Tumor microvasculature and microenvironment: targets for anti-angiogenesis and normalization

Abstract

A solid tumor forms an organ-like entity comprised of neoplastic cells and non-transformed host stromal cells embedded in an extracellular matrix. Similar to normal tissues, blood vessels nourish cells residing in tumors. However, unlike normal blood vessels, tumor vasculature has abnormal organization, structure, and function. Tumor vessels are leaky and blood flow is heterogeneous and often compromised. Vascular hyperpermeability and the lack of functional lymphatic vessels inside tumors cause elevation of interstitial fluid pressure in solid tumors. Each of these abnormalities forms a physiological barrier to the delivery of therapeutic agents to tumors. Furthermore, elevated tumor interstitial fluid pressure increases fluid flow from the tumor margin into the peri-tumor area and may facilitate peri-tumor lymphatic hyperplasia and metastasis. Abnormal microcirculation in tumors also leads to a hostile microenvironment characterized by hypoxia and acidosis, which hinder the effectiveness of anti-tumor treatments such as radiation therapy and chemotherapy. In addition, host-tumor interactions regulate expression of pro- and anti-angiogenic factors and hence contribute to their imbalance and resulting pathophysiological characteristics of the tumor. Restoration of pro- and anti-angiogenic balance in tumors may "normalize" tumor vasculature and thus improve its function. Indeed, anti-angiogenic treatments directly targeting angiogenic signaling pathways as well as indirectly modulating angiogenesis show normalization of tumor vasculature and microenvironment at least transiently in both preclinical and clinical settings. Combination of cytotoxic therapy and anti-angiogenic treatment during the vascular normalization exhibits synergistic effect.

Figures

Figure 1. Abnormal vasculature and microenvironment in tumors

(A) Multiphoton laser-scanning microscopy image 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. Images are 550 μm across. (B) Microvascular permeability determined by intravital microscopy. Left, vascular permeability to dextran of 150,000 molecular weight in non-maligant (mature granulation) and neoplastic (VX2 carcinoma) tissues grown in the rabbit ear chamber. Right, vascular permeability to liposome with diameter between 86 and 90 nm in normal subcutaneous tissues (not detectable) and LS174T tumor tissues in the dorsal skin chambers. (C) Blood flow determined by intravital microscopy in normal pial vessels (maximum bead velocities, left) and tumor vessels in MCaIV murine breast cancer and U87 human glioma (RBC velocities, right) in mouse cranial windows. (D) Fluorescence microlymphangiography with FITC-dextran in mouse tail 28 days after the implantation of FSaII murine fobrosarcoma (left part of the image). The bar indicates 400 μm. (E) Mean interstitial pH and pO2 profiles in LS174T tumors in the dorsal skin chambers as one moves away from the nearest blood vessels. Tissue pO2 and pH were determined by phosphorescence quenching microscopy with a porphyrin probe and fluorescence ratio-imaging microscopy with BCECF, respectively. Open symbols, pH; closed symbols, pO2. Scale corresponds to distance from the vessel wall. (F) VEGF promoter activity (green), tissue pO2 (blue) and pH (red) in U87 tumors. Left, intravital microscopy image of GFP driven by VEGF promoter. The three parameters are determined along the yellow line. Center, this tumor is well oxygenated and there is no correlation between tissue pO2 and VEGF promoter activity. Right, on the other hand, the peak of VEGF promoter activity is observed in acidic pH region. A, courtesy of Dr. Edward Brown; B, data from (Gerlowski and Jain, 1986; Yuan et al., 1994a); C, adapted from (Yuan et al., 1994b); D, adapted from (Leu et al., 2000); E, adapted from (Helmlinger et al., 1997); F, adapted from (Fukumura et al., 2001).

Figure 2. Steps of lymphatic metastasis and the effect of anti-lymphangiogenic treatment

Elevated tumor IFP increases interstitial fluid flow at the tumor margin (Jain et al., 2007). The exudates, which contain fluid, protein, and cells from tumors, are collected in the peritumoral lymphatic vessels. Although the mechanical signals that could trigger the lymphangiogenic switch are not known, hydrostatic pressure is likely to be such trigger (Boardman and Swartz, 2003). Furthermore, many tumors express lymphangiogenic factors such as VEGF-C (Alitalo et al., 2005). (A) FITC-dextran microlymphangiography of peritumor and normal lymphatics in mouse ears. Bar, 850 μm. MT, mock transduced. VEGF-C, VEGF-C overexpressing. Lymphatic vessels are hyperplastic in the peritumor region and ear base (further down stream) of the T241 fibrosarcomas. VEGF-C expression further increases lymphatic vessel diameter. Number of lymphatic vessels is also increased in the tumor periphery especially in the presence of excess VEGF-C (Isaka et al., 2004). Malformed lymphatic valves allow retrograde flow in these lymphatic vessels and facilitate transfer of metastasizing tumor cells, which can be visualized by intravital microscopy after the transfection of GFP expression vector (Hoshida et al., 2006). (B) Then GFP-positive tumor cells (green) are observed entering the cervical lymph node from afferent lymphatic (red, arrow) by MPLSM. Bar, 100 μm. VEGF-C overexpression significantly increases arrival of tumor cells to the lymph node. Macroscopic lymph node metastasis increases with the increase of tumor cell arrival to the lymph node (Hoshida et al., 2006). (C) The treatment with anti-VEGF receptor 3 antibody (mF4-31C1) significantly reduces peritumor and ear base lymphatic vessel size. (D) Tumor cells cannot reach to the cervical lymph node under the anti-VEGFR3 treatment. As a result, macroscopic lymph node is significantly reduced by anti-VEGFR3 treatment if it is given before the arrival of tumor cells to the lymph node (Hoshida et al., 2006). A–C, adapted or reproduced from (Hoshida et al., 2006).

Figure 3. Role of host-tumor interaction in angiogenesis and vessel function

(A) Imaging of VEGF promoter activity in host stromal cells. MCaIV murine breast tumor is grown in the dorsal skin chamber of a transgenic mouse expressing GFP under the control of VEGF promoter. Left, activated fibroblasts exhibit strong VEGF promoter activity (green) at the host-tumor interface. Right, VEGF expressing host cells associate with blood vessels (red) inside tumors. Images are 733 μm across. (B) Microangiography of B16F10 murine melanoma grown in the cranial window (left) and dorsal skin chamber (right). Images are 3.75 mm across. (C) Vessel morphology in B16 melanomas grown in the dorsal skin chamber (DSC) and cranial window (CW) determined by intravital microscopy. (D) Pore cut-off size of tumor vessels in MCaIV tumors and murine hepatoma HCaI grown in the dorsal skin chamber (S.C.) and cranial window (Cranial) are determined by intravital microscopy after the injection of different size fluorescent nanoparticles. Filled and open circles indicate negative and positive extravasation, respectively. (E) Vascular permeability to BSA in normal blood vessels before (Pre) and after (Post) superfusion with VEGF. A, adapted from (Brown et al., 2001a); C adapted from (Kashiwagi et al., 2005); D, adapted from (Hobbs et al., 1998); E, adapted from (Monsky et al., 1999).

Figure 4. Normalization of tumor vasculature and microenvironment by anti-VEGFR2 treatment

(A) Normal microvessels (left) and U87 tumor vessels pre (center) and 2 days after anti-VEGFR2 antibody (DC101) treatment (right) in the mouse brain visualized by MPLSM through cranial window. (B) U87 tumor vessel morphology and function during anti-VEGFR2 treatment determined by intravital microscopy. (C) Immunohistochemical analysis of NG2-positive perivascular cell (red) coverage of U87 tumor vessels (green) during anti-VEGFR2 treatment. (D) Hypoxic fraction of U87 tumor tissues determined by a redox marker (pimonidazole) staining. (E) U87 tumor growth delay by anti-VEGFR2 (DC101), radiation (RT), and the combination (RT1-RT5) treatments compared to control tumor growth (C). Inset shows schedules of the combined treatments. Fractionate radiation (8Gy × 3 days) is given starting at 9 days before (RT1), 2 days before (RT2), 1 day after (RT3), 4 days after (RT4), or 7 days after (RT5) the start of DC101 treatment. A–E, adapted or reproduced from (Winkler et al., 2004).

Figure 5. Tumor vascular normalization by indirect anti-angiogenesis

(A) Microangiography in control and Herceptin treated MDA-MB-361HK tumors grown in the mouse cranial window. The bar represents 100 μm. (B) MDA-MB-361HK tumor vessel morphology and function during Herceptin treatment determined by intravital microscopy. (C) Macroscopic images of androgen-dependent Shionogi tumors grown in the mouse dorsal skin chamber. Castration or sham operation is performed 15 days after the tumor implantation. (D) Changes in tumor vasculature during the course of anti-angiogenic therapy. Compared to the well-organized structure of normal vessels (Normal, far left), tumor vasculature is structurally and functionally abnormal (Abnormal, middle left). Anti-angiogenic therapies initially alleviate these abnormalities (Normalized, middle right). However, sustained or excessive anti-angiogenic treatments result in significant vessel reduction and thus, inadequate delivery of drugs or oxygen (Inadequate, far right). A, B adapted from (Izumi et al., 2002a); C, reproduced from (Jain et al., 1998); D, adapted from (Jain, 2001).