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Cell Growth & Differentiation Vol. 13, 387-395, August 2002
© 2002 American Association for Cancer Research

Vascular Endothelial Growth Factor and Kaposi’s Sarcoma Cells in Human Skin Grafts1

Felipe Samaniego2, Daniel Young, Cara Grimes, Vanessa Prospero, Melpo Christofidou-Solomidou, Horace M. DeLisser, Om Prakash, Aysegul A. Sahin and Suizhao Wang

Department of Lymphoma/Myeloma [F. S., C. G., V. P.], Clinical Cancer Prevention, Departments of Clinical Cancer Prevention [F. S., C. G., V. P., D. Y., S. W.] and Pathology [A. S.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 [M. C-S., H. M. D.]; and Ochsner Clinic Foundation, New Orleans, Louisiana 70121 [O. P.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 
Human cancer cells often produce tumors in animal models that incompletely reproduce the histology of the parental tumor. Kaposi’s sarcoma (KS) cells, in particular, have not produced durable angiogenic lesions in animal models that resemble those of KS in humans. We investigated the contribution of transformed KS cells, vascular endothelial growth factor (VEGF), and human skin tissue on tumor development in a human skin graft/mouse model. High levels of serum VEGF (322 pg/ml) were seen in HIV-1-infected persons with KS compared with HIV-1-infected persons without KS (115 pg/ml). Human KS lesions expressed VEGF in the spindle cells. Transformed KS cells expressed the mitogenically active 121-amino acid and 165-amino acid isoforms of VEGF. Tumors induced by KS cells implanted in the SCID mice grew preferentially in human skin grafts rather than in ungrafted murine skin. Tumors induced in the presence of human skin grafts developed numerous lumens expressing {alpha}vß3 integrin. KS cells inoculated with neutralizing anti-VEGF antibody did not form tumors. This study supports an important role for VEGF in tumor development and shows how a human tissue can preferentially promote tumor growth.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 
KS3 is an angioproliferative disease that frequently appears in persons infected with HIV-1, particularly homosexual men. Although many infectious agents have been associated with the emergence of KS, only HIV-1 and HHV-8 (also known as KS-associated herpesvirus) have been strongly implicated in the induction of KS (1, 2, 3) . All clinical epidemiological forms of KS contain HHV-8 in tumor tissue. HHV-8 is present in most cells of KS lesions with the majority of cells containing the virus in latent form. The cells containing HHV-8 share marker expression of endothelial and macrophage/monocyte cell lineages. The association of HHV-8 with all of the clinical-epidemiological types of KS (HIV-1-associated, those affecting solid organ transplant recipients, classical, and African) and their common histopathological appearance suggest a common pathogenesis but disparate rates of clinical progression (4) . In addition, KS has been regarded as an infrequent and indolent disorder that in the last decades has been shown to be a very frequently occurring and progressive disorder in individuals with HIV-1 infection (5, 6, 7) .

We and others have suggested that, at least in the early stages of KS, tissue inflammation drives cellular proliferation to produce the characteristic hyperplastic and angiogenic features of KS lesions (8 , 9) . KS lesions are composed of a mixed-cell infiltrate with hyperplastic spindle-shaped cells that resemble cytokine-activated ECs and monocytes/macrophages by their expression of tissue-specific markers (CD34, vascular-endothelial cadherin, endothelial leukocyte adhesion molecule type 1, CD4, CD14, CD68, and PECAM-1; Refs. 9, 10, 11, 12 ). The most common cell type isolated from HIV-1-associated KS (AIDS-KS) has been the spindle-shaped endothelial-like cells that proliferate in response to inflammatory cytokines and have a limited replicating life span. The highly angiogenic tissue of AIDS-KS lesions is believed to be mediated by bFGF, VEGF (13) , and (in HIV-1-related KS) the transactivating protein HIV-1 Tat (14 , 15) . VEGF and bFGF coupled with integrins interact synergistically to promote angiogenesis. An angiogenic factor paired with an anchorage signal through integrins are prerequisites for angiogenesis.

AIDS-KS spindle cells, cell isolates from KS lesions of HIV-1-infected individuals, are hyperplastic nontransformed cells that proliferate in culture with inflammatory cytokines. These cell isolates initially contain HHV-8 that is rapidly lost as the cells are passaged in culture. AIDS-KS spindle cells are characterized by morphological characteristics, marker expression, and angiogenic factor production (16, 17, 18) . Endothelial cells treated with inflammatory cytokines acquired many of the features of AIDS-KS spindle cells, suggesting that endothelial lineage may give rise to the KS tumor cells.

Cell lines composed of transformed cells have also been isolated from advanced KS lesions. KS cell lines have been isolated from KS lesions (KS SLK and KS IMM) from two HIV-1 noninfected renal transplant recipients. These cell lines, unlike AIDS-KS spindle cells, are transformed cells that grow in the absence of inflammatory cytokines, contain cytogenetic abnormalities, and induce durable tumor lesions when inoculated in nude mice (19, 20, 21, 22, 23) . Similar to the hyperplastic KS spindle cells, KS transformed cells also lack HHV-8.

Although xenograft models have shown some of these in vivo effects, they are still considered inadequate for studying tumorigenesis, in part because of anticipated limited interspecies cell-cell communication (24) . This can be particularly critical in studies when attempting to reproduce histological features of tumors such as KS that are largely represented by nontransformed stromal cells, such as endothelial and mononuclear cells. An animal model would theoretically be optimized by testing of human tumor cells in human tissue (24, 25, 26, 27) . Testing of KS cell growth in human tissue would offer additional insights into its pathogenesis.

Thus, we undertook this study to gain further insights into possible contributory mechanisms of transformed KS cells and human skin tissue to KS histopathology. We studied whether: (a) VEGF is produced in human KS lesions; (b) the levels of VEGF in the serum of HIV-1-infected individuals reflect the presence of KS; (c) two transformed KS cell lines (KS SLK and KS IMM) that represent nodular or late-stage KS also express VEGF and the {alpha}vß3 integrins critical for angiogenesis; (d) KS SLK cells implanted in xenografted human skin tissue cooperate in generating KS-like lesions that better mimic human KS; and (e) blockage of VEGF can interfere with tumor lesion formation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 
Sera VEGF Levels Are Elevated in HIV-1-infected Individuals with KS.
The levels of VEGF in serum have been linked to the presence and stage of tumors (28 , 29) . We reasonably anticipated that individuals with a highly angiogenic cancer such as KS would exhibit circulating concentrations of this cytokine, reflecting tumor presence and burden. By measuring serum VEGF levels in HIV-1-infected individuals with KS (n = 28) or without KS (n = 24), we found that VEGF concentrations ranged from 25 to 2100 pg/ml, with individuals with KS having the seven highest values. The data did not appear to be normally distributed, and a nonparametric statistical analysis was therefore applied (Fig. 1)Citation . A Mann-Whitney test comparing the two groups found statistically significant higher VEGF levels in the patients with KS (P = 0.02). The individuals with the highest VEGF levels did not exhibit particularly extensive KS, inflammation, or altered immunity. The difference in VEGF levels from individuals with or without KS may be better reflected in plasma versus serum (30 , 31) , but only sera was available in this retrospective analysis. The levels of IL-12 (15 pg/ml) were not different in the sera of HIV-1-infected individuals with or without KS.



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Fig. 1. VEGF concentrations in serum from HIV-1-infected individuals with or without KS. VEGF was measured in duplicate 100 µl samples of serum by ELISA. Values are the means of duplicate samples. VEGF concentrations were significantly higher in persons with KS than in persons without KS (P = 0.02, Mann-Whitney test). Serum levels of IL-12 (15 pg/ml) were not different in individuals with or without KS.

 
VEGF is composed of 121, 165, 189 and 206 amino acid isoforms with variable cellular localizations and cellular effects. To characterize the isoforms of VEGF produced by transformed KS cells, we selected representative cells of KS that also produce tumors in human skin-SCID mice, i.e., KS SLK cells, for analysis after these cells were metabolically labeled with [S35]methionine and [S35]cysteine. The combined cell extracts and cell supernatants were immunoprecipitated with anti-VEGF polyclonal antibody and visualized by SDS-PAGE (Fig. 2A)Citation . The immunoprecipitant of anti-VEGF antibody that was preabsorbed with saturating levels of targeted synthetic VEGF peptide showed the disappearance of three bands (Fig. 2ACitation , Lane +, arrows) compared with immunoprecipitant with anti-VEGF antibody alone (Fig. 2ACitation , Lane -). The three bands (Mr 16,000, 18,000, and 24,000), therefore, represented proteins related to VEGF. The Mr 16,000 and 18,000 monomers are known products of the 121-amino acid form of VEGF, and the Mr 18,000 and 24,000 monomers are known products of the 165-amino acid isoform of VEGF, depending on glycosylation modifications. Thus, the results of this radioimmunoprecipitation assay agree with coexpression of 121- and 165-amino acid isoforms of VEGF, which are detected in cell supernatants by ELISA (see below) and also present in the serum of individuals (see above; Ref. 32 ). The 121- and 165-amino acid isoforms of VEGF are also produced at high levels by hyperplastic AIDS-KS cells and are believed to be the isoforms that contribute to KS-like lesion formation (13 , 33 , 34) .



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Fig. 2. KS SLK and KS IMM cells express VEGF isoforms and {alpha}vß3 integrin receptor. KS SLK and KS IMM produced 1520 and 1150 pg/ml of VEGF, respectively (see "Results"). Given this similar production, we selected the KS SLK cells to identify VEGF isoforms. A, KS SLK cells were metabolically labeled with [S35]methionine and [S35]cysteine. Cell extracts and supernatants were combined and immunoprecipitated with anti-VEGF polyclonal antibodies as described in "Patients and Methods." KS SLK cells showed three bands of Mr ~16,000, 18,000, and 24,000 (arrows) that were absent in the immunoprecipitant of antibody preabsorbed with immunogen VEGF peptide. Lane +, immunoprecipitant using anti-VEGF antibody that has been preabsorbed with immunizing peptide. Lane -, labeled proteins immunoprecipitated with anti-VEGF-antibody alone. B and C, KS SLK and KS IMM cells express the {alpha}vß3 integrin receptor. One million cells were fixed and labeled with anti-{alpha}vß3 integrin antibody and then examined by flow cytometry. B, the boldface tracing on the right represents the signal with anti-{alpha}vß3 integrin antibody of KS SLK cells; the tracing on the left represents the signal of the control isotype antibody. C, anti-{alpha}vß3 integrin antibody staining of KS IMM cells using similar methods. D, KS SLK, KS IMM, and dermal microvascular endothelial cells (DMEC) expressed the ß3 subunit of vitronectin receptor (arrow), which is expressed at lower levels in peripheral blood lymphocytes (PBL).

 
Having identified the major isoforms of VEGF produced by KS cells, we then quantified the levels of VEGF in cell culture supernatants by ELISA. The supernatants of the transformed KS cell lines KS SLK and KS IMM contained 1520 and 1150 pg/ml of VEGF, respectively. The cell extracts did not contain VEGF, indicating that the cytokine is primarily secreted. In comparison, cell supernatants and extracts of dermal microvascular ECs and their supernatants did not contain VEGF, as noted previously (35) . Localization of the VEGF in the extracellular non-cell-associated compartment is in agreement with the cellular localization pattern of the 121- and 165-amino acid isoforms of VEGF. Thus, transformed KS cells were found to produce VEGF in isoforms and at levels that are known to stimulate EC proliferation and may contribute to the characteristically angiogenic appearance of KS lesions (13 , 34) .

The {alpha}vß3 Integrin Receptor Is Expressed by Transformed KS Cells.
Angiogenic factors such as VEGF and bFGF cooperate with specific integrins in angiogenesis and tumor development. VEGF is known to cooperate with the {alpha}vß3 integrin to induce a broad spectrum of cellular responses (36 , 37) . To determine whether the transformed type of KS cells expresses {alpha}vß3 integrin, we analyzed KS SLK and KS IMM cells for {alpha}vß3 integrin expression (Fig. 2, B and C)Citation . KS SLK cells spontaneously expressed {alpha}vß3 integrin (boldface tracing) compared with signal from the same cells stained with isotype antibody (non-boldface tracing; Fig. 2BCitation ). KS IMM cells analyzed by similar methodology showed a more intense level of {alpha}vß3 integrin expression (Fig. 2C)Citation . To demonstrate expression of this integrin receptor by a second method, cell extracts of these cells were subjected to immunoblot analysis. Filters blotted with anti-ß3 antibody showed the presence of a Mr 105,000 band for KS SLK, KS IMM, and dermal microvascular endothelial cells and at lower levels in peripheral blood lymphocytes (Fig. 2DCitation , arrow). Thus, the two transformed KS cell lines showed evidence of {alpha}vß3 integrin expression, which is known to be expressed in situ by KS cells and is essential for proliferation and angiogenesis (38, 39, 40) .

Induction of KS-like Tumors with Transformed KS Cells Inoculated into Human Skin Grafts.
The potential contributions of transformed KS cells and of human skin tissue to KS development have heretofore not been examined. To determine whether KS cells in human skin tissue would generate KS-like lesions and to determine whether the histological features of KS pathology could be produced in this model, we inoculated KS SLK cells under engrafted human skin on SCID mice. Because of the limited resources, only one cell line (both KS SLK and KS IMM cells produce comparable levels of VEGF) was selected for these studies (22 , 41) . Inoculation of KS SLK cells under human grafts produced substantially larger tumors, whereas sites inoculated under mouse skin showed small lesions, if any (Fig. 3)Citation . The mean volumes of tumors under neonatal and adult skin xenografts (180 ± 120 mm3 and 170 ± 121 mm3, respectively) were significantly larger (P < 0.005 and P < 0.005, respectively) than the mean volume of tumors grown under endogenous murine skin (15 ± 1 mm3). Thus, KS SLK cells showed preferential tumor growth when inoculated in human skin.



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Fig. 3. Human skin grafts on SCID mice promote the growth of tumors induced by KS SLK cells, and anti-VEGF antisera blocks tumor development. Two million KS SLK cells were inoculated under a human (neonatal) foreskin graft, a human adult skin graft, or nongrafted skin of SCID mice and observed for tumor growth. After 3 weeks, the sites were examined macroscopically for lesions; tumor volume was measured, and lesions were resected. The mean tumor volumes present under both the human neonatal and adult skin grafts (180 ± 120/mm3 and 170 ± 121/mm3, respectively) were significantly larger (P < 0.0005 and P < 0.0005) than the volume of tumors under the endogenous mouse skin (15 ± 1/mm3). In another set of experiments, 2 million KS SLK cells were combined with 50 µg of control antiserum or anti-VEGF antisera, and the combination was inoculated under human skin grafts. After 3 weeks, the sites were examined macroscopically for lesions; tumor volume was measured, and lesions were resected. The mean tumor volume of sites inoculated with control antisera (170 ± 58 mm3) was significantly larger (P < 0.0005) than the volume of tumors from sites inoculated with anti-VEGF antisera (10 ± 1/mm3). Neonatal skin was derived from resected foreskin; adult skin was from mastectomy resection. Each column represents the volume of one tumor lesion from one mouse. Inoculation sites under murine skin were made under skin contralateral to skin grafts.

 
VEGF Antiserum Prevented Tumor Induction in Human Skin Graft.
VEGF is known to cooperate with integrins to induce a broad spectrum of cellular responses (37) . To examine the role of VEGF in tumor development, we performed cell culture experiments that showed that anti-VEGF blocked the proliferation of ECs but not of KS SLK cells (see below). To determine whether VEGF also contributes to KS-like lesion formation, we inoculated, under the human skin grafts, KS SLK cells (2 x 106) that were mixed ex vivo with 50 µg of anti-VEGF antibody or control antibody. Because of the costly expense of long-term animal experiments in these studies, we used only the KS SLK cell line, which has consistently produced tumors in mice. Four grafts inoculated with KS SLK cells and control antibody showed palpable lesions after 2 weeks and at pathological examination had a mean lesion size of 170 ± 58 mm3 (Fig. 3)Citation . The grafts inoculated with anti-VEGF antibody and KS SLK cells did not contain palpable lesions after 1 and 2 weeks. Mice were sacrificed and evaluated by pathological inspection at four grafts inoculated with anti-VEGF antisera KS SLK cells showed minute lesions (Fig. 3)Citation . Thus, VEGF may be required for the development of tumors induced by KS SLK cells in human skin.

Immunohistochemistry of Human KS Lesions.
To examine the role of VEGF in human KS, we stained sections of four human patch-stage KS lesions with anti-VEGF antibody. Approximately 10–30% of the cells in these lesions expressed VEGF (Fig. 4, A–C)Citation . The cells demonstrating VEGF staining were cells with spindle morphology, consistent with descriptions of spindle cells in KS lesions (35 , 42) . Anti-VEGF antibody also stained some mononuclear cells; morphologically, however, the predominant VEGF-expressing cell type was spindle shaped. Staining of tissues using this same methodology but replacing anti-VEGF antibody with PBS showed no marker staining. Lymphoid tissue (lymph node) did not show staining for VEGF except for rare cells that were morphologically similar to lymphocytes. Thus, the expression of VEGF in AIDS-KS lesions was found primarily in the spindle cells, which correspond to the CD34+ and CD68+ cells of endothelial and monocyte/macrophage lineages, respectively (43) .



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Fig. 4. Human KS lesions express VEGF, and human skin graft promotes the development of KS-like lesions with vascular lumens expressing {alpha}vß3 integrin. A, representative section of human KS tumor from an HIV-1-infected individual; the tumor was stained with antibody-targeting VEGF and developed with alkaline phosphatase anti-phosphatase/Fast Red system. The section also demonstrates vascular structures of KS tissues and cells expressing VEGF (red indicator). Approximately 10–30% of cells in human KS lesions express VEGF protein. B and C, high power images of KS lesion stained with anti-VEGF antibody. D, representative section stain with human-specific anti-PECAM antibody of skin graft on SCID mouse showing fusion of mouse (M) and human (H) skin tissue. Tumor induced by inoculation of KS SLK cells grew under human skin graft (T; see also Fig. 3Citation ). Antihuman PECAM-1 staining selectively localized to cells in tumor (arrow) and was absent in mouse tissue (M). Morphology and distribution of PECAM-1-staining cells were consistent with those EC of tumor tissue. E, representative section of tumor induced by KS SLK under human skin stained with anti-vitronectin antibody highlight cells in vessel-like structure ({alpha}vß3 integrin +, PECAM-1 -). Shown is one (arrow) of many vessel-like structures in tumor tissue having apparent lumens and cells heavily stained with the antivitronectin receptor {alpha}vß3. F, a tumor induced by SLK cells under a human skin graft stained with anti-PECAM-1 antibody. Arrow, PECAM-1 staining of cells in tumor vessels. The KS SLK tumor cells lack PECAM-1 staining.

 
KS SLK Cells Induce Lesions in Human Skin Grafts with Blood Vessels of Human Origin.
Microscopic examination of tumors in the human skin showed that lesions developed directly under the human skin grafts (Fig. 4D)Citation . The bulk of the tumor volume originated with tumor cells directly under human skin grafts, with smaller lesions growing under murine skin (Fig. 3)Citation . There was no evidence of abundant growth of inflammatory tissue or stromal tissue to explain the larger size of tumors under human skin. Thus, the larger tumor volumes are directly related to higher tumor cell masses. The junction of the thinner mouse skin tissue (Fig. 4D, M)Citation and thicker human skin (Fig. 4D, H)Citation overlay the tumor mass (Fig. 4D, T)Citation . The dashed line separates the mouse tissue from human tissue and tumor (Fig. 4D)Citation . Importantly, human-specific anti-PECAM-1 antibody localized to cells (Fig. 4DCitation , arrow) that were present in tumor tissue (Fig. 4D, TCitation , arrow) and in human skin but were entirely absent under mouse skin (Fig. 4D, M)Citation . This is species-specific antibody reactivity indicates that tumor tissue contains vessels of human tissue origin. When anti-PECAM-1 was not added, there was no evidence of marker staining. The tumors induced by KS SLK cells were angiogenic, as shown by numerous PECAM-1-positive cells. Thus, human tissue is associated with enhanced tumor lesion development and the tumors that develop a vasculature system of human origin.

The tumor cells in tumor lesions stained lightly positive for {alpha}vß3 integrin. KS SLK cells in culture show abundant receptor expression by flow cytometry and immunoblots (Fig. 2, B and D)Citation . Most of the cells in the background in Fig. 4ECitation are tumor cells, as verified in H&E-stained sections, and they also stain lightly positively for {alpha}vß3 integrin, whereas cells in vascular structures (4E, arrow) are frankly positive. This discrepancy may be based on tumor cells having {alpha}vß3 integrin expression susceptible to in vivo modulation. Less likely is that the tumor cells have another origin. We doubt this because skin grafts without inoculated tumor cells did not show any tumor lesion formation, and tumor explants placed in culture grow cells identical to KS SLK cells. Overall, the tumor cells express {alpha}vß3, albeit at lower levels, and tumors developing in human skin grafts produce lesions with blood vessels of human origin.

Tumors also regularly exhibited, aside from PECAM-1-positive blood vessels, structures suggestive of vascular lumens ({alpha}vß3 integrin +, PECAM-1 -) of variable sizes and lined with variable cell layers (Fig. 4ECitation , arrow). Tissue sections stained with anti-{alpha}vß3 antibody showed that the cells lining these apparent vascular structures uniformly expressed {alpha}vß3 integrin, which was absent or of low level expression in the surrounding tumor cells (Fig. 4E)Citation . We anticipate that these structures are vascular lumens, given their cell arrangement forming a lumen and high level of {alpha}vß3 integrin expression restricted to these cells (44) . At odds with this concept is the negative staining of the apparent vasculature lumen cells for PECAM-1. The PECAM-1 antibody detected its targets without difficulty in endothelial cells of capillaries of the same tumor tissue (Fig. 4FCitation , arrow). Thus, we found that human skin grafts allow the formation of tumors with numerous apparent vascular structures lined with {alpha}vß3 integrin-expressing cells. When KS SLK cells were injected into murine skin, the smaller tumor lesions that developed lacked these vascular structures. These vascular structures have not appeared in other in vivo models using the same cells or in lesions induced by inoculation in nude mice of the hyperplastic type of KS cells (9) . The channels were also absent in transient lesions induced by the inoculation of hyperplastic AIDS-KS3, AIDS-KS4, and AIDS-KS6 cells in nude mice (9) . These results provide evidence that human tissue supports enhanced tumor development and contributes to the formation of apparent vascular structures that are not possible in murine tissues.

The primary targets of anti-VEGF antiserum in blocking tumor development can be any number of cells in tumor lesions, including tumor cells, stromal fibroblasts, and blood vessel endothelial cells. To identify possible target cells of anti-VEGF antiserum in cell proliferation, we tested anti-VEGF antiserum on dermal microvascular ECs and KS SLK cells in culture. We incubated KS SLK cells (0.9 x 105 cells/well) in growth medium with buffer or anti-VEGF antiserum (10 µg/ml) on days 1 and 3. On day 6, cells were counted by trypan blue exclusion. Buffer-treated cells contained 4.4 x 105 ± 28,700 cells/well, and anti-VEGF antiserum-treated wells contained 4.3 x 105 ± 26,500 cells/well, indicating no difference in cell proliferation. Dermal microvascular ECs analyzed in parallel showed 4.1 x 104 ± 7067 cells/well in buffer-treated wells and 1.4 x 104 ± 2663 cells/well in anti-VEGF antiserum-treated wells, indicating blockage of cell proliferation with anti-VEGF antiserum. Thus, it is likely that the antitumor effect of anti-VEGF antiserum in vivo stems from a direct antiserum action on stroma compartment cells and not KS SLK cells.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 
In this study, we showed that transformed KS cells produce abundant levels of mitogenically active forms of VEGF and that this cytokine is elevated in serum of HIV-1-infected individuals with KS. Anti-VEGF antiserum blocked tumor development induced by the inoculated KS SLK cells in human skin grafts. The antitumor effect of anti-VEGF antiserum appears to act through the stromal cell compartment because in culture the antiserum blocked the proliferation of ECs but not of KS SLK. Tumors induced by KS SLK cells in human skin grafts formed larger tumors that were angiogenic and also contained numerous vessel-like structures that expressed vitronectin integrin. The tumors grown in human tissue develop blood vessels of human cell origin. These results suggest that the human tissue microenvironment may offer support for human tumor cells that exceed that of animal tissues.

We reasonably anticipated that transformed KS cells such as KS SLK and KS IMM cells might have a central but underappreciated role in late-stage KS. Prior to these studies, transient KS-like lesions with some microvascular formation could be produced in nude mice models following inoculation of hyperplastic AIDS-KS cells. Here we show that in addition to supporting tumor development, human skin grafts promoted formation of larger tumors (Fig. 3)Citation . The basis for the selective tumor growth under human skin is unknown. Enhanced tumor growth may be related to the development of a human tissue-derived stroma, including angiogenesis. The blood vessels in tumors in human skin grafts were of human origin, and this in part may be the basis for selective growth of tumors.

The preferential tumor growth in human tissue suggests that human tissue might better respond to the growth requirements of human tumor cells. A selective protumor response of human tissue to VEGF alone is unlikely, given that mouse tissues readily support robust angiogenesis in responses to human-derived VEGF (35) . Angiogenic factors such as VEGF and bFGF are known to cooperate with specific integrins in angiogenesis and tumor development. VEGF cooperates with the {alpha}vß3 integrin to induce a broad spectrum of cellular responses including cell survival and proliferation (37) . Integrins such as {alpha}vß3 serve as coreceptors for the VEGF receptor-2. Integrin {alpha}vß3 forms complexes with VEGF receptor-2 and facilitates its signaling (38) . Activation of {alpha}vß3 by plating cells vitronectin induces VEGFR-2 phosphorylation and its downstream mitogenic effects (45) . Whether murine {alpha}vß3 can substitute for human {alpha}vß3 in tumor development remains to be seen. The results of this study suggest that human tissue may better support human tumor development, and further studies in this human stromal tissue/human tumor cell model may illustrate how some of these factors come together in tumor formation.

Tumors in skin grafts formed structures suggestive of vascular lumens (Fig. 4E)Citation . These apparent vascular structures were lined with {alpha}vß3 integrin-expressing cells that could represent attempts at forming blood vessels. {alpha}vß3 is a marker for newly formed blood vessel endothelium. These vascular structures were found only in tumor tissue under the human skin xenografts and not under murine skin. They have not been observed previously in lesions induced by hyperplastic KS cells or transformed cells in nude mice (16 , 35) . Clearly, the human skin-SCID mouse model may produce new histological features in tumor development that have not been possible in conventional immunodeficient mouse models (24, 25, 26, 27) .

Little is known on how stromal tissue may better promote the development of KS lesions and other tumors. Stromal tissue plays an active role in supporting tumor growth and can at times have an overriding decisive effect on tumor development (46 , 47) . The network of infiltrating stromal cells in tumors suggests that tumor-stromal cell interactions are pervasive, and virtually every tumor cell maintains contact with stromal cells. Stromal cells provide blood vessels, collagen scaffolds, and even some protumorigenic functions such as exaggerated proliferation, elaboration of cytokines, and lowered requirements for cytokines (48 , 49) . Stromal cells that have been primed with inflammatory cytokines have played a decisive role in determining whether tumor cells or carcinogens ultimately produce tumors (48 , 49) .

Transformed KS SLK cells produced mitogenically active isoforms of VEGF, and blockage with anti-VEGF antibody prevented the subsequent development of tumors in human skin grafts. These studies show that transformed KS cells contain abundant presence of VEGF without having to account for the role of HHV-8. HHV-8 infection may further contribute to the VEGF growth stimulation by inducing cellular VEGF production as well as inducing VEGF receptor expression. HHV-8 infection and some of its genes have enhanced the survival of endothelial cells through VEGF (50, 51, 52) . In other tumor models, blockage of VEGF at the level of ligand, receptor binding, or receptor kinase activity has ultimately inhibited or stabilized tumor growth (53, 54, 55) . Thus, this study highlights the contribution of transformed KS cells, human tissue, and VEGF to KS cell-induced lesion formation. Further studies using this model should elucidate how human tissue may support human tumor cell development into tumors that better mimic human cancers.


    Patients and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 
VEGF and VEGF Isoforms.
Informed consent was obtained from all HIV-1-infected individuals under the guidelines of the Institutional Review Board. Blood was collected from consecutive patients who were scheduled for routine blood analysis, and serum was collected and stored at -80°C. Serum VEGF and IL-12 concentrations were measured for 52 HIV-1-infected individuals, 28 of whom also had KS, by a two-antibody sandwich ELISA using 100 µl of serum in duplicate wells (R&D Systems, Minneapolis, MN). Two transformed KS cell lines were studied for VEGF production. KS SLK cells were originally derived from an oral lesion of an HIV-1-seronegative individual who had undergone a kidney transplantation (41) . KS IMM cells were derived from a lesion of an HIV-1-seronegative individual who had undergone a kidney transplantation (56) . Both transformed cell lines differ from hyperplastic AIDS-KS cells by being immortalized and having the ability to grow in conventional medium without added cytokines. The cells grow as adherent cells and share some endothelial cell markers and chromosomal breakpoints. Dermal microvascular ECs (Clonetics Walkersville, MD) were plated at 20% density in medium with cytokine supplementation as recommended by the manufacturer. Cell passages 4–6 were used in our experiments. The cells were analyzed for production of VEGF and their respective isoforms. KS cells were plated in culture at 20% density for 24 h. Cells were washed and then incubated in RPMI 1640 with reduced (5%) FBS for 24 h. Cell supernatant, supernatants of trypsin incubations of cells, and cell extracts were prepared (9) and subjected to measurement of VEGF by ELISA. Cell extracts were diluted in 1% NP40 buffer. To characterize the VEGF isoforms produced by the transformed KS cells, we metabolically labeled KS SLK cells (one cell line was selected because both cell lines produce equivalent levels of VEGF; see "Results") in culture with 100 µl Ci/ml each of [35S]methionine and [35S]cysteine (Amersham, Piscataway, NJ) in RPMI 1640 with reduced (5%) methionine-free and cysteine-free FBS for 12 h (32) . Cell extracts and supernatants were precleared with protein A-Sepharose. Proteins were immunoprecipitated with anti-VEGF polyclonal (5 µg) antibody or anti-VEGF polyclonal antibody preabsorbed with saturating levels of targeted VEGF peptide and incubated with 50 µl of protein-Sepharose at 4°C for 12 h (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitated protein was size fractionated by 16% SDS-PAGE and exposed on autoradiographic film.

Cell Proliferation Antibody Blockage.
To identify a possible VEGF mitogenic pathway, we tested whether neutralizing VEGF antibody would block the proliferation of EC and KS SLK cells in culture. Dermal microvascular ECs and KS SLK cells were plated in culture wells at 20% density and allowed to rest for 1 day in medium containing reduced (5%) FBS and lacking mitogens (VEGF, hepatocyte growth factor, or epidermal growth factor supplementation to EC medium). After 24 h in culture, the medium was replaced with fresh medium with either anti-VEGF antiserum (10 µg/ml Prepro Tech) or control antibody (10 µg/ml). The anti-VEGF antibody used in these studies was generated by using 165-amino acid recombinant VEGF as an immunogen, which should target all isoforms of VEGF. Fresh medium with antibodies was replaced after 3 days; after 6 days, the cells were counted.

Tissue Staining.
Frozen tumor tissue of patch-stage KS and control tissue (lymphoid tissue) were fixed in cold acetone for 10 min, air-dried, washed in TBS, and stained by the alkaline phosphatase anti-alkaline phosphatase method. Slides were incubated with affinity-purified monoclonal anti-VEGF antibodies (R&D Systems) at room temperature for 30 min. The slides were then rinsed in TBS and incubated with (1:25) rabbit anti-mouse antibody (DAKO, Carpinteria, CA). The slides were washed again in TBS, and the alkaline phosphatase anti-alkaline phosphatase complex (1:25) was applied for 20 min at room temperature and developed with the Fast Red substrate system (DAKO). The percentage of positive cells in duplicate samples and in at least four fields/slide was counted after counterstaining with Mayer’s hematoxylin solution (Sigma Chemical Co., St. Louis, MO). Tumors induced under the human skin grafts and under mouse skin were resected, and thin sections were stained with: (a) murine antihuman PECAM-1/CD31 (4G6) (AMAC, Westbrook, ME); (b) rat antimouse murine PECAM-1 (antibody 390; Ref. 57 ); and (c) antihuman {alpha}vß3 integrin antibody (anti-vitronectin receptor; LM609, Chemicon, Temeculah, CA; Ref. 58 ). Each section was also stained using PBS in lieu of the primary antibody to determine background staining.

Detection of {alpha}vß3 Integrin Receptor Expression in Transformed KS Cells.
For analysis of protein expression, 106 cells were washed in PBS containing 2% FBS and stained with antibody-targeting {alpha}vß3 (anti-CD59/CD61 FITC-conjugated antibody; PharMingen, San Diego, CA) or with isotype antibody (PharMingen) for 1 h and then washed two times with PBS-2% FBS. Cells were fixed in 2% paraformaldehyde. Protein expression was analyzed on a FACScan flow cytometer (Becton Dickinson, San Diego, CA) using CellQuest software. To confirm protein expression, cell extracts (100 µg) diluted in 1% NP40 buffer were subjected to immunoblot analysis using an antibody (PharMingen) targeting ß3 of the integrin receptor.

Skin Transplantation and Tumor Induction.
The protocols for skin transplantation have been described previously in detail (24, 25, 26 , 59) . Briefly, male SCID mice, 3–4 weeks of age, from Taconic (Germantown, NY) were used for these studies. Human neonatal foreskins obtained from circumcisions and human adult skin tissue obtained from mastectomy resections were trimmed to a diameter of 1–1.5 cm. Full-thickness human skin grafts were transplanted into size-matched wound beds prepared on each flank of the mice. To secure engraftment, mice were used for experiments only within the second month after human skin transplantation, and only mice whose grafts showed no gross signs of inflammation or rejection were used. Grafts were examined by immunohistochemical assay to demonstrate persistence of the human versus murine cells.

Two million KS SLK cells were injected subdermally into the grafts of mice bearing human neonatal (foreskin) and adult skin from mastectomy resections (20) . Cells were also injected subdermally on the contralateral flank to allow comparison. The resultant tumors were allowed to grow for 3 weeks or until they completely involved the skin graft. The mice were then sacrificed, and the skin grafts were carefully dissected from the mice. The grafts were snap-frozen for later immunohistochemical analysis and counterstained with hematoxylin (24 , 26) or fixed in formalin for histological analysis. Tumor volume was determined as described previously (59) .

To determine whether VEGF is essential for formation of the lesions induced by the KS SLK cells, we coinoculated under the human skin grafts 2 x 106 KS SLK cells combined with 50 µg of either neutralizing anti-VEGF antiserum or control preimmune rabbit serum (Pepro Tech, Rocky Hill, NJ). The skin graft sites were examined, and estimates were made of lesion size at weeks 1 and 2 and at pathological examination at 3 weeks.

Statistical Analysis.
The Mann-Whitney test was used to compare means of two samples. Results are shown as means with SDs. Student’s t test was used to estimate the statistical significance of differences between means (60) . All Ps were for two-sided test statistics.


    Acknowledgments
 
We thank Maria Montelongo for expert assistance with the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Concern Foundation and NIH Grant K08 CA808915-02 (to F. S.) and Core Grant CA16672. Back

2 To whom requests for reprints should be addressed, at Department of Lymphoma/Myeloma, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 429, Houston, TX 77030. Phone: (713) 792-2860; Fax: (713) 794-5656; E-mail: fsamaniego{at}mdanderson.org Back

3 The abbreviations used are: KS, Kaposi’s sarcoma; EC, endothelial cell; HHV, human herpes virus; PECAM-1, platelet endothelial cell adhesion marker-1; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; IL, interleukin; FBS, fetal bovine serum; SCID, severe combined immunodeficient; TBS, Tris-buffered saline. Back

Received for publication 4/ 5/02. Revision received 6/20/02. Accepted for publication 6/20/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Patients and Methods
 References
 

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