A solid tumor is an organ composed of neoplastic cells and host stromal cells nourished by a 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 pathophysiological characteristics of the tumor. Tumor pathophysiology governs not only tumor growth, invasion, and metastasis but also the response to various therapies. In this chapter we will discuss various pathophysiological 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;67:239–48.)

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 200 µm from a blood capillary so 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 transparent window techniques were developed in the 1940s.^2–5 The possibility that tumors produce an “angiogenic” substance was suggested in 1968.⁶ The hypothesis that blocking angiogenesis should block tumor growth and metastasis was proposed shortly thereafter in 1971.⁷ 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 1978.⁸ The first antiangiogenic agent approved for patients with cancer was bevacizumab, an antibody specific to vascular endothelial growth factor (VEGF), on the basis of the increased survival seen in patients with metastatic colorectal cancer with the combination of bevacizumab and standard chemotherapy in a pivotal randomized placebo-controlled phase III trial.⁹ At present, various antiangiogenesis and proangiogenesis strategies are being evaluated clinically to prevent or treat a large number of diseases, including cancer.^12–12 Both normal and pathological angiogenic processes are governed by the net balance between proangiogenic and antiangiogenic factors.^13,14 This balance is spatially and temporally regulated under physiological conditions, so that the “angiogenic switch” is “on” when needed (e.g., during embryonic development, wound healing, and 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 because of 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.^15–18 “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 anastomosis of sprouts to permit blood flow; and finally, these immature vessels are invested in basement membrane and pericytes. During physiological angiogenesis, these vessels differentiate into mature arterioles, capillaries, and venules, whereas in tumors they remain largely immature.^5,11,12,19 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—also can contribute to neovascularization (a process referred to as “postnatal” vasculogenesis) in tumors and other tissues.^22–22 In addition, recent reports demonstrated that a fraction of endothelial cells in tumor vessels may be derived via transdifferentiation from cancer cells,²³ or from cancer stemlike cells (e.g., in glioblastomas).^11,24–26 In this larger context, the term “vasculogenesis” might be invoked to describe neovascularization in tumors from tumor stem cells.²⁷ Tumor vasculogenesis also may occur from EPCs of non–bone marrow origin that are recruited from adjacent tissues and/or circulation.^28,29 The current challenge is to discern the relative contribution of each of the these mechanisms of neovascularization during tumor growth and during and after treatment of tumors.^27,30

Molecular Mechanisms

Various proangiogenic and antiangiogenic molecules that orchestrate different steps in vessel formation, along with their functions, are listed in Table 8-1. VEGF currently is 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.^{15,22,31–33} During tumor progression, or with treatment, the number of distinct angiogenic molecules produced by a tumor can increase.^36–36 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 growth factor [PlGF], or interleukin-8 [IL-8]).³⁷ 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–derived VEGF).^19,38,39 Angiogenesis inhibitors include endogenous soluble receptors of various proangiogenic ligands (e.g., sVEGFR1/sFLT1) 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.¹³ Other endogenous inhibitors include fragments of various plasma or matrix proteins (e.g., angiostatin, a fragment of plasminogen; endostatin, a fragment of collagen XVIII; and tumstatin, a fragment of collagen IV).^42–42 Neither the mechanisms of action of the matrix-derived inhibitors nor their physiological role are well understood.⁴³ The generation of proangiogenic and antiangiogenic molecules can be triggered by metabolic stress (e.g., low po₂, low pH, or hypoglycemia), mechanical stress (e.g., shear stress or 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).^{13,14,44–47} These molecules can emanate from cancer cells, endothelial cells, stromal cells, blood, and extracellular matrix (Fig. 8-3).^48–51 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.^52–59 Furthermore, because the tumor microenvironment is likely to change during tumor growth, regression, and relapse, profiles of proangiogenic and antiangiogenic molecules are likely to change with time and space.^60,61 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.^37,62–64

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 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 α_vβ₃, α_vβ₅, 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 pathological 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 Maspin Canstatin, proliferin-related protein, restin	Interfere with integrin signaling Protease inhibitor mechanisms unknown

COX-2, Cyclooxygenase-2; EG-VEGF, endocrine gland–derived vascular endothelial growth factor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; IFN, interferon; IL, interleukin; IP, interferon-inducible protein; MCP-1, monocyte chemotactic protein-1; MMP, metalloproteinase; NRP-1, neuropilin-1; NOS, nitric oxide synthase; PAI-1, plasminogen activator inhibitor-1; PDGF-BB, platelet-derived growth factor-BB; PECAM, platelet endothelial cell adhesion molecule; PEX, hemopexin fragment of MMP-2; PlGF, placental growth factor; SPARC, secreted protein acidic and rich in cysteine; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TSP, thrombospondin; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; VEGI, vascular endothelial growth inhibitor.

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

^†Also present in or affecting nonendothelial cells.

^‡See reference 19.

^§Opposite effect in some contexts.

Figure 8-3 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 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 co-localize 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;94:715–25. **C-E** 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 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.15,65–68 In general, tumor vessels are dilated, saccular, tortuous, and chaotic in their patterns of interconnection.⁶⁹ For example, whereas normal vasculature is characterized by dichotomous branching, tumor vasculature has many trifurcations and branches with uneven diameters.^70,71 The fractal dimensions and minimum path lengths of tumor vasculature are different from those of normal host vasculature.^67–67 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.^19,72 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.^{61,63,73–76} Mechanical solid stress (i.e., physical forces exerted by solid tissue components) generated by proliferating cancer and stromal cells and their excessive interstitial matrix production also can lead to the partially compressed or totally collapsed vessels often found in tumors (Fig. 8-5).^64,77,78 The decompression of blood vessels observed after induction of apoptosis in perivascular cells or stromal cells, or enzymatic degradation of matrix hyaluronan, supports this mechanical hypothesis.^78–81 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 anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med 2001;7:987–89; Jain RK. Angiogenesis and lymphangiogenesis in tumor: insights from intravital microscopy. Cold Spring Harbor Symp Quant Biol [The Cardiovascular System] 2002;67:239–48; Jain RK, Carmeliet PF. Vessels of death or life. Sci Am 2001;285:38.)

Figure 8-5 Vascular compression in tumors. **Middle**, In solid tumors, proliferating cancer cells and activated fibroblasts deform the interstitial matrix, resulting in stretched collagen fibers, compressed hyaluronan, and deformed cells—all storing solid stress, a type of mechanical force transmitted by solid tissue components. This stress compresses intratumor blood and lymphatic vessels. Potential strategies to alleviate solid stress and decompress vessels involve depleting these components. Depleting cancer cells (*top left*) or fibroblasts (*top right*) relaxes collagen fibers, hyaluronan, and the remaining cells, alleviating solid stress. Depleting collagen (*bottom left*) alleviates the stress that was held within these fibers while also relaxing stretched/activated fibroblasts and compressed cancer cells within nodules. Finally, depleting hyaluronan (*bottom right*) alleviates the stored compressive stress, allowing nearby components to decompress. (Note that other stromal cells, such as pericytes, macrophages, and various immune cells that might also control production of collagen or hyaluronan, are not shown to simplify the schematic. Lymphatic vessels also are not shown for the same reason.) (Courtesy Dr. Lance L. Munn, PhD, Massachusetts General Hospital, Boston; reproduced from Stylianopoulos T, Martin JD, Chauhan VP, et al. Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors. Proc Natl Acad Sci USA 2012;109(38):15101–8.)

Blood Flow and Microcirculation

Blood flow in a vascular network, whether normal or abnormal, is governed by the arteriovenous 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).⁶⁹ Abnormalities in both vasculature and viscosity increase the resistance to blood flow in tumors.^71,82–84 As a result, overall perfusion rates (i.e., blood flow rate per unit volume) in tumors are lower than in many normal tissues.^85,86 Both macroscopically and microscopically, tumor blood flow is temporally and spatially chaotic. Macroscopically, four spatial regions can be recognized in a tumor (Fig. 8-6):

Figure 8-6 The tumor microenvironment is heterogeneous with proliferative, quiescent, and necrotic regions. These regions can be characterized in terms of various physiological 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;98:14748–50.)

1. An avascular necrotic region

2. A seminecrotic region

3. A stabilized microcirculation region

4. An advancing front 87,88

At the microscopic level, in normal tissues, erythrocyte velocity is dependent on vessel diameter, but there is no such dependence in most tumors.54,59,89 Furthermore, the average erythrocyte velocity can be an order of magnitude lower in some tumors compared with that of normal host tissue (Fig. 8-7).⁵⁹ In a given vessel within a tumor, blood flow fluctuates with time and can reverse its direction.^87,89,90 In addition to the elevated geometric and viscous (rheologic) resistance, other molecular and mechanical factors contribute to this spatial and temporal heterogeneity. These factors include imbalance between proangiogenic and antiangiogenic molecules, “solid stress” generated by proliferating tumor cells and matrix production, vascular remodeling by intussusception, and coupling between luminal and interstitial fluid pressure via hyperpermeability of tumor vessels.^{6,16,64,65,77–79,91–93} 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. Increased perfusion also correlates with a positive response to radio/chemotherapy and patient survival.^94,95 Increased perfusion has been difficult to achieve reproducibly, because tumor vasculature consists of both vessels co-opted from the preexisting 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.^48,69,96 Presumably as a result, efforts to increase tumor blood flow by pharmacologic or physical agents have not always been reproducible or successful.^48,69,85,96 On the other hand, the strategy of decreasing or shutting down 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.^{85,86,97–99} 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.¹⁰⁰

Figure 8-7 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. *RBC,* Red blood cell. (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;54:4564–8.)

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.100,101 The effective 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).^15,102 Some of these anatomic pathways may be lined with glycocalyx on ECs, 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.^{15,51,96,102,103} 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.^59,104–108 Furthermore, unlike normal vessels in the surrounding tissue, tumor vessels partially lose molecular size–based selectivity in permeability to different molecules.¹⁰⁹ Despite increased overall permeability, not all blood vessels of a tumor are leaky (Fig. 8-8, A). Even the leaky vessels have a finite pore size that is tumor dependent (Fig. 8-8, B), and ultrastructural studies show that the larger pore size in tumors represents wide interendothelial junctions.^56,103 Thus permeability is inversely related to molecular size due to steric and hydrodynamic hindrance from these pores, and very large molecules, such as nanoparticles, do not penetrate tumors efficiently.^110,111 Molecular shape also influences penetration of these pores, such that rod-shaped molecules have a higher permeability than do spherical molecules.¹¹² Similarly, positively charged molecules have a higher affinity for the negatively charged glycocalyx in the pores of these angiogenic tumor vessels, reducing this hindrance and giving them a higher permeability.^113–116 Not only do the vascular permeability and pore size vary from one tumor to the next (see Fig. 8-8, A-C), but within the same tumor they vary spatially and temporally, as they do during tumor growth, regression, and relapse.^56,60,61 The local microenvironment plays an important role in controlling vascular permeability (Fig. 8-8, D). 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.^59,72 Such site-dependent differences for other tumors have been observed in other orthotopic sites.^54,57,58 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).^{19,39,72,117,118} 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 8-8 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 (Sc) 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;54:3352–6. 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;95:4607–12.)

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 pathways (through ECs) and periendothelial pathways (between ECs) 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.119–122 Recently, it was demonstrated that metastatic cells could bring their own stroma from the primary tumor in the form of hereotypic cell clumps.¹²³ More is known about the molecular and cellular mechanisms of extravasation.¹²⁴ 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.^125–129 Deformability of cells affects both types of forces.¹³⁰ In addition, cancer cells may grow intravascularly at a distant site (e.g., in the lungs).¹³¹

Rolling of endogenous leukocytes is generally low in tumor vessels, whereas stable adhesion (≥30 sec) is comparable between normal vessels and tumor vessels.¹³² 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.¹³³ 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 currently is not known. The age of the animal also plays an important role in leukocyte-endothelial interactions.¹³⁴ 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.^135,136 After systemic injection, these cells localized primarily in the lungs immediately after injection and could not be detected in the tumor.¹³⁵ Increased rigidity caused by IL-2 activation might contribute to the mechanical entrapment of these cells in the lung microcirculation.^137,138 Constitutive expression of certain adhesion molecules in the lung vasculature also might facilitate their retention in the lungs.¹²⁴ One approach to reducing lung entrapment is to reduce the rigidity of these cells.^112,117 Alternatively, the lung can be circumvented by injecting A-NK cells directly into the blood supply of tumors. In this case, A-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 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.^{33,136,139–142} 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.^{55,125,143–147} Surprisingly, the proangiogenic VEGF also can upregulate these molecules, whereas another proangiogenic molecule, bFGF, can downregulate these molecules.^{33,51,61,76,148} Inflammatory cells such as monocyte/macrophages or neutrophils also are recruited by VEGF and may play important roles in promoting matrix remodeling and angiogenesis.¹⁴⁹ 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 and 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 and melanomas) or as clumps, sheets, or nests (e.g., carcinomas). More than 80% of tumors are carcinomas arising from epithelial cells. The remaining cancers 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.¹⁵ 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.¹²⁴ Other host cells, such as fibroblasts, might proliferate and migrate from the adjacent connective tissue.^19,48,150 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 previously discussed, is defective in tumors.¹⁹ In addition, functional lymphatics might be confined to the tumor margin.^151,152 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 current understanding of blood vessel formation, 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.¹⁵ 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, also are synthesized by the stromal cells, albeit in a form that differs from that in the plasma or normal tissues.¹⁵ Tumor interstitial fluid also can contain various growth factors that facilitate stroma formation. For example, in vitro studies suggest that platelet-derived growth factor-BB (PDGF-BB) is involved in the recruitment of fibroblasts to tumors, and TGF-β induces the production of collagen and other matrix molecules in tumors.^19,153 With the increasing interest in using the fragments of matrix constituents for controlling angiogenesis, increased understanding of the molecular and cellular mechanisms of stroma generation in tumors will increase.⁴³

Interstitial Transport

Once a molecule has extravasated, its movement through the interstitial space occurs by diffusion and convection.101,154 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 fluid 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.^155–165 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.^167–167 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. 8-9, A).^157,168 Collagen content and structure have a significant effect on D in tumors.^{159,163,166,169–173} This finding 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. 8-9, A). Thus agents that interfere with collagen synthesis and/or organization (e.g., relaxin, losartan, and bacterial collagenase) might increase interstitial transport in tumors^156,169,170 (Fig. 8-9, B). The time constant for a molecule with diffusion coefficient D to 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. Thus for a 1-mm distance in tumor, diffusional transport would take days, and for a 1-cm distance in tumor, it would take months. Furthermore, the path through the interstitium is tortuous over large length scales, greatly increasing the distance a molecule must travel.¹⁵⁸ If the central vessels have collapsed completely as a result of 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.^6,79 Binding of a low- or high-molecular-weight drug to plasma proteins and various tissue components could further retard their transport in tumors.^{168,174–180} The role of binding is clearly illustrated in Figure 8-9, C, which compares the rate of fluorescence recovery of a photobleached spot in tumor tissue injected with a nonspecific versus 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.¹⁶⁸