Keehoon Jung

Profound but Dysfunctional Lymphangiogenesis via Vascular Endothelial Growth Factor Ligands from CD11b+ Macrophages in Advanced Ovarian Cancer

Severe ascites is a hallmark of advanced ovarian cancer (OVCA), yet the underlying mechanism that creates an imbalance between peritoneal vascular leakage and lymphatic drainage is unknown. Here, we identified and characterized peritoneal lymphatic vessels in OVCA mice, a model generated by implantation of human OVCA cells into athymic nude mice. The OVCA mice displayed substantial lymphangiogenesis and lymphatic remodeling, massive infiltration of CD11b(+)/LYVE-1(+) macrophages and disseminated carcinomatosis in the mesentery and diaphragm, and progressive chylous ascites formation. Functional assays indicated that the abnormally abundant lymphatic vessels in the diaphragm were not conductive in peritoneal fluid drainage. Moreover, lipid absorbed from the gut leaked out from the aberrant mesenteric lymphatic vessels. Our results indicate that vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF-A from CD11b(+) macrophages are responsible for producing OVCA-induced dysfunctional lymphangiogenesis, although other cell types contribute to the increased ascites formation. Accordingly, the combined blockade of VEGF-C/D and VEGF-A signaling with soluble VEGF receptor-3 and VEGF-Trap, respectively, markedly inhibited chylous ascites formation. These findings provide additional therapeutic targets to ameliorate chylous ascites formation in patients with advanced OVCA.

Endoscopic time-lapse imaging of immune cells in infarcted mouse hearts.

Rationale: High-resolution imaging of the heart in vivo is challenging owing to the difficulty in accessing the heart and the tissue motion caused by the heartbeat. Objective: Here, we describe a suction-assisted endoscope for visualizing fluorescently labeled cells and vessels in the beating heart tissue through a small incision made in the intercostal space. Methods and Results: A suction tube with a diameter of 2 to 3 mm stabilizes the local tissue motion safely and effectively at a suction pressure of 50 mm Hg. Using a minimally invasive endoscope integrated into a confocal microscope, we performed fluorescence cellular imaging in both normal and diseased hearts in live mice for an hour per session repeatedly over a few weeks. Real-time imaging revealed the surprisingly rapid infiltration of CX3CR1+ monocytes into the injured site within several minutes after acute myocardial infarction. Conclusions: The time-lapse analysis of flowing and rolling (patrolling) monocytes in the heart and the peripheral circulation provides evidence that the massively recruited monocytes come first from the vascular reservoir and later from the spleen. The imaging method requires minimal surgical preparation and can be implemented into standard intravital microscopes. Our results demonstrate the applicability of our imaging method for a wide range of cardiovascular research.

Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Significance Improving survival of patients with glioblastoma (GBM) using antiangiogenic therapy remains a challenge. In this study we show that dual blockade of angiopoietin-2 and vascular endothelial growth factor delays tumor growth and enhances survival benefits through reprogramming of tumor-associated macrophages toward an antitumor phenotype as well as by pruning immature tumor vessels. The antitumor immunomodulatory potential of this dual blockade supports clinical testing of this approach for GBM with other immunotherapeutic approaches such as checkpoint blockers. Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Significance Inhibition of the VEGF/VEGF receptor (VEGFR) pathway has failed to increase overall survival in phase III trials in patients with glioblastoma (GBM). Previously we identified the angiopoietin-2 (Ang-2)/TEK receptor tyrosine kinase (Tie-2) pathway as a potential driver of resistance to VEGF inhibition in GBM. Here we show that dual inhibition of VEGFRs and Ang-2 inhibits tumor growth and prolongs vessel normalization compared with VEGFR inhibition alone, resulting in improved survival in murine GBM models. Furthermore, by blocking macrophage recruitment, we demonstrate that macrophages contribute to the beneficial effects of dual therapy. Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti–Ang-2–neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti–colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy

UNLABELLED It remains unclear how obesity worsens treatment outcomes in patients with pancreatic ductal adenocarcinoma (PDAC). In normal pancreas, obesity promotes inflammation and fibrosis. We found in mouse models of PDAC that obesity also promotes desmoplasia associated with accelerated tumor growth and impaired delivery/efficacy of chemotherapeutics through reduced perfusion. Genetic and pharmacologic inhibition of angiotensin-II type-1 receptor reverses obesity-augmented desmoplasia and tumor growth and improves response to chemotherapy. Augmented activation of pancreatic stellate cells (PSC) in obesity is induced by tumor-associated neutrophils (TAN) recruited by adipocyte-secreted IL1β. PSCs further secrete IL1β, and inactivation of PSCs reduces IL1β expression and TAN recruitment. Furthermore, depletion of TANs, IL1β inhibition, or inactivation of PSCs prevents obesity-accelerated tumor growth. In patients with pancreatic cancer, we confirmed that obesity is associated with increased desmoplasia and reduced response to chemotherapy. We conclude that cross-talk between adipocytes, TANs, and PSCs exacerbates desmoplasia and promotes tumor progression in obesity. SIGNIFICANCE Considering the current obesity pandemic, unraveling the mechanisms underlying obesity-induced cancer progression is an urgent need. We found that the aggravation of desmoplasia is a key mechanism of obesity-promoted PDAC progression. Importantly, we discovered that clinically available antifibrotic/inflammatory agents can improve the treatment response of PDAC in obese hosts. Cancer Discov; 6(8); 852-69. ©2016 AACR.See related commentary by Bronte and Tortora, p. 821This article is highlighted in the In This Issue feature, p. 803.

Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals

Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, owing to their size, the objective lenses required have limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes to minimally invasive imaging of internal organs. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope to enable visualization of fluorescent cells and microvasculature in various mouse organs. Some experience in building an optical setup is required to complete the protocol. We also present longitudinal imaging of immune cells in renal allografts and tumor development in the colon. Fabrication and integration can be completed in 5–7 h, and a typical in vivo imaging session takes 1–2 h.

The lymph node microenvironment and its role in the progression of metastatic cancer

Lymph nodes are initial sites for cancer metastasis in many solid tumors. However, their role in cancer progression is still not completely understood. Emerging evidence suggests that the lymph node microenvironment provides hospitable soil for the seeding and proliferation of cancer cells. Resident immune and stromal cells in the lymph node express and secrete molecules that may facilitate the survival of cancer cells in this organ. More comprehensive studies are warranted to fully understand the importance of the lymph node in tumor progression. Here, we will review the current knowledge of the role of the lymph node microenvironment in metastatic progression.

Solid stress and elastic energy as measures of tumour mechanopathology

Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that two-dimensional spatial mappings of solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and wide size ranges can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that: (i) solid stress depends on both cancer cells and their microenvironment; (ii) solid stress increases with tumour size; and (iii) mechanical confinement by the surrounding tissue significantly contributes to intratumoural solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to significant discoveries and new therapies.

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases

Anti-VEGF therapy enhances mechanical barriers to therapy in colorectal cancer liver metastases by increasing hyaluronan deposition. Stiff resistance to cancer therapy Antiangiogenic therapy with drugs that block vascular endothelial growth factor (VEGF) signaling to inhibit formation of new blood vessels in tumors is commonly used in colorectal cancer. Unfortunately, the effects of this therapy usually do not last for long, and a study by Rahbari et al. shows why this might be the case. The authors found that VEGF inhibition increased the stiffness of colorectal cancer liver metastases, making them more difficult to treat with chemotherapy. In a mouse model, the researchers were able to overcome this difficulty by using an enzyme to degrade a component of the extracellular matrix in liver metastases, suggesting that the matrix may be a target for future cancer therapies. The survival benefit of anti–vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.

In vivo imaging of tracheal epithelial cells in mice during airway regeneration.

Many human lung diseases, such as asthma, chronic obstructive pulmonary disease, bronchiolitis obliterans, and cystic fibrosis, are characterized by changes in the cellular composition and architecture of the airway epithelium. Intravital fluorescence microscopy has emerged as a powerful approach in mechanistic studies of diseases, but it has been difficult to apply this tool for in vivo respiratory cell biology in animals in a minimally invasive manner. Here, we describe a novel miniature side-view confocal probe capable of visualizing the epithelium in the mouse trachea in vivo at a single-cell resolution. We performed serial real-time endotracheal fluorescence microscopy in live transgenic reporter mice to view the three major cell types of the large airways, namely, basal cells, Clara cells, and ciliated cells. As a proof-of-concept demonstration, we monitored the regeneration of Clara cells over 18 days after a sulfur dioxide injury. Our results show that in vivo tracheal microscopy offers a new approach in the study of altered, regenerating, or metaplastic airways in animal models of lung diseases.