Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models

Abstract

Intravital imaging techniques have provided unprecedented insight into tumor microcirculation and microenvironment. For example, these techniques allowed quantitative evaluations of tumor blood vasculature to uncover its abnormal organization, structure and function (e.g., hyper-permeability, heterogeneous and compromised blood flow). Similarly, imaging of functional lymphatics has documented their absence inside tumors. These abnormalities result in elevated interstitial fluid pressure and hinder the delivery of therapeutic agents to tumors. In addition, they induce a hostile microenvironment characterized by hypoxia and acidosis, as documented by intravital imaging. The abnormal microenvironment further lowers the effectiveness of anti-tumor treatments such as radiation therapy and chemotherapy. In addition to these mechanistic insights, intravital imaging may also offer new opportunities to improve therapy. For example, tumor angiogenesis results in immature, dysfunctional vessels--primarily caused by an imbalance in production of pro- and anti-angiogenic factors by the tumors. Restoring the balance of pro- and anti-angiogenic signaling in tumors can "normalize" tumor vasculature and thus, improve its function, as demonstrated by intravital imaging studies in preclinical models and in cancer patients. Administration of cytotoxic therapy during periods of vascular normalization has the potential to enhance treatment efficacy.

Figure 1

Imaging of tumor microvasculature and microenvironment. (A) Schematic of intravital microscopy set-up. An appropriate animal/tumor model, imaging probe(s), microscope, and image acquisition and analysis system are essential requirement of intravital microscopy. (B) OFDI angiography of mouse brain harboring U87 human glioma xenograft showing the depth-projected vasculature within the first 2 mm of mouse brain and tumor (upper left). Depth variation is denoted by color: yellow (superficial) to red (deep). Scale bar, 500 µm. (C) Multiphoton laser-scanning microscopy images of normal blood vessels (left) and tumor vessels in LS174T human colon cancer xenografts (right) in mouse dorsal skin chambers. Blood vessels are contrast enhanced by FITC-dextran. The bar indicates 100 µm. (D) Schematic of the composition of solid tumor. Tumors consist not only of cancer cells but also of host stromal cells—non-malignant cells in tumors which include endothelial cells, peri-vascular cells, fibroblasts, and multiple immune cell types. These cells, embedded within a protein-rich extracellular matrix, face a hostile metabolic microenvironment characterized by hypoxia and acidosis. Each of these cells is capable of producing positive and negative regulators of angiogenesis such as vascular endothelial growth factors (VEGFs); angiopoietins (Angs) and nitric oxide (NO) in response to the exposed microenvironment. These local interactions vary with tumor type and site of tumor growth (host organ), and may change during the course of tumor growth and treatment. B: reproduced from [148]; C: courtesy of Dr. Edward Brown; D: reproduced from ref. [37].

Figure 2

Regulation of angiogenesis by tumor microenvironment. (A) Relationship between VEGF promoter activity and local pO₂ or pH in U87 glioma xenografts. Tumor cell VEGF promoter activity was determined by the intensity of GFP (which is driven by the VEGF promoter). Tissue pO₂ and pH were determined by phosphorescence quenching microscopy with a porphyrin probe and fluorescence ratio-imaging microscopy with BCECF, respectively. Tissue pO₂ level inversely correlated with VEGF promoter activity especially in the region with neutral pH (left) and hypoxic region (not shown). On the other hand, tissue pH level inversely correlated with VEGF promoter activity in the oxygenated area (right). (B) Imaging of VEGF promoter activity in host stromal cells: MCaIV murine breast cancer grown in the dorsal skin chamber of a transgenic mouse expressing GFP under the control of VEGF promoter. Left: high density of activated fibroblasts exhibiting strong VEGF promoter activity (in green) at the host-tumor interface. Blood vessels are contrast enhanced by tetramethylrhodamine-dextran. Right: in contrast, deeper (200 µm) inside the tumor the VEGF expressing host stromal cells (in green) closely associate with blood vessels (in red). The bar indicates 100 µm. (C) Angiogenesis and tissue NO level in B16F1 and F10 tumors grown in the dorsal skin chamber and the cranial window. Vessel density was determined by intravital microscopy and tissue NO level was measured by an NO sensitive recessed microelectrode. (D) Microangiography of B16F1 and F10 murine melanomas grown in the cranial window. A: adapted from ref. [44]; B, adapted from ref. [9]; C, D: adapted from ref. [84].

Figure 3

Imaging therapeutic responses. (A) Simultaneous OFDI angiography and lymphangiography of control IgG and anti-VEGFR2 antibody treated MCaIV murine tumors. The images are taken 5 days after the initiation of treatment. For angiography depth-projected images are shown and the depth is denoted by color: yellow (superficial) to red (deep). The lymphatic vascular networks are presented (blue) for both tumors. The bar indicates 500 µm. (B, C) Normalization of U87 tumor vasculature by restoration of perivascular NO gradients. Tissue distribution of NO in U87 tumors grown in the cranial window was visualized by means of DAF-2T fluorescence imaging using MPLSM (B). The NO-sensitive fluorescence probe DAF-2 is converted to DAF-2T in the presence of NO, increasing fluorescence by a factor of 200. Control U87 tumors are shown in top row and nNOS-shRNA58-transfected-U87 tumors are shown in bottom row. Left: microangiography using tetramethylrhodamine-dextran (MW 2000 kDa). Middle: representative image of DAF-2T microfluorography captured 60 min after the loading of DAF-2 in tumors. Right: pseudocolor representation of DAF-2T microfluorographs. Color bar in the right shows calibration of the fluorescence intensity with known concentrations of DAF-2T. The bar indicates 100 µm. (C) Effects of nNOS silencing in U87 tumor cells on tumor tissue oxygenation. Top row: confocal laser-scanning microscopy images of hypoxyprobe-1 pimonidazole adduct–stained hypoxic cells (in red), lectin-bound perfused blood vessels (in green) and DAPI-stained nuclei (in blue). Bottom row: binarized images of blood vessels (in green) and hypoxic cells (in red). Scale bar, 100 µm. A, reproduced from ref. [148]; B, C: reproduced from ref. [85].