Clin. Cancer Res., 9: 1639 −1647, 2003

Tumors and Their Microenvironments: Tilling the Soil : Commentary re: A. M. Scott et al., A Phase I Dose-Escalation Study of Sibrotuzumab in Patients with Advanced or Metastatic Fibroblast Activation Protein-positive Cancer. Clin. Cancer Res., 9: 1639 −1647, 2003.

Jonathan D. Cheng and Louis M. Weiner

Clin Cancer Res 2003;9:1590-1595.

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The Biology Behind Tumors and Their Microenvironments: Tilling the Soil Commentary re: A. M. Scott et al., A Phase I Dose-Escalation Study of Sibrotuzumab in Patients with Advanced or Metastatic Fibroblast Activation Protein-positive Cancer. Clin. Cancer Res., 9: 1639–1647, 2003.

Jonathan D. Cheng,1 and Louis M. Weiner “seminal influence” on the metastatic microenvironment, and Fox Chase Cancer Center, Department of Medical Oncology, thereby act together with the distant organ to effect tumor Philadelphia, Pennsylvania 19111 metastases. The identity of these seminal influences is a subject of active investigation but remains elusive. However, The Shifting Paradigm the importance of tumor-associated fibroblasts, the predom- The conventional concept of tumor-host interactions is inant cell in the microenvironment, has become more evident. rooted in the realm of parasitology. A tumor invades an unsus- pecting host, and in response to this assault, the host attempts to Tumor Stromal Fibroblasts: Contracted Farmers resist the advances of the tumor by calling on the armamentar- Both in vivo and in vitro studies have demonstrated that ium of the immune system. This implies a fundamental oppo- fibroblasts contribute to tumor formation and growth rates sition between the tumor and host in purpose and intent. How- (6), and can be thought of as “contracted farmers” used by ever, an accumulation of evidence points to an alternative tumors to prepare the microenvironment. Fibroblasts coin- paradigm, where the tumor microenvironment is not an idle oculated with breast or bladder tumor cell lines in nude mice bystander, but actively participates in tumor progression and shorten tumor latency and increase tumor growth (7). Fibro- metastasis (1, 2). In fact, stromal cells and their cytokines blasts cultured from malignant tumors have stimulatory ef- coordinate critical pathways that exert important roles in the fects on MCF-7 cells, whereas fibroblasts cultured from ability of tumors to invade and metastasize (3). normal tissue are inhibitory (8). Phenotypic differences In this issue, Scott et al. (4) describe a Phase I clinical trial among tumor-associated fibroblasts have also been seen. using the radiolabeled antibody sibrotuzumab to target the tu- Fibroblasts with smooth muscle differentiation, termed myo- mor stromal antigen FAP2 in metastatic lung and colon cancers. fibroblasts, are abundant in the stromal cells of malignant Human FAP is unique in its selective expression by tumor breast tissue but are rarely seen in normal breast tissue (9). stromal fibroblasts in epithelial carcinomas, but not by epithelial These findings suggest that tumor-associated fibroblasts are carcinoma cells, normal fibroblasts, or other normal tissues. functionally distinct compared with fibroblasts that are not in Therefore, FAP is an attractive target for the study of tumor the tumor microenvironment, and subpopulations of fibro- stromal cell biology and provides valuable insights into the roles blast may perform specialized functions to coordinate events of the tumor microenvironment. FAP may exemplify pathways required for tumor invasion and metastasis. Thus, fibroblasts by which the microenvironment soil is “tilled” in preparation for provide critical frameworks for tumor growth and metastasis. tumor invasion and metastasis. The microenvironment can also influence tumorigenic transformation. Kinzler and Vogelstein (10) proposed the The Tumor Microenvironment Soil concept of landscaper defects to explain the increased inci- The process of tumor metastasis was described using the dence of colorectal cancer in patients with juvenile polyposis “seed and soil” analogy by Paget (5) more than 100 years syndrome or ulcerative colitis. In this situation, the prolifer- ago. His landmark paper in 1889 examined 735 patients with ating stromal cells predispose the normal epithelial cells to breast cancer at autopsy and showed that the distribution of undergo malignant transformation. Thus, stromal inflamma- metastases did not occur by chance, but was regulated by the tion and proliferation eventually lead to the development of predisposition of congenial soil. For example, the liver had a malignant epithelial transformation because of an altered 14-fold higher incidence of metastases compared with the microenvironment terrain. Thus, the gene expression pattern spleen, although these organs roughly possess the same cir- of the fibrotic stroma is distinct from that of the carcinoma culatory volume. Paget (5) proposed that tumor cells, or cells. Iacobuzio-Donahue et al. (11, 12) used serial analysis “seeds,” were randomly scattered by vascular routes, but of gene expression techniques in breast cancer to show that could only form metastatic deposits if they landed in conge- five regions of invasive tumors can be identified, each with a nial territory, or “soil.” He hypothesized that tumors have a distinct gene expression profile. These regions consist of the neoplastic epithelium, angioendothelium, inflammatory stroma, panstroma, and juxtatumoral stroma. Furthermore, they identified receptor-ligand pairs between the neoplastic epithelium and the surrounding stroma, implying significant Received 3/25/03; accepted 3/28/03. communication between host and tumor compartments. In 1 To whom requests for reprints should be addressed, at Fox Chase addition, loss of heterozygosity has been identified in the Cancer Center, Department of Medical Oncology, 7701 Burholme Av- enue, Philadelphia, PA 19111. Phone: (215) 728-7083; Fax: (215) 728- fibrotic reaction surrounding pancreas cancer that is distinct 3639; E-mail: [email protected]. from the loss of heterozygosity pattern in pancreas adenocar- 2 The abbreviations used are: FAP, fibroblast activation protein. cinoma (13). A number of these genes identified in the Downloaded from clincancerres.aacrjournals.org on November 12, 2012 Copyright © 2003 American Association for Cancer Research Clinical Cancer Research 1591

stroma had not been associated previously with pancreatic ing wounds, can provide fertile soil for malignant cell “hom- adenocarcinoma. These studies imply that tumor stroma may ing.” Experimental animal models demonstrate that tumor me- contribute independently to tumor development through tastases arise preferentially in healing wounds (25, 26). The mutation-inducing phenotypes. Such considerations chal- incidence of wound recurrences is proportional to the extent of lenge preexisting notions of a passive stroma that is permis- wound trauma, as larger wounds and wounds that are not sur- sive of local tumor growth. gically repaired exhibit greater frequencies of metastasis. This has been confirmed clinically in the occurrences of surgical FAP: A Tractor wound and port site metastases (27, 28). Given the ability of the The current study by Scott et al. (4) demonstrates the wound healing environment to promote tumor cell proliferation ability to safely administer the humanized anti-FAP antibody (29), tumors may subvert normal wound healing machinery to sibrotuzumab to patients with FAP-positive malignancies with activate factors necessary for tumor metastases. Therefore, tu- tumor-specific uptake as seen by biodistribution images. This mor cells may initiate a permissive microenvironment by induc- clinical trial is an extension of the groundbreaking work by ing expression of permeability factors, procoagulants, or che- Garin-Chesa et al. in discovering the unique distribution of FAP motactic and mitogenic factors. on tumor stromal fibroblasts (14), and has been confirmed by Similarly, the fibrotic reaction seen in desmoplasia tends to Welt et al. (15) as a clinical target. FAP, also known as seprase confer a worse prognosis and may represent a histopathologic marker of fertilized soil. Invasive ductal carcinoma of the breast (16), is a Mr 95,000 cell surface glycoprotein expressed transiently in healing wounds, and abundantly on reactive stromal with a fibrotic focus has more aggressive clinical characteristics fibroblasts in Ͼ90% of human epithelial carcinomas (breast, than carcinoma without fibrosis (30, 31). The identification of lung, colorectal, and ovary; Ref. 14). Neural and lymphoid cells, fibrotic foci predicts worse overall survival, a higher incidence as well as surrounding normal tissue are FAP-negative. Bone of lymph node and distant organ metastases, greater angiogenic and soft tissue sarcomas occasionally express FAP (17), con- activity, and a higher tumor cell proliferation rate. Similar sistent with the mesenchymal origin of FAP. FAP expression in observations have correlated desmoplasia of colorectal carcino- normal tissue has only been seen in a subset of pancreatic mas with liver metastases (32) and worse prognosis (33). In endocrine cells and transiently in healing wounds. The specific- addition, focal fibrosis correlates with the proliferative activity ity of FAP for tumor fibroblasts but not tumor cells has been of tumor-associated fibroblasts (34). These studies suggest that confirmed by immunohistochemical staining in colorectal proliferating fibroblasts in the tumor microenvironment gener- cancer patients (15, 18), breast cancer patients (19), reverse ate fibrotic foci and increase the invasive characteristics of transcription-PCR of pancreas, lung, and renal cell xenografts carcinomas. The mechanisms by which this occurs are un- (20), mixed hemadsorption rosetting assays from breast cancer- known, although the many similarities of tumor stroma desmo- derived cultured fibroblasts (21), and renal cancer-derived plasia with wound healing pathways have been noted (29). fibroblasts (22). FAP exhibits both dipeptidyl peptidase and Because fibroblasts are responsible for the desmoplastic collagenase proteolytic activity (18). Thus, FAP can cleave reaction (35), as well as the production of the majority of proteinases in the tumor microenvironment (36, 37), it is rea- NH2-terminal dipeptides from polypeptides with L-proline or L-alanine in the penultimate position (23), as well as degrade sonable to hypothesize that the desmoplastic reaction is a his- both gelatin and native type I collagen (23). Mutation of the topathologic marker of proteinase activity by the tumor fibro- catalytic serine residue abolishes FAP dipeptidyl peptidase and blasts. In such a scenario, tumor cells induce surrounding collagenase activity (18). The clinical antibody sibrotuzumab fibroblasts to express a number of proteases to facilitate tumor does not appear to affect FAP enzymatic function, one of the invasion by remodeling extracellular matrix, inducing angiogen- most intriguing aspects of this target. esis, and promoting motility. This protease milieu initiates a We have demonstrated that FAP overexpression in an fibrotic reaction in the tumor microenvironment, with subse- animal model results in a greater propensity for tumor develop- quent scarring that is manifested histologically as the fibrosis of ment, a shortened latency period, and an enhanced growth rate desmoplasia. Thus, the tumor stroma provides critical factors, compared with mock-transfected controls (24). Because FAP is distinct from those produced by the neoplastic cells, that are not expressed by epithelial carcinomas, but only by tumor required for tumor invasion and metastases. fibroblasts, this provides the proof-of-principle that this stroma- specific protein can alter tumor growth properties. If tumor FAP and Frogs fibroblasts can be thought of as contracted farmers hired by FAP has been identified not only in wound healing and tumors to prepare the microenvironment soil, then FAP would desmoplasia, but also in amphibian metamorphosis. Amphibian be analogous to the tractors the fibroblasts use to till the soil so tail resorption in tadpoles induces a set of complex develop- tumors can flourish. mental changes similar to those seen with tumor invasion and metastasis. These changes include epithelial proliferation, apo- Fallow Ground of Wound Healing and Desmoplasia ptosis, and tissue remodeling with redistribution. The invasion FAP is also expressed in healing wounds and in desmopla- of the collagen basement lamella by mesenchymal cells during sia. The congenial territory that Paget (5) described may be tail resorption is strikingly similar to tumor invasion. Proteolytic demonstrated clinically in the fallow ground of wound healing enzymes that are commonly seen in the tumor stroma have been and desmoplasia (Fig. 1B). An activated environment in the identified as part of the tail resorption program. These include absence of malignant transformation, such as that seen in heal- collagenase 3, stromelysin 3, and FAP (38). In fact, FAP ex-

Fig. 1 FAP: a model for tumor-stromal epithelial interactions.

pression is highest within the subepidermal fibroblast layer of growth potentiating factors. Therapeutic interventions directed the resorbing tail (39). This is consistent with the tumor fibro- against the stroma are more likely to have clinical benefit by blast localization of FAP in the tumor microenvironment. Thus, altering dormant metastasis progression into metastatic storm, there is a striking convergence of FAP expression in the tumor rather than by arresting metastases once the microenvironment microenvironment, in healing wounds, and in physiological has been activated. Indeed, we have shown that only moderate organ remodeling. tumor growth attenuation can be achieved in an animal model using FAP inhibitory antibodies (24). Many clinical trials that have targeted the tumor microenvironment, including large ran- Not All Seeds Germinate domized Phase III clinical trials testing both antiangiogenic Multiple tumor-stromal interactions and extensive cross- agents (45) and matrix metalloproteinase inhibitors (46), have talk are required to coordinate the multiple events of the meta- resulted in limited clinical success. This may be partially ex- static process. As shown in Fig. 1, epithelial cells in the normal plained by the redundancy of tumor:microenvironment interac- quiescent state are not transformed, and the stromal compart- tions, as the inhibition of any single protease family or pathway ment is in homeostasis. Through the accumulation of multiple is diluted by the ability of tumors to use other mechanisms to mutations, malignant epithelial transformation occurs. How- accomplish similar ends. ever, cancer cells require assistance to accomplish the complex series of events required for invasion and subsequent metastasis. One approach to overcoming this obstacle is to inhibit Malignant cells usurp their microenvironments to synthesize multiple stations along any given pathway or to target mul- necessary growth factors, cytokines, angiogenesis molecules, tiple discrete pathways. This strategy has been used in a wide adhesion molecules, and proteases to break down the extracel- assortment of clinical treatments, such as antibiotics (e.g., lular matrix. If malignant cells are unable to activate these trimethoprim-sulfamethoxazole), HIV disease (e.g., protease determinants of metastases, clinical metastases do not occur, inhibitor mixtures), and some combination chemotherapy and these cells remain in a dormant condition. This setting is programs for prostate cancer (e.g., estramustine/paclitaxel). akin to metastases before initiation of the “angiogenic switch” These approaches use noncross-resistant treatments targeting (40, 41) and can be observed clinically in autopsy findings of the same mechanistic pathway, such as folic acid synthesis malignant cells lodged in the pulmonary vasculature, but with- for trimethoprim-sulfamethoxazole or microtubule formation out frank metastasis (42). for prostate cancer treatment. Clinical efficacy can be dem- Experience with peritoneovenous shunts has provided clin- onstrated if the pathway targeted is of sufficient centrality to ically relevant confirmation that metastases are actually rare the disease process. Therefore, identification of the crucial events. Animal studies have demonstrated that large numbers of stations for targeting tumor-stromal interactions is critically tumor cells are shed from primary tumors into the vascular important. system (43, 44). However, only a fraction of these circulating In conclusion, tumor invasion and metastasis is a complex tumor cells form metastatic tumor deposits. Peritoneovenous process requiring the coordination of multiple events. Given the shunts alleviate abdominal distension from malignant ascites by complexity of events required for clinical metastases to develop, returning fluid from the peritoneal cavity into the venous circu- cancers cells cannot metastasize without assistance from host lation through one-way valves, infusing large numbers of ma- elements. Therefore, cancers induce stromal elements, in par- lignant tumor cells directly into the venous circulation. In a ticular tumor fibroblasts, as active participants in the process of study of 29 patients with locally advanced abdominal tumors invasion and metastasis by expressing stromal proteins such as requiring peritoneovenous shunts for malignant ascites (42), Ͼ100 million malignant cells were infused into the circulating FAP. 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