Tumor Microvessel Research Articles

An-te-xiao capsule inhibits tumor growth in non-small cell lung cancer by targeting angiogenesis

An-te-xiao capsule consists of total alkaloids from the dried whole plantof Solanum lyratum, and showed antitumor effects in our previous study. However, its inhibitory effect on multiple non-small cell lung cancer (NSCLC) cell lines and the underlying mechanisms have not been elucidated clearly. This study sought to investigate the inhibitory effects of An-te-xiao capsule on three main types of NSCLC cell lines (A549, NCI-H460, and NCI-H520) in vitro and in vivo and the underlying mechanisms of action including its potential anti-angiogenesis effects. An-te-xiao capsule showed no acute oral toxicity in mice, and significantly prolonged survival time in a mouse model of Lewis tumor xenograft. The inhibition of A549, NCI-H460, and NCI-H520 cells by An-te-xiao capsule was reflected in its effects on tumor growth, histopathological changes, tumor microvessel density (MVD), cell cycle regulatory proteins, and cell apoptosis. In vitro, An-te-xiao capsule repressed migration, invasion, and tube formation of tumor-derived vascular endothelial cells (Td-ECs), which were obtained using a co-culture system, in the presence or absence of vascular endothelial growth factor (VEGF) at safe concentrations selected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, An-te-xiao capsule inhibited the secretion of VEGF by A549 cells in the co-culture system and suppressed the phosphorylation of VEGF receptor 2 (VEGFR2). Taken together, An-te-xiao capsule has potential for treating NSCLC.

CCL19 suppresses angiogenesis through promoting miR-206 and inhibiting Met/ERK/Elk-1/HIF-1\u03b1/VEGF-A pathway in colorectal cancer

The mechanisms underlying the role of chemokines in tumor angiogenesis is still not fully understood. In this study, we detected the influence of CCL19 on colorectal cancer (CRC) angiogenesis. The expression of CCL19 and CD31 in CRC tissues were detected by immunohistochemistry. Human CRC cell lines SW1116 and SW620 stably transfected with CCL19 lentivirus and CCL19 shRNA, and HUVEC stably transfected with CCR7 shRNA were used in our study. Our study showed that CCL19 was significantly low-expressed in CRC tissues and positively related to highly tumor microvessel density. In vitro, we observed that CCL19 high-expressed SW1116 supernatant was able to inhibit proliferation, migration, and sprouting responses of HUVEC, whereas CCL19 low-expressed SW620 supernatant can promote HUVEC angiogenesis. Additionally, we further demonstrated that these functions maybe achieved through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway in a CCR7-dependent manner. Mice angiogenesis model also confirmed that elevated expression of CCL19 inhibit the angiogenesis of CRC in vivo. In summary, our results supported that CCL19 can inhibit CRC angiogenesis through promoting miR-206 thus inhibiting Met/ERK/Elk-1/HIF-1α/VEGF-A pathway. This may be a novel therapeutic option for anti-vascular treatment in CRC.

P04.35 Preoperative tumor growth and microvessel density measurements in patients with glioblastoma

P04.35 Preoperative tumor growth and microvessel density measurements in patients with glioblastoma

Abstract A069: Combination of immune status and tumor microvascularization provides strong prognostic markers for prostate cancer recurrence prediction

Abstract Introduction: The composition of different immune cell populations in the tumor microenvironment plays an important role for tumor progression in various cancer types. In particular, tumor-associated macrophages (TAMs) and tumor-infiltrating T cells (TILs) are relevant players. On the other hand, structural changes such as neoangiogenesis triggered by cancerous tumors to increase their nutrient supplies have also been associated with tumor progression. In this work we systematically quantified these factors in prostate cancer (PCa) and correlated them with clinical outcome data using a Tissue Phenomics approach. By investigating the prognostic relevance of TAMs (CD68/CD163), TILs (CD3/CD8), and microvessels (CD34) in the tumor, tumor microenvironment (TME), and stroma we identified strong prognostic markers for PCa recurrence prediction in patients after radical prostatectomy. Methods: In this study, we analyzed a cohort of 90 PCa patients, of whom 40 suffered from tumor progression measured by prostate cancer antigen (PSA) recurrence after prostatectomy. The cohort comprised low- and intermediate-risk PCa patients (Gleason-Score≤7b) since providing a reliable prognosis is particularly difficult for such grades. Tissue sections were immunohistochemically stained using the duplex stains CD68/CD163 for TAMs, CD3/CD8 for TILs, and CK18/p63 to identify and characterize glands as cancerous vs. noncancerous based on their expression level of p63 (in cancerous glands p63 is not expressed). To quantify tumor neoangiogenesis microvessels were stained by CD34. All sections were geometrically aligned per case (virtual multiplexing) to enable coanalysis of stains, and quantified within relevant regions-of-interest (tumor, TME, stroma) using fully automated computational methods (1, 2). In particular, we determined region-specific densities and average distances of TAMs, TILs, and microvessels, as well as ratios of all measures. We systematically analyzed the prognostic power of each measure by optimizing a cutoff with respect to the disease-free survival statistic (log-rank test) using cross-validation to avoid for overfitting. Results: The top-ranking prognostic markers regarding robustness and prediction performance were related to microvessel density combined with immune cell densities. In particular, we found that within the TME, a coverage of CD8(+) cytotoxic T cells larger than 10% of the coverage of CD34(+) microvessels is correlated with a good prognosis and long-term disease-free survival (cross-validated p <3.1•10-7, accuracy=83%). This corresponds to high densities of CD8(+) cells and/or low microvessel densities, which both have been shown to be associated with good prognosis in prostate cancer. In addition, we found that a larger average distance of CD68(+) M1 macrophages to CD34(+) microvessels above 75.7µm in the tumor region is associated with good prognosis (cross-validated p <2.9•10-8, accuracy=82%). Again, low tumor microvessel density seems to be beneficial as well as high densities of CD68(+) macrophages. The CD68(+) M1-polarized phenotype is associated with tumor-suppressing properties, indicating that a higher density of this population compared to the tumor-promoting M2-polarized phenotype fosters disease-free survival. Conclusion: Our results indicate a considerable prognostic potential of markers combining microvessel density with measures of TAMs and TILs to predict PSA recurrence in PCa. This application shows that systematic analysis as performed by Tissue Phenomics enables discovery of non-obvious combined prognostic markers characterizing the tumor landscape with high potential to improve patient treatment. In future work we aim to validate our findings on additional data from other clinical sites.

Heterogeneous blood flow in microvessels with applications to nanodrug transport and mass transfer into tumor tissue.

Nanodrug transport in tumor microvasculature and deposition/extravasation into tumor tissue are an important link in the nanodrug delivery process. Considering heterogeneous blood flow, such a dual process is numerically studied. The hematocrit distribution is solved by directly considering the forces experienced by the red blood cells (RBCs), i.e., the wall lift force and the random cell collision force. Using a straight microvessel as a test bed, validated computer simulations are performed to determine blood flow characteristics as well as the resulting nanodrug distribution and extravasation. The results confirm that RBCs migrate away from the vessel wall, leaving a cell-free layer (CFL). Nanodrug particles tend to preferentially accumulate in the CFL, leading to increased concentration near the endothelial surface layer. However, shear-induced NP diffusion is diminished within the CFL, causing to a much slower lateral transport rate into tumor tissue. These competing effects determine the NP deposition/extravasation rates. The present modeling framework and NP flux results provide new physical insight. The analysis can be readily extended to simulations of NP transport in blood microvessels of actual tumors.

STK405759 as a combination therapy with bortezomib or dexamethasone, in in vitro and in vivo multiple myeloma models.

Multiple myeloma (MM) remains an incurable hematological malignancy. Combination regimens of conventional and novel drugs have improved patient’s survival. However, most patients inevitably relapse and become refractory to the current therapeutic armamentarium.We investigated the efficacy of combining the microtubule-targeting agent STK405759 with dexamethasone or bortezomib in vitro and in vivo.STK405759 combined with dexamethasone or bortezomib had synergistic cytotoxic activity in RPMIS, CAG and MM1.S human MM cell lines through activation of caspase 2, 3, 8, 9 and PARP. These treatments remained cytotoxic in the presence of bone marrow stroma cells. In other MM cells, including cells resistant to vincristine, melphalan, mitoxantrone or dexamethasone, these combinations decreased significantly survival as compared to single agents.In in vivo studies, STK405759 disrupted existing blood vessels in xenograft tumors, acting not only as a cytotoxic agent but also as an anti-angiogenic drug. Mice treated with STK405759 in combination with dexamethasone or bortezomib resulted in greater tumor growth inhibition, increased overall response and prolonged survival as compared to as compared to BTZ or DEXA alone. Their anticancer activity was mediated by activation of apoptosis and reduction of tumor microvessel density.These preclinical studies provide the rationale for future clinical trials of STK405759, dexamethasone and bortezomib combinations to improve the outcome of multiple myeloma patients.

Abstract 5016: A unique ex vivo drug evaluation model: 3D co-cultured system with tumor, stroma and blood microvessels

Abstract Background; Two dimensional single-layer culture still remains the preferred platform for most laboratory preclinical studies, although interaction between cancer and stromal cells has been well reported to be important in tumor progression and resistance against therapies. In order to mimic the patient tumor tissues, ex vivo model which recaptures the tumor microenvironment is required. Methods; Layered 3D stromal tissues were produced by culturing normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs) coated with extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and overlaid tumors were morphologically characterized by HE stain, immunohistochemistry (IHC) and immunofluorescence (IF). Furthermore, drug sensitivity assays were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in the laboratory of Japanese Foundation for Cancer Research. Cancer cell viability was evaluated by fluorescent labeling, enzymatic dissociation and cell counting analysis. IF with cancer specific markers and imaging analyses were also performed. Results; The 3D stromal tissues including CD31 positive luminal structure were multi-layered (approximately 20 layers), and formation of microvascular network was observed within several days. In comparison with 2D mono-culture or 3D mono/co-cultured spheroid model, decreased drug sensitivities were represented in our 3D co-cultured model. In the simultaneous treatment with cytotoxic anticancer agents and molecular targeted drugs, dose-responses were significantly different between the2D and 3D models. Conclusion; We developed the layered 3D stromal tissue culture system including blood micro-vessels. Drug evaluation with the co-cultured tumors may reflect the drug sensitivity of cancer cells in vivo. Our unique 3D ex vivo model represents a valuable tool for drug development in a fully human cell and matrix microenvironment, and thus testing patient-derived cells and approved compounds also enable better prediction their efficacy. Citation Format: Yuki Takahashi, Kei Tsukamoto, Shiro Kitano, Shinji Irie, Michiya Matsusaki, Satoshi Nagayama, Ryohei Katayama, Eiji Shinozaki, Naoya Fujita. A unique ex vivo drug evaluation model: 3D co-cultured system with tumor, stroma and blood microvessels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5016.

Abstract 11: Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor

Abstract Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, especially in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). However, benefits of anti-VEGF therapies are still limited because of the intrinsic/acquired resistance to the treatment. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib has been approved as a monotherapy for the treatment of radioiodine-refractory differentiated thyroid cancer and in combination with everolimus for the treatment of patients with RCC treated with one prior anti-VEGF therapy in the US and EU. In this study, we established an anti-VEGF treatment resistant preclinical mouse tumor model. We also examined whether lenvatinib, which targeted multiple receptor tyrosine kinases, showed anti-tumor activity in this model. To establish model resistant to anti-VEGF treatment, an expression vector encoding VEGFR-Fc protein (VEGF decoy) was introduced into a mouse RCC cell line RENCA. First, we confirmed the RENCAVEGFR-Fc transfectants secreted VEGF decoy, which selectively interacted with recombinant VEGF protein but not with ligands to other RTKs in in vitro. Tumor growth in vivo of RENCAVEGFR-Fc was significantly slower than that of RENCAMock. RENCAMock transfectants took 16 days to reach a mean tumor volume (TV) of 500 mm3 whereas RENCAVEGFR-Fc transfectants required 72 days to reach same TV. Furthermore, 72 days after inoculation of RENCAVEGFR-Fc transfectants, we resected tumor tissues and confirmed the expression of VEGF decoy protein. Tumor microvessel density was decreased in resected RENCAVEGFR-Fc tumor compared to the same size of RENCAMock tumor. From the above results, we characterized RENCAVEGFR-Fc tumor was resistant to VEGF decoy. Finally, anti-tumor activity of lenvatinib was challenged against RENCAMock or RENCAVEGFR-Fc tumors. When tumor volume reached approximately 300mm3, 10 mg/kg of lenvatinib was orally administered once daily for 14 days. Treatment of lenvatinib led to almost complete inhibition of tumor growth in both RENCAMock and RENCAVEGFR-Fc models. In conclusion, we developed a mouse RENCAVEGFR-Fc model resistant to anti-VEGF treatment, and lenvatinib showed anti-tumor activity in the resistant model as with RENCAMock model. Additional preclinical studies will be investigated to elucidate the signaling pathways associated with resistance to anti-VEGF treatment, in which lenvatinib may inhibit such as FGFR1-4, PDGFRα, KIT and RET. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui. Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 11.

Abstract 6: Role of Notch signaling in bevacizumab-induced vascular normalization in glioblastoma

Abstract Tumor angiogenesis occurs in the setting of a defective vasculature, which is associated with increased vascular permeability and enhanced tumor permeability. Bevacizumab is used to treat malignant glioma and was found to reduce microvascular density and prune abnormal tumor microvessels. Using electron microscopic observation of two autopsy cases, we investigated the effects of blood vessel normalization in glioblastomas treated with bevacizumab. Notch-1 and SMA immunostaining were used to compare initial surgical specimens with postmortem specimens obtained after bevacizumab treatment. Postmortem samples showed marked proliferation of SMA-positive cells (pericytes) in tumor vessels and marked proliferation of Notch-1–positive cells around vessels. Electron microscopic images confirmed the presence of pericytes surrounding the vascular endothelium. These findings suggest that bevacizumab treatment promotes vascular normalization by recruiting mature pericytes. Next, we investigated the effects of bevacizumab on VEGF inhibition in glioma stem cells. Bevacizumab treatment attenuated activation of VEGFR2 and increased Notch signaling expression. VEGF inhibition by bevacizumab treatment attenuated proliferation and self-renewal of glioma stem cells and induced endothelial and pericyte differentiation. In tumor angiogenesis, vascular endothelial growth factor induces sprouting angiogenesis and recruitment of vascular endothelial cells such as tip cells, stalk cells, and phalanx cells. Fully mature phalanx cells are in close contact with pericytes. Our results suggest that bevacizumab treatment induces glioma stem cells to differentiate to endothelium and pericytes. These mechanisms might be important in normalizing tumor vasculature after bevacizumab treatment and could be useful in improving the effectiveness of current glioma therapy. Citation Format: Norihiko Saito, Kazuya Aoki, Nozomi Hirai, Satoshi Fujita, Haruo Nakayama, Morito Hayashi, Takatoshi Sakurai, Satoshi Iwabuchi. Role of Notch signaling in bevacizumab-induced vascular normalization in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 6.

Abstract 3704: Efficacy of DR5-targeted immunoliposomes in pancreatic cancer models primed with SMO inhibitors

Abstract Background: Pancreatic cancer (PaCA) is a deadly disease with 5-year survival of 6%. Poor prognosis is attributed to low tumor microvessel density, poor perfusion, and amplified stroma, constituting a drug delivery barrier. Tumor priming using NVP-LDE225, an inhibitor of sonic hedgehog (sHH) signaling, can compromise the stromal drug delivery barrier by mediating stromal thinning, and discharge of tumor interstitial pressure (IFP) and decompression of tumor vasculature, resulting in increased perfusion and deposition of drug-containing nanoparticles into tumors. However, convective fluid efflux may increase as interstitial tumor pressure declines during priming, increasing drug washout. We hypothesize that a targeting nanoparticle delivery system will increase drug retention during priming, thereby increasing efficacy. Approach: Patient-derived xenograft (PDX) PaCA tumors possessing varied stromal architecture and degrees of desmoplasia were implanted s.c. in SCID mice. They ranged from low- to high desmoplasia and varied in microvessel density. The sHH inhibitor (sHHI) NVP-LDE225 was administered p.o. for 10 days, and fluorescent 80nm sterically-stabilized liposomes (SSL) were administered at intervals to probe tumor vascular perfusion/permeability. Efficacy was investigated with SSL liposomes containing doxorubicin (SSL-DXR), optionally coupled covalently with Death Receptor 5 monoclonal antibody AMG655 (SSL-DXR-DR5), which binds the pro-apoptotic DR5 receptor and initiates the extrinsic apoptotic pathway. Results: Quantitative fluorescence microscopy showed that sHH inhibitor treatment mediated a temporal window of enhanced permeability/perfusion and deposition of SSL that varied with the amount of desmoplasia in PDX models, which are otherwise highly impermeant to SSL. Quantitative IFP measurements using the “wick and needle” technique showed reduction of IFP paralleled establishment of the priming window. In the absence of sHH inhibitor priming, SSL-DXR (2 mg/kg), SSL-DXR-DR5 (2 mg/kg of DXR), and free DR5 (dose-matched to SSL-DXR-DR5 protein) were administered to evaluate the effect of tumor priming on therapeutic efficacy. Any DR5-containing treatment was curative for MiaPaCA-2 tumors (high DR5 expression). The response of Panc-1 tumors (low DR5 expression) was greatest with SSL-DXR-DR5. Tumor responses with the PDX model #12424, (high DR5 expression) were greater in sHHI-treated animals compared to controls. SSL-DXR + free DR5 mediated a transient inhibition of tumor progression, whereas a durable inhibition of tumor progression was observed with SSL-DXR-DR5 immunoliposomes. Conclusions: Efficacy of SSL-DXR-DR5 immunoliposomes was superior to other treatments in two cell line-based tumors, and tumor response varied with DR5 receptor density. Efficacy in low permeability/perfusion PDX tumors was greatest following tumor priming by sHH inhibitors. Citation Format: Darren Chan, Charlene Minx, Jun Wang, Arindam Sen, Michael Johnston, Wen Wee Ma, Christopher J. Scott, Robert M. Straubinger. Efficacy of DR5-targeted immunoliposomes in pancreatic cancer models primed with SMO inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3704.

Abstract 4822: Anti-MMP9 antibody in combination with standard cytotoxic therapy in preclinical models of pancreatic cancer

Abstract BACKGROUND: Matrix metalloproteinase 9 (MMP9) is involved in the proteolysis of extracellular proteins and is reported to play a key role in development, invasion and metastasis of many cancers including pancreatic ductal adenocarcinoma (PDAC). Andecaliximab is a potent and highly selective monoclonal antibody inhibitor of MMP9 that has shown antitumor activity in combination with chemotherapy in gastric cancer. The therapeutic potential of targeted MMP9 inhibition in combination with cytotoxic therapy in preclinical models of PDAC was evaluated through the use of an αMMP9 antibody. METHODS: Animal survival and qualitative analysis were performed in the peritoneal dissemination model in mice using 7.5x105 AsPC-1 cells. Tumor growth study was performed in subcutaneous xenografts in mice using 7.5x105 AsPC-1 cells. The mechanistic evaluation involved RNASeq, Luminex, IHC and Immunoblot analysis in tumor samples. RESULTS: Median animal survival compared to controls (17 days) was increased after 2-week therapy with NPT (27 days, a 59% increase), Gem (22 days, +29%) and NPT+Gem (30 days, +76%). Addition of αMMP9 antibody increased survival as follows: NPT+MMP9 (30 days, +76%), Gem+MMP9 (25 days, +47%) and NPT+Gem+MMP9 (33 days, +94%). Evaluation of efficacy of maintenance therapy (6-weeks) revealed that median animal survival (controls: 22 days) was increased after NPT+Gem therapy (63 days, +186%) and further improved by addition of αMMP9 antibody (70 days, +218%). Qualitative assessment of mice after 2-week therapy revealed that αMMP9 therapy led to reduction in jaundice, bloody ascites and metastatic burden, both alone and in combination with NPT+Gem. Tumor lysates demonstrated changes in several proteins in chemotherapy groups including IP-10, MDC, PAI-1, GM-CSF, MIP-1b and IL-12b. αMMP9 therapy increased IL-28 (1.5 fold, p = 0.016). IHC analysis revealed decreased tumor microvessel density based on endomucin staining in αMMP9 therapy groups (59.9%, p<0.0001). Immunoblot analysis showed decreased vimentin expression in αMMP9 therapy groups (42.5%, p =0.03). In tumor growth study, NPT+Gem significantly decreased tumor growth (72%, p=0.0005) compared to controls. αMMP9 therapy caused a non-significant decrease in tumor growth (17%, p=0.24). Tumor tissues revealed low, but detectable levels of MMP9 mRNA in all therapy groups but no difference in MMP9 expression. αMMP9 monotherapy resulted in more gene expression changes in the mouse stromal than the human tumor compartment compared to other treatments. However, NPT+Gem+MMP9 combination therapy resulted in greater numbers of changes in gene expression compared to the other treatments in the tumor compartment. CONCLUSION: These findings suggest that MMP9 inhibition can augment the effects of standard cytotoxic therapy and support the potential of this combination therapeutic strategy for clinical PDAC therapy. Citation Format: Niranjan Awasthi, Amanda Mikels-Vigdal, Erin Stefanutti, Margaret A. Schwarz, Sheena Monahan, Victoria Smith, Roderich E. Schwarz. Anti-MMP9 antibody in combination with standard cytotoxic therapy in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4822.

Abstract 5767: Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy

Abstract Background Photoimmunotherapy (PIT) is a new class of molecular targeted cancer theranostics, which employs monoclonal antibody (mAb) conjugated to a photosensitizer, IR700, that is activated by focal near-infrared (NIR) light irradiation leading to necrotic cell death by cell membrane rapture where mAb-IR700 conjugates binds to target membrane proteins specifically. We have previously demonstrated HER2-targeted PIT employing trastuzumab-IR700 conjugates for HER2 expressing cancers. However, some cancer cells were survived partly because of tumor heterogeneity and inhomogeneous micro-distribution of mAb-IR700 conjugates. In this study, we developed a new type of PIT agents targeting VEGFR-2 expressed on vascular endothelium in a tumor and evaluated the feasibility by comparing conventional PIT in vitro and in vivo. Methods HER2-positive human gastric cancer cells, NCI-N87, were used for the experiments. HER2-targeting trastuzumab and VEGFR-2-targeting ramucirumab were conjugated to IR700. Cells were treated with mAb-IR700 conjugates followed by NIR light irradiation after washing the cells. LIVE/DEAD assay was performed to assess short-term phototoxicity and trypan blue dye exclusion assay to assess long-term phototoxicity. Next, mouse tumor xenograft models were created for in vivo PIT. Tumor-bearing mice were randomized and treated with mAb-IR700 conjugates followed by NIR light irradiation under anesthesia. Antitumor effects were monitored by measuring tumor diameters with a caliper. Results PIT utilizing ramucirumab-IR700 conjugates did not induce phototoxic effect in vitro because of the absence of membranous expression of VEGFR-2 in NCI-N87 cells, while PIT utilizing trastuzumab-IR700 conjugates induced rapid phototoxic effect because of the strong membranous expression of HER2 in NCI-N87 cells. By contrast, antitumor effects were observed in NCI-N87 xenograft tumors in vivo utilizing both ramucirumab- and trastuzumab-IR700 conjugates followed by NIR light irradiation. Microscopic analysis of the harvested tumors showed decreasing tumor micro-vessel densities only when tumors were treated with PIT utilizing ramucirumab-IR700 conjugates but not trastuzumab-IR700, which represents a different mechanism than that of conventional PIT targeting antigens expressed on the tumor cell membrane. Conclusion We demonstrated a new mechanism of PIT utilizing ramucirumab-IR700 conjugates. As VEGFR-2 is upregulated in many types of solid cancers, this method may be considered as being applicable to various types of cancers in future clinical settings. Citation Format: Makoto Mitsunaga, Takashi Nishimura, Kimihiro Ito. Vascular endothelial growth factor receptor 2 targeted photoimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5767.

Characterisation of tumour microvessel density during progression of high-grade serous ovarian cancer: clinico-pathological impact (an OCTIPS Consortium study).

BackgroundHigh-grade serous ovarian cancer (HGSOC) intratumoural vasculature evolution remains unknown. The study investigated changes in tumour microvessel density (MVD) in a large cohort of paired primary and recurrent HGSOC tissue samples and its impact on patients’ clinico-pathological outcome.MethodsA total of 222 primary (pOC) and recurrent (rOC) intra-patient paired HGSOC were assessed for immunohistochemical expression of angiogenesis-associated biomarkers (CD31, to evaluate MVD, and VEGF-A). Expression profiles were compared between pOCs and rOCs and correlated with patients' data.ResultsHigh intratumoural MVD and VEGF-A expression were observed in 75.7% (84/111) and 20.7% (23/111) pOCs, respectively. MVDhigh and VEGF(+) samples were detected in 51.4% (57/111) and 20.7% (23/111) rOCs, respectively. MVDhigh/VEGF(+) co-expression was found in 19.8% (22/111) and 8.1% (9/111) of pOCs and rOCs, respectively (p = 0.02). Pairwise analysis showed no significant change in MVD (p = 0.935) and VEGF-A (p = 0.121) levels from pOCs to rOCs. MVDhigh pOCs were associated with higher CD3(+) (p = 0.029) and CD8(+) (p = 0.013) intratumoural effector TILs, while VEGF(+) samples were most frequently encountered among BRCA-mutated tumours (p = 0.019). Multivariate analysis showed VEGF and MVD were not independent prognostic factors for OS.ConclusionsHGSOC intratumoural vasculature did not undergo significant changes during disease progression. High concentration of CD31(+) vessels seems to promote recruitment of effector TILs. The study also provides preliminary evidence of the correlation between VEGF-positivity and BRCA status.

DCE-MRI of Sunitinib-Induced Changes in Tumor Microvasculature and Hypoxia: A Study of Pancreatic Ductal Adenocarcinoma Xenografts

The purpose of this study was dual: to investigate (a) whether sunitinib may induce changes in tumor microvasculature and hypoxia in pancreatic ductal adenocarcinoma (PDAC) and (b) whether any changes can be detected by DCE-MRI. Sunitinib-treated and untreated control tumors of two PDAC xenograft models (BxPC-3 and Panc-1) were subjected to DCE-MRI before the imaged tumors were prepared for quantitative analysis of immunohistochemical preparations. Pimonidazole was used as a hypoxia marker, and fraction of hypoxic tissue (HFPim), density of CD31-positive microvessels (MVDCD31), and density of αSMA-positive microvessels (MVDαSMA) were measured. Parametric images of Ktrans and ve were derived from the DCE-MRI data by using the Tofts pharmacokinetic model. BxPC-3 tumors showed increased HFPim, decreased MVDCD31, unchanged MVDαSMA, and increased vessel maturation index (VMI = MVDαSMA/MVDCD31) after sunitinib treatment. The increase in VMI was seen because sunitinib induced selective pruning rather than maturation of αSMA-negative microvessels. Even though the microvessels in sunitinib-treated tumors were less abnormal than those in untreated tumors, this microvessel normalization did not improve the function of the microvascular network or normalize the tumor microenvironment. In Panc-1 tumors, HFPim, MVDCD31, MVDαSMA, and VMI were unchanged after sunitinib treatment. Median Ktrans increased with increasing MVDCD31 and decreased with increasing HFPim, and the correlations were similar for treated and untreated BXPC-3 and Panc-1 tumors. These observations suggest that sunitinib may induce significant changes in the microenvironment of PDACs, and furthermore, that Ktrans may be an adequate measure of tumor vascular density and hypoxia in untreated as well as sunitinib-treated PDACs.

Blockade of Myeloid-Derived Suppressor Cell Expansion with All-Trans Retinoic Acid Increases the Efficacy of Antiangiogenic Therapy.

Intrinsic and adaptive resistance hampers the success of antiangiogenic therapies (AAT), especially in breast cancer where this treatment modality has proven largely ineffective. Therefore, novel strategies to improve the efficacy of AAT are warranted. Solid tumors such as breast cancer are characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of resistance to AAT. Therefore, we hypothesized that all-trans retinoic acid (ATRA), which induces differentiation of MDSC into mature cells, could improve the therapeutic effect of AAT. ATRA increased the efficacy of anti-VEGFR2 antibodies alone and in combination with chemotherapy in preclinical breast cancer models. ATRA reverted the anti-VEGFR2-induced accumulation of intratumoral MDSC, alleviated hypoxia, and counteracted the disorganization of tumor microvessels. Mechanistic studies indicate that ATRA treatment blocked the AAT-induced expansion of MDSC secreting high levels of vessel-destabilizing S100A8. Thus, concomitant treatment with ATRA holds the potential to improve AAT in breast cancer and possibly other tumor types.Significance: Increasing the therapeutic efficiency of antiangiogenic drugs by reducing resistance-conferring myeloid-derived suppressor cells might improve breast cancer treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/12/3220/F1.large.jpg Cancer Res; 78(12); 3220-32. ©2018 AACR.

Lenvatinib inhibits angiogenesis and tumor fibroblast growth factor signaling pathways in human hepatocellular carcinoma models.

Unresectable hepatocellular carcinoma (uHCC) is one of the most lethal and prevalent cancers worldwide, and current systemic therapeutic options for uHCC are limited. Lenvatinib, a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs), recently demonstrated a treatment effect on overall survival by statistical confirmation of noninferiority to sorafenib in a phase 3 study of uHCC. Here, we investigated mechanisms underlying the antitumor activity of lenvatinib in preclinical HCC models. In vitro proliferation assay of nine human HCC cell lines showed that lenvatinib selectively inhibited proliferation of FGF signal‐activated HCC cells including FGF19‐expressing Hep3B2.1‐7. Lenvatinib suppressed phosphorylation of FRS2, a substrate of FGFR1–4, in these cells in a concentration‐dependent manner. Lenvatinib inhibited in vivo tumor growth in Hep3B2.1‐7 and SNU‐398 xenografts and decreased phosphorylation of FRS2 and Erk1/2 within the tumor tissues. Lenvatinib also exerted antitumor activity and potently reduced tumor microvessel density in PLC/PRF/5 xenograft model and two HCC patient‐derived xenograft models. These results suggest that lenvatinib has antitumor activity consistently across diverse HCC models, and that targeting of tumor FGF signaling pathways and anti‐angiogenic activity underlies its antitumor activity against HCC tumors.

Ultrasound-mediated microbubbles destruction for treatment of rabbit VX2 orthotopic hepatic tumors

The aim of this study was to determine whether ultrasound (US) mediated microbubbles (MBs) destruction (UMMD) could inhibit VX2 orthotopic hepatic tumor growth in rabbit models. Twenty-four VX2 orthotopic hepatic tumor rabbit models were randomly divided into four groups (n = 6 each): saline group, SonoVue alone group, US alone group, US + SonoVue group. Tumor volume (TV), peak intensity (PI) of contrast enhanced ultrasound (CEUS) in US + SonoVue group were significantly lower than that in other groups after treatment (P < 0.05, for all). Immunohistochemical analysis of tumor tissue showed that microvascular density (MVD) in US + SonoVue group was significantly lower than that in other groups (P < 0.05, for all). Extensive interstitial hemorrhage, intravascular thrombosis, destroyed nucleus membrane, mitochondria vacuolation and chromatin condensation were observed in US + SonoVue group, whereas these changes were rarely appeared in other groups. Our study indicated that UMMD could inhibit the growth of VX2 hepatic tumors in rabbits by irreversible destroying tumor microvessel and tumor cells.

Intratumoral evaluation of 3D microvasculature and nanoparticle distribution using a gadolinium-dendron modified nano-liposomal contrast agent with magnetic resonance micro-imaging

The enhanced permeability and retention (EPR) effect is variable depending on nanoparticle properties and tumor/vessel conditions. Thus, intratumoral evaluations of the vasculature and nanoparticle distribution are important for predicting the therapeutic efficacy and the intractability of tumors. We aimed to develop a tumor vasculature evaluation method and high-resolution nanoparticle delivery imaging using magnetic resonance (MR) micro-imaging technology with a gadolinium (Gd)-dendron assembled liposomal contrast agent. Using the Gd-liposome and a cryogenic receiving coil, we achieved 50-μm isotropic MR angiography with clear visualization of tumor micro-vessel structure. The Gd-liposome-enhanced MR micro-imaging revealed differences in the vascular structures between Colon26- and SU-DHL6-grafted mice models. The vessel volumes and diameters measured for both tumors were significantly correlated with histological observations. The MR micro-imaging methods facilitate the evaluation of intratumoral vascularization patterns, the quantitative assessment of vascular-properties that alter tumor malignancy, particle retentivity, and the effects of treatment.

Evaluation of three-dimensional arterial spin labeling perfusion imaging for the pathological investigation of musculoskeletal tumors.

The present study aimed to assess the clinical utility of three-dimensional arterial spin labeling (3D-ASL) perfusion imaging in discriminating between benign, intermediate and malignant musculoskeletal tumors, as well as to analyze the correlation between tumor blood flow (TBF) and microvessel density (MVD). 3D-ASL was performed on 44 patients with musculoskeletal tumor using a 3.0-T magnetic resonance system to obtain TBF values prior to surgery. TBF was independently measured by two radiologists. The TBF values of different groups were compared by one-way analysis of variance. A receiver operating characteristic (ROC) curve was applied to assess the threshold and diagnostic reliability of TBF. Immunohistochemical staining of tumor specimens was performed using a cluster of differentiation 34 monoclonal antibody to calculate MVD counts. The correlation between TBF and MVD counts was analyzed using correlative analysis. Pathology results for a total of 44 cases were obtained by surgery. Good interobserver agreement was found for the TBF values independently determined by the two radiologists (intra-class correlation coefficient test, 0.891; P<0.05). TBF and MVD values in the malignant group were significantly higher compared with that of the benign, and intermediate groups. No significant difference was found between the TBF and MVD values of the benign, and intermediate groups. According to the ROC analysis, the area under the curve was largest (0.951) when 45.5 ml/min/100 g was considered as the TBF cut-off value in the diagnosis. The diagnostic sensitivity and specificity were 90.5 and 100%, respectively. Additionally, a significant positive correlation was found between TBF and MVD (r, 0.784; P<0.05). The results of the present study suggest that 3D-ASL is valuable in discriminating between benign, intermediate and malignant musculoskeletal tumors. 3D-ASL may be utilized to evaluate angiogenesis in musculoskeletal tumors in vivo.

Chemorepulsion as a novel therapeutic concept to inhibit pancreatic cancer metastasis

Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate primarily due to the absence of effective pharmacologic treatments and the limited effects of surgical resection in the early stages of the disease. The most frequent cause of death from the disease is due to its tendency to metastasize to the liver, at which point the chances of long‐term survival drop significantly. Therefore, there is an urgent need to find a drug therapy that not only affects the primary tumor, but perhaps more importantly, prevents the formation of metastases in the liver. Neuropilin 2 (Nrp2) is a transmembrane co‐receptor protein that binds a secreted protein called semaphorin 3F (S3F). Nrp2 is not expressed in the liver but is highly overexpressed in PDAC cells and PDAC‐associated endothelial cells. Based on these observations, we hypothesized that S3F expression in the liver could repel metastasizing PDAC cells. As part of a series of prevention trials, we performed an IV injection of S3F expressing adenovirus in mice and thereby induced the expression of the protein in the liver. Three days later we performed an orthotopic injection of syngeneic PDAC cells. For our intervention trials, we inverted the sequence of tumor and virus injections. Our results showed a significant reduction in tumor metastases in the experimental SEMA3F‐treated group compared to control virus treated mice. But the actions of Nrp2 are not limited to its ability to mediate repulsion. The tumor microvessel density and hence the overall PDAC tumor size was also reduced, thereby illustrating the systemic anti‐angiogenic effects of S3F‐mediated signaling via Nrp2. The action of Nrp2 on tumor growth was further elucidated by repeating the same experimental procedure in Nrp2‐deficient mice. Again, we saw a reduction in size and microvessel density within the primary tumor, which validates our initial hypothesis that Nrp2 is necessary for tumor growth and angiogenesis in PDAC. In summary the therapeutic effects of inhibiting Nrp2 are twofold: First and foremost, we have shown that S3F expression in the liver will repel Nrp2‐positive PDAC cells, as well as reduce tumor growth in the pancreas compared to the control. Finally, we have also demonstrated the anti‐angiogenic effects of S3F.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.