Fighting vessel dysmorphia to improve glioma chemotherapy

High-grade gliomas are aggressive and abundantly vascular tumors, and as in most cancer types, blood vessels in advanced lesions are highly abnormal. Poor perfusion and vascular leakage in tumor tissue resulting in hypoxia, necrosis, and high interstitial fluid pressure can hamper the efficient delivery of chemotherapy. Tumor angiogenesis is known to be supported by host leukocytes recruited to the tumor microenvironment, but the mechanisms leading to dysfunctional vascular network formation are incompletely understood. In this issue of EMBO Molecular Medicine, Mathivet et al (2017) present an elegant study, where longitudinal intravital imaging gives new insight on how recruitment and polarization of tumor-associated macrophages regulate aberrant angiogenesis in experimental gliomas. They show that macrophage targeting results in vessel normalization and improved chemotherapy response, suggesting that the combination of these therapeutic modalities could improve the outcome of glioma treatment in the clinic.Non peer reviewe

Effects of Angiopoietin-2-Blocking Antibody on Endothelial Cell–Cell Junctions and Lung Metastasis

Background: Angiopoietin-2 (Ang2), a ligand for endothelial TEK (Tie2) tyrosine kinase receptor, is induced in hypoxic endothelial cells of tumors, where it promotes tumor angiogenesis and growth. However, the effects of Ang2 on tumor lymphangiogenesis and metastasis are poorly characterized. Methods: We addressed the effect of Ang2 on tumor progression and metastasis using systemic Ang2 overexpression in mice carrying tumor xenografts, endothelium-specific overexpression of Ang2 in VEC-tTA/Tet-OS-Ang2 transgenic mice implanted with isogenic tumors, and administration of Ang2-blocking antibodies to tumor-bearing immunodeficient mice. Fisher's exact test was used for analysis of metastasis occurrence, and repeated measures one-way analysis of variance was used for the analysis of primary tumor growth curves. Unpaired t test was used for all other analyses. All statistical tests were two-sided. Results: Adenoviral expression of Ang2 increased lymph node and lung metastasis in tumor xenografts. The metastatic burden in the lungs was increased in transgenic mice in which Ang2 expression was induced specifically in the vascular endothelium (tumor burden per grid, VEC-tTA/Tet-OS-Ang2 mice [n = 5] vs control mice [n = 4]: 45.23 vs 12.26 mm[superscript 2], difference = 32.67 mm[superscript 2], 95% confidence interval = 31.87 to 34.07, P < .001). Ang2-blocking antibodies reduced lymph node and lung metastasis, as well as tumor lymphangiogenesis, and decreased tumor cell homing to the lungs after intravenous injection. In the lung metastases, Ang2 overexpression decreased endothelial integrity, whereas the Ang2-blocking antibodies improved endothelial cell–cell junctions and basement membrane contacts of metastasis-associated lung capillaries. At the cellular level, the Ang2-blocking antibodies induced the internalization of Ang2-Tie2 receptor complexes from endothelial cell–cell junctions in endothelial–tumor cell cocultures. Conclusion: Our results indicate that blocking Ang2 inhibits metastatic dissemination in part by enhancing the integrity of endothelial cell–cell junctions

VEGF-C and angiopoietins in lymphangiogenesis

Angiogenesis and lymphangiogenesis occur during development as the result of tightly coordinated signalling programs to generate two hierarchically organised vascular systems. All tissues and organs are dependent on a functional blood vasculature for oxygen and nutrients, whereas the lymphatic vasculature functions to collect excess tissue fluid, passing it through lymph nodes for immune surveillance, and returning it to the blood circulation. Effectors that control developmental angiogenesis and lymphangiogenesis are also involved in pathological settings, and therefore potential targets for therapy. Vascular endothelial growth factor (VEGF) and angiopoietin (Ang) growth factors, signalling through endothelial VEGFR and Tie receptors, have been established as key regulators of angiogenic and lymphangiogenic processes in development and disease. In this study, we aimed to obtain a clearer understanding of the vascular effects of stimulation by VEGF-C, Ang1 and Ang2, all known to be involved in lymphangiogenesis. In cell culture models, we found that both intrinsic and microenvironmental regulatory mechanisms are involved in the regulation of endothelial cell phenotypes, and distinct responses to VEGF signalling are induced by specific receptor pathways in different endothelial cell types. Surprisingly, we also found that Ang1 induces sprouting lymphangiogenesis in vivo by a VEGFR-3 dependent mechanism, establishing Ang1 as a novel lymphangiogenic factor. Using inducible transgenic mouse models, we found that VEGF-C-induced lymphatic hyperplasia persisted independently of the growth factor, indicating that short pro-lymphangiogenic therapy could lead to lasting improvements in tissue oedema. While VEGF-C had blood vessel effects in embryos, no angiogenic side effects were observed in adult tissues. Furthermore, inducible transgenic expression of Ang2 during embryonic development confirmed Ang2 as an important regulator of lymphatic remodelling and mural cell contacts. The unexpected similarity of the lymphatic maturation defects caused by excess Ang2 to those observed in Ang2 deficient mice demonstrated that correct doses of Ang2 are crucial for the control of lymphatic development. Unlike Ang1, Ang2 did not induce lymphatic sprouting. Although Ang1 has been shown to be able to substitute for Ang2 during developmental lymphangiogenesis, their lymphatic effects are not identical. These findings further our understanding of the basic mechanisms of angiogenesis and lymphangiogenesis, important for the future development of targeted therapies for vascular diseases such as cancer, inflammation, lymphoedema and ischemia. VEGF-C and Ang1 especially emerged as promising candidates for pro-lymphangiogenic therapy.Kaikki kudokset ja elimet tarvitsevat toimivan verisuoniverkoston saadakseen happea ja ravinteita. Imusuonten tehtävä puolestaan on kerätä verisuonista kudoksiin vuotava neste ja valkuaisaineet, ja kuljettaa ne immuunipuolustusta valvovien imusolmukkeiden kautta takaisin verenkiertoon. Verisuonten uudismuodostusta (angiogeneesia) tapahtuu normaalisti sikiönkehityksen aikana ja haavojen parantuessa, mutta angiogeneesi on välttämätöntä myös syöpäkasvainten kehitykselle, ja verisuonten liikakasvua ilmenee myös esimerkiksi silmäsairauksissa. Imusuonten uudismuodostus (lymfangiogeneesi) voi edistää etäpesäkkeiden muodostumista. Toisaalta imusuonten vajaatoiminta aiheuttaa kudosturvotusta (lymfödeemaa), ja angiogeneesi olisi hyödyksi veritulppien aiheuttaman lihasten hapenpuutteen korjaamiseksi. Siksi sekä suonten kasvun pysäyttäminen että kasvun edistäminen ovat mahdollisia hoitomuotoja eri sairauksiin. Sikiönkehityksen aikana veri- ja imusuoniston muodostumista säätelee joukko kasvutekijöitä, joiden toiminta on keskeisessä roolissa myös useissa sairauksissa. Verisuonen endoteelin kasvutekijöiden (VEGF) sekä angiopoietiinien (Ang) signaalinvälitys suonten sisäseinän endoteelisoluissa on olennaisen tärkeää suonten uudismuodostukselle. Tässä työssä tutkimme VEGF-C-, Ang1- ja Ang2-kasvutekijöiden vaikutuksia erityisesti imusuonten kehitykseen ja uudismuodostukseen. Tutkimuksen ensimmäisessä osatyössä havaitsimme, että erilaisten suonten endoteelisoluissa VEGF-kasvutekijöiden vaikutukset olivat erilaisia, ja selviteltiin mihin prosesseihin eri signaalinvälitysreitit vaikuttavat. Totesimme myös, että solun seinän sileälihassolut voivat muokata VEGF-tekijöiden vaikutuksia endoteelisoluihin. Useat mekanismit siis säätelevät myös VEGF-signaalinvälitystä. Toinen osatyö osoitti, että Ang1-kasvutekijä aiheuttaa lymfangiogeneesiä VEGFR-3-reseptorin signaalinvälityksen kautta. Kolmannessa osatyössä selvitimme imusuonten kasvutekijä VEGF-C:n vaikutusta, ja totesimme että lyhytaikainen VEGF-C-terapia voi johtaa pysyvän uuden imusuoniston muodostumiseen ilman sivuvaikutuksia verisuoniin. Nämä rohkaisevat tulokset luovat pohjaa kasvutekijähoitojen kehittämiselle lymfödeemaan. Neljännessä osatyössä havaitsimme, että liian suuri pitoisuus Ang2-kasvutekijää sikiönkehityksen aikana estää imusuonten normaalin kehityksen viimeisen vaiheen, jossa yksinkertaisesta suoniverkostosta muokataan hierarkkinen järjestelmä. Yllätykseksemme huomasimme, että liiallinen Ang2 aiheuttaa samankaltaisen ilmiasun kuin Ang2:n täydellinen puutos. Tämä tulos osoittaa, että Ang2:n pitoisuuden tarkka säätely on ensiarvoisen tärkeää imusuonten kehitykselle

Real-time Imaging of Myeloid Cells Dynamics in <em>Apc^Min/+</em> Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc(Min)(/+) mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment

Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc(Min/+) mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment

Delineating CSF-1-dependent regulation of myeloid cell diversity in tumors

Myeloid cells contribute to increased malignancy and poor prognosis in breast cancer. We demonstrate that anti-CSF-1R therapy depletes a cell population sharing characteristics of tumor-associated macrophages (TAMs) and dendritic cells (DCs). Intravital imaging combined with cellular characterization has refined our understanding of anti-CSF-1R therapy on the tumor microenvironment

Delineating CSF-1-dependent regulation of myeloid cell diversity in tumors.

Myeloid cells contribute to increased malignancy and poor prognosis in breast cancer. We demonstrate that anti-CSF-1R therapy depletes a cell population sharing characteristics of tumor-associated macrophages (TAMs) and dendritic cells (DCs). Intravital imaging combined with cellular characterization has refined our understanding of anti-CSF-1R therapy on the tumor microenvironment

Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R-dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti-CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1(+) neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment