Molecular Pathophysiology of Tumors - Perez & Brady's Principles and Practice of Radiation Oncology (Perez and Bradys Principles and Practice of Radiation Oncology), 6 Ed.

Perez & Brady's Principles and Practice of Radiation Oncology (Perez and Bradys Principles and Practice of Radiation Oncology), 6 Ed.

Chapter 4. Molecular Pathophysiology of Tumors

Rakesh K. Jain and Dan G. Duda

A solid tumor is an organlike structure containing neoplastic and stromal cells nourished by the tumor vasculature composed of endothelial cells, basement membrane, and perivascular cells. All of these components are embedded in an extracellular matrix (Fig. 4.1). The interactions between these cells, their surrounding matrix, and their local microenvironment influence the expression of various genes. The products encoded by these genes, in turn, control the pathophysiologic characteristics of the tumor. The tumor pathophysiology affects tumor growth, invasion, and metastasis, as well as the response to radiation and other therapies. In this chapter, we will discuss various pathophysiologic parameters that characterize the vascular and extravascular compartments of a tumor as well as the molecular players involved in the formation and function of these compartments. Finally, we will point out some clinical implications of the findings and present a future perspective.

FIGURE 4.1. Schematic representation of a solid tumor—an organlike structure. The key components include cancer cells, host cells, and blood vasculature made of endothelial and perivascular cells—all embedded in a matrix bathed in interstitial fluid. The lymphatic vasculature, present in most normal tissues, is often lacking or is dysfunctional in solid tumors.

VASCULAR COMPARTMENT

Neoplastic cells, similar to normal cells, need oxygen and other nutrients for their survival and growth. Every reproductively intact normal cell in our body is located within 100 to 200 μm from a blood capillary so that it can receive adequate levels of oxygen and other nutrients by the process of diffusion. Likewise, cells undergoing neoplastic transformation depend on nearby capillaries for growth. These preneoplastic (i.e., dysplastic or hyperplastic) cells can grow as spherical or ellipsoidal cellular aggregates. However, once the size of the cellular aggregate reaches the diffusion limit for critical nutrients, the aggregate may become dormant. Indeed, human tumors may remain dormant for a number of years despite active cell proliferation because of a balance between proliferation and cell death. However, once they have access to new blood vessels, the tumor may grow and metastasize. What triggers the growth of new vessels? What molecular and cellular players are involved? How do these vessels compare with normal vessels with respect to their structure and function?

Angiogenesis

The fact that the vascular system is associated with tumor growth in animals and humans has been known for more than a century.¹ Ide et al.² and Algire and Chalkley³ provided powerful insight into the neovascularization of transplanted tumors using transparent window techniques (for reviews on this subject, see ⁴^,⁵). In 1968 Rijhsinghani et al.⁶ and Ehrmann and Knoth⁷ suggested the possibility that tumors produce an “angiogenic” substance. In 1971, Folkman⁸^,⁹ proposed the hypothesis that blocking angiogenesis should block tumor growth and metastasis. In 1978, Gullino¹⁰ demonstrated that a tissue acquires angiogenic capacity during neoplastic transformation and proposed that antiangiogenesis approaches be used to prevent cancer. Both of these hypotheses have been validated in a number of preclinical studies.¹¹^,¹² A wide range of antiangiogenic strategies are currently being evaluated in the clinic to prevent or treat a large number of diseases, including cancer.¹¹ Most importantly, antiangiogenic strategies have yielded overall survival benefits in patients with advanced colorectal cancer, non–small-cell lung cancer, renal cell cancer, hepatocellular carcinoma, and gastrointestinal stromal tumors and have shown increased response rates and progression-free survival benefits in advanced breast, medullary thyroid, and ovarian cancer; pancreatic neuroendocrine tumors (pNETs); and glioblastoma.¹³^–²¹^,²² This has led so far to U.S. Food and Drug Administration (FDA) approval for eight antiangiogenic drugs in the treatment of these diseases (Table 4.1).

The net balance between pro- and antiangiogenic factors governs both normal and pathologic angiogenic processes.¹¹ 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 corpus luteum) and “off” otherwise. During neoplastic transformation and tumor progression, this regulation is deranged, which results in ectopically formed blood vessels to support the growing mass.¹¹

Cellular Mechanisms

Several cellular mechanisms have been described in the vascularization of tumors: (a) co-option, (b) intussusception, (c) sprouting (angiogenesis), (d) vasculogenesis from endothelial precursors, (e) cancer cell lining of vessels (vascular mimicry), or (f) transdifferentiation to the endothelial cell (Fig. 4.2).¹¹ Tumor cells can co-opt and grow around the existing vessels to form “perivascular” cuffs. However, as stated earlier, these cuffs cannot grow beyond the diffusion limit of critical nutrients, and they actually may cause the collapse of the vessels as a result of the growth pressure (referred to as “solid stress”).²³^,²⁴^,²⁵ 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.²⁶^,²⁷

“Sprouting” angiogenesis is the most widely studied mechanism of vessel formation. During sprouting angiogenesis, the existing vessels become leaky in response to growth factors released by cancer or stromal cells; the basement membrane and the interstitial matrix dissolve; the pericytes dissociate from the vessel; endothelial cells (ECs) migrate and proliferate to form an array/sprout; a lumen is formed in the sprout (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 may remain immature.⁵^,¹¹^,²⁸

During mammalian embryonic development, a primitive vascular plexus is formed from angioblasts or endothelial precursor cells (EPCs) by a process referred to as vasculogenesis. Distinct signals specify arterial or venous differentiation.²⁹ Several studies showed that circulating EPCs mobilized from the bone marrow or peripheral blood also can contribute to postnatal vasculogenesis in tumors and other tissues.³⁰ Although debated, the repair of healthy adult vessels or the expansion of pathologic vessels can be aided by the recruitment of bone marrow–derived cells (BMDCs) and/or EPCs to the vascular wall.³¹ The progenitor cells then become incorporated into the endothelial lining in a process known as postnatal vasculogenesis. Collateral vessels, which bring bulk flow to ischemic tissues during revascularization, enlarge in size by distinct mechanisms, such as the attraction and activation of myeloid cells.³¹^,³² However, this process appears to be of rather limited importance in tumor neovascularization.³³^–³⁷

Tissues can also become vascularized by other mechanisms, but the relevance of these processes is not well understood. For example, tumor cells can line vessels—a phenomenon known as vascular mimicry. Putative cancer stemlike cells can even generate tumor endothelium.³⁸^,³⁹^–⁴¹

The challenge now is to discern the relative contribution of each of these mechanisms of new vessel formation in tumors to optimize antiangiogenic treatment of cancer.¹⁶^,⁴¹^,⁴²

TABLE 4.1 FDA-APPROVED ANTI-ANGIOGENIC DRUGS

FIGURE 4.2. Modes of vessel formation in solid tumors. There are several known methods of blood vessel formation in normal tissues and tumors. A–C: Vessel formation can occur by sprouting angiogenesis (A), by the recruitment of bone marrow–derived and/or vascular wall–resident endothelial progenitor cells (EPCs) that differentiate into endothelial cells (ECs; B), or by a process of vessel splitting known as intussusception (C). D–F: Tumor cells can co-opt pre-existing vessels (D), or tumor vessels can be lined by cancer cells (vascular mimicry; E) or by endothelial cells, with cytogenetic abnormalities in their chromosomes, derived from putative cancer stem cells (F). Unlike normal tissues, which use sprouting angiogenesis, vasculogenesis, and intussusception (A–C), tumors can use all six modes of vessel formation (A–F). (Reproduced from Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011;473:298–307, with permission.)

FIGURE 4.3. Molecular basis of vessel branching. The consecutive steps of blood vessel branching are shown, with the key molecular players involved denoted in parentheses. A: After stimulation with angiogenic factors, the quiescent vessel dilates and an endothelial-cell tip cell is selected (DLL4 and JAGGED1) to ensure branch formation. Tip-cell formation requires degradation of the basement membrane, pericyte detachment, and loosening of endothelial cell junctions. Increased permeability permits extravasation of plasma proteins (such as fibrinogen and fibronectin) to deposit a provisional matrix layer, and proteases remodel pre-existing interstitial matrix, all enabling cell migration. For simplicity, only the basement membrane between endothelial cells and pericytes is depicted, but in reality, both pericytes and endothelial cells are embedded in this basement membrane. B: Tip cells navigate in response to guidance signals (such as semaphorins and ephrins) and adhere to the extracellular matrix (mediated by integrins) to migrate. Stalk cells behind the tip cell proliferate, elongate, and form a lumen, and sprouts fuse to establish a perfused neovessel. Proliferating stalk cells attract pericytes and deposit basement membranes to become stabilized. Recruited myeloid cells such as tumor-associated macrophages (TAMs) and TIE-2–expressing monocytes (TEMs) can produce proangiogenic factors or proteolytically liberate angiogenic growth factors from the extracellular matrix. C: After fusion of neighboring branches, lumen formation allows perfusion of the neovessel, which resumes quiescence by promoting a phalanx phenotype, re-establishment of junctions, deposition of basement membrane, maturation of pericytes, and production of vascular maintenance signals. Other factors promote transendothelial lipid transport. (Reproduced from Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011;473:298–307, with permission.)

Molecular Mechanisms

Various pro- and antiangiogenic molecules orchestrate different steps in vessel formation. Vascular endothelial growth factor (VEGF) is, perhaps, the most critical angiogenic molecule. Originally discovered in 1983 as the vascular permeability factor by Dvorak et al. and cloned in 1989 by Ferrara et al., VEGF increases vascular permeability, promotes migration and proliferation of ECs, serves as an EC survival factor, and is known to up-regulate leukocyte adhesion molecules on ECs.⁴³^–⁴⁴^,⁴⁵ During tumor progression, the variety and concentration of angiogenic molecules produced by a tumor can increase. Thus, if VEGF were blocked, tumor growth might continue as a result of the action of other angiogenic molecules (e.g., basic fibroblast growth factor [bFGF], interleukin-8 [IL-8], stromal cell–derived factor 1α[SDF1α]).⁴²^,⁴⁶^,⁴⁷ Other positive regulators include angiopoietins that are involved in blood vessel maturation;⁴⁸ various proteases involved in extracellular matrix remodeling and growth factor release;¹¹^,²⁸ and organ-specific angiogenic stimulators such as endocrine gland VEGF⁴⁵ (Fig. 4.3).

Angiogenesis inhibitors include soluble receptors of various proangiogenic ligands as well as molecules that down-regulate stimulator expression (e.g., interferons), interfere with stimulator release, or block binding of stimulators to their receptors (e.g., platelet factor 4). Thrombospondins are among the first and best characterized endogenous inhibitors that interfere with the growth, adhesion, migration, and survival of ECs. Other endogenous inhibitors include fragments of various plasma or matrix proteins, for example, angiostatin—fragment of plasminogen;⁴⁹ endostatin—fragment of collagen XVIII;⁵⁰ and tumstatin—fragment of collagen IV.⁵¹

The generation of pro- and antiangiogenic molecules can be triggered by injury, metabolic stress (e.g., low partial pressure of oxygen [Po₂], low pH, or hypoglycemia), mechanical stress (e.g., shear stress, solid stress), immune/inflammatory responses (e.g., 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).⁵²^–⁵⁵^,⁵⁶^–⁵⁷ These molecules can emanate from cancer cells, endothelial cells, stromal cells, blood, and extracellular matrix⁵⁸^,⁵⁹^,⁶⁰ (Fig. 4.4). Because the host cells differ among organs, angiogenesis depends on host–tumor interactions.⁵⁹^,⁶¹^–⁶⁴^,⁶⁵ Furthermore, because the tumor microenvironment is likely to change during tumor growth, regression, and relapse after treatment, profiles of pro- and antiangiogenic molecules are likely to change with time and space.⁶⁶^–⁷⁵ The challenge now is to develop a unified theoretic framework to describe the temporal and spatial profiles of this increasing number of angiogenesis regulators to develop effective therapeutic strategies.⁴²^,⁷⁶

FIGURE 4.4. Tumor induction of host promoter activity in stromal cells. The expression of vascular endothelial growth factor (VEGF) in host cells can be examined using transgenic mice expressing 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. (From Fukumura D, Xavier R, Sugiura T, et al. Tumor induction of VEGF promoter activity in stromal cells. Cell 1998;94:715–725, with permission). C: A GFP-expressing layer of host cells can be seen at the tumor–host interface. D, E: The VEGF-expressing host cells colocalize with the angiogenic tumor vessels. (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;7:866–870, with permission.)

Vascular Architecture

In normal tissue, blood flows in a closed circuit from an artery to an arteriole 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 be completely different depending on the tumor type, its location, and whether it is growing, regressing, or relapsing.⁷⁷^,⁷⁸^–⁷⁹ In general, tumor vessels are dilated, saccular, tortuous, and chaotic in their patterns of interconnection. For example, whereas the normal vasculature is characterized by dichotomous branching, the tumor vasculature has many trifurcations and branches with uneven diameters.⁸⁰^,⁸¹ The fractal dimensions and minimum path lengths of tumor vasculature are different from those of the normal host vasculature.⁷⁸^,⁷⁹^,⁸²

The molecular mechanisms of this abnormal vascular architecture are not entirely understood, but it seems reasonable to hypothesize that an imbalance of pro- and antiangiogenic molecules is a key contributor.¹¹^,⁸³ By extension, modulation of this angiogenic imbalance may allow for the correction of the tumor vascular abnormalities, leaving behind a more structurally and functionally normal vascular bed. Several observations support this “normalization” hypothesis.⁸³ Normalization of tumor xenograft vasculature is observed during therapies that lower VEGF (e.g., hormone withdrawal from a hormone-dependent tumor⁸⁴), that interfere with VEGF signaling (e.g., treatment with anti-VEGF or anti-VEGF receptor-2 antibody or tyrosine kinase inhibitor⁸⁵^,⁸⁶^,⁸⁷^–⁹⁰) (Fig. 4.5), or that mimic an antiangiogenic cocktail (e.g., trastuzumab treatment of a HER2 overexpressing tumor⁷⁰). Emerging clinical data from cancer patients treated with bevacizumab, an anti-VEGF antibody, or pan-vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors AZD2171 (cediranib) or sunitinib lend even more compelling support to this hypothesis.¹⁶^,⁴¹^,⁶⁶^,⁶⁷^,⁶⁹^,⁷¹^,⁷²^,⁷³^,⁷⁵^,⁸³^,⁹¹^,⁹² More crucially, the extent of vascular normalization directly correlates with the survival of patients with recurrent glioblastoma.⁹² Moreover, the patients whose tumor blood flow increased most after cediranib treatment had the longest overall survival.⁹³^,⁹⁴

Mechanical stress generated by proliferating tumor cells also may lead to partially compressed or totally collapsed vessels often found in tumors.²³^,⁹⁵ Decompression of blood vessels by depleting cells or matrix supports this mechanical hypothesis.²⁴^,²⁵^,⁹⁶ Perhaps the combination of both molecular and mechanical factors renders the tumor vasculature abnormal; thus, both must be taken into account when designing novel strategies for cancer treatment.

FIGURE 4.5. Normalization of tumor vasculature. A: Normal vessels are well organized with even diameters. B: In contrast, tumor vessels are tortuous with increased vessel diameter, length, density, and permeability. C: Antiangiogenic therapies “normalize” the tumor vascular network and ultimately may reduce the vasculature to the point that it provides inadequate support for tumor growth. (From Jain RK. Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med 2001;7:987–989; and Tong RT, et al. Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res 2004;64:3731–3736, with permission.)

Blood Flow and Microcirculation

Blood flow in a vascular network—whether normal or abnormal—is governed by arteriovenous pressure difference and flow resistance. The flow resistance is a function of the vascular architecture and the blood viscosity.⁹⁷Abnormalities in both the vasculature and blood viscosity increase the resistance to blood flow in tumors.⁸⁰^,⁸¹^,⁹⁸^–¹⁰⁰ As a result, overall perfusion rates (blood flow rate per unit volume) in tumors are lower than in many normal tissues.¹⁰¹^–¹⁰³

Macroscopically and microscopically, tumor blood flow is temporally and spatially heterogeneous. Macroscopically, four spatial regions can be recognized in a tumor: an avascular necrotic region, a seminecrotic region, a stabilized microcirculation region, and an advancing front¹²^,¹⁰⁴ (Fig. 4.6A). Microscopically, in normal tissues red blood cell (RBC) velocity is dependent on vessel diameter, but there is no such dependence in most tumors.⁶¹^,⁶⁵^,¹⁰⁵Furthermore, the average RBC velocity may be an order of magnitude lower in some tumors compared to the host tissue.⁶⁵ In a given tumor vessel, blood flow fluctuates with time and can even reverse its direction.¹⁰⁴^,¹⁰⁵^–¹⁰⁶

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,¹⁰⁵ solid stress generated by proliferating cancer cells,²³^,²⁴^,²⁵^,⁹⁵^,⁹⁶ vascular remodeling by intussusception, and coupling between luminal and interstitial fluid pressure via hyperpermeability of tumor vessels.⁷⁸^,¹⁰⁸^–¹¹⁰ As discussed later, this heterogeneity contributes to both acute and chronic hypoxia in tumors—a potential cause of resistance to radiation and other therapies and increased metastatic potential.

Considerable effort has gone into modulating tumor blood flow to improve cancer treatment. This has been difficult to achieve reproducibly because the 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 either lack perivascular cells or are abnormally invested.¹¹¹^–¹¹³ As a result, efforts to increase tumor blood flow and the delivery of cytotoxins, by pharmacologic or physical agents, have not always been successful.⁹⁷^,¹¹⁴ In contrast, efforts to “starve” tumors by decreasing or shutting down tumor blood flow by “stealing” blood away from the passive component of the tumor vasculature by vasodilators¹¹⁴ as well as by vascular targeting or intravascular coagulation have shown promise in experimental systems.¹¹⁵ It also appears that judiciously applied antiangiogenic therapy may “normalize” the abnormal tumor vasculature and the resulting “normalized” vessels might be more responsive to vasoactive agents¹²^,¹⁶^,⁴¹^,⁸³^,¹¹⁶ (Fig. 4.5).

FIGURE 4.6. The tumor microenvironment is heterogeneous with proliferative, quiescent, and necrotic regions. A: These regions can be characterized in terms of various physiologic parameters. Decreasing magnitude of these parameters is indicated as + + +, + +, +, +/–, and – in the adjoining table. (From Jain RK, Forbes NS. Commentary–can engineered bacteria help control cancer? Proc Natl Acad Sci U S A 2001;98:14748–14750, with permission.) B: 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. (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;3:177–182, with permission.)

Vascular Permeability

Once a bloodborne molecule has reached an exchange vessel, its extravasation occurs by diffusion, convection, and, to some extent, presumably transcytosis.¹¹⁷ The diffusive permeability of a molecule depends on the size, shape, charge, and flexibility of the molecule as well as the size, shape, charge, and dynamics of the transvascular transport pathway. In normal vessels, these pathways include diffusion through 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 organelles [VVOs]).¹¹⁸ Some of these pathways may be lined with glycocalyx on ECs, thus effectively reducing the size of the pathway. A basement membrane may retard further the movement of molecules. Ultrastructural studies show widened interendothelial junctions; an increased number of fenestrations, vesicles, and VVOs in tumor vessels; and a lack of normal basement membrane and pericytes.⁴³^,⁶³^,¹¹⁸^,¹¹⁹^–¹²⁰

In concert with these ultrastructural findings, both vascular permeability to solutes and water permeability (referred to as hydraulic conductivity) of tumors, in general, are significantly higher than that of various normal tissues.⁶⁵^,¹⁰⁰^,¹²¹^–¹²⁴ Furthermore, unlike normal vessels, tumor vessels lack selectivity for the size of extravasating molecules.¹²⁵ However, positively charged molecules have a higher affinity for the negatively charged angiogenic tumor vessels.¹²⁶ Despite increased overall permeability, not all blood vessels of a tumor are leaky. Even the leaky vessels have a finite pore size that is tumor dependent, and ultrastructural studies show that the larger pore size in tumors represents wide interendothelial junctions.⁶³^,¹²⁰ Not only do the vascular permeability and pore size vary from one tumor to the next, but also within the same tumor they vary both spatially and temporally as well as during tumor growth, regression, and relapse.⁶³^,⁷⁰^,⁸⁴

The local microenvironment plays an important role in controlling vascular permeability. For example, a human glioma (HGL21) is fairly leaky when grown subcutaneously in immunodeficient mice, but it exhibits blood–brain barrier properties in the cranial window.⁶⁵ Such site-dependent differences for other tumors have been observed in other orthotopic sites.⁶¹^,⁶⁴^,¹²⁷ 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).¹²^,²⁸^,⁴⁸^,⁹⁰^,¹²⁸ 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.¹²⁹

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 and periendothelial pathways have been proposed as a route for intravasation and extravasation of cells. Very little is known about intravasation, except that a tumor may shed more than a million cells per gram per day and most of these are not clonogenic, and that some may be shed as fragments along with stromal cells.¹⁰³^,¹³⁰^–¹³² More is known about the molecular and cellular mechanisms of extravasation.¹³³^–¹³⁵ A cell within a blood vessel may continue to move with the flowing blood, collide with the vessel wall, adhere transiently or stably, and finally extravasate. These interactions are governed by both 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 binding between adhesion molecules and by the surface area of contact.¹³³^,¹³⁶^,¹³⁷^–¹⁴⁰ Deformability of cells affects both types of forces.¹⁴¹

Rolling of endogenous leukocytes is generally low in tumor vessels, whereas stable adhesion (≥30 seconds) is comparable between normal and tumor vessels.¹⁴² However, 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. Whether the latter is due to a low flux of leukocytes into angiogenic vessels and/or down-regulation of adhesion molecules in these immature vessels is currently not known. Age may also play an important role in leukocyte–endothelial interactions.¹⁴³

Further insight into the biology 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.¹⁴⁴^,¹⁴⁵ Immediately following systemic injection, these cells localized primarily in the lungs, whereas a nondetectable number of cells arrived in the tumor.¹⁴⁴ Increased rigidity caused by IL-2 activation may contribute to the mechanical entrapment of these cells in the lung microcirculation.¹⁴⁶^,¹⁴⁷ Constitutive expression of certain adhesion molecules in the lung vasculature also may facilitate their localization in the lungs.¹³³ One approach to reducing lung entrapment is to reduce the rigidity of these cells.¹⁴¹^,¹⁴⁵ Alternatively, entrapment in lung vasculature can be circumvented by injecting A-NK cells directly into the blood supply of tumors. In this case, A-NK cells, both xenogeneic and syngeneic, adhered to some blood vessels in three different tumor models¹⁴⁵^,¹⁴⁸^,¹⁴⁹ via CD18 and very late antigen-4 (VLA-4) on the A-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.⁴⁴^,¹⁵⁰^,¹⁵¹

These molecules can be up-regulated by a number of cytokines, including tumor necrosis factor-α (TNF-α) and a protein of 90 kD molecular weight (p90); secreted by some neoplastic cells;⁴⁴^,¹³⁷ and down-regulated by others, for example, transforming growth factor-β (TGF-β) also, presumably, secreted by cancer cells.⁶²^,¹⁵² Surprisingly, the proangiogenic VEGF also can up-regulate these molecules, whereas another proangiogenic molecule, bFGF, can down-regulate these molecules.⁴⁴^,⁵⁹^,⁸⁶^,¹⁵³ The challenge now 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. Judicious doses of anti-VEGF agents that “normalize” tumor vasculature can potentially realize this goal.¹⁵⁴^,¹⁵⁵

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 subcompartment bathed by the interstitial fluid (Fig. 4.1). Depending on the tumor type and its stage of differentiation, neoplastic cells may be dispersed in the matrix as individual cells (e.g., lymphomas, melanomas) or as clumps or nests (e.g., carcinomas). More than 80% of tumors are carcinomas arising from epithelial cells. The remaining 20% 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 endothelial cells. In a poorly differentiated carcinoma, the cancer cells may be loosely packed in clumps, whereas in a well-differentiated carcinoma, the cells may be connected with intercellular junctions and tightly packed in a nest enveloped by a basement membrane. With tumor progression, cancer cells may invade the basement membrane and spread to other regions.⁴³

Unlike cancer cells, host cells must migrate into the tumor from normal tissue. Inflammatory cells may enter the tumor via blood vessels or may infiltrate from the adjacent tissue.¹³¹ Other host cells, such as fibroblasts, may proliferate and migrate from the adjacent connective tissue²⁸^,⁵⁸^,⁶⁰^,¹⁵⁶^,¹⁵⁷ or from primary tumor to the metastatic site.¹³¹ Increasingly, infiltrating host cells are being recognized as critical modulators of the tumorigenic process. For example, mesenchymal cells generically termed carcinoma-associated fibroblasts have been shown to promote tumor growth, metastasis, and angiogenesis, potentially through the secretion of stromal cell–derived factor-1α.¹³¹^,¹⁵⁸^–¹⁶¹Furthermore, tumor-associated immune/inflammatory cells such as tumor-associated macrophages (TAMs), Tie2-expressing monocytes (TEMs), or Gr-1+ myeloid-derived suppressor cells (MDSCs) have been linked to both cancer immunosurveillance and suppression as well as to tumor promotion.⁵⁴^,¹⁵⁹^,¹⁶²^,¹⁶³^,¹⁶⁴ The challenge now is to establish approaches for skewing the TAMs toward a phenotype that promotes antitumor immune responses in cancer patients.⁵⁴

The interstitial compartment 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 is defective in tumors (see the Vascular Permeability section).²⁸^,⁸³ In addition, functional lymphatics may be confined to the tumor margin (see the Lymphatic Transport section).¹⁶⁵^,¹⁶⁶ Similar to normal tissues, the interstitial space of tumors 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 [PGs]), which form a hydrophilic gel.

Compared with our understanding of blood vessel formation, our understanding of stroma generation is minimal. Dvorak has proposed that the extravasated plasma protein fibrinogen, a key component of the tumor interstitial fluid (TIF), clots to form fibrin, which serves as a major component of the provisional stroma.⁴³ This provisional stroma eventually is replaced by more mature connective tissue stroma. The TIF also contains several proteins including fibronectin, vitronectin, osteopontin, thrombospondin, decorin, and tenascin. These proteins are present in both free and bound forms and contain the amino acid sequence arg-gly-asp (RGD). The RGD 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 PGs, also are synthesized by the stromal cells, albeit in a form that is different from that in the plasma or normal tissues.⁴³ TIF also may contain various growth factors that facilitate stroma formation. For example, in vitro studies suggest that platelet-derived growth factor-βis involved in the recruitment of fibroblasts to tumors, and TGF-β controls the production of collagen and other matrix molecules in tumors.²⁸^,¹⁶⁷ 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 is likely to increase.¹⁵⁶

FIGURE 4.7. Schematic of lymphatics in low (left) versus high (right) vascular endothelial growth factor-C (VEGF-C)—expressing tumors. VEGF-C secreted from tumor cells stimulates vascular endothelial growth factor receptor-3 (VEGFR-3) expressed in lymphatic endothelial cells, inducing peritumor lymphatic hyperplasia (top right). An increase in lymphatic surface area may increase the opportunity for tumor cell entry into lymphatic vessels. Augmented lymph flow enhances tumor cell delivery to draining lymph nodes (bottom right). In the absence of VEGF-C overexpression, peritumor lymphatic hyperplasia is less pronounced and fewer tumor cells are delivered to draining lymph nodes (left). Anti-VEGF-C/anti-VEGFR-3 treatment inhibits VEGF-C–induced lymphatic hyperplasia and tumor cell delivery to draining lymph nodes (bottom left). (From Hoshida T, Isaka N, Hagendoorn J, et al. Imaging steps of lymphatic metastasis reveals that vascular endothelial growth factor-C increases metastasis by increasing delivery of cancer cells to lymph nodes: therapeutic implications. Cancer Res2006;66:8065–8075, with permission.)

Interstitial Transport

Once a molecule has extravasated, its movement through the interstitial space occurs by diffusion and convection.¹¹⁸ 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/mm Hg · s), relates the interstitial velocity to the pressure gradient.¹¹⁸ Values of these transport coefficients are governed by the structure and composition of the interstitial compartment as well as by the physicochemical properties of the solute molecule.¹²⁶^,¹⁶⁸^,¹⁶⁹^–¹⁷²

The value of K (interstitial hydraulic conductivity) for a human colon carcinoma xenograft (LS174 T), measured using two different methods,¹⁷³^,¹⁷⁴ was found to be higher than that of a hepatoma,¹⁷² which, in turn, was higher than that of the normal liver. Using fluorescence recovery after photobleaching, the diffusion coefficient (D) of various molecules in tumors was found to be about one-third that in water¹⁷⁵ and higher than the values in the host tissue.¹⁶⁹ Collagen content and structure have a significant effect on D in tumors.¹⁷¹^,¹⁷³^,¹⁷⁶ This is surprising because hyaluronan and proteoglycans, and not collagen, account for most of the resistance to transport in normal tissues. Because collagen is produced by the host-derived cells (e.g., fibroblasts), the penetrability of macromolecules into a tumor will depend on the host–tumor interaction. Thus, agents that interfere with collagen synthesis and/or organization (e.g., relaxin, bacterial collagenase, losartan) may increase interstitial transport in tumors.¹⁶⁸^,¹⁷⁷^,¹⁷⁸

The time constant for a molecule with diffusion coefficient D to diffuse across a distance L is approximately L²/4D. For diffusion of IgG (immunoglobulin G) 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 tumor, diffusional transport across the tumor would take days, and for a 1-cm tumor, it would take months. If the central vessels have collapsed completely as a result of cellular proliferation²³^,²⁴^,⁹⁶ and interstitial matrix rearrangement, the reduced delivery of macromolecules by blood flow would make diffusion the primary mechanism of delivery to this hypoxic center. Binding may further retard the transport in tumors.¹⁷⁵^,¹⁷⁹^,¹⁸⁰ The role of binding is illustrated clearly by comparing the rate of fluorescence recovery of a photobleached spot in tumor tissue injected with a nonspecific versus 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.¹⁷⁵ 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 using viruses, nano-therapeutics) in tumors, and strategies are needed to overcome this barrier.¹¹⁷^,¹²⁶^,¹⁷⁷^,¹⁷⁸^,¹⁸¹

Lymphatic Transport

In most normal tissues, extravasated plasma and macromolecules are taken up by the lymphatics and returned to the central circulation. Although it is widely accepted that lymphatic vessels are present in the tumor margin and the peritumoral tissue, 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.¹⁶⁶^,¹⁸² Currently available immunohistochemical markers stain for structures in some tumors that resemble lymphatic vessels. However, because many of these markers lack specificity,¹⁶⁵^,¹⁶⁶^,¹⁸³ it is not clear whether they stain functional lymphatic vessels, endothelial cells from remnant lymphatic vessels, or some other structures or cell types (e.g., preferential fluid channels¹⁷³). It is likely that the stress induced by proliferating cancer cells compresses and impairs lymphatic vessels that are co-opted or formed in a tumor²³^,²⁴ and/or lymphatic valves are impaired by tumor growth.¹⁸⁴ The impaired lymphatic vessels, in turn, may contribute to the interstitial hypertension characteristic of animal and human tumors (see the Interstitial Hypertension section). In addition, invasion of the functional peritumoral lymphatics is considered to be a poor prognostic factor for a number of tumors, and lymphatic metastasis is a major cause of morbidity and mortality.

Our understanding of the mechanisms of lymphangiogenesis lags behind our understanding of the mechanisms of angiogenesis. However, considerable progress has recently been made toward identifying molecular players responsible for lymphangiogenesis. VEGF-C, acting through VEGFR-3, appears to play a central role in tumor-associated lymphangiogenesis. Several experimental¹⁸⁵^–¹⁸⁶^,¹⁸⁷^–¹⁸⁹ and clinical¹⁹⁰ studies have demonstrated a positive correlation between VEGF-C expression and peritumoral lymphatic vessel density, lymphatic metastasis, and, in some cases, poor clinical outcomes. Like in vascular angiogenesis, other positive and negative regulators, such as VEGF,¹⁹¹^,¹⁹² VEGF-D,¹⁸⁶ hepatocyte growth factor, platelet-derived growth factor-BB, and angiopoietins are involved in lymphangiogenesis.¹¹^,¹⁹⁰ Furthermore, mechanisms analogous to co-option, intussusception, sprouting, and vasculogenesis may operate in lymphatic growth¹¹ (see the Angiogenesis: Cellular Mechanisms section). Similar to organ-specific angiogenic molecules (e.g., EG-VEGF)⁴⁵ and blood vascular endothelial precursor cells,³⁰^,¹⁹³ there may be organ-specific lymphangiogenic molecules and lymphatic endothelial precursor cells that contribute to tumor-associated lymphangiogenesis.¹⁹⁴ Moreover, the proteolytic processing of lymphangiogenesis molecules, as well as the phenotype and function of the resulting lymphatics, may depend on the tumor type as well as on the host organ in which the tumor is growing.²⁸^,⁸⁶^,¹⁶⁵^,¹⁹⁰

The precise roles for these lymphangiogenic molecules in the induction of lymphatic metastasis are imperfectly understood. Recent data demonstrate that tumor VEGF-C overexpression induces peritumoral lymphatic hyperplasia through activation of VEGFR-3. Consequently, lymph fluid volumetric flow increases.¹⁸⁵ This results in increased tumor cell delivery to lymph nodes and a higher rate of lymphatic metastasis¹⁸⁵ (Fig. 4.7). It remains unclear how VEGF-C overexpression impacts tumor cell entry into lymphatic vessels; however, an attractive hypothesis is that the increased lymphatic surface area simply increases the probability of tumor cell entry and dissemination. Alternatively, VEGF-C may stimulate the release of a chemotactic factor that recruits tumor cells into lymphatic vessels. Of potential clinical importance, VEGFR-3 blockade was shown to inhibit VEGF-C–induced lymphatic hyperplasia, tumor cell delivery to draining lymph nodes, and lymphatic metastasis when treatment was started at the time of tumor initiation. However, lymphatic metastases were not significantly reduced if VEGFR-3 blockade was started after tumor cell seeding of draining lymph nodes.¹⁸⁵^,¹⁹⁵ These data suggest that anti-VEGFR-3 therapy may be effective in preventing, but not treating, lymphatic metastases—the more common clinical imperative—except in cases where a significant fraction of vascular endothelial cells and/or cancer cells express VEGFR-3. The challenge now is to identify alternative strategies for treating lymphatic metastases, either through combination therapy (e.g., anti-VEGFR-2/anti-VEGFR-3) or modulation of other pathways.

Mechanical signals that trigger the lymphangiogenic switch are unknown. Because lymphatic vessels help maintain the balance of fluid in tissues, hydrostatic pressure is a likely trigger.¹⁹⁵^,¹⁹⁷ Whether the lymphatic hyperplasia seen in tumor margins is, in part, a response to the elevated hydrostatic pressure in tumors and whether the newly formed lymphatics remain open and relieve this pressure is an open question. Techniques such as microlymphangiography,⁴^,¹⁶⁵^,¹⁶⁶^,¹⁸⁵^,¹⁸⁸^,¹⁹⁸^,¹⁹⁹ fluorescence photobleaching lymph flow quantitation,¹⁸²^,¹⁸⁵^,¹⁹⁸^,²⁰⁰^–²⁰² and optical frequency domain imaging (OFDI)²⁰³ will allow us to answer these important questions.

Interstitial Hypertension

Unlike normal tissues, where the interstitial fluid pressure (IFP) is around 0 mm Hg, both animal and human tumors exhibit interstitial hypertension.⁶⁹^,⁷¹^,⁷³^,¹¹⁸^,¹⁶⁶^,¹⁷⁴^,²⁰⁴^,²⁰⁵^,²⁰⁶^,²⁰⁷^–²¹¹^,²¹²^,²¹³^–²¹⁵ The tumor IFP begins to increase as soon as the host vessels become leaky in response to angiogenic molecules such as VEGF.²¹⁶ Thus, IFP can be lowered by inhibiting the VEGF pathway using blocking antibodies.⁶⁹^,⁷¹^,⁷²^,⁷³^,⁸⁸^,²¹⁷ The IFP increases with tumor size in some tumors²⁰⁴^,²⁰⁶^,²¹⁰ and remains independent of tumor size in others.²¹¹

Three mechanisms contribute to the interstitial hypertension in tumors. In normal tissues, lymphatics maintain fluid homeostasis; thus, the lack of functional lymphatics within tumors is a key contributor. Indeed, DiResta et al. have shown that one could lower the IFP by placing “artificial lymphatics” in tumors.²¹⁸ The second contributor is the leaky nature of tumor vessels. As a result, the hydrostatic and oncotic (colloid osmotic) pressures become almost equal between the intravascular and extravascular space.⁸²^,⁸⁸^,²⁰⁵^,²¹⁹ At least two pieces of evidence support this hypothesis. First, lowering permeability by blocking VEGF signaling lowers IFP.⁷¹^,⁸⁸^,²¹⁷ Second, IFP goes up and down with the microvascular pressure within seconds.²²⁰^–²²² The two mechanisms described so far can only explain hypertension up to 20 to 30 mm Hg—the microvascular pressure of most exchange vessels in our body—but IFPs as high as 94 mm Hg have been measured in human tumors.²¹⁴ Because the microvascular pressure (MVP) is the driving force for IFP in tumors, these tumors must have a high MVP. Indeed, this is the case.²⁰⁵ There are two possible explanations for the elevated MVP in tumors: (a) the tumor vessels have reduced arterial resistance so that the MVP becomes closer to arterial pressure, and/or (b) the tumor vessels have increased venous resistance as a result of compression and tortuosity so that the whole vascular network is under hypertension. Indirect evidence for the latter comes from the decrease in IFP following decompression of tumor vessels by drug-induced apoptosis of perivascular cancer cells.²⁴^,⁹⁶

The elevated pressure can compromise the tumor microcirculation and delivery of therapeutics in three ways. First, reduced transmural pressure gradients resulting from equilibrium between MVP and IFP reduce convection across tumor vessels and thus compromise the transport of macromolecules.⁸²^,⁸⁸^,⁹⁷^,²⁰⁵^,²²¹ Second, 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.¹⁵^,³⁵^,¹³⁰ Finally, transmural coupling between IFP and MVP as a result of high permeability of tumor vessels can lead to blood flow stasis in tumors without physically occluding the vessels.¹⁰⁸^–¹¹⁰ Thus, decreasing vascular leakiness might restore the transmural pressure gradients and potentially resume/re-establish blood flow in the nonperfused regions of tumors. Some direct and indirect antiangiogenic therapies may “normalize” the tumor vasculature through this mechanism¹²^,¹⁶^,⁴¹^,⁶⁷^,⁷⁰^,⁷¹^,⁸³^,¹¹⁶^,²²³ (Tables 4.2 and 4.3 and Fig. 4.5).

TABLE 4.2 STUDIES REPORTING ANTIANGIOGENIC THERAPY–INDUCED IMPROVEMENT IN TUMOR OXYGENATION

Metabolic Environment

Hypoxia

A key function of the vasculature is to provide adequate levels of nutrients 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 to 200 μm nearly a century ago.²²⁴ This unit of tissue—a single capillary surrounded by a 100 to 200 μm 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 μm away from blood vessels, presumably due to a lack of oxygen.²²⁵ 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 Po₂gradients, as well as perivascular pH gradients, became possible only with the development of phosphorescence quenching microscopy²³^,²²⁶ (Fig. 4.6B).

As discussed earlier, blood flow in tumor vessels is intermittent, and, thus, some regions of a tumor are periodically starved for oxygen. The resulting hypoxia is referred to as acute hypoxia or perfusion-limited hypoxia. 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 ischemia-reperfusion injury or reoxygenation injury; thus, applying additional selection pressure on cancer cells can cause them to become more locally aggressive, metastatic, and resistant to therapy.²²⁷

TABLE 4.3 STUDIES REPORTING THE IMPACT OF ANTIANGIOGENIC/VASCULAR NORMALIZATION STRATEGIES UPON DELIVERY OF THERAPEUTIC COMPOUNDS/SYSTEMICALLY ADMINISTERED MOLECULES INTO TUMORS

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.²²⁸ The former results from glycolysis, and the latter results from conversion of CO₂ and H₂O via carbonic anhydrase. However, the intracellular pH of cancer cells remains neutral or alkaline (≥7.2) despite the acidic extracellular pH. Because carbonic anhydrase-9 and various glucose transporters (GLUT-1, -3) and enzymes in the glycolytic pathway are up-regulated by hypoxia,²²⁷ one would expect low extracellular pH and hypoxia to track each other and to colocalize with regions of low blood flow. It is surprising that there is a lack of spatial correlation among these parameters—a discovery made possible by recent developments in optical techniques that permit the simultaneous high-resolution mapping of multiple physiologic parameters.²³ A potential explanation for this lack of concordance is that some perfused tumor vessels carry hypoxic blood.²³ Thus, although they may not be able to deliver enough oxygen to the surrounding cells, they may be able to carry away the waste products (e.g., lactic acid).

Therapeutic Consequences

The presence of molecular oxygen during irradiation can “fix” biologic (e.g., DNA) free radicals, making radiation-induced damage irreparable (oxygen fixation hypothesis). Thus, hypoxia reduces the radiation sensitivity of neoplastic and normal cells both in vitro and in vivo. Similarly, hypoxia can compromise the efficacy of some chemotherapeutics. Independently, hypoxia can increase the metastatic potential of cancer cells.²²⁷ Therefore, for nearly half a century considerable preclinical and clinical effort has been focused on alleviating hypoxia through a multitude of interventions such as improving tumor perfusion with mild hyperthermia or drugs, increasing oxygen content of the blood via hyperbaric oxygenation, and increasing hemoglobin/hematocrit by transfusion or exogenous erythropoietin. Unfortunately, the clinical outcomes have not met expectations. Although early studies suggested a marked benefit of transfusion in anemic cervical cancer patients undergoing definitive radiotherapy, careful analysis suggests that these studies are confounded by selection biases that preclude the conclusion that anemia correction by transfusion impacts outcome.²²⁹^,²³⁰ Furthermore, erythropoietin (or analog) treatment showed encouraging survival results in anemic cancer patients receiving nonplatinum chemotherapy and in anemic lung cancer patients receiving chemotherapy.²³¹^,²³² However, subsequent trials in anemic head and neck cancer patients and mainly nonanemic metastatic breast cancer patients actually suggested outcomes may be impaired by erythropoietic agents.²³³^,²³⁴ There are multiple possible reasons such interventions have yielded mixed results. These include the inability to increase tumor Po₂ as markedly as systemic Po₂,²³⁵ the inability to increase Po₂ in all areas of a tumor to optimal levels due to abnormal vasculature,⁸²^,¹¹⁶ and undesired “off-target” effects of interventions (e.g., immunosuppression with transfusion²³⁶^–²³⁸). Furthermore, tumors may reoxygenate during radiation therapy with standard fractionation, potentially minimizing the impact of providing additional oxygen to the target tissue.

Similarly, low extracellular pH can adversely (or favorably) affect the uptake and cytotoxicity of some therapeutics. The pH gradient difference between tumor and normal tissue may offer a tumor-specific target for weak acid chemotherapeutics for the treatment of cancer.²³⁹^,²⁴⁰ The development of specific drugs that exploit this pH difference and strategies to modulate pH in tumors have not yet reached the clinic but are anticipated.²²⁷

Two broad strategies targeting the unique tumor metabolic environment are emerging: (a) exploit hypoxia to activate drugs or attract tumoricidal anaerobic bacteria and (b) dissect hypoxia-induced pathways to identify novel targets for drug development. The first strategy has led to the development of drugs such as tirapazamine and to renewed interest in bacteriolytic therapy;²⁴¹ both approaches are in clinical trials, but promising data are yet to emerge²⁴² (see Trial Identifier NCT00358397 at ClinicalTrials.gov). The second strategy has revealed several molecular players in the physiologic and pathophysiologic response to hypoxia.²²⁷^,²⁴³^,²⁴⁴ 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 selects for tumor cells that are more malignant, more invasive, and genetically unstable, rendering them resistant to various therapies. Therefore, certain players in the hypoxia-induced pathways now are being targeted in the development of diagnostic and therapeutic agents. Hypoxia-induced pathways include genes involved in oxygen delivery, glycolysis and glucose uptake, pH control, stress-response pathways, growth factor signaling, angiogenesis, transcription, apoptosis, growth inhibition, and invasion and metastasis (Fig. 4.3 and Box 4.1).¹¹^,²⁴³

Of the various molecular players involved in sensing and responding to hypoxia, hypoxia-inducible factor-1 a (HIF-1α) has received the most attention. This transcription factor is up-regulated in a number of human tumors.²⁴³^,²⁴⁵Regulated by proline and asparagine hydroxylases, HIF-1α activates genes involved in an array of physiologic responses including angiogenesis, vasodilation, glycolysis, and RBC production by binding to the hypoxia-response element (HRE). Although HIF-1α is an attractive therapeutic target, its pleiotropic action may prove to be a major challenge for clinical exploitation. For example, teratomas arising from HIF-1α(–/–) embryonic cells grow more rapidly despite lower levels of VEGF and angiogenesis.²⁴⁶ This counterintuitive finding may be a result of the ability of HIF-1α(–/–) cells to survive under hypoxic conditions, instead of undergoing apoptosis.⁶⁰ HIF-1α has also been shown to play an important role in determining tumor radioresponsiveness through the regulation of multiple, and sometimes opposing, processes.²⁴⁷ Under some circumstances, HIF-1α inhibition reduces tumor cell radiosensitivity by protecting hypoxic cells from radiation-induced apoptosis and enhancing clonogenic survival potentially through reductions in adenosine triphosphate metabolism, cellular proliferation, and p53 activation.²⁴⁷ Furthermore, HIF-1αserves a key function in inflammatory cell energy metabolism, and its inhibition results in profound immunodeficiency.²⁴⁸ Consequently, molecular therapies that target HIF-1α or HRE, as well as more selective therapies that target key downstream effectors of HIF-1α, are under intensive investigation for cancer detection and treatment.²²⁷^,²⁴³^,²⁴⁹

Box 4.1

Hypoxia and Epigenetic Regulation of Angiogenesis

The prolyl hydroxylase domain (PHD) proteins PHD1–3 are oxygen-sensing enzymes that hydroxylate the hypoxia-inducible factor (HIF) proteins HIF-1α and HIF-2α when sufficient oxygen is available. Once hydroxylated, HIFs are targeted for proteasomal degradation.²⁶⁸ Under hypoxia, PHDs become inactive, and HIFs initiate broad transcriptional responses to increase the oxygen supply by angiogenesis, through the up-regulation of angiogenic factors such as vascular endothelial growth factor (VEGF).²⁶⁹ HIFs are also activated in nonhypoxic conditions by oncogenes and growth factors, allowing tumor cells to stimulate angiogenesis before they become deprived of oxygen. In general, HIF-1α promotes vessel sprouting, whereas HIF-2α mediates vascular maintenance.²⁶⁹ Reduced HIF-1α levels in mice impair embryonic vascular development, revascularization of ischemic tissues, and angiogenesis in injured tissues and tumors.²⁶⁹ The use of HIF-1α inhibitors to block tumor or ocular angiogenesis has therefore received attention. Conversely, Hif1 α gene transfer in mice or activation of HIF-1α by pharmacologic blockade of PHDs promotes ischemic tissue revascularization.

HIF-1α also regulates tumor angiogenesis indirectly, by releasing chemoattractants such as stromal cell–derived factor 1α (SDF-1α) to recruit proangiogenic bone marrow–derived cells (BMDCs).²⁷⁰ Gene silencing of Phd2 in mouse tumor cells enhances vessel growth by similar mechanisms. Hypoxia also regulates the polarization and proangiogenic activity of tumor-associated macrophages (TAMs) by means of HIF-1α and HIF-2α with different effects.²⁶⁸ That hypoxia and inflammation are closely intertwined is illustrated by the finding that signaling by HIF-1α and nuclear factor-κB cross-activate each other. In certain cases, hypoxic up-regulation of VEGF occurs independently of HIF-1α and is mediated by the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α in preparation for oxidative metabolism once the ischemic tissue is revascularized.²⁷¹Because HIF signaling contributes to acquired resistance against anti-VEGF therapy, the combined blockade of VEGF and HIF-1α is being explored as a cancer treatment strategy.

There is increasing evidence for epigenetic control of angiogenesis, particularly by noncoding microRNAs (miRNAs),²⁷² which induce messenger RNA degradation or block translation. Because miRNAs target multiple genes, they are well positioned to regulate complex processes such as angiogenesis. Endothelial cells express several miRNAs that are induced by hypoxia or VEGF. Most of those stimulate angiogenesis by hijacking proangiogenic cascades while suppressing angiostatic pathways.²⁷³ The expression of miR-126 is induced by the mechanosensitive transcription factor KLF2A and integrates the mechanosensory stimulus of blood flow to shape the vascular system.²⁷⁴ Endothelial cell–specific loss of DICER, an exonuclease involved in miRNA biogenesis, impairs pathologic angiogenesis. Angiogenic miRNAs seem to offer significant pro- or antiangiogenic potential.

Reproduced with permission from Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011; 473:298–307.

CLINICAL IMPLICATIONS

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 radiation sensitizer) at effective concentrations to all cancer cells.⁴¹^,⁸³^,²⁵⁰ Second, inherent or acquired resistance resulting from genetic and epigenetic mechanisms reduces the effectiveness of conventional as well as novel therapies.²⁵¹ Can we take advantage of the unique pathophysiology of tumors to overcome these problems for better management of cancer? As discussed next, recent clinical data offer some hope.

Prognostic/Predictive Biomarker Implications

Multiple indices of tumor pathophysiology have been evaluated as potential predictors of treatment outcome including vessel density (reviewed in ⁴²^,²⁵²), oxygen level (reviewed in²⁴³^,²⁵³), interstitial pressure,⁴²^,²¹²^,²¹⁴^,²³⁰ and blood or urine circulating molecules⁷³^,⁹¹ (reviewed in⁴²). Vessel density can be evaluated in biopsies and is measured either in “hot spots” (i.e., regions of most active angiogenesis) or in the tissue as a whole. The former presumably provides a measure of a tumor’s aggressiveness, and the latter reflects the status of global oxygenation. Most studies to date show that poor outcome of radiation therapy correlates with high vessel density in “hot spots” and/or low overall microvessel density. There are, however, several studies showing a lack of correlation or an opposite correlation. This discrepancy may be the result of the morphometric techniques used or of differences in tumor types or treatment schedules.

The oxygen level in a tumor also has a potential prognostic value, and it can be directly measured with microelectrodes. Alternatively, immunohistochemical analysis of tumor tissue for endogenous or exogenous hypoxic markers (e.g., HIF-1α, glucose transporter-1, carbonic anhydrase-9, pimonidazole) can be used as a surrogate for tumor oxygenation status. However, immunohistochemical assessments of hypoxia do not necessarily correlate with oxygen status measured directly with microelectrodes.²⁵⁴ A concerted effort is under way to assess hypoxia using novel, noninvasive imaging techniques.²⁵⁵^,²⁵⁶ Several studies have shown that tumor hypoxia is a predictor of a poor outcome of radiation therapy when used alone or in combination with other therapies. These findings are consistent with in vitro and in vivo preclinical studies showing the adverse effect of hypoxia on radiation responses.

Because the IFP is a reflection of the global physiology of tumors, a correlation between tumor IFP and the response to radiation therapy has been suggested. One cervical cancer study has shown that elevated tumor IFP can, indeed, independently predict a poor outcome of radiation therapy.²³⁰ Further studies are needed to evaluate the prognostic significance of IFP in tumors. However, one potential application of the steep rise of pressure at the tumor periphery is improved localization of tumors before their removal.

Finally, circulating biomarkers may provide information about tumor pathophysiology and its changes after treatment. Of note, some of the emerging biomarkers—such as circulating collagen IV or soluble VEGFR-1—may represent biomarkers of vascular normalization and, if validated, could be useful in treatment decisions.⁹¹^,⁹²

Although each of these approaches has advantages, key disadvantages include their invasiveness and their potential for sampling error. With rapid developments in the field of noninvasive imaging, it is likely that the measurement of various physiologic and molecular parameters in tumors will become more refined and convenient for patients. Examples of such imaging approaches include blood oxygen level–dependent magnetic resonance imaging (BOLD MRI), electron paramagnetic resonance spectroscopy/imaging, and [¹⁸F]-misonidazole positron emission tomography (FMISO-PET).²⁵⁵^–²⁵⁶^,²⁵⁷^–²⁵⁹ The promise of such imaging approaches has just started to be realized. FMISO-PET has been evaluated in a substudy of patients with stage III or IV squamous cell carcinoma of the head and neck randomized to concurrent radiotherapy with either tirapazamine and cisplatin or infusional fluorouracil and cisplatin. Pretreatment FMISO-PET–detected hypoxia was associated with a higher risk of locoregional recurrence among patients who did not receive the tirapazamine-containing regimen compared to patients who did receive tirapazamine.²⁵⁹ This study suggests that FMISO-PET can provide clinically meaningful information about tumor physiology and simultaneously provides evidence that tirapazamine acts by specifically targeting hypoxic tumor cells. Such progress will continue and physiologic/molecular profiles of patients’ tumors will yield improved and better-tailored therapies for individual patients.

FIGURE 4.8. Vascular “normalization” in rectal cancer patients following treatment with the anti–vascular endothelial growth factor (VEGF) antibody, bevacizumab. A–C: Tumor vessel “normalization” following a single injection of bevacizumab is suggested by the reduction of tumor microvessel density (A), by the increase in fraction of tumor vessels with pericyte coverage (B), and by the drop in interstitial fluid pressure (C). 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 residual tumor blood vessels after bevacizumab treatment is improved. (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, with permission.)

Therapeutic Implications

Given the physiologic barriers to the delivery and effectiveness of various therapeutics, a strategy that is gaining increasing interest is targeting the tumor vasculature. This strategy has the advantage of targeting ECs that are easily accessible to a bloodborne drug and are presumably genetically stable. In addition, each EC supports multiple cancer cells, thus providing “therapeutic amplification.” However, the inability to target all ECs in a tumor can reduce the effectiveness of antivascular therapy. Similarly, the dependence of ECs on multiple angiogenic molecules can limit the effectiveness of various antiangiogenic therapies when used alone.⁴¹^,⁸³ These challenges may explain why currently available antiangiogenic agents, although demonstrating biologic activity, are unable to provide durable tumor control when used as monotherapy.

Although of limited utility when used alone, the judicious combination of antiangiogenic therapies with conventional cytotoxic therapies has led to improved tumor control in mice and lengthened survival in certain types of human tumors¹⁴^,¹⁶^,¹⁷^,²⁴⁰^,²⁶⁰^–²⁶³ (see Table 4.1). For example, in two human tumor xenograft models, a VEGFR-2–blocking antibody decreased the dose of fractionated radiation required to control 50% of tumors (TCD₅₀) by 11 to 27 Gy without modifying in-field skin reactions.²⁴⁰ Thus, to maximize clinical gains, these agents must be employed in combination with radiation and chemotherapy. The challenge now is to optimally combine these therapies in patients. Destruction of tumor vasculature by antiangiogenic agents should antagonize chemo- and radiotherapy by compromising the delivery of therapeutics and oxygen, respectively. However, judiciously applied antiangiogenic therapy can prune inefficient tumor vessels and render the remaining vasculature more efficient (Fig. 4.5).¹²^,⁴¹^,⁸³^,¹¹⁶^,²²³ This “normalization” of tumor vasculature has been demonstrated in various preclinical models (reviewed in⁴¹^,⁸³) and in rectal cancer, hepatocellular carcinoma, ovarian carcinoma, and glioblastoma patients⁶⁶^,⁶⁷^,⁶⁹^,⁷¹^,⁷³^,⁷⁵ (Fig. 4.8). Vascular “normalization” should result in improved delivery of cytotoxic chemotherapy and radiosensitizing oxygen, thereby improving tumor control. This principle has been rigorously tested in animal models⁸³^,⁸⁹^,²⁶⁰^,²⁶⁴^–²⁶⁶ and confirmatory data on the impact of vascular “normalization” on patient outcomes await mature results of ongoing and future clinical trials.

ACKNOWLEDGMENTS

This chapter is an update of the chapter published in the fifth edition of Principles and Practice of Radiation Oncology and based on a review article by Carmeliet and Jain, Nature 2011.¹¹ The work summarized here was supported by continuous support from the National Cancer Institute since 1980 to RKJ. We want to acknowledge the support through grants P01CA80124, R01CA115767, R01CA85140, R01CA126642, and Federal Share/NCI Proton Beam Program Income (RKJ), and R21CA139168, R01CA159258, and Federal Share/NCI Proton Beam Program Income (DGD), as well as the National Foundation for Cancer Research Grant and Department of Defense Breast Cancer Research Innovator Award W81XWH-10-1-0016 (RKJ), and the American Cancer Society Research Grant RSG-11-073-01-TBG (DGD).

DISCLOSURE

R.K.J. has research grants from Dyax, MedImmune, and Roche; serves as a consultant to Noxxon Pharmaceuticals; serves on the Scientific Advisory Board of Enlight and SynDevRx; serves on the Board of Directors of XTuit; serves on the Board of Trustees of H&Q Healthcare Investors and H&Q Life Sciences Investors; and has equity in Enlight, SynDevRx and XTuit Pharmaceuticals.

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