been studied.MethodsWe examined human HCCs in settings that selected for tumor formation from functionally defined TICs, and in which the origin of intratumoral ECs from TICs as opposed to host tissues could be clearly distinguished. We generated HCC nodules in the livers of immunodeficient mice by intrasplenic injection of HCC cells from cell lines and patient specimens and studied the tumor ECs by immunohistochemistry for mouse and human markers. We then used immunohistochemistry for EC markers in combination with fluorescence in situ hybridization (FISH) for X and Y chromosomes to study the endothelium of recurrent HCC specimens resected from sex-mismatched liver allografts of patients who had undergone liver transplantation for HCC.ResultsWe observed that all ECs in intrahepatic human HCC xenografts expressed mouse rather than human CD31. FISH analysis of recurrent HCCs resected from patients with sex-mismatched liver allografts revealed that all CD31+ and CD34+ intratumoral ECs originated from the donor allograft rather than the tumor.ConclusionsThese observations suggest that the vasculature of human HCC arises from normal host tissues rather than from TICs, supporting ongoing efforts to target angiogenesis in HCC as it is currently understood, and suggesting that the contribution of TICs to the vasculature of other cancers is disease-specific.

Quantification in Molecular Ultrasound Imaging

The purpose of this study was to quantitatively assess the contrast kinetics of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted microbubbles (BR55; Bracco Suisse, Geneva, Switzerland) compared to clinically used microbubbles (SonoVue; Bracco SpA, Milan, Italy) in both normal liver and human hepatocellular carcinoma xenograft tumors in mice. Microbubbles were injected intravenously into healthy mice (n = 5) and mice bearing hepatocellular carcinoma xenograft tumors (n = 10). Cine loops of contrast enhancement in normal liver and the tumors were acquired for 10 minutes. Quantitative perfusion parameters were derived by fitting time-intensity curves using a dedicated mathematical model combining a bolus function and a ramp function. Immunohistochemical examinations were also performed for normal liver and tumor specimens to determine the level of VEGFR2 expression. The peak contrast enhancement observed in normal liver with BR55 was comparable to that with SonoVue, whereas a significant difference was observed in latephase enhancement at 10 minutes (ramp slope: P < .01). In the tumor model, SonoVue was rapidly cleared from the circulation, without any noticeable binding in the tumor, whereas BR55 showed a gradual decline, resulting in a longer wash-out period (mean transit time: P < .01). Immunohistochemical examinations showed that intratumoral vascular endothelial cells had sparse and weak VEGFR2 expression, whereas the sinusoidal capillaries in normal liver had much more diffuse and much stronger expression. Our results suggest that BR55 accurately reflects the VEGFR2 status in human hepatocellular carcinoma xenograft tumors. We showed that quantification applied to molecular ultrasound imaging may provide an objective method for measuring the degree of microbubble binding.

Blocking Fibroblast Growth Factor Receptor Signaling Inhibits Tumor Growth, Lymphangiogenesis, and Metastasis

Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DN-expressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of pro-lymphangiogenic molecules such as VEGFR-3, integrin α9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread.

Abstract IA5: Developing therapeutics in mice with ductal pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common malignancy, with little improvement in patient outcomes over the past 40 years. PDAC frequently harbors somatic mutations in 4 genes (KRAS, P16, Tp53, and SMAD4), and recent exomic and whole genome sequencing efforts have identified a number of less frequent events. Accurate mouse models of PDAC were generated over the last decade, confirming the relevance of these 4 genes and enabling the investigation of fundamental aspects of PDAC tumor biology and therapeutic response. The poor response of pancreatic cancer patients to systemic agents is not predicted by xenograft and other transplanted model systems. An evaluation of this discordant behavior revealed that transplanted tumor models have superior tissue perfusion and delivery of the chemotherapeutic gemcitabine compared to primary murine PDAC, and that this inversely correlates with stromal content. Human PDAC was subsequently confirmed to phenocopy the mouse model as it is also profoundly hypovascular. Stromal depletion with a hedgehog pathway inhibitor increased vascular density and gemcitabine delivery, and prolonged survival to demonstrate a causative role of the hypovascular state in drug responsiveness in PDAC3. The clinical translation of this observation has been encouraging, with several dramatic responses in patients with metastatic PDAC. A randomized trial is currently underway. Alternative methods that target the tumor stroma may also be beneficial in PDAC, including the use of nab-paclitaxel (Abraxane), an albumin-paclitaxel formulation proposed to be sequestered in the intratumoral stroma. Similar to the hedgehog inhibitor, nab-paclitaxel also elevated the gemcitabine levels in the mouse PDAC tumors. However, the mechanism employed was distinct as nab-Paclitaxel did not deplete the stroma and increase the vascular density. Rather, paclitaxel elicits the liberation of reactive oxygen species (ROS), and ROS destabilize and induce the destruction of cytidine deaminase, the major pathway of gemcitabine inactivation in cells4. This novel method of drug synergy was overlooked in the PDX (patient derived xenograft) models, as the chimeric murine stroma is only loosely associated with the human neoplastic cells and they therefore may provide misleading results5. Nab-Paclitaxel treatment with gemcitabine is active in early clinical trials, and genetic variants affecting gemcitabine metabolism may modify this outcome. PDAC is notoriously resistant to VEGF targeted therapeutics despite numerous preclinical experiments that predicted efficacy, and recent approaches in murine PDAC confirmed the prior clinical data6. The hypovascular content of PDAC suggests that the intratumoral endothelial cells are poorly responsive to the available VEGF ligand, and therefore additional pathways may play a more dominant role in vessel proliferation and maintenance. The notch pathway has previously been implicated in vessel morphogenesis, and can be chemically inhibited with gamma secretase inhibitors (GSI). GSI treatment induced the rapid destabilization of the PDAC vasculature, by promoting the death of intratumoral endothelial cells. These effects were exacerbated by concomitant exposure to gemcitabine, resulting in hypoxic necrosis and death of both endothelial cells and neoplastic cells7. A clinical trial has recently begun to evaluate this approach. Although the unique attributes of the PDAC microenvironment participate in the resiliency of PDAC to therapeutics, they also serve as vulnerabilities to exploit for clinical benefit.

Expression of Vascular Notch Ligand Delta-Like 4 and Inflammatory Markers in Breast Cancer

Delta-like ligand 4 (Dll4) is a Notch ligand that is predominantly expressed in the endothelium. Evidence from xenografts suggests that inhibiting Dll4 may overcome resistance to antivascular endothelial growth factor therapy. The aims of this study were to characterize the expression of Dll4 in breast cancer and assess whether it is associated with inflammatory markers and prognosis. We examined 296 breast adenocarcinomas and 38 ductal carcinoma in situ tissues that were represented in tissue microarrays. Additional whole sections representing 10 breast adenocarcinomas, 10 normal breast tissues, and 16 angiosarcomas were included. Immunohistochemistry was then performed by using validated antibodies against Dll4, CD68, CD14, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), CD123, neutrophil elastase, CD31, and carbonic anhydrase 9. Dll4 was selectively expressed by intratumoral endothelial cells in 73% to 100% of breast adenocarcinomas, 18% of in situ ductal carcinomas, and all lactating breast cases, but not normal nonlactating breast. High intensity of endothelial Dll4 expression was a statistically significant adverse prognostic factor in univariate (P = 0.002 and P = 0.01) and multivariate analyses (P = 0.03 and P = 0.04) of overall survival and relapse-free survival, respectively. Among the inflammatory markers, only CD68 and DC-SIGN were significant prognostic factors in univariate (but not multivariate) analyses of overall survival (P = 0.01 and 0.002, respectively). In summary, Dll4 was expressed by endothelium associated with breast cancer cells. In these retrospective subset analyses, endothelial Dll4 expression was a statistically significant multivariate prognostic factor.

Genetic and epigenetic inactivation of T‐cadherin in human hepatocellular carcinoma cells

T-cadherin is an atypical cadherin and growing evidence has indicated that T-cadherin exerts tumor-suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T-cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T-cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T-cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT-PCR analysis also showed that the T-cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32-38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5-aza-2'-deoxycytidine or histone deacetylase inhibitor trichostatin A, T-cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T-cadherin induced G(2)/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage-independent growth in soft agar and increased sensitivity to TNFalpha-mediated apoptosis in HCC cells. Intriguingly, we found that T-cadherin significantly suppressed the activity of c-Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T-cadherin was differentially expressed in human HCCs. The underexpression of T-cadherin in HCC cells suggests it may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC development.

Anti‐VEGF‐A therapy reduces lymphatic vessel density and expression of VEGFR‐3 in an orthotopic breast tumor model

Because metastasis contributes significantly to cancer mortality, understanding its mechanisms is crucial to developing effective therapy. Metastasis is facilitated by lymphangiogenesis, the growth of new intratumoral or peritumoral lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor A (VEGF-A) is a well-known angiogenic factor. Increasing evidence implicates VEGF-A in lymphangiogenesis, although the mechanism of its pro-lymphangiogenic effect is poorly understood. We examined the effect of the anti-VEGF-A neutralizing antibody 2C3 on tumor lymphangiogenesis and metastasis in an orthotopic breast carcinoma model using MDA-MB-231 cells and its luciferase-tagged derivative, 231-Luc(+) cells. Anti-VEGF-A antibody therapy reduced blood and lymphatic vessel densities by 70% and 80%, respectively, compared with the control antibody. Treatment with 2C3 antibody also decreased incidence of lymphatic and pulmonary metastases by 3.2- and 4.5-fold, respectively. Macrophage infiltration was reduced in 2C3-treated tumors by 32%, but VEGF-C expression was unchanged. In contrast, neoplastic cells and blood vessels in tumors from 2C3-treated mice expressed significantly less angiopoietin-2 (Ang-2) than tumors from control mice. The reduction in Ang-2 was associated with inhibition of VEGFR-3 expression in intratumoral lymphatic endothelial cells. Both VEGF-A and Ang-2 upregulated the expression of VEGFR-3 in cultured lymphatic endothelial cells. VEGF-A induced proliferation of lymphatic endothelial cells was reduced by 50% by soluble Tie-2, suggesting that Ang-2 is an intermediary of the pro-lymphangiogenic VEGF-A effect. These results suggest a novel mechanism by which anti-VEGF-A therapy may suppress tumor lymphangiogenesis and subsequent metastasis supporting the use of anti-VEGF-A therapy to control metastasis clinically.

Human brain tumors: multidrug-resistance P-glycoprotein expression in tumor cells and intratumoral capillary endothelial cells

Malignant brain tumor is a lethal disease with currently available treatment options having a limited impact on outcome. Nevertheless, novel therapeutic approaches combined with genetic prediction of chemosensitivity have, in the last decade, significantly improved clinical benefit for the treated patients. The fine characterization of the MDR1 gene encoding for P-glycoprotein (MDR1-Pgp) in brain tumors may be a crucial determinant for evaluating the long-term efficiency of specific anti-cancer compounds. By using a very high specific monoclonal antibody, the MDR1-Pgp was immunodetected in 34 out of 43 grade IV, 6 out of 10 grade III, 4 out of 7 grade II, and 1 out 3 grade I brain tumors. MDR1-Pgp resulted hyper-expressed, both in vessels and in neoplastic cells from the majority of tumors examined, compared to normal parenchyma. This study demonstrates that the MDR1 gene can be detected in all grade tumor brain malignancies and in endothelial cells of newly formed capillaries, thus, impairing drug access at the tumor cell level. Although the role of MDR1-Pgp in tumor blood vessels needs to be further examined and more clearly defined, drug resistance in malignant brain tumors may result from characteristics not only of tumor vasculature but also of neoplastic cells.