Abeloff's Clinical Oncology, 4th Edition

Part I – Science of Clinical Oncology

Section A – Biology and Cancer

Chapter 8 – Vascular and Interstitial Biology of Tumors

Rakesh K. Jain,Dan G. Duda

SUMMARY OF KEY POINTS

  

   

A solid tumor is an organ composed of neoplastic cells and host stromal cells nourished by the vasculature made of endothelial cells—all embedded in an extracellular matrix. The interactions among these cells and between these cells, their surrounding matrix, and their local microenvironment control the expression of various genes. The products encoded by these genes, in turn, control the pathophysiologic characteristics of the tumor. The tumor pathophysiology governs not only tumor growth, invasion, and metastasis but also the response to various therapies.

  

   

Tumor vasculature is made of host vessels co-opted by tumor cells and by new vessels formed by the processes of vasculogenesis and angiogenesis. A constellation of positive and negative regulators of angiogenesis governs the process of neovascularization.

  

   

Tumor vessels are abnormal in terms of their organization, structure, and function. These abnormalities contribute to heterogeneous vascular permeability, blood flow, and microenvironment.

  

   

Tumor interstitial matrix is formed by proteins secreted by host and tumor cells and by those leaked from the nascent blood vessels.

  

   

Tumor interstitium is heterogeneous, with some regions fairly permeable and others difficult to penetrate. Modification of the collagen matrix can improve penetration of large-molecular-weight therapeutics.

  

   

Interstitial hypertension is a hallmark of solid tumors and results from vessel leakiness, lack of functional lymphatics, and compression of vessels by proliferating cancer cells.

  

   

Judicious application of angiogenic therapy can normalize the tumor vessels and make them more effcient for delivery of oxygen (a known radiosensitizer) and drugs. Antiangiogenic agents can prune tumor vessels, induce cancer cell apoptosis, reduce the number of blood circulating endothelial cells and progenitor cells, and lower interstitial hypertension in tumors.

  

   

Thus far, three antiangiogenic agents have been approved for patients with certain types of cancer. Based on these successes, antiangiogenic therapy is expected to make a difference in many other tumor types. Two main hurdles to further development of antiangiogenic agents are the better understanding of the mechanisms of action of these agents and developing biomarkers to monitor their effects.

INTRODUCTION

A solid tumor is an organ composed of neoplastic cells and host stromal cells nourished by the vasculature made of endothelial cells—all embedded in an extracellular matrix ( Fig. 8-1 ). The interactions among these cells and between these cells, their surrounding matrix, and their local microenvironment, control the expression of various genes. The products encoded by these genes, in turn, control the pathophysiologic characteristics of the tumor. The tumor pathophysiology governs not only the tumor growth, invasion, and metastasis but also the response to various therapies. In this chapter we will discuss various pathophysiologic parameters that characterize the vascular and extravascular compartments of a tumor and the mechanisms governing the formation and function of these compartments.

 
 

Figure 8-1  Schematic representation of a solid tumor. The key components include cancer cells, host cells, and vasculature made of endothelial cells—all embedded in a matrix bathed in interstitial fluid. Arrows indicate interactions between the components.  (Adapted from Jain RK: Angiogenesis and lymphangiogenesis in tumor: insights from intravital microscopy. Cold Spring Harbor Symp Quant Biol [The Cardiovascular System] 2002;67:239–248.)

 

 

 

VASCULAR COMPARTMENT

Neoplastic cells, like normal cells, need oxygen and other nutrients for their survival and growth. Every normal cell in our body is located within 100 to 200μm from a blood capillary so that it can receive oxygen and other nutrients by the process of diffusion. Likewise, cells undergoing neoplastic transformation depend on nearby capillaries for growth. These preneoplastic (i.e., hyperplastic or dysplastic) cells can grow as a spherical or ellipsoidal cellular aggregate. Once the size of the cellular aggregate reaches the diffusion limit for critical nutrients and oxygen, however, the aggregate as a whole can become dormant. Indeed, human tumors can remain dormant for many years because of a balance between neoplastic cell proliferation and apoptosis. However, once they have access to new blood vessels they may grow and metastasize. What triggers the growth of new vessels? What molecular and cellular mechanisms are involved? How do these vessels compare with normal vessels with respect to structure and function? Can we prevent or delay tumor progression only by interfering with the neovascularization process?

New Vessel Formation

It has been known for nearly a century that the vascular system is associated with tumor growth in animals and humans.[1] Powerful insights into the neovascularization of transplanted tumors using the transparent window techniques were developed in the 1940s. [2] [3] [4] [5] The possibility that tumors produce an “angiogenic” substance was suggested in 1968. [6] [7] The hypothesis that blocking angiogenesis should block tumor growth and metastasis was proposed shortly thereafter in 1971.[8] The concept that a tissue acquires angiogenic capacity during neoplastic transformation—and, by extension, that antiangiogenesis could be used to prevent cancer—was put forward in 1976.[9] The first antiangiogenic agent approved for cancer patients was bevacizumab, an antibody specific to vascular endothelial growth factor (VEGF), on the basis of the increased survival seen in metastatic colorectal cancer patients with the combination of bevacizumab with standard chemotherapy in a pivotal randomized placebo-controlled phase III trial.[10] At present, various anti- and proangiogenesis strategies are being evaluated clinically to prevent or treat a large number of diseases, including cancer. [11] [12] [13] Both normal and pathologic angiogenic processes are governed by the net balance between pro- and antiangiogenic factors. [14] [15] This balance is spatially and temporally regulated under physiologic conditions, so that the “angiogenic switch” is “on” when needed (e.g., during embryonic development, wound healing, formation of the corpus luteum) and “off” at other times. During neoplastic transformation and tumor progression, this regulation is deranged, and blood vessels form ectopically to support a growing tumor mass.

Cellular Mechanisms

At least four cellular mechanisms are involved in the vascularization of tumors: co-option, intussusception, sprouting (angiogenesis), and vasculogenesis ( Fig. 8-2 ).[11] Tumor cells can co-opt and grow around existing vessels to form “perivascular” cuffs. However, as stated earlier, these cuffs cannot grow beyond the diffusion limit of critical nutrients and may actually cause the collapse of the vessels due to the growth pressure (referred to as “solid stress”). Alternatively, an existing vessel may enlarge in response to the growth factors released by tumors, and an interstitial tissue column may grow in the enlarged lumen and partition the lumen to form an expanded vascular network. This mode of intussusceptive microvascular growth has been observed during tumor growth, wound healing, and gene therapy. [16] [17] [18] [19] “Sprouting” angiogenesis is perhaps the most widely studied mechanism of vessel formation. During sprouting angiogenesis, the existing vessels become leaky in response to growth factors released by normal cells or cancer cells; the basement membrane and the interstitial matrix dissolve; pericytes dissociate from the vessel; endothelial cells (ECs) migrate and proliferate to form an array/sprout; a lumen is formed in the sprout (a process referred to as canalization); branches and loops are formed by confluence and anastomoses of sprouts to permit blood flow; and finally, these immature vessels are invested in basement membrane and pericytes. During physiologic angiogenesis, these vessels differentiate into mature arterioles, capillaries, and venules, whereas in tumors they remain largely immature. [5] [11] [12] [20] During embryonic development, a primitive vascular plexus is formed from endothelial precursor cells (EPCs, also known as angioblasts) by a process referred to as vasculogenesis. In adults, EPCs—mobilized from bone marrow niches into the peripheral blood circulation—can also contribute to neovascularization (process referred to as “postnatal” vasculogenesis) in tumors and other tissues. [21] [22] [23] The current challenge is to discern the relative contribution of each of the four mechanisms of neovascularization during the growth and/or during treatment of tumors.[24]

 
 

Figure 8-2  Cellular mechanisms of vascularization in tumors. At least four mechanisms are involved: (1) intussusception, where tumor vessels enlarge and an interstitial tissue column grows in the enlarged lumen, expanding the network; (2) vasculogenesis, where endothelial precursor cells mobilized from the bone marrow or peripheral blood contribute to the endothelial lining of tumor vessels; (3) “sprouting” angiogenesis, where the existing vascular network expands by forming sprouts or bridges; and (4) co-option (not shown), where tumor cells grow around existing vessels to form “perivascular” cuffs.  (Adapted from Jain RK, Carmeliet PF: Angiogenesis in cancer and other diseases. Nature 2000;407:249–257.)

 

 

 

Molecular Mechanisms

Various pro- and antiangiogenic molecules that orchestrate different steps in vessel formation, along with their functions, are listed in Table 8-1 . VEGF is currently considered the most critical proangiogenic molecule. Originally discovered in 1983 as the vascular permeability factor and cloned in 1989, VEGF increases vascular permeability, promotes migration and proliferation of ECs, serves as an EC survival factor, can mobilize EPC populations from the bone marrow, and is known to upregulate leukocyte adhesion molecules on ECs. [16] [22] [25] [26] [27] During tumor progression, or with treatment, the number of distinct angiogenic molecules produced by a tumor can increase. [28] [29] [30] Thus, after VEGF signaling is blocked, a tumor might rely on other, alternative angiogenic molecules (e.g., basic fibroblast growth factor [bFGF], stromal-derived factor 1α [SDF1α], placental-derived growth factor [PlGF], or interleukin-8 [IL-8]).[31] Other positive regulators of angiogenesis include the angiopoietins that are involved in stabilizing vessels and controlling vascular permeability; various proteases involved in dissolving/remodeling matrix and releasing growth factors; and recently discovered organ-specific angiogenic stimulators (e.g., endocrine gland VEGF). [20] [32] [33] Angiogenesis inhibitors include endogenous soluble receptors of various proangiogenic ligands (e.g., sVEGFR1) and molecules that downregulate the expression of stimulators (e.g., interferons) or that interfere with the release of the stimulators or binding with their receptors (e.g., platelet factor 4). Thrombospondins are among the first and most well-characterized endogenous inhibitors that interfere with the growth, adhesion, migration, and survival of ECs.[14] Other endogenous inhibitors include fragments of various plasma or matrix proteins (e.g., angiostatin, a fragment of plasminogen; endostatin, a fragment of collagen XVIII; tumstatin, a fragment of collagen IV). [34] [35] [36] Neither the mechanisms of action of the matrix-derived inhibitors nor their physiologic role are well understood.[37] The generation of pro- and antiangiogenic molecules can be triggered by metabolic stress (e.g., low pO2, low pH, or hypoglycemia), mechanical stress (e.g., shear stress, solid stress), immune/inflammatory cells that have infiltrated the tissue, and genetic mutations (e.g., activation of oncogenes or deletion of suppressor genes that control the production of angiogenesis regulators). [14] [15] [38] [39] [40] [41] These molecules can emanate from cancer cells, endothelial cells, stromal cells, blood, and extracellular matrix ( Fig. 8-3). [42] [43] [44] [45] Because the normal host cells differ among organs, the detailed mechanisms of angiogenesis might depend on the specific host-tumor interactions operating within a given tissue. [46] [47] [48] [49] [50] [51] [52] [53] Furthermore, because the tumor microenvironment is likely to change during tumor growth, regression, and relapse, profiles of pro- and antiangiogenic molecules are likely to change with time and space. [54] [55] The challenge currently is to develop a unified conceptual framework to describe the temporal and spatial profiles of this increasingly diverse array of angiogenesis regulators with the aim of developing effective therapeutic strategies. [56] [57]


Table 8-1   -- Angiogenesis Activators and Inhibitors[*]

Activators

Function

Inhibitors

Function

VEGF family members[†][‡]

Stimulate angio/vasculogenesis, permeability, leukocyte adhesion

VEGFR-1; soluble VEGFR-1; soluble neuropilin-1 (NRP-1)

Sink for VEGF, VEGF-B, PlGF

VEGFR[‡], NRP-1, NRP-2

Integrate angiogenic and survival signals

Ang 2[†][‡]

Antagonist of Ang 1

EG-VEGF

Stimulate growth of endothelial cells derived from endocrine glands

TSP-1,2

Inhibit endothelial migration, growth, adhesion, and survival

Ang 1 and Tie 2[†][‡]

Stabilize vessels

Angiostatin and related plasminogen kringles

Inhibit endothelial migration and survival

PDGF-BB and receptors

Recruit smooth muscle cells

Endostatin (collagen XVIII fragment)

Inhibit endothelial survival and migration

TGF-β1[§], endoglin, TGF-β receptors

Stimulate extracellular matrix production

Tumstatin (collagen IV fragment)

Inhibit endothelial protein synthesis

FGF, HGF, MCP-1

Stimulate angio/arteriogenesis

Vasostatin; calreticulin

Inhibit endothelial growth

Integrins αvβ3, αvβ5, α5β1

Receptors for matrix macromolecules and proteinases

Platelet factor-4

Inhibit binding of bFGF and VEGF

VE-cadherin; PECAM (CD31)

Endothelial junctional molecules

Tissue-inhibitors of MMP (TIMPs); MMP-inhibitors; PEX

Suppress pathologic angiogenesis

Ephrins[‡]

Regulate arterial/venous specification

Meth-1; Meth-2

Inhibitors containing MMP, TSP-, and disintegrin domains

Plasminogen activators, MMPs

Remodel matrix, release growth factor

IFN-α, -β, -γ; IP-10, IL-4, IL-12, IL-18

Inhibit endothelial migration; downregulate bFGF

PAI-1

Stabilize nascent vessels

Prothrombin kringle-2; anti-thrombin III fragment

Suppress endothelial growth

NOS; COX-2

Stimulate angiogenesis and vasodilation

16 kD-prolactin

Inhibit bFGF/VEGF

AC133

Regulate angioblast differentiation

VEGI

Modulate cell growth

Chemokines[§]

Pleiotropic role in angiogenesis

Fragment of SPARC

Inhibit endothelial binding and activity of VEGF

Id1/Id3

Inhibit differentiation

Osteopontin fragment

Interfere with integrin signaling

 

 

Maspin

Protease inhibitor

 

 

Canstatin, proliferin-related protein, restin

Mechanisms unknown

See text for explanation of abbreviations.

 

*

Selected list updated from ref. 11; for complete function and references, see supplementary information (http://steele.mgh.harvard.edu).

Also present in or affecting nonendothelial cells.

See ref. 20.

§

Opposite effect in some contexts.

 

 
 

Figure 8-3  Tumor induction of host promoter activity in stromal cells. The expression of VEGF in host cells can be examined using transgenic mice expressing a green fluorescent protein (GFP) under the control of the VEGF promoter. A, A murine mammary carcinoma xenograft shows host cell VEGF expression mainly at the periphery of the tumor after 1 week. B, After 2 weeks, the VEGF-expressing host cells have infiltrated the tumor. C, A GFP-expressing layer of host cells can be seen at the tumor-host interface. D and E, The VEGF-expressing host cells colocalize with the angiogenic tumor vessels.  (A and B, From Fukumura D, Xavier R, Sugiura T, et al: Tumor induction of VEGF promoter activity in stromal cells. Cell 1998;94:715–725. CE, From Brown EB, Campbell RB, Tsuzuki Y, et al: In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat Med 2001;7:1069.)

 

 

 

Vascular Architecture

In a normal tissue, blood flows from an artery to arterioles to capillaries to venules to a vein. Although the tumor vasculature originates from these host vessels and the mechanisms of angiogenesis are similar, its organization may differ dramatically, depending on the tumor type, its location, and whether it is growing, regressing, or relapsing. [16] [58] [59] [60] [61] In general, tumor vessels are dilated, saccular, tortuous, and chaotic in their patterns of interconnection.[62] For example, whereas normal vasculature is characterized by dichotomous branching, tumor vasculature has many trifurcations and branches with uneven diameters. [63] [64] The fractal dimensions and minimum path lengths of tumor vasculature are different from those of normal host vasculature. [58] [59] [60] The molecular mechanisms of this abnormal vascular architecture are not understood, but it seems reasonable to hypothesize that the imbalance of VEGF and angiopoietins is a key contributor. [20] [65] In mice, “normalization” of the tumor vasculature observed during therapies that reduce VEGF (e.g., hormone withdrawal from a hormone-dependent tumor), interfere with VEGF signaling (e.g., treatment with anti-VEGF or anti-VEGFR2 antibody; Fig. 8-4 ), or mimic an antiangiogenic cocktail (e.g., trastuzumab [Herceptin] treatment of a HER2-overexpressing tumor) is in concert with this molecular hypothesis. [12] [54] [55] [56] [66]Mechanical stress generated by proliferating tumor cells also can lead to the partially compressed or totally collapsed vessels often found in tumors. [67] [68] The decompression of blood vessels observed after induction of apoptosis in perivascular cells supports this mechanical hypothesis. [69] [70] Perhaps the combination of both molecular and mechanical factors renders the tumor vasculature abnormal, and, thus, both types of factors must be taken into account when designing novel strategies for cancer treatment.

 
 

Figure 8-4  Normalization of tumor vasculature. Normal vessels are well organized with even diameters. In contrast, tumor vessels are tortuous, with increased vessel diameter, length, density, and permeability. Antiangiogenic therapies “normalize” the tumor vascular network and could ultimately reduce the vasculature to the point at which it provides inadequate support for tumor growth.  (Adapted from Jain RK: Normalizing tumor vasculature with antiangiogenic therapy: a new paradigm for combination therapy. Nat Med 2001;7:987–989; Jain RK: Angiogenesis and lymphangiogenesis in tumor: insights from intravital microscopy. Cold Spring Harbor Symp Quant Biol [The Cardiovascular System] 2002;67:239–248; Jain RK, Carmeliet PF: Vessels of death or life. Sci Am 2001;285:38.)

 

 

 

Blood Flow and Microcirculation

Blood flow in a vascular network, whether normal or abnormal, is governed by the arterio-venous pressure difference and flow resistance. Flow resistance is a function of the vascular architecture (referred to as geometric resistance) and of the blood viscosity (rheology, referred to as viscous resistance).[62] Abnormalities in both vasculature and viscosity increase the resistance to blood flow in tumors. [64] [71] [72] [73] As a result, overall perfusion rates (blood flow rate per unit volume) in tumors are lower than in many normal tissues. [74] [75] [76] Both macroscopically and microscopically, tumor blood flow is temporally and spatially chaotic. Macroscopically, four spatial regions can be recognized in a tumor ( Fig. 8-5 ):

  

1.   

An avascular necrotic region

  

2.   

A semi-necrotic region

  

3.   

A stabilized microcirculation region

  

4.   

An advancing front [77] [78]

 
 

Figure 8-5  The tumor microenvironment is heterogeneous with proliferative, quiescent, and necrotic regions. These regions can be characterized in terms of various physiologic parameters. Decreasing magnitude of these parameters is indicated as +++, ++, +, +/-. and -. (From Jain RK, Forbes NS: Can engineered bacteria help control cancer? Proc Natl Acad Sci USA 2001;98:14748–14750.)

 

 

 

At the microscopic level, in normal tissues, erythrocyte velocity is dependent on vessel diameter, but there is no such dependence in most tumors. [47] [52] [79] Furthermore, the average erythrocyte velocity can be an order of magnitude lower in some tumors as compared with that of normal host tissue ( Fig. 8-6 ).[52] In a given vessel within a tumor, blood flow fluctuates with time and can reverse its direction.[77] [79] [80] In addition to the elevated geometric and viscous (rheologic) resistance, other molecular and mechanical factors contribute to this spatial and temporal heterogeneity. These include imbalance between pro- and antiangiogenic molecules, “solid stress” generated by proliferating cancer cells, vascular remodeling by intussusception, and coupling between luminal and interstitial fluid pressure via hyperpermeability of tumor vessels. [17] [57] [58] [67] [68] [69] [81] [82] [83] As we will learn later, this heterogeneity contributes to both acute and chronic hypoxia in tumors—a major cause of resistance to radiation and other therapies. Considerable effort has gone into increasing tumor blood flow for improving radiation therapy, or decreasing tumor perfusion in the case of hyperthermia. This has been difficult to achieve reproducibly, because tumor vasculature consists of both vessels co-opted from the pre-existing host vasculature and vessels resulting from the angiogenic response of host vessels to cancer cells. The former are invested in normal contractile perivascular cells, whereas the latter lack these perivascular cells or these cells are abnormal. [42] [62] [84] Presumably as a result, efforts to increase the tumor blood flow by pharmacologic or physical agents have not always been reproducible or successful. [62] [75] On the other hand, the strategy of decreasing or shutting down the tumor blood flow—by “stealing” blood away from the “passive component” of the tumor vasculature by vasodilators, by vascular targeting, or by intravascular coagulation—has shown promise in experimental systems. [75] [76] [85] [86] [87] It also appears that judiciously applied antiangiogenic therapy could “normalize” the abnormal tumor microcirculation by pruning the immature vessels (see Fig. 8-4 ), thus rendering the remaining vasculature more responsive to vasoactive agents.[88]

 
 

Figure 8-6  Blood velocity as a function of vessel diameter. A, Normal pial vessels. B, MCaIV (mammary carcinoma) and U87 (glioma) tumors xenografted on the pial surface. The measured tumor blood velocities are an order of magnitude lower than the velocities in the normal host tissue and are not related to the vessel density.  (From Yuan F, Salehi HA, Boucher Y, et al: Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows. Cancer Res 1994;54:4564–4568.)

 

 

 

VASCULAR PERMEABILITY

Once a blood-borne molecule has reached an exchange vessel, its extravasation occurs by diffusion, convection, and, to some extent, presumably by transcytosis.[88] The diffusive permeability, P, of a molecule depends on the size, shape, charge, and flexibility of the molecule, and on the size, shape, charge, and dynamics of the transvascular transport pathway. In normal vessels, these pathways include diffusion along the EC membrane (for lipophilic solutes), trans-EC diffusion, interendothelial junctions (<7 nm), open or closed fenestrations (<10 nm), and transendothelial channels (including vesicles or vesicovacuolar channels). [16] [89] Some of these anatomical pathways may be lined with glycocalyx on EC, thus effectively reducing the size of the pathway. A basement membrane may further retard the movement of molecules. Ultrastructure studies show widened interendothelial junctions, increased numbers of fenestrations, vesicles, and vesicovacuolar channels in tumor vessels, and a lack of normal basement membrane and pericytes. [16] [49] [84] [89] [90] In concert with these ultrastructural findings, both vascular permeability and hydraulic conductivity (a measure of water movement by pressure gradient) of tumors, in general, are significantly higher than those for various normal tissues. [52] [91] [92] [93] [94] [95] Furthermore, unlike normal vessels, tumor vessels lack selectivity in permeability to different molecules.[96] Positively charged molecules have a higher affinity for the negatively charged angiogenic tumor vessels. [97] [98] [99] Despite increased overall permeability, not all blood vessels of a tumor are leaky ( Fig. 8-7A ). Even the leaky vessels have a finite pore size that is tumor dependent ( Fig. 8-7B ), and ultrastructural studies show that the larger pore size in tumors represents wide interendothelial junctions. [49] [90] Not only do the vascular permeability and pore size vary from one tumor to the next (see Fig. 8-7A–C ), but within the same tumor they vary spatially and temporally, as they do during tumor growth, regression, and relapse. [49] [54] [55] The local microenvironment plays an important role in controlling vascular permeability ( Fig. 8-7D ). For example, a human glioma (HGL21) has fairly leaky vessels when grown subcutaneously in immunodeficient mice, but it exhibits blood-brain barrier properties in the brain. [52] [65] Such site-dependent differences for other tumors have been observed in other orthotopic sites. [47] [50] [51] One possible explanation is that the host-tumor interactions control the production and secretion of cytokines associated with permeability increase (e.g., VEGF) and decrease (e.g., angiopoietin 1). [20] [33] [65] [100] [101] A better understanding of the molecular mechanisms of permeability regulation in tumors is likely to yield strategies for improved delivery of molecular medicine to tumors.

 
 

Figure 8-7  Heterogeneous permeability of tumor vessels. Vessel permeability varies spatially within a given tumor, between tumors of identical type implanted in different host organ environments, and between different tumor types in the same organ environment. A,Vessels within a given tumor are leaky in some areas and relatively impermeable in others. B and C, Tumors of different types grown in the same environment show variations in both vessel pore size and permeability. Pore sizes are measured by the largest tracer particle able to permeate the vessel wall. D, MCaIV and HCaI tumors implanted in two different sites (subcutaneous and cranial) have vessels with different pore sizes. For both tumor types, larger pore sizes are observed in the subcutaneous (S.C.) tumors compared with the cranial tumors.  (A and C, From Yuan F, Leunig M, Huang SK, et al: Microvascular permeability and interstitial penetration of sterically stabilized [stealth] liposomes in a human tumor xenograft. Cancer Res 1994;54:3352–3356. B and D, From Hobbs SK, Monsky WL, Yuan F, et al: Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci USA 1998;95:4607–4612.)

 

 

 

Movement of Cells across Vessel Walls

Both cancer cells and immune cells frequently move across the walls of blood vessels—the former in the process of metastasis and the latter during immune response or cell-based immunotherapy. Both transendothelial (through ECs) and periendothelial (between ECs) pathways have been proposed as a route for intravasation and extravasation of cells. Very little is known about intravasation except that tumors might shed more than a million cells per gram per day and most of these are not clonogenic. [102] [103] [104] [105] More is known about the molecular and cellular mechanisms of extravasation.[106]When a cell enters a blood vessel, it can continue to move with the flowing blood, collide with the vessel wall, adhere transiently or stably, and finally extravasate. These interactions are governed both by local hydrodynamic forces and adhesive forces. The former are determined by the vessel diameter and fluid velocity and the latter by the expression, strength, and kinetics of bond formation between adhesion molecules and by the surface area of contact. [107] [108] [109] [110] [111] Deformability of cells affects both types of forces.[112] In addition, cancer cells may grow intravascularly at the distant site (e.g., in the lungs).[113]

Rolling of endogenous leukocytes is generally low in tumor vessels, whereas stable adhesion (≥30 sec) is comparable between normal vessels and tumor vessels.[114] On the other hand, both rolling and stable adhesion are nearly zero in angiogenic vessels induced in collagen gels by bFGF or VEGF, two of the most potent angiogenic factors.[46] Whether this observation is due to a low flux of leukocytes into angiogenic vessels and/or to downregulation of adhesion molecules in these immature vessels is currently not known. The age of the animal also plays an important role in leukocyte-endothelial interactions.[115] Further insight into the types of cells that adhere to tumor vessels comes from studies on the localization of IL-2–activated natural killer (A-NK) cells in normal and tumor tissues in mice using positron emission tomography. [116] [117] After systemic injection, these cells localized primarily in the lungs immediately after injection and could not be detected in the tumor.[116] Increased rigidity caused by IL-2 activation might contribute to the mechanical entrapment of these cells in the lung microcirculation. [118] [119] Constitutive expression of certain adhesion molecules in the lung vasculature might also facilitate their retention in the lungs.[106] One approach to reducing lung entrapment is to reduce the rigidity of these cells. [112] [117] Alternatively, the lung can be circumvented by injecting α-NK cells directly into the blood supply of tumors. In this case, α-NK cells, both xenogenic and syngeneic, adhered to some blood vessels in three different tumor models via CD18 and very large antigen-4 (VLA-4) on the α-NK cells and intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on the activated endothelium of angiogenic vessels. [27] [117] [120] [121] [122] [123] These molecules can be upregulated by tumor necrosis factor-α (TNF-α) and a protein of 90 kd molecular weight (p90) that is secreted by some neoplastic cells and downregulated by transforming growth factor-β (TGF-β)—also, presumably, secreted by cancer cells. [48] [107] [124] [125] [126] [127] [128] Surprisingly, the proangiogenic VEGF also can upregulate these molecules, presumably via VEGFR1, whereas another proangiogenic molecule, bFGF, can downregulate these molecules. [27] [45] [55] [66] [129] On the other hand, inflammatory cells such as monocyte/macrophages or neutrophils are also recruited by VEGF and may play important roles in promoting matrix remodeling and angiogenesis.[130] The challenge currently is to decrease nonspecific entrapment of immune cells in normal vessels and to increase their delivery to tumor vessels to improve various cell-based therapies, including gene therapy.

EXTRAVASCULAR COMPARTMENT

Composition and Origin

The extravascular compartment of a solid tumor consists of neoplastic cells (parenchyma) and host cells (e.g., inflammatory cells, fibroblasts) residing in an interstitial matrix bathed by the interstitial fluid (see Fig. 8-1 ). Depending on the tumor type and its stage of differentiation, neoplastic cells might be dispersed in the matrix as individual cells (e.g., lymphomas, melanomas) or as clumps, sheets, or nests (e.g., carcinomas). More than 80% of tumors are carcinomas arising from epithelial cells. The remaining include sarcomas arising from mesenchymal cells (e.g., bone or muscle cells), lymphomas arising from lymphoid tissue, leukemias arising from hematopoietic cells, and hemangiomas arising from ECs. In a poorly differentiated carcinoma, the cancer cells might be packed loosely in clumps, whereas in a well-differentiated carcinoma, the cells might be connected with intercellular junctions and tightly packed in a nest enveloped by a basement membrane. With tumor progression, cancer cells can invade the basement membrane and spread to other regions.[16] These various types of normal host cells must migrate into the tumor from normal tissue. Inflammatory cells might enter the tumor via blood vessels or might infiltrate from the adjacent tissue or lymphatics.[106] Other host cells, such as fibroblasts, might proliferate and migrate from the adjacent connective tissue. [20] [42] [43] The interstitial subcompartment of a tumor is bounded by the walls of the blood vessels on one side and by the membranes of cancer and stromal cells on the other. In normal tissues, the blood vessels are surrounded by a basement membrane, which, as discussed before, is defective in tumors.[20] In addition, functional lymphatics might be confined to the tumor margin. [131] [132] The interstitial space of tumors, like that of normal tissues, is composed of a collagen and elastin fiber network that provides structural support to the tissue. Interdispersed in this cross-linked structure are the interstitial fluid and macromolecular constituents (polysaccharides, hyaluronan, and proteoglycans), which form a hydrophilic gel. Compared with our understanding of blood vessel formation, our understanding of stroma generation is minimal. Dvorak and coworkers have proposed that the extravasated plasma protein fibrinogen, a key component of the tumor interstitial fluid, clots to form fibrin, which serves as a major component of the provisional stroma. This provisional stroma eventually is replaced by more mature connective tissue stroma. The tumor interstitial fluid also contains several other Arg-Gly-Asp (RGD)-containing proteins, including fibronectin, vitronectin, osteopontin, thrombospondin, decorin, and tenacin.[16] These proteins are present in both free and bound forms. Their Arg-Gly-Asp sequence provides a binding site for adhesion that assists in the migration of various cells, including stromal cells. In addition to extravasating from the leaky tumor vessels, these proteins, along with collagen and various proteoglycans, are also synthesized by the stromal cells, albeit in a form that differs from that in the plasma or normal tissues.[16] Tumor interstitial fluid also can contain various growth factors that facilitate stroma formation. For example, in vitro studies suggest that platelet-derived growth factor-ββ (PDGF-ββ) is involved in the recruitment of fibroblasts to tumors, and TGF-β induces the production of collagen and other matrix molecules in tumors. [20] [133] With the increasing interest in using the fragments of matrix constituents for controlling angiogenesis, our understanding of the molecular and cellular mechanisms of stroma generation in tumors will increase.[37]

Interstitial Transport

Once a molecule has extravasated, its movement through the interstitial space occurs by diffusion and convection.[134] Diffusion is proportional to the concentration gradient in the interstitium, and convection is proportional to the interstitial fluid velocity, which, in turn, is proportional to the pressure gradient in the interstitium. Just as the interstitial diffusion coefficient D (cm2/s) relates the diffusive flux to the concentration gradient, the interstitial hydraulic conductivity K (cm2/mmHg/sec) relates the interstitial velocity to the pressure gradient.[134] Values of these transport coefficients are governed by the structure and composition of the interstitial compartment and by the physicochemical properties of the solute molecule. [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] The value of K for a human colon carcinoma xenograft (LS174T), measured using two different methods, was found to be higher than that of a hepatoma, which, in turn, was higher than that of the normal liver. [140] [145] [146] Using fluorescence recovery after photobleaching, D of various molecules in tumors was found to be about one-third that in water and higher than the values in the host tissue ( Fig. 8-8A ). [136] [147] Collagen content and structure have a significant effect on D in tumors. [142] [143] [145] [148] [149] [150] This is surprising because hyaluronan and proteoglycans, not collagen, account for most of the resistance to transport in normal tissues. Because collagen is produced by host cells (e.g., fibroblasts), the penetrability into a tumor depends on the host-tumor interaction (see Fig. 8-8A ). Thus, agents that interfere with collagen synthesis and/or organization (e.g., relaxin, bacterial collagenase) might increase interstitial transport in tumors [144] [150] ( Fig. 8-8B ). The time constant for a molecule with diffusion coefficient Dto diffuse across a distance L is approximately L2/4D. For diffusion of immunoglobulin G (IgG) in tumors, this time constant is on the order of 1 hour for a 100-μm distance, days for a 1-mm distance, and months for a 1-cm distance. So for a 1-mm distance in tumor, diffusional transport would take days, and for a 1-cm distance in tumor, it would take months. If the central vessels have collapsed completely due to cellular proliferation and interstitial matrix rearrangement, the reduced delivery of macromolecules by blood flow would make diffusion the primary mechanism of delivery to this necrotic center. [67] [69] Binding of a low- or high-molecular-weight drug to plasma proteins and various tissue components could further retard their transport in tumors. [147] [151] [152] [153] [154] [155] [156] [157] The role of binding is clearly illustrated in Figure 8-8C , which compares the rate of fluorescence recovery of a photobleached spot in tumor tissue injected with a nonspecific vs. a specific IgG. In addition to the heterogeneity of D in tumors, the most unexpected result of these photobleaching studies was the large extent (30% to 40%) of nonspecific binding.[147] These results collectively suggest that the interstitial compartment of a tumor can be a formidable barrier to the uniform delivery of therapeutic macromolecules (e.g., antibodies, genes) in tumors, and strategies are needed to modify this barrier.

 
 

Figure 8-8  Interstitial transport in tumors. Transport of molecules through a tumor is affected by several factors. A, Diffusivity of a molecule in a tumor decreases with increasing molecular weight and is dependent on the host-tumor interaction. The diffusivity of macromolecules is lower in subcutaneous (sc) tumors in dorsal chamber (DC, octagons-U87 glioblastoma and diamonds-Mu89 melanoma) than in the same tumors grown in cranial windows (CW, squares-U87 and triangles-Mu89), and both are less than in phosphate-buffered saline (PBS). B, A representative model of improvement in oncolytic viral distribution and tumor cell infection by collagenase treatment. Following direct intratumor injection, viral spread (red area) is limited by fibrillar collagen (red lines) and results in a cluster of infected cells (light green). The collagen network also restricts the distribution of subsequent viral progeny, and tumor cell infection beyond the initial injection site is not achieved. In contrast, injection of virus together with collagenase results in a more diffuse distribution of viral particles and a greater number of initially infected cells (light green). Viral particles released by these cells have greater access to neighboring uninfected cells. This process results in more widespread secondary infection (dark green) and ultimately greater therapeutic efficacy. C, Interstitial transport is also reduced by binding. The fluorescence of a photobleached spot recovers much more slowly with a specific antibody than with a nonspecific antibody. The binding of the specific antibody hinders the transport of the molecule into the photobleached spot, slowing the fluorescence recovery.  (A, From Pluen A, Boucher Y, Ramanujan S, et al: Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc Natl Acad Sci USA 2001;98:4628–4633. B,McKee TD, Grandi P, Mok W, et al: Degradation of fibrillar collagen in a human melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector. Cancer Res 2006;66:2509–2513. C, Adapted from Berk DA, Yuan F, Leunig M, Jain RK: Direct in vivo measurement of targeted binding in a human tumor xenograft. Proc Natl Acad Sci USA 1997;94:1785–1790.)

 

 

 

Lymphangiogenesis and Lymphatic Transport

In most normal tissues, extravasated plasma and macromolecules are taken up by the lymphatics and returned to the blood circulation. It is widely accepted that lymphatic vessels are present in the tumor margin and the peritumoral tissue ( Fig. 8-9A ). Indeed, invasion of peritumoral lymphatics is considered to be a poor prognostic factor for several tumors (e.g., breast, colorectal, and endometrial cancers), and lymphatic metastasis is a major cause of morbidity and mortality for others (e.g., melanoma, head and neck cancer, lung cancer, and cervical cancer). The hotly debated issue for nearly a century has been whether anatomically defined lymphatic vessels are present within solid tumors and, if so, whether they function (see Fig. 8-9A ). [74] [158] Currently available immunohistochemical markers stain for structures in some tumors that resemble lymphatic vessels. Because many of these markers lack specificity, however, it is not clear whether they stain functional lymphatic vessels, ECs from remnant lymphatic vessels, or some other structures (e.g., preferential fluid channels). [132] [145] [159] [160] It is likely that the “mechanical” stress induced by proliferating cancer cells compress and impair lymphatic vessels that are co-opted or formed within a tumor. [67] [70] The impaired lymphatic vessels, in turn, contribute to the interstitial hypertension characteristic of animal and human tumors (to be discussed shortly). Embryonic lymphatic vessels originate primarily from blood vessels according to the following process ( Fig. 8-9B ). [161] [162] [163]

  

1.   

In the early embryo, endothelial cells of the cardinal vein express lymphatic vascular endothelial receptor-1 (LYVE-1) and VEGFR3, molecules observed primarily (but not exclusively) on lymphatic vessels in normal adult tissues.

  

2.   

A yet unknown signal triggers the expression of the homeobox gene Prox-1 so that the protein is displayed in a polarized fashion in the endothelial cells of the cardinal vein. This marks the first stage of commitment to the lymphatic lineage.

  

3.   

These LYVE-1+VEGFR3+Prox-1+ cells then start to bud, again in a polarized fashion.

  

4.   

At this stage, these early lymphatic endothelial cells start expressing secondary lymphoid chemokine and increased levels of VEGFR3, markers of mature lymphatic ECs.

  

5.   

They then begin to form the lymphatic system.

 
 

Figure 8-9  A, Schematic of lymphatics in tumors. It is widely accepted that peritumoral lymphatics exist and that metastasis can occur via these lymphatic vessels. Recent evidence shows that structures within tumors that stain for lymphatic markers are not functional. The lower-left insert shows the molecular players in lymphangiogenesis. B, Mechanisms of lymphatic vessel formation and separation from blood vessels. C, The tumor is modeled as a sphere of radius R, embedded in a body fluid (e.g, peritoneal cavity, pleural cavity) or host tissue. The interstitial fluid oozing from the tumor periphery carries therapeutics (e.g, monoclonal antibodies), growth factors (e.g, VEGF-A, -B, -C, and -D), and cells (e.g., metastatic cells) into the surrounding fluid/tissue. The result may be higher fluxes of cancer cells and growth factors from the tumor into the surrounding tissue or body fluid. The growth factors (including VEGF-A, B, C, and D) can induce angiogenesis and lymphangiogenesis, and the cancer cells can contribute to metastatic dissemination via the lymphatic or blood vessels. Fluid seeping from the tumor surface can also cause edema (e.g., around brain tumors) and ascites formation (e.g., ovarian cancer). Antiangiogenic therapy can “normalize” the tumor vasculature, leading to lower interstitial fluid pressure (IFP) at the center, less steep IFP gradients and lower fluid flow rates at the tumor margin, thus potentially reducing peritumor edema, ascites formation, and lymphatic metastasis. Furthermore, the normalized vessels may be more resistant to cancer cell intravasation, a prerequisite for hematogenous metastasis.  (A, From Jain RK, Fenton BT: Intratumoral lymphatic vessels: a case of mistaken identity or malfunction? J Natl Cancer Inst 2002;94:417–421. B, From Jain RK, Padera TP: Lymphatics make the break. Science 2003;299:209–210. Illustration: Katherine Sutliff. Reprinted with permission from AAAS. C, From Jain RK, Tong RT, Munn LL: Effect of vascular normalization by antiangiogenic therapy on interstitial hypertension, peritumor edema and lymphatic metastasis: insights from a mathematical model. Cancer Res 2007;67:2729–2735).

 

 

 

Members of the angiopoietin family (Ang-2) and its receptor (Tie-2), as well as podoplanin are presumably involved in the maturation and patterning of these nascent lymphatic vessels.[164] The hematopoietic signaling pathway, Syk/SLP-76, contributes to the separation of lymphatics from blood vessels. The molecules involved in angiogenesis are also involved in lymphangiogenesis. For example, VEGF-C and -D can induce both angiogenesis and lymphangiogenesis and are associated with lymphogenic metastasis in a variety of tumors.[131] Their receptor VEGFR3 is present in both lymphatic and selected vascular endothelium. As is the case with vascular angiogenesis, other positive and negative regulators (e.g., angiopoietins) and other receptors (e.g., chemokine receptors and neuropilins) could be involved in lymphangiogenesis, and as discussed already, mechanisms analogous to co-option, intussusception, sprouting, and vasculogen esis might operate in lymphatic growth. [11] [164] Similar to the recently discovered organ-specific angiogenic molecule (EG-VEGF) and endothelial precursor cells, there could be organ-specific lymphangiogenic molecules and lymphatic EPCs that contribute to tumor-associated lymphangiogenesis. [22] [32] [163] Moreover, the proteolytic processing of lymphangiogenic molecules and the phenotype and function of the resulting lymphatics might depend not only on the tumor type but also on the host organ in which the tumor is growing. [66] [159] [161] [163]

The mechanical and/or molecular signals that could trigger the lymphangiogenic switch are unknown. Because lymphatic vessels help maintain the balance of fluid in tissues, hydrostatic pressure is a probable trigger. Whether the hyperplasia and the increased density of lymphatic vessels seen in the tumor margins are a response to elevated hydrostatic pressure in tumors and whether the newly formed lymphatics are able to remain open and carry cancer cells are open questions. Techniques such as microlymphangiography, reagents that block signaling of VEGF-C and -D, and lymphangiogenic factors yet to be discovered will allow us to answer these important questions. [4] [159] [163] [165] [166] [167] [168] [169] [170]

Interstitial Hypertension

Unlike normal tissues, in which the interstitial fluid pressure (IFP) is around 0μmHg, both animal and human tumors exhibit interstitial hypertension ( Table 8-2 ). [132] [134] [146] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] [181] [182] The tumor IFP begins to increase as soon as the host vessels become leaky in response to permeability factors such as VEGF, and, thus, IFP can be lowered by antibodies against VEGF or VEGFR2. [183] [184] [185] The IFP increases with tumor size in some tumors and remains independent of tumor size in others. [171] [172] [175] [176] Three mechanisms contribute to interstitial hypertension in tumors. In normal tissues, the lymphatics maintain the fluid homeostasis; thus, the lack of functional lymphatics in tumors is a key contributor. Indeed, DiResta and colleagues have been able to lower the IFP by placing “artificial lymphatics” in tumors.[186] The second contributor is the high permeability of tumor vessels. As a result, the hydrostatic and oncotic (colloid osmotic) pressures become almost equal between the intravascular and extravascular spaces. [174] [187] At least two pieces of evidence support this hypothesis. First, reducing permeability by blocking VEGF signaling lowers IFP. [184] [185] [188] [189] Second, IFP increases and decreases with the microvascular pressure within seconds. [190] [191] [192] The two mechanisms described thus far can only explain interstitial hypertension up to 20–30μmHg, the microvascular pressure of most exchange vessels (the hydrostatic pressure within the lumina of capillaries) in our bodies, but IFPs as high as 94 mmHg have been measured in human tumors.[173] Because microvascular pressure (MVP) is the driving force for IFP in tumors, these tumors must have a high MVP. Indeed, this is the case.[174] There are two possible explanations for elevated MVP in tumors: the tumor vessels have reduced arterial resistance so that the MVP becomes closer to arterial pressure, and/or the tumor vessels have increased venous resistance due to compression and tortuousity so that the whole intratumor vascular network is under hypertension. Indirect evidence for the latter comes from the decrease in IFP after decompression of tumor vessels by taxol-induced apoptosis of perivascular cells.[69]


Table 8-2   -- Interstitial Fluid Pressure (mmHg) in Normal and Neoplastic Tissues in Patients

Tissue Type

N

Mean

Range

References

Normal skin

5

0.4

- 1.0–3.0

( 166 )

Normal breast

8

0.0

- 0.5–3.0

( 166 )

Head and neck carcinomas

27

19.0

1.5–79.0

( 165 )

Cervical carcinomas

12

15.7

10.0–26.0

( 163 )

Cervical carcinomas

102

19.0[†]

- 3.0–48.0

( 172 )

Lung carcinomas

26

10.0

1.0–27.0

( 123 )

Metastatic melanomas

14

21.0

0.0–60.0

( 167 )

Metastatic melanomas

12

14.5

2.0–41.0

( 162 )

Breast carcinomas

13

29.0

5.0–53.0

( 169 )

Breast carcinomas

8

15.0

4.0–33.0

( 166 )

Brain tumors[*]

17

7.0

2.0–15.0

( 168 )

Brain tumors[*]

11

1.0

- 0.5–8.0

( 171 )

Colorectal liver metastasis

8

21.0

6.0–45.0

( 166 )

Lymphomas

7

4.5

1.0–12.5

( 167 )

Renal cell carcinoma

1

38.0

( 166 )

*

Patients were treated with anti-edema therapy.

IFP given is a median value.

 

The elevated pressure can compromise the tumor microcirculation and delivery of therapeutics in three ways:

  

1.   

Reduced transmural pressure gradients due to equilibrium between MVP and IFP reduce convection across tumor vessel walls and thus compromise the transport of macromolecules. [174] [192]

  

2.   

Because IFP is nearly uniform throughout a tumor and drops precipitously in the tumor margin, the interstitial fluid oozes out of the tumor into the surrounding normal tissue, carrying macromolecules with it ( Fig. 8-10 ). [171] [193]

  

3.   

Finally, transmural coupling between IFP and MVP due to high permeability of tumor vessels can lead to blood flow stasis in tumors without physically occluding the vessels. [81] [82] [83]

 
 

Figure 8-10  Interstitial fluid pressure (IFP) profile in a tumor and its potential application. A, IFP is elevated and nearly uniform in the bulk of the tumor and drops precipitously in the tumor margin. B, This elevated pressure can be exploited to determine the location of a tumor precisely.  (A, Adapted from Boucher Y, Baxter LT, Jain RK: Interstitial pressure gradients in tissue-isolated and subcutaneous tumors: implications for therapy. Cancer Res 1990;50:4478–4484. B, From Jain RK, Boucher Y, Stacey-Clear A, et al: Method for locating tumors prior to needle biopsy. United States patent US 19955396897. 1995.)

 

 

 

The enhanced permeability can also facilitate lymphatic metastasis ( Fig. 8-9C ). Thus, decreasing vascular permeability might restore the transmural pressure gradients and potentially resume/reestablish blood flow in the nonperfused regions of tumors. Some direct and indirect antiangiogenic therapies might “normalize” the tumor vasculature through this mechanism (see Fig. 8-4 ).[88] This normalization can also reduce the number of cells shed into peritumor lymphatics and alleviate peritumor edema (see Fig. 8-9C ).[194]

METABOLIC ENVIRONMENT

Hypoxia

A key function of the vasculature is to provide adequate levels of nutrients and oxygen to the parenchymal cells and to remove waste products. Based on the anatomy of the capillary bed and a mathematical model of oxygen diffusion and consumption, the Nobel laureate August Krogh introduced the concept of a diffusion limit for oxygen of 100–200 mm nearly a century ago.[195] This unit of tissue—a single capillary surrounded by a 100- to 200-mm-radius cylinder—is referred to as a “Krogh cylinder” in physiology. Nearly 50 years later, Thomlinson and Gray identified similar “cords” in human lung cancer and found necrotic cells beyond 180 mm away from blood vessels, presumably as a result of lack of oxygen.[196] This is referred to as “chronic hypoxia” or “diffusion-limited” hypoxia. Although various hypoxia markers and microelectrodes have suggested these gradients, the first direct measurements of these perivascular gradients, along with pO2 and blood flow rate of the same vessels, became possible only recently with the development of phosphorescence quenching microscopy ( Fig. 8-11A ). [197] [198] As discussed previously, blood flow in tumor vessels is intermittent, and, thus, some regions of a tumor are starved for oxygen periodically. The resulting hypoxia is referred to as “acute hypoxia” or “perfusion-limited hypoxia.” [199] [200] A necessary consequence of intermittent blood flow is the resumption of blood flow after shutdown, and the resulting production of free radicals can lead to “reperfusion injury” or “reoxygenation injury,” applying additional selection pressure on cancer cells.

 
 

Figure 8-11  A, pH and pO2 as a function of distance from a blood vessel in a tumor. The tumor environment becomes progressively more hypoxic and acidic farther away from a blood vessel. B, Lack of correlation between pH and pO2 in tumors.  (From Helmlinger G, Yuan F, Dellian M, et al: Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med 1997;3:177.)

 

 

 

Low pH

Another consequence of the abnormal microcirculation of the tumor is low extracellular pH. There are at least two sources of H+ ions in tumors—lactic acid and carbonic acid.[201] The former results from glycolysis and the latter from conversion of CO2 and H2O via carbonic anhydrase. The intracellular pH of cancer cells remains neutral or alkaline (pH 7.4), however, despite the acidic extracellular pH. Because carbonic anhydrase-9, various glucose transporters (GLUT1, GLUT3), and enzymes in the glycolytic pathway are upregulated by hypoxia, one would expect low extracellular pH and hypoxia to track each other and to colocalize with regions of low blood flow.[202] Surprisingly, there is a lack of spatial correlation among these parameters ( Fig. 8-11B ), a discovery made possible by recent developments in optical techniques that permit the simultaneous high-resolution mapping of multiple physiologic parameters.[198] A potential explanation for this lack of concordance is that some perfused tumor vessels carry hypoxic blood.[198] Thus, although they might not be able to deliver adequate oxygen to the surrounding cells, they may be able to carry away the waste products (e.g., lactic acid).

Molecular, Cellular, and Therapeutic Consequences

The presence of oxygen during irradiation makes the damage to DNA produced by radiation-induced free radicals permanent, whereas such damage can be repaired under hypoxic conditions.[203]Therefore, hypoxia in solid tumors significantly reduces their radiation sensitivity. Tumor hypoxia is also associated with resistance to some chemotherapeutics.[203] Similarly, low extracellular pH can affect the cellular uptake and cytotoxicity of some therapeutics adversely or favorably. [204] [205] [206] As a result, for nearly half a century, considerable preclinical and clinical efforts have been focused on alleviating hypoxia by improving tumor perfusion with various therapies, including the following:

  

   

Mild hyperthermia or drugs

  

   

Increasing oxygen content of the blood (via hyperbaric oxygenation, for example)

  

   

Increasing hemoglobin/hematocrit (via erythropoietin, for example)

  

   

Developing radiation sensitizers

Unfortunately, the clinical outcome has not met expectations for multiple reasons. These include the inability to increase pO2 in all regions of tumors to optimal levels and/or to deliver radiation sensitizers or chemotherapeutic drugs to all regions of a tumor at therapeutically effective levels. As a result, three broad strategies are emerging:

  

1.   

Use hypoxia and/or low pH to activate drugs or to attract anaerobic bacteria.

  

2.   

Dissect hypoxia-induced pathways to identify novel targets for drug development.

  

3.   

Normalize tumor vasculature.[207]

The first strategy has led to the development of drugs such as tirapazamine and to the rejuvenation of interest in bacteriolytic therapy; both approaches are in clinical trials. [78] [203] The second strategy has revealed several molecular determinants of the physiologic and pathophysiologic responses to hypoxia.[202] The balance between hypoxia-induced apoptosis/necrosis on one hand, and the increased resistance to cell death mediated by various hypoxia-induced pathways on the other, determines whether a tumor can survive and even grow under hypoxic conditions. Ultimately, hypoxia might select for tumor cells that are more malignant, more invasive and genetically unstable, and less susceptible to apoptosis, thus rendering them resistant to various therapies. Therefore, several molecules in the hypoxia-induced pathways are now being targeted in the development of diagnostic and therapeutic agents. Finally, normalization of tumor vessels by antiangiogenic agents may reduce hypoxia within tumor environment and synergize with radiation therapy.[207]

Hypoxia-induced pathways include genes involved in the following processes:

  

   

Oxygen delivery (e.g., heme oxygenase 1, erythropoietin)

  

   

Glycolysis and glucose uptake (e.g., GLUT1 and GLUT3, hexokinase-1 and -2)

  

   

pH control (e.g., carbonic anhydrase-9 and -12)

  

   

Stress-response pathways (e.g., growth arrest- and DNA damage-induced gene GADD153)

  

   

Growth factor signaling (e.g., IL-6, IL-8, insulin-like growth factor-2 [IGF-2])

  

   

PDGF-β

  

   

Angiogenesis (e.g., VEGF-A, VEGFR1, Ang-2, Tie-2, FGF-3, TGF-β, nitric oxide synthase [NOS], cyclooxygenase-2 [COX-2], hepatocyte growth factor [HGF])

  

   

Transcription (e.g., HIF1α and HIF2α, JUN, FOS, nuclear factor kB [NF-κB])

  

   

Apoptosis (e.g., BCL-interacting killer [BIK], annexin V, 19-kd interacting protein-3 [NIP3], NIP3-like protein X [NIX])

  

   

Growth inhibition signaling factors (e.g., p21, p27, GADD153)

  

   

Invasion and metastasis (e.g., metalloproteinases [MMPs], MMP-13, plasminogen activator inhibitor-1 [PAI-1])[202]

Of the various molecules involved in sensing and responding to hypoxia, HIF1α has received the most attention. This transcription factor is upregulated in several human tumors.[202] Regulated by a proline hydroxylase, HIF1α can activate the genes for angiogenesis, vasodilation, glycolysis, and erythrocyte production by binding to the hypoxia-response element. Surprisingly, teratomas arising from HIF1α-/-embryonic stem cells grow more rapidly despite lower levels of VEGF and angiogenesis. [45] [208] This counterintuitive finding could be a result of the ability of HIF1α-/- cells to survive under hypoxic conditions instead of undergoing apoptosis.[42] Interestingly, other HIF1α-/- cancer cells lead to slowly growing tumors. As a result, molecular therapies that target HIF1α or hypoxia-response element are under intensive investigation for cancer detection and treatment.[202]

ANTIANGIOGENIC AGENTS IN THE CLINIC

Two major problems currently plague the nonsurgical treatment of malignant solid tumors. First, physiologic barriers within tumors impede the delivery of therapeutics and oxygen (a key sensitizer to ionizing radiation) at effective concentrations to all cancer cells. [209] [210] Second, inherent or acquired resistance resulting from genetic and epigenetic mechanisms reduces the effectiveness of both conventional and novel therapies.[211] Can we take advantage of the unique pathophysiology of tumors to overcome these problems for better management of cancer?

Mechanisms of Action

As discussed next, recent clinical data offer great hope, with three VEGF-blocking antiangiogenic agents (bevacizumab, sorafenib, and sunitinib) already in clinical use.[212] Our studies in rectal cancer and glioblastoma patients have largely confirmed the hypotheses that anti-VEGF therapy has antivascular effects and can induce vascular normalization in cancer patients. [31] [188] [189] [212] In brief, bevacizumab alone reduced the tumor tissue vascular density by approximately half at day 12 after first infusion, reduced significantly the tumor blood flow evaluated on computed tomography scans and the number of viable circulating endothelial cells and progenitor cells. Clinically, both the low-dose-bevacizumab and the high-dose-bevacizumab infusion showed a less hyperemic/hemorrhagic appearance, but no significant tumor regression after bevacizumab treatment at flexible sigmoidoscopy. [188] [189] Although the significant pruning of tumor vasculature led to a significant increase in cancer cell apoptosis, it also led to a more mature (pericyte-covered) tumor vasculature, and a stable or increased cancer cell proliferation ( Fig. 8-12A–D ). [188] [189] Whether the increase in tumor cell apoptosis was due to a direct or indirect effect of bevacizumab is currently unclear. Similarly, the role of VEGF blockade on immune or stromal cells in patients is not yet understood. After bevacizumab therapy alone, the tumor IFP was consistently decreased, particularly in the patients with high baseline values. [188] [189] This suggested that in human tumors, similar to mouse models, the tumor microenvironment was normalized by the reduction of the excessive vascularization and potentially sensitized the tumor to the subsequent cytotoxic therapy within the “normalization window.” [185] [207] Imaging studies landed more supportive data for the normalization hypothesis: despite the significant reduction in vessel density and blood flow, the fluorodeoxyglucose uptake measured on positron emission tomography scans (a measure of tumor metabolic activity) and the P · S product (proportional to the penetration of tracer in tumor) evaluated on computed tomography scans did not significantly change at day 12. [188] [189] A recent study of antiangiogenic therapy in recurrent glioblastoma demonstrated the existence of a window of vascular normalization in cancer patients ( Fig. 8-12E and F ).[31]

 
 

Figure 8-12  Vascular “normalization” in cancer patients. In rectal cancer patients, tumor vessel “normalization” following a single injection of bevacizumab is suggested by the (A) reduced tumor microvessel density, (B) increased fraction of tumor vessels with pericyte coverage, and (C) reduced interstitial fluid pressure. (D) Positron emission tomography reveals no change in 18-fluorodeoxyglucose (FDG) uptake after a single dose of bevacizumab and complete resolution of FDG uptake following neoadjuvant chemoradiation (bevacizumab, 5-fluorouracil, pelvic external beam radiation therapy). The stability of FDG uptake following bevacizumab monotherapy, despite marked reductions in microvessel density, suggests the efficiency of persistent tumor blood vessels after bevacizumab treatment is improved. E and F, Changes in magnetic resonance imaging parameters in glioblastoma patients receiving AZD2171 over time. E, Median values for contrast-enhancement T1-weighted tumor volume (CE-T1), vessel size (VS), and permeability (P) of the tumor over time as measured by an independent expert. Day -1 was set as 100% in all lesions, and changes during AZD2171 treatment were plotted for all 16 patients. Note the rebound of volume and vessel size after day 28, which indicates a partial closure of the normalization window. F,Median values of T2-weighted abnormality volume measured in fluid-attenuated inversion recovery images (FLAIR), apparent diffusion coefficient (ADC), and extracellular-extravascular volume fraction (Ve) before and during treatment showing a sustained decrease of edema while taking AZD2171. (*P < 0.05 for values compared with day -1, whereas # represents P < 0.05 for values compared with day +1.)  (AD, From Willett CG, Boucher Y, di Tomaso E, et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004;145–147. E and F, From Batchelor TT, Sorensen AG, di Tomaso E, et al: AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer Cell 2007;11:83–95.)

 

 

 

Biomarkers

Biomarker identification and validation for this novel type of therapy are also facing important hurdles. Unlike preclinical models, the phase III bevacizumab experience in metastatic colorectal cancer patients did not identify p53, k-ras, or b-raf status, VEGF or thrombospondin-2 (TSP2) expression, or microvascular density at baseline as predictive markers of response. [213] [214]

Our results in rectal cancer patients demonstrated that bevacizumab decreased tumor microvascular density and the number of viable circulating ECs, consistent with an antivascular effect. [188] [189] [215]Whether these changes have predictive value is currently being investigated in an ongoing phase II trial. Plasma angiogenic proteins have also been investigated in multiple phase I–II and in some phase III trials. We reported that the plasma levels of VEGF and PlGF are significantly increased in cancer patients receiving bevacizumab.[189] Other groups have reported similar observations with a variety of anti-VEGF agents, and have also found a decrease in soluble VEGFR2 levels in plasma. [216] [217] These data strongly suggest a potential “pharmacodynamic biomarker” value for these three plasma markers. Of great interest for the field would be to identify biomarkers that predict disease progression through anti-VEGF therapy. Although in the rectal cancer patients we were unable to identify significant changes in bFGF, our data from a recently completed phase II trial of AZD2171 in recurrent glioblastoma patients showed a highly significant correlation between bFGF and SDF1α and tumor progression.[31] These differences may be due to the excellent clinical response in rectal cancer patients, or to disease or agent specificity. In addition, we discovered that viable circulating ECs correlate with progression of glioblastoma during antiangiogenic therapy, whereas circulating progenitor cells predicted relapse after drug interruptions.[31] With the development and improved flow cytometry and protein array analysis techniques and subsequent standardization, circulating cell and plasma protein measurements hold great promise for biomarker validation for antiangiogenic therapy.

Toxicity

Experimental studies have shown that, in 11 of the 17 healthy organs studied, VEGF blockade can significantly decrease the number of normal capillaries.[218] In cancer patients, most anti-VEGF agents often induce proteinuria, hypertension, thyroid-stimulating hormone elevation, and gastrointestinal toxicity, but agent-specific toxicities have also been reported.[13] In addition, the long-term effects of antiangiogenic therapies in patients with less advanced lesions remain to be established.

Perspective

The major directions for the immediate future are further understanding of the mechanisms of action and identification of the first biomarkers for anti-VEGF therapy.[212] Achieving these goals would allow optimization of treatment protocols and reduction of the adverse effects.

First, identifying the vascular “normalization window” in patients would allow synergistic combinations with chemotherapeutics or radiotherapy. Second, understanding the mechanisms of vessel pruning and cancer cell apoptosis induced by anti-VEGF therapy, and tumor escape from it, may allow further sensitization of tumor cells to cytotoxic therapies. Third, characterization of the effect of anti-VEGF therapy on bone marrow-derived cells’ contribution to tumor growth and relapse would allow judicious and more effective approaches to therapy involving these cells. Finally, clarification of other mechanisms involving the immune system or the stromal and interstitial matrix compartments would contribute to establishing more efficacious combinatorial strategies.

In this respect, new biomarkers and improved imaging techniques will play a major role in monitoring the effects and stratifying the patients with the ultimate goal of individualized therapy.

CONCLUSION

The recent successes of the anti-VEGF agents have raised great hope and have taught us important lessons about the significance of the target, timing, and dosage of each agent[212]:

  

   

According to the results of the phase III trials completed to date, bevacizumab can increase median survival when combined with standard chemotherapy, but not when used as monotherapy.

  

   

Anti-VEGF therapy with bevacizumab can increase overall survival and/or progression-free survival in colorectal, breast, and lung cancer patients when combined with cytotoxic agents.

  

   

Improved survival has been observed with broad-spectrum multitargeted tyrosine kinase inhibitors (e.g. sorafenib, sunitinib) when used in monotherapy.

  

   

In colorectal cancer patients, vatalanib, a VEGF receptor–selective tyrosine kinase inhibitor, does not confer the same survival advantage as bevacizumab when combined with chemotherapy.

  

   

Anti-VEGF therapy can prune and “normalize” tumor vasculature, and decrease the number of circulating endothelial cells and progenitor cells.

  

   

There is an urgent need to identify biomarkers to guide anti-VEGF therapy and combination therapies using anti-VEGF agents.

Antiangiogenic agents are now expected to make a difference in cancer patients with a wide variety of tumor types. With the advent of specific and potent new agents—approved or in the process of being approved—oncologists have a variety of direct and indirect antiangiogenic agents to choose from when designing therapy protocols. Determining whether the regimens used in the successful trials are optimal, however, and whether antiangiogenic agents will work in patients outside the rigorous inclusion criteria used for those trials, will be critical for deciding the standard of care for different malignancies. Establishing the most advantageous combinations will require a better understanding of the mechanisms of action of each anti-VEGF agent and the sensitivity of each tumor type, as well as development of robust biomarkers and imaging techniques to guide patient selection and protocol design. A deeper understanding of the mechanisms of antitumor activity of specific and multitargeted antiangiogenic agents in patients, how they can best be combined with other treatment approaches such as chemotherapy and radiation therapy, and how optimization of these effects can be monitored clinically, should contribute to significantly improved cancer treatment and extend survival of cancer patients in the near future, as well as enhance the prospects of developing curative treatment for different cancers in the more-distant future.

ACKNOWLEDGMENTS

We would like to thank Kevin Kozak for his input on updating this chapter. This chapter is an updated and expanded version of a chapter by R.K. Jain entitled “Molecular Pathophysiology of Tumors (In Perez CA, Brady LW, Halperin EC, Schmidt-Ullrich R [eds]: Principles and Practice of Radiation Therapy. New York, Lippincott, Williams & Wilkins, 2003, pp 163–179). The work summarized here has been supported continuously by the National Cancer Institute since 1980 with funding to R.K. Jain.

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