Steps in Tumor Stroma Generation

Studies of transplantable tumors have yielded important information concerning the pathogenesis of tumor stroma generation (Figure 35-2).^{3,4 9,10} Among the earliest steps in this process is local vascular hyperpermeability to circulating macromolecules. Increased vascular permeability is attributable to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A), a multifunctional cytokine that is synthesized and secreted by the great majority of animal and human tumors.^10–13

Figure 35-2

Schematic diagram of tumor stroma generation.

Among other activities, VEGF-A renders the microvasculature hyperpermeable to plasma and plasma proteins with a potency some 50,000 times that of histamine and ranks among the most powerful vascular permeabilizing substances known (Figure 35-3).^10–13When injected into skin or other normal tissues, VEGF-A, like mediators of inflammation, such as histamine, provokes the extravasation of a protein-rich plasma exudate; also like histamine, the primary target of VEGF-A action is postcapillary venules and small veins whose lining endothelial cells express the two VEGF-A tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR, flk-1

Figure 35-3

Lewis lung carcinoma growing in flank of a syngeneic C57BI/6 mouse that had received a macromolecular tracer, 70 kD fluoresceinated dextran, 15 minutes previously. Bright-staining apple-green fluorescence forming a rim around the tumor represents extensive (more...)

An important and almost immediate consequence of VEGF-A action is leakage of plasma proteins, including fibrinogen and other clotting factors. Vascular hyperpermeability and extravasation of plasma proteins leads to activation of the coagulation system by a tissue factormediated mechanism.¹⁴ As a result, extravasated plasma fibrinogen is rapidly clotted to form an extravascular gel of crosslinked fibrin (see Figure 35-2). Extravascular fibrin deposits are important because they dramatically alter the local microenvironment, transforming the erstwhile inert extravascular matrix of normal adult tissues into a proangiogenic provisional matrix that favors and apparently stimulates inward migration of host mesenchymal cells.^10,13 Indeed, fibrin implanted in animals without tumor cells induces the invasion of new blood vessels and fibroblasts, resulting in a vascularized connective tissue that is not dissimilar in appearance or composition to tumor stroma. Other plasma proteins (eg, plasma fibronectin), as well as locally synthesized structural proteins (eg, cellular fibronectins, tenascin), hyaluronan, and proteoglycans (eg, chondroitin sulfate-rich proteoglycan and decorin) also contribute to this new tissue.^9,10,13

The fibrin gel deposited by tumors is modulated by proteases (see Figure 35-2) and is gradually replaced by the ingrowth of fibroblasts and new blood vessels which give rise to loose connective tissue, similar to the “granulation tissue” of healing wounds. After an additional period of time, this granulation tissue is further transformed into the poorly vascularized, densely collagenous scar-like connective tissue characteristic of tumor desmoplasia. Simultaneously, of course, other tumor cells have broken away from the original tumor site and have begun to recapitulate at nearby sites and particularly at the tumor's growing edge the same sequence of events—increased vascular permeability and new fibrin deposition. Thus, at any one time, growing desmoplastic tumors consist of older, generally more centrally placed portions comprising tumor cells that are encased in poorly vascularized, dense collagenous stroma and a more active, newer, fibrin-rich peripheral zone that interfaces with the surrounding host tissue.

VEGF-A: A Multifunctional Cytokine Critical to Tumor Angiogenesis and Stroma Formation

At this point, something more needs to be said about VEGF-A because of its central role in tumor angiogenesis and stroma generation (for more detailed accounts, see recent reviews^10–12). VEGF-A is expressed in several different isoforms as the result of alternative splicing of a single, highly conserved gene. VEGF-A is the founding member of a family of proteins whose members include placenta growth factor and VEGF B, C, D, and E.¹¹ At present, much more is known about VEGF-A than about other, more recently discovered family members. In addition to its potent function as an effector of vascular hyperpermeability, VEGF-A has other important actions that contribute importantly to angiogenesis and stroma generation. Thus, VEGF-A stimulates endothelial cell division and migration. It also induces endothelial cells to express increased amounts of tissue factor, urokinase, tissue plasminogen activator and matrix metalloproteases.^10–12 Collectively, these endothelial cell products induce clotting and initiate fibrinolysis and degradation of collagen and other elements of preexisting matrix, all-important steps in angiogenesis and stroma generation.

A number of factors serve to regulate VEGF-A expression in tumor cells. VEGF-A expression is greatly stimulated by tissue hypoxia and, perhaps independently, by low tissue pH, conditions that are commonly present in the tumor microenvironment.^10–12 However, it would be a mistake to think that hypoxia is the only factor responsible for VEGF-A overexpression by tumor cells. Many tumor cells make substantial amounts of VEGF-A under normoxic or even hyperoxic conditions. Other factors that induce overexpression include cytokines (eg, epidermal growth factor, basic fibroblast growth factor), certain hormones (eg, thyroglobulin), and, perhaps of more general interest, various oncogenes (eg, src, ras) and tumor suppressor genes including the von Hippel Lindau protein.^10–12

Relation of Tumor Stroma Generation to Wound Healing and Other Examples of Pathologic and Physiologic Angiogenesis and Stroma Generation

The events of stroma generation in transplantable tumors closely mimic those of normal wound healing.^4,15 As in tumors, VEGF-A expression is strikingly upregulated in healing wounds as well as in a variety of analogous pathologic and physiologic processes that involve new blood vessel and stroma formation; these include rheumatoid arthritis, psoriasis, delayed hypersensitivity, diabetic retinopathy, and corpus luteum formation.^10,15 In all these processes, the initial event is a local increase in vascular permeability, followed, in turn, by extravascular clotting, fibrin deposition, and infiltration of new blood vessels and connective tissue cells, leading to the development of granulation tissue and finally of dense fibrous connective tissue (termed “desmoplasia” in tumors and “scars” or fibroplasia in the other entities). It would seem, therefore, that tumors have preempted and subverted, for their own purposes, a fundamental host mechanism, the wound healing response, as the means to acquire the stroma they need to grow and spread.⁴ Of course, there are some differences. Platelets, which play several critical roles in wound healing, seem not to participate to any great extent in tumor stroma generation; however, many platelet functions can be subsumed by tumor cells, which express similar or analogous cytokines and growth factors. Tumors differ from healing wounds in another important respect. At wound sites, overexpression of VEGF-A and consequent vascular hyperpermeability are limited to a period of a few days, presumably until oxygen tension has returned to normal.¹⁵ By contrast, VEGF-A expression and vascular hyperpermeability are not limited in tumors and persist indefinitely. Thus, tumors behave in some sense as wounds that do not heal.⁴

The analogy between wound healing and tumor stroma generation may be taken one step further. Except in lower vertebrates that are capable of regenerating normal tissues, wound healing does not recapitulate ontogeny but, instead, replaces injured parenchyma and stroma with connective tissue whose functional capacities fall well short of the original normal tissue. In the same manner, the stroma of malignant tumors is generally a disorganized and poorly supportive parody of normal connective tissue. The vascular supply is often marginal. Tumor blood vessels are generally poorly differentiated, unevenly spaced, and often unequal to the task of supporting the growth and even the life of rapidly metabolizing tumor cells.¹⁶ The result is irregular blood flow, uneven perfusion, shifting zones of anoxia, low pH, and, commonly, necrosis and apoptosis.¹⁷ In fact, the presence of necrosis is sometimes helpful to the pathologist in distinguishing malignant tumors from their benign counterparts and certain nonneoplastic processes.

Stroma Generation in Autochthonous Human Tumors

Detailed, interventional studies of the type required to elucidate the pathogenesis of tumor stroma generation in animal tumors are not ethically feasible in patients. Nonetheless, there is good reason to believe that similar mechanisms are involved in human malignancy. First, VEGF-A is overexpressed at both the mRNA and protein levels in the great majority of primary and metastatic human tumors that have been studied; these include carcinomas arising in the gastrointestinal tract, pancreas, stomach, breast, ovary, kidney and bladder, as well as glioblastomas.^10,12 Second, both specific, high-affinity receptors for VEGF-A are also overexpressed in the microvascular endothelial cells that supply these tumors. Finally, many human tumors exhibit evidence of vascular hyperpermeability to plasma proteins, including spillage of fibrinogen with deposition of crosslinked extravascular fibrin as observed in animal tumors.^4,12 Taken together, there is strong evidence that the pathogenesis of stroma formation in human tumors closely follows that in animal tumors, although allowance must be made for species differences and for the generally slower growth rate of autochthonous human tumors.