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Establishment and characterization of two new Kaposi's sarcoma cell cultures from an AIDS and a non-AIDS patient
Res. ViroL
© INSTITUTPASTEUR/ELSEVIER Paris 1994
1994, 145, 251-259
Establishment and characterization of two new Kaposi's sarcoma cell cultures from an A I D S and a non-AIDS patient R. Benelli 0), L. R e p e t t o 0), S. C a r l o n e (1), C. Parravicini (2) and A. Albini (i) (*) (1) Istituto Nazionale per ia Ricerca sul Cancro, IST, 16132 Geneva (Italy), and (2) Ospedale "L. Sacco'" 20157 Milano (Italy)
SUMMARY We have established and characterized t w o new Kaposi's sarcoma (KS) cell lines derived from skin biopsies: AIDS-KStsTIV (from an AIDS-associated KS) and KSIsTVIII (from a sporadic KS). AIDS-KSIsTIV and KSisTVIII are composed mostly of spindleshaped cells. They show similar patterns of immunohistochemical staining and are positive for smooth muscle (smooth muscle ¢-actin) and fibroblastoid (TE7) markers. Neither of these lines express the endothelial marker von Willebrand factor VIII. These immunohistochemical patterns are similar to numerous other KS lines that we and others have established. When seeded on a reconstituted basement membrane ("Matriger'), AIDSKSjsTIV and KStsTVIII cells form branching colonies and invade into the Matrigel, as do other KS cultures that we have previously examined. This behaviour on Matrigel is similar to that of malignant sarcoma cells of different origin. The expression of vimentin and the morphology of the invasive colonies on Matrigel suggest that KS-derived cells are poorly differentiated mesenchymal cells. KS lesions are characterized by a conspicuous neovascularization, which appears to be derived from host cell recruitment. We tested the capability of the KS-cell supernatents to induce an angiogenic response in vitro. The new lines are able to stimulate human endothelial cell chemotaxis and invasion through Matrigel-coated filters. No differences in angiogenic potential in vitro were observed between the AIDS and the non-AIDS case, as we previously noted for other established cultures. Our new lines have the properties of true KS cells and confirm that KS spindle cells from HIV-positive or -negative patients have identical phenotypic and behavioural characteristics in vitro. Key-words: Kaposi's sarcoma, AIDS; Cell cultures.
INTRODUCTION The first sporadic cases o f Kaposi's sarcoma (KS) were described over one century ago (Kaposi, 1872)
Received February 23, 1994. (*) Corresponding author.
and, until the eighties, this pathology remained unc o m m o n and little studied. With the spread o f AIDS this sarcoma is now found as an opportunistic lesion present in about one out o f three HIV + patients
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(Armes, 1989; Friedman-Kien et al., 1982; Safai et al., 1985). Kaposi's sarcoma has been described in four different forms (Armes, 1989). KS as a rare sporadic form is generally found in elderly individuals as small, isolated cutaneous lesions that often occur in the lower leg; like most Kaposi's forms, this form has the property of being more frequent in male than in female patients, with ratios varying from 4:1 to 10:1. As an endemic form, KS is frequent in subSaharan Africa (where it shows three different subforms : localized, locally aggressive and disseminated); the African endemic form also includes a childspecific lymphadenopathic form, which is the only KS form that occurs with a 1:1 sex ratio. The epidemic KS form linked to the immune deficiency vires infection is the most common form in the US and Europe and is usually aggressive, with multiple lesions in the skin, lymph nodes and gastrointestinal tract (Safai, Johnson et al., 1985); the most acute form of epidemic KS is found in regions where the endemic form is also diffuse. Iatrogenic KS is associated with immunodepression during immunosuppressive therapy after organ transplants (Siegal et al., 1990); this form frequently regresses when the immunosuppressive therapy is suspended. Due to its linkage with HIV, the epidemic form of KS is the most studied. Although the HIV virus is not able to infect KS cells (Peterman et al., 1991 ; Werner et al., 1989), one of its products, the transactivator protein (tat), has been shown to act as a growth factor for the spindle cells derived from KS (Ensoli et al., 1990). However, tat expression is not sufficient to explain the high incidence of epidemic KS among HIV-infected homosexuals, since the pathology is more common among homosexual and bisexual HIV-affected patients than in intravenous drug abusers. It is probable that opportunistic pathogens (Friedman-Kien et al., 1982) associated with the sexual behavior of the high-risk category cause an initial onset of inflammation; the combination of a cascade reaction from the cytokines and lymphokines secreted by the inflammatory cells (Buonaguro et al., 1992; Ensoli et al., 1992) and of immunodepression creates a microenvironment that leads to the formation of KS lesions. Histologically, the KS lesion is characterized by an initial leukocyte-macrophage infiltration, spindleshaped cells and vascular cells forming thin, dilated vessels that leak erythrocytes (Armes, 1989; Peterman et al., 1991). The highly vascularized nature of KS has led pathologists to assume that this is ale-
BSA FCS HI HUVEC KS
= = = =
bovineserum albumin. foetalcalf serum. heat-inactivated. humanumbilicalvein endothelialcell.
ffi
Kaposi's
sarcoma.
sion of endothelial cells. However, there is a large body of evidence indicating that the vascular cells present in the KS lesion are normal vascular cells recruited from the surrounding host tissue. Cultures from KS biopsies have consistently yielded a population of spindle-shaped cells and more rarely, endothelial-like cells (Albini et al., 1992; Corbeil et al., 1991; Nakamura et al., 1988; Nickoioff and Griffiths, 1989; Werner et al., 1989). The KS spindle cells appear to be the original cells of the lesion which are able to recruit normal vascular cells from the host tissue (Adatia et al., 1992; Salahuddin et al., 1988). KS cells cultured from various laboratories release a number of angiogenic factors (Ensoli et al., 1992). KS cultures have also been shown to be able to stimulate endothelial cells in vitro (Corbeil et al., 1991 ; Werner et al., 1989) and in vivo (Salahuddin et al., 1988). In our laboratory we have tested the supernatants of KS-cell lines using migration (Albini et al., 1987a) and invasion (Albini et al., 1987b) assays in the Boyden chamber system. We have shown that KS-cell supernatants can induce both the migration and invasion of endothelial and smooth muscle cells (Adatia et al., 1992; Thompson et al., 1991). In vivo, the injection of AIDS-KS spindle-shaped cells into nude mice gives rise to a Kaposi-like lesion which contains only host endothelial cells (Salahuddin et al., 1988). We have recently shown that cellfree supernatants from KS spindle cells can reproduce a KS-like lesion in vivo in mice (Albini et al., submitted). Although the KS spindle cell is now considered the "neoplastic cell" of the KS lesion, the study of the characteristics and origins of these cells has been made difficult by the limited number of KS-cen lines available. The nature of the spindle cell is still controversial: it was first described as having an endothelial origin; however, more recent studies define it as an immature smooth muscle cell (Albini et al., 1988 ; Weich et al., 1991 ; Wittek et al., 1991) or a myofibroblast. Growth of KS spindle-shaped cells is linked to autocrine and paracrine stimulation: it has already been observed that the supernatants of HTLV-II-activated T lymphocytes are able to support the proliferation of the KS cells in vitro (Salahuddin et al., 1988); this mechanism probably involves the large amounts of TGF~ (Roth, 1993) and Oncostatin-M (Miles et ai., 1992) produced by the activated T cells. Spindleshaped cells themselves are able to produce, among others, ILl, IL6, bFGF, kFGF (FGF type 5), PDGF,
KSCM mAb PBS SFM TBS
= KS-conditionedmedium. = monoclonalantibody. = phosphate-buffered saline. -- serum-freemedium. = Tris-bufferedsaline.
NEW KAPOSrS
SARCOMA
CELL CULTURES
GMCSF, TGF[3, TNF0t (Barillari et al., 1992; Corbeil et al., 1991; Ensoli et al., 1989; Roth, 1993; Sturzl et al., 1992; Xerri et al., 1991), P A F (Sciacca et ai., submitted) and their related receptors, showing a complex autocrine stimulation pathway. O f course many other growth factors and cytokines may be involved in the onset and progression o f the KS lesion. For this reason, the presence and availability o f well-characterized KS-cell lines is important in order to enable the investigation of the nature of this complex pathology. We have previously established, characterized and provided to other laboratories two continuously growing KS-derived ceils (Albini et al., 1992). Here we demonstrate (1) that two cultures, from an A I D S and a non-AIDS patient, have characteristics similar to those of previously established lines and (2) that the protocol that we established is reliable.
MATERIALS A N D METHODS Patients
A sporadic KS-cell culture was derived from the biopsy of a KS lesion on the arch of the left foot of a 78-yearold male. The patient also had an arterial fistula. An epidemic KS-cell culture was derived from a biopsy taken from an AIDS KS lesion behind the ear of a 40-year-old HIVpositive homosexual male having multiple lesions on the face. This patient also had an opportunistic tuberculosis infection. Establishment of KS-cell cultures
Biopsies were immediately put in RPMI medium supplemented with glutamine (300 ~g/ml), amphotericin B (2.5 ~g/ml), penicillin (100U/ml) and streptomycin (50 i~g/ml). The fragments were rapidly washed in absolute ethanol, rinsed with PBS and transferred to a Petri dish with PBSw/o Ca,Mg for mechanical removal of the connective and fat tissue. The lesion was then minced in small pieces, some of which were used to obtain explants. The rest of the biopsy was enzymatically digested, alternating trypsin and collagenase as previously described (Albini et al., 1992). Cells freed were removed, and the remaining pieces were further digested overnight in collagenase. The cells obtained from enzymatic digestion were centrifuged and plated in gelatin-coated 25-ml flasks. Cells were cultured using RPMI-1640 medium with 10 or 15 070 heat-inactivated (HI) FCS, glutamine, amphotericin B and antibiotics as above. Sometimes cells were cultured with the addition of 5 ~.I/ml "Ultroser G" (Sepracor) to the normal medium. The line from the epidemic case, named AIDSKSIsTIV, was obtained from the enzymatic digestions, while the sporadic cell line, KSxsTVIII, was derived from a single colony from the explant cultures. Neither activated lymphocyte supernatants nor "Nutridoma Hu" (Boehringer Mannheim) were used for either of these cultures. Trypsinization was used for the subculture of primary cells (0.05 07o trypsin, 0.02 °7o EDTA in PBS, pH 7.4).
FROM AIDS AND NON-AIDS
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Other cells
Human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection (ATCC), grown on gelatin (Difco, 1.5 °70in distilled water) -coated flasks in M199, 20 07oFCS HI supplemented with heparin (100 ~g/ml) and "Endothelial Cell Growth Supplement" (Sigma; 30 ~g/ml).
Immunohistochemistry
Antibodies The monoclonal antibodies (mAb) employed were MNF116 and LP34 (Dakopatts) against epithelial cytokeratins; clone V9 (Dakopatts) against vimentin; clone IA4 (Bio-Makor) against smooth-muscle-specific alpha-actin ; and clone F8/86 and a polyclonal rabbit antiserum (Dakopatts) against vimentin. Antibodies against yon Willebrand factor VIII (factor VIII) for endothelial cells, clone TE7 (Seralab Ltd) recognizing reticular fibroblasts, clone 9.4 (New England Nuclear) for the CD45 panleukocytic antigen and "QB END 10" (Melgriff) for the CD34 haematopoietic stem cell antigen were also used for cell type identification.
Cells Cells were seeded in "Lab-tek" chamber slides and grown for 24-48 hours. They were then rinsed twice in PBS and air-dried for 2 hours. Glass slides were timed in cold acetone and dried. Fixed cells were washed in TBS (Trisbuffered saline pH 7.4) 0.05 M and preincubated for 10 rain in TBS containing 0.5 07oBSA; an overnight incubation (4°C) with the various mAb appropriately diluted was followed by multiple washings in TBS. The primary antibodies were revealed with anti-m"ouse peroxidaseconjugated rabbit antiserum (Dakopatts) diluted 1:100 in TBS-BSA (1 hour at room temperature). Cells were then washed in Tris and stained using DAB (Sigma) as chromogen. Growth on Matrigel
Matrigel, a mixture of basement membrane components extracted from the EHS tumour, was prepared according to Kleinman et al. (1986) and stored at -20°C. For morphological assays, Matrigel was thawed in a 0°C ice-water bath, diluted to a final concentration of 10 mg/ml, and 0.4 ml was gently pipetted into each 13-mm diameter tissue culture well. After 1 h at 37°C, Matrigel was completely polymerized, and 5 x 1 0 4 cells in 1 ml of normal medium were carefully seeded on top of the Matrigel layer. The seeded plates were incubated at 37°C in a 5 % CO2 humidified atmosphere and photographed at various intervals. Chemotaxis assay
Chemotaxis assays were performed in Boyden's chambers according to Postlethwaite et al. (1976) as modified (Albini et al., 1987a). Briefly, the lower compartment was filled (200 ~l of total volume) with the KS-conditioned
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medium (KSCM) as chemoattractant (or with serum-free medium as controls) and covered with a polycarbonate filter (12-1an pore size; Nucleopore) coated with type IV collagen (5 ~g/filter; Sigma) to support cell adhesion. KSCM was collected after incubating KS cells in serum-free medium (SFM) for 24 hours. The medium was then centrifuged and stored frozen at -20°C. HUVEC were harvested with trypsin, then centrifuged, washed and resuspended in SFM. The cells were brought to a final concentration of 250 x 10a cells/mis~, and 400 ~l of the suspension were placed in the upper compartment. After a 6-h incubation (37°C, 5 % CO2), filters were taken, and the cells present on the upper side were removed. The migrated cells were fixed in absolute ethanol, stained with toluidine blue and counted (5 unit fields per filter) at a 160x magnification with a "Zeiss" microscope. One field represents 1/160 of the entire area of the migrated cells.
normal culture medium. After the 8th passage they began a slow process o f regression. The growth of KSIsTVIII cultures could be supported by the addition of Ultroser-G (similarly to the previously established AIDS-KSIsTIII), while AIDS-KSIsTIV cells did not respond to Ultroser-G. Ultroser-G was able to p r o m o t e AIDS-KSIsTIII and KSIsTVIII cell proliferation until 15-16 passages in culture. We phenotyped our cultures using a large panel o f antibodies (table I). Like AIDS-KSIsTIII cells, both AIDS-KSIsTIV and KSIsTVIII cell showed a smooth-muscle/fibroblast-like phenotype, being positive for smooth-muscle-specific alpha-actin, desmin (not shown) and TE7. A mesenchymal origin o f these cells was confirmed by strong positivity for vimentin (fig. 1). In contrast, both AIDS-KSIsTIV and KSIsTVIII were negative for the endothelial marker factor VIII, the pan-leukocytic marker CD45 and for epithelial cytokeratins. When these cells were cultured in Ultroser-G enriched medium, the pattern o f ch~acterization changed; in fact, after a few passages in Ultroser-G the smooth muscle actin staining almost disappeared in the KSls-rVIII cultures. However, this was reversible: the immunochemical staining for a-actin returned to the previous state after withdrawal o f the supplement. This change occurred without apparent alterations in the morphology and the staining for other markers, such as TE7 and vimentin.
Chemoinvasion assay The chemoinvasion assay (Albini et al., 1987b) was performed as for chemotaxis, except that the collagen-IVcoated polycarbonate filter was overcoated with 25 ~g of Matdgel, dried and reconstituted with SFM prior to use.
RESULTS Establishment histochemistry
of
KS
cultures
and
immuno-
Both KS primary cultures, AIDS-KSIsTIV and KSlsrVIII had the same spindle-shaped m o r p h o l o gy previously described for other KS cultures (Albini et al., 1992; Corbeil et al., 1991 ; N a k a m u r a et ai., 1988; Salahuddin et al., 1988; Wittek et al., 1991). Similarly to previous cell cultures that we have obtained f r o m KS biopsies, AIDS-KSxsTIV and KSIsTVIII cells proliferated with a doubling time o f 72 h until the 8th passage, without the addition o f supernatants o f activated P B L or N u t r i d o m a to the
Growth o f KS-derived cultures on Matrigel When grown on a three-dimensional Matrigel substrate, 24 hours after seeding, the new cell lines established showed the typical invasive branching morphology of neoplastic mesenchymal cells (fig. 2). This m o r p h o l o g y has been described previously for other KS cell lines (Albini et al., 1992) and for primary malignant sarcoma cells (Nicolb et ai., 1991).
Table I. Immunohistochemistry of KSIsT cells and relative controls. Marker Keratin Vimentin a-Actin VW-Fact. TE7 CD34 CD45
KSIsTIV
KSIsTVIII
SMC
Fb
HUVEC
+ + + .
+ + + -
nd + + + / _ (a) nd .
+ + nd
+ + _ nd
.
.
.
Cells grown on chamber slides were fixed and stained as described in "Materials and Methods". nd= not done; (a)=TE7 positive for SMC in vivo and detected in vitro in cultures of SMC derived from human carotide artery. SMC = smooth musclecell. Fb = fibroblast.
.!.J
a
¢
b
d
Fig. 1. Immunohistochemistry of KS-derived cells. a = expression of TE7 (KS~sTVIII), b = expression o f smooth muscle isoform of ~-actin (KSIsTIV), ¢ = lack of CD34 expression (KSIsTVIII) and d = expression of vimentin (KSIsrVIII); detected with immunoperoxidase method. ( × 685).
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y
a
e
b
d
Fig. 2. Growth on Matrigel. a = HUVEC form tube-like structures as previously described, b = fibroblasts form small colonies, c = AIDS-KSIsTIVand d = KSIsTVIIIshowing the typical invasive branching colonies exhibited by tumours of mesenchymal origin (photograhs taken 48 hours after plating; a and b x 270; c and d x 274.
Induction of endothelial cell chemotaxis and invasiveness by KS cultures
We tested the in vitro angiogenic properties of these KS-derived cells by utilizing KSCM as an endothelial cell activator in the Boyden's chamber chemotaxis and chemoinvasion assays. The HUVEC chemotactic response to conditioned medium of both AIDS-KStsrlV and KS~-rVIII was much higher than that to SFM controls (fig. 3). KSCM also induced a three-fold increase in endothelial cell invasion through a thin layer of Matrigel. DISCUSSION The newly established AIDS-KSIsTIV and KSxsT_ VIII Kaposi's sarcoma-derived ceils show morphology, immunohistological staining, invasive behaviour and in vitro angiogenic properties similar to those seen for the previously obtained KStsTII and AIDSKSmTIII lines (Albini et al., 1992). Their morphological appearance is also similar to that described
for most established KS-cell cultures. The culture procedure appears to select for the "neoplastic" cells found in KS. The histopathological analysis of KS biopsies has always demonstrated abundant expression of endothelial markers in the lesions (Armes, 1989); however, very rarely have cells with endothelial markers been obtained from these biopsies. This is probably due to the fact that most of the endothelial cells are " n o r m a l " cells recruited from the host, and these do not survive well in culture. Immunohistochemistry also shows conflicting information between the in vivo lesion and the cultured cells. Only a few of the poorly differentiated spindle cells proliferating inside the KS lesion express smooth muscle actin; however, cultured cells have a much higher frequency of positively stained cells (20-50 %). We hypothesize that the KS spindle cell can assume more smooth-muscle-like or more fibroblasticlike (or perhaps even more endothelial-like) characteristics under different conditions or stimulation. This hypothesis is supported by the different expression of smooth muscle a-actin demonstrated by
N E W K A P O S I ' S S A R C O M A CELL CU LTU R E S F R O M A I D S A N D N O N - A I D S 100'~1
Chemotaxis and chemolnvasion of AIDS KS IST IV-stimulated HUVE cells
80 • [] _=
chemotaxis invasion
6O
257
polyclonal KS lesions. Under appropriate conditions, it is possible that some undifferentiated mesenchymal/vascular precursor cells normally present in the organism begin to proliferate but do not differentiate, giving rise to KS lesions. This hypothesis could explain the evident discrepancies in the immunohistochemical distribution of markers in vivo and in vitro. In KS lesions, the presence of activated lymphocytes, macrophages, "spindle-shaped" cells and endothelial cells indicates the presence of a very complex network of autocrine and paracrine stimulation. It would be of interest, although likely very difficult, to detect the true plasticity of spindleshaped KS cells inside the lesion. It would be of even greater interest to discover the cytokine(s) able to induce a definitive differentiation of KS spindle cells that could end the progression of the lesion.
o}
0
40,
=E z 20,
KS IV CM
SFM
Acknowledgements
I O0
We would like to thank Dr. Douglas Noonan (IST, Genova) for critical reading and for revision of the English. This work was financed by grants from the Italian Ministero della Sanit/l, VI progetto AIDS. R. BeneUi is an AIRC fellow.
80 tcJ ~t o "ID
60
Caract~risation de deux nouveHes cultures cellulaires de sarcome de Kaposi ~tablies k partir d'un cas de S I D A et d'un cas de sarcome sporadique
E 0
40 E
20
KS VIII CM
SFM
Fig. 3. Chemotaxis (black) and invasion (grey) of HUVEC in response to AIDS-KSIsTIV (top) and KSIsTVIII (bottom) conditioned media. SFM = serum-free medium controls.
KSmTVIII cells cultured with or without Ultroser G, It is probable, as has been shown for other systems, that cytokines or components of the extracellular matrix are able to induce pluripotential differentiation of vascular precursor cells. Another phenomenon which supports the emergence of an undifferentiated mesenchymal cell in the KS pathology is the frequent observation of multifocal and
A partir de biopsies cutan6es, deux nouvelles ligndes cellulaires de sarcome de Kaposi (KS= Kaposi's sarcoma) ont ~t6 &ablies: AIDS-KSxsTIV darts un cas de KS associ6 au SIDA et KS[sTVIII dans un cas de KS sporadique. Ces deux lign6es sont principalement compos6es de cellules fusiformes, elles ont des diagrammes de coloration immunohistochimique semblables et elles sont positives pour les marqueurs des muscles lisses (actine-~ de muscle lisse) et les marqueurs fibroblastoides (TE7). Aucune de ces lign6es n'exprime le marqueur endoth61ial du facteur VIII de yon WiIlebrand. Ces profils immunohistochimiques sont semblables A ceux d'autrcs ligndes cellulaires KS d6j/t &ablies. Quand elles sont ensemenc6es sur une membrane basale reconstitu6e (>), les cellules AIDS-KSxsTIV et KSmTVIII forment des colonies ramifi~'es et envahissent le Matrigel. Ce comportement sur Matrigel est semblable ~ celui des ceUules sarcomateuses malignes de diff&entes origines. L'expression de vimentine et la morphologie des colonies invasives sur Matrigel nous sugg~rent que les lign6es cellulaires d&iv6es de KS sont des cellules m6senchymateuses peu diff6renci6es.
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Les 16sions du KS sont caract6ris~es par une n6ovascularisation tr~s 6vidente, d6riv~e des cellules h6tes. Nous avons test6 la capacit6 des surnageants des cellules KS d'induire une r6ponse angiog~nique in vitro. Les nouvelles lign6es sont capables de stimuler le chimiotactisme des cellules endoth61iales humaines et l'invasion ~ travers les filtres couverts de Matrigel. Aucune diff6rence darts le potentiel angiog6nique in vitro n'a 6t6 observ6 entre les cellules du sujet VIH + et celles du sujet V I H - comme nous l'avions d6j/l not~ pour d'autres cultures. Les nouvelles lign~es cenulaires 6tudi6es ici ont les propd6t6s des cellules du sarcome de Kaposi et elles confirment que les cellules fusiformes du KS de patients VIH + ou V I H - ont des ph6notypes et des caract6dstiques de comportement in vitro identiques. Mots-clds: Sarcome de Kaposi, SIDA; Cultures cellulaires.
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tein on the expression of inflammatory cytokines. J. Virol., 66, 7159-7167. Corbeil, J., Evans, L.A., Vasak, E., Cooper, D.A. & Penny, R. (1991), Culture and properties of cells derived from Kaposi sarcoma. J. Immunol., 146, 2972-76. Ensoli, B., Barillari, G. & Gallo, R. (1992), Cytokines and growth factors in the pathogenesis of AIDS-associated Kaposi's sarcoma, lmmunol. Rev., 127, 147-155. Ensoli, B., Barillari, G., Salahuddin, S.Z., Gailo, R. & Wong-Staal, F. (1990), Tat protein of HIV-I stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients. Nature (Lond.), 345, 84. Ensoli, B., Nakamura, S., Salahuddin, S.Z., Biberfeld, P., Larsson, L., Beaver, B., Wong-Staal, F. & Gallo, R.C. (1989), AIDS-Kaposi's sarcoma-derived cells express cytokines with autocrine and paracrine growth effects. Science, 243, 223-226. Friedman-Kien, A.E., Laubenstein, M., Rubinstein, P., Buimovici-Klein, E., Marmot, M., Stahl, R., Spigland, I., Kim, K. & Zolla-Pazner, S. (1982), Disseminated Kaposi's sarcoma in homosexual men. Ann. Intern. Med., 96, 693-700. Kaposi, M. (1872), Idiopathisches multiples Pigment Sar~:om der Haut. Arch. Dermatol. Syph., 4, 265-273. Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Star, V.L., Cannon, F., Laurie, G.W. & Martin, G.R. (1986), Basement membrane complexes with biological activity. Biochemistry, 25, 312-318. Miles, S.A., Martinez-Maza, O., Rezai, A., Magpantay, L., Kishimoto, T., Nakamura, S., Radka, S.F. & Linsley, P.S. (1992), Oncostatin M as a potent mitogen for AIDS-Kaposi's sarcoma-derived cells. Science, 255, 1432-1434. Nakamura, S., Salahuddin, S.Z., Biberfeld, P., Ensoli, B., Markham, P.D., Wong-Staal, F. & Gallo, R.C. (1988), Kaposi's sarcoma cells: long-term culture with growth factor from retrovirus-infected CD4 + T cells. Science, 242, 426-429. Nickoloff, B.J. & Griffiths, C.E.M. (1989), The spindleshaped cells in cutaneous Kaposi's sarcoma. Am. J. Pathol., 135, 793. Nicol6, G., Albini, A., Orengo, G., Margallo, E., Margiocco, M., Palumbo, R., Leonessa, F., Parodi, S. & Toma, S. (1991), Relationship between DNA ploidy cytological grade of malignancy and in vitro invasiveness for human sarcoma. Eur. J. Cancer, 27, S 35. Peterman, T., Jaffe, H., Friedman-Kien, A. & Weiss, R. (1991), The aetiology of Kaposi's sarcoma. Cancer. HIV and AIDS. Imperial Cancer Research Fund. Postlethwalte, A.E., Snyderman, R. & Kang, A.H. (1976), The chemotactic attraction of human fibroblasts to a lymphocyte-derived factor. J. Exp. Med., 144, 1188. Roth, W. (1993), TGF-~ and FGF-like growth factors involved in the pathogenesis of AIDS-associated Kaposi's sarcoma. Res. Virol., 144, 105-109. Safal, B., Johnson, K.G., Myskowski, P.L., Koziner, B., Yang, S.Y., Cunningham, R.S., Godbold, J.H. & Dupont, B. (1985), The natural history of Kaposi's sarcoma in the acquired immunodeficiency syndrome. Ann. Intern. Med., 103, 744-750. Salahuddin, S.Z., Nakamura, S., Biberfeld, P., Kaplan, M.H., Markham, P.D., Larsson, L. & Gallo, R.C. (1988), Angiogenic properties of Kaposi's sarcomaderived cells after long-term culture in vitro. Science, 242, 430.
NEW KAPOSI'S SARCOMA CELL CULTURES FROM AIDS AND NON-AIDS
Siegal, B., Levington-Kriss, S., Schiffer, A., Sayar, J., Engelberg, I., Vonsover, A., Ramon, Y. & Rubinstein, E. 0990), Kaposi's sarcoma in immunosuppression. Possibly the result of a dual viral infection. Cancer, 65,492-498. Sturzl, M., Roth, W., Brockmeyer, N., Zietz, C., Speiser, B. & Hofschneider, P. (1992), Expression of platelet-derived growth factor and its receptor in AIDS-related Kaposi sarcoma in vivo suggests paracrine and autocrine mechanisms of tumor maintenance. Proc. Natl. Acad. Sci. USA, 89, 7046-7050. Thompson, E., Nakamura, S., Shima, T., Melchiori, A., Martin, G., Salahuddin, S., Gallo, R. & Albini, A. (1991), Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness. CancerRes., 51, 2670-2671. Weich, A.W., Zaki Salauddin, S., Parkash, G., Nakamura, S., Gallo, R.C. & Folkman, J. (1991), AIDSassociated Kaposi's sarcoma-derived cells in long-term
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culture express and synthesize smooth muscle alphaactin. Am. J. Pathoi., 139, 1251-1258. Werner, S., Hofschneider, P.H. & Roth, W.K. (1989), Cells derived from sporadic and AIDS-related Kaposi's sarcoma reveal identical cytochemicai and molecular properties in vitro. Int. J. Cancer, 43, 1137-1144. Wittek, A.E., Mitchell, C.D., Armstrong, G.R., Albini, A., Martin, G.R., Seeman, R., Levenbook, I.S., Wierenga, D.E., Ridge, J., Dunlap, R.C., Lundquist, M.L., Steis, R.G., Longo, D.L., Muller, J. & Quinnan, G.V.J. (1991), Propagation and properties of Kaposi's sarcoma-derived cell lines obtained from patients with AIDS: similarity of cultured cells to smooth muscle cells. AIDS, 5, 1485-1493. Xerri, L., Hassoun, J., Planchet, J., Guigou, V., Grob, J.J., Parc, P., Birnbaum, D. & deLapeyriere, O. (1991), Fibroblast growth factor gene expression in AIDS-Kaposi's sarcoma detected by in situ hybridization. Am. J. Pathol., 138, 9-15.
© INSTITUTPASTEUR/ELSEVIER Paris 1994
1994, 145, 251-259
Establishment and characterization of two new Kaposi's sarcoma cell cultures from an A I D S and a non-AIDS patient R. Benelli 0), L. R e p e t t o 0), S. C a r l o n e (1), C. Parravicini (2) and A. Albini (i) (*) (1) Istituto Nazionale per ia Ricerca sul Cancro, IST, 16132 Geneva (Italy), and (2) Ospedale "L. Sacco'" 20157 Milano (Italy)
SUMMARY We have established and characterized t w o new Kaposi's sarcoma (KS) cell lines derived from skin biopsies: AIDS-KStsTIV (from an AIDS-associated KS) and KSIsTVIII (from a sporadic KS). AIDS-KSIsTIV and KSisTVIII are composed mostly of spindleshaped cells. They show similar patterns of immunohistochemical staining and are positive for smooth muscle (smooth muscle ¢-actin) and fibroblastoid (TE7) markers. Neither of these lines express the endothelial marker von Willebrand factor VIII. These immunohistochemical patterns are similar to numerous other KS lines that we and others have established. When seeded on a reconstituted basement membrane ("Matriger'), AIDSKSjsTIV and KStsTVIII cells form branching colonies and invade into the Matrigel, as do other KS cultures that we have previously examined. This behaviour on Matrigel is similar to that of malignant sarcoma cells of different origin. The expression of vimentin and the morphology of the invasive colonies on Matrigel suggest that KS-derived cells are poorly differentiated mesenchymal cells. KS lesions are characterized by a conspicuous neovascularization, which appears to be derived from host cell recruitment. We tested the capability of the KS-cell supernatents to induce an angiogenic response in vitro. The new lines are able to stimulate human endothelial cell chemotaxis and invasion through Matrigel-coated filters. No differences in angiogenic potential in vitro were observed between the AIDS and the non-AIDS case, as we previously noted for other established cultures. Our new lines have the properties of true KS cells and confirm that KS spindle cells from HIV-positive or -negative patients have identical phenotypic and behavioural characteristics in vitro. Key-words: Kaposi's sarcoma, AIDS; Cell cultures.
INTRODUCTION The first sporadic cases o f Kaposi's sarcoma (KS) were described over one century ago (Kaposi, 1872)
Received February 23, 1994. (*) Corresponding author.
and, until the eighties, this pathology remained unc o m m o n and little studied. With the spread o f AIDS this sarcoma is now found as an opportunistic lesion present in about one out o f three HIV + patients
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(Armes, 1989; Friedman-Kien et al., 1982; Safai et al., 1985). Kaposi's sarcoma has been described in four different forms (Armes, 1989). KS as a rare sporadic form is generally found in elderly individuals as small, isolated cutaneous lesions that often occur in the lower leg; like most Kaposi's forms, this form has the property of being more frequent in male than in female patients, with ratios varying from 4:1 to 10:1. As an endemic form, KS is frequent in subSaharan Africa (where it shows three different subforms : localized, locally aggressive and disseminated); the African endemic form also includes a childspecific lymphadenopathic form, which is the only KS form that occurs with a 1:1 sex ratio. The epidemic KS form linked to the immune deficiency vires infection is the most common form in the US and Europe and is usually aggressive, with multiple lesions in the skin, lymph nodes and gastrointestinal tract (Safai, Johnson et al., 1985); the most acute form of epidemic KS is found in regions where the endemic form is also diffuse. Iatrogenic KS is associated with immunodepression during immunosuppressive therapy after organ transplants (Siegal et al., 1990); this form frequently regresses when the immunosuppressive therapy is suspended. Due to its linkage with HIV, the epidemic form of KS is the most studied. Although the HIV virus is not able to infect KS cells (Peterman et al., 1991 ; Werner et al., 1989), one of its products, the transactivator protein (tat), has been shown to act as a growth factor for the spindle cells derived from KS (Ensoli et al., 1990). However, tat expression is not sufficient to explain the high incidence of epidemic KS among HIV-infected homosexuals, since the pathology is more common among homosexual and bisexual HIV-affected patients than in intravenous drug abusers. It is probable that opportunistic pathogens (Friedman-Kien et al., 1982) associated with the sexual behavior of the high-risk category cause an initial onset of inflammation; the combination of a cascade reaction from the cytokines and lymphokines secreted by the inflammatory cells (Buonaguro et al., 1992; Ensoli et al., 1992) and of immunodepression creates a microenvironment that leads to the formation of KS lesions. Histologically, the KS lesion is characterized by an initial leukocyte-macrophage infiltration, spindleshaped cells and vascular cells forming thin, dilated vessels that leak erythrocytes (Armes, 1989; Peterman et al., 1991). The highly vascularized nature of KS has led pathologists to assume that this is ale-
BSA FCS HI HUVEC KS
= = = =
bovineserum albumin. foetalcalf serum. heat-inactivated. humanumbilicalvein endothelialcell.
ffi
Kaposi's
sarcoma.
sion of endothelial cells. However, there is a large body of evidence indicating that the vascular cells present in the KS lesion are normal vascular cells recruited from the surrounding host tissue. Cultures from KS biopsies have consistently yielded a population of spindle-shaped cells and more rarely, endothelial-like cells (Albini et al., 1992; Corbeil et al., 1991; Nakamura et al., 1988; Nickoioff and Griffiths, 1989; Werner et al., 1989). The KS spindle cells appear to be the original cells of the lesion which are able to recruit normal vascular cells from the host tissue (Adatia et al., 1992; Salahuddin et al., 1988). KS cells cultured from various laboratories release a number of angiogenic factors (Ensoli et al., 1992). KS cultures have also been shown to be able to stimulate endothelial cells in vitro (Corbeil et al., 1991 ; Werner et al., 1989) and in vivo (Salahuddin et al., 1988). In our laboratory we have tested the supernatants of KS-cell lines using migration (Albini et al., 1987a) and invasion (Albini et al., 1987b) assays in the Boyden chamber system. We have shown that KS-cell supernatants can induce both the migration and invasion of endothelial and smooth muscle cells (Adatia et al., 1992; Thompson et al., 1991). In vivo, the injection of AIDS-KS spindle-shaped cells into nude mice gives rise to a Kaposi-like lesion which contains only host endothelial cells (Salahuddin et al., 1988). We have recently shown that cellfree supernatants from KS spindle cells can reproduce a KS-like lesion in vivo in mice (Albini et al., submitted). Although the KS spindle cell is now considered the "neoplastic cell" of the KS lesion, the study of the characteristics and origins of these cells has been made difficult by the limited number of KS-cen lines available. The nature of the spindle cell is still controversial: it was first described as having an endothelial origin; however, more recent studies define it as an immature smooth muscle cell (Albini et al., 1988 ; Weich et al., 1991 ; Wittek et al., 1991) or a myofibroblast. Growth of KS spindle-shaped cells is linked to autocrine and paracrine stimulation: it has already been observed that the supernatants of HTLV-II-activated T lymphocytes are able to support the proliferation of the KS cells in vitro (Salahuddin et al., 1988); this mechanism probably involves the large amounts of TGF~ (Roth, 1993) and Oncostatin-M (Miles et ai., 1992) produced by the activated T cells. Spindleshaped cells themselves are able to produce, among others, ILl, IL6, bFGF, kFGF (FGF type 5), PDGF,
KSCM mAb PBS SFM TBS
= KS-conditionedmedium. = monoclonalantibody. = phosphate-buffered saline. -- serum-freemedium. = Tris-bufferedsaline.
NEW KAPOSrS
SARCOMA
CELL CULTURES
GMCSF, TGF[3, TNF0t (Barillari et al., 1992; Corbeil et al., 1991; Ensoli et al., 1989; Roth, 1993; Sturzl et al., 1992; Xerri et al., 1991), P A F (Sciacca et ai., submitted) and their related receptors, showing a complex autocrine stimulation pathway. O f course many other growth factors and cytokines may be involved in the onset and progression o f the KS lesion. For this reason, the presence and availability o f well-characterized KS-cell lines is important in order to enable the investigation of the nature of this complex pathology. We have previously established, characterized and provided to other laboratories two continuously growing KS-derived ceils (Albini et al., 1992). Here we demonstrate (1) that two cultures, from an A I D S and a non-AIDS patient, have characteristics similar to those of previously established lines and (2) that the protocol that we established is reliable.
MATERIALS A N D METHODS Patients
A sporadic KS-cell culture was derived from the biopsy of a KS lesion on the arch of the left foot of a 78-yearold male. The patient also had an arterial fistula. An epidemic KS-cell culture was derived from a biopsy taken from an AIDS KS lesion behind the ear of a 40-year-old HIVpositive homosexual male having multiple lesions on the face. This patient also had an opportunistic tuberculosis infection. Establishment of KS-cell cultures
Biopsies were immediately put in RPMI medium supplemented with glutamine (300 ~g/ml), amphotericin B (2.5 ~g/ml), penicillin (100U/ml) and streptomycin (50 i~g/ml). The fragments were rapidly washed in absolute ethanol, rinsed with PBS and transferred to a Petri dish with PBSw/o Ca,Mg for mechanical removal of the connective and fat tissue. The lesion was then minced in small pieces, some of which were used to obtain explants. The rest of the biopsy was enzymatically digested, alternating trypsin and collagenase as previously described (Albini et al., 1992). Cells freed were removed, and the remaining pieces were further digested overnight in collagenase. The cells obtained from enzymatic digestion were centrifuged and plated in gelatin-coated 25-ml flasks. Cells were cultured using RPMI-1640 medium with 10 or 15 070 heat-inactivated (HI) FCS, glutamine, amphotericin B and antibiotics as above. Sometimes cells were cultured with the addition of 5 ~.I/ml "Ultroser G" (Sepracor) to the normal medium. The line from the epidemic case, named AIDSKSIsTIV, was obtained from the enzymatic digestions, while the sporadic cell line, KSxsTVIII, was derived from a single colony from the explant cultures. Neither activated lymphocyte supernatants nor "Nutridoma Hu" (Boehringer Mannheim) were used for either of these cultures. Trypsinization was used for the subculture of primary cells (0.05 07o trypsin, 0.02 °7o EDTA in PBS, pH 7.4).
FROM AIDS AND NON-AIDS
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Other cells
Human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection (ATCC), grown on gelatin (Difco, 1.5 °70in distilled water) -coated flasks in M199, 20 07oFCS HI supplemented with heparin (100 ~g/ml) and "Endothelial Cell Growth Supplement" (Sigma; 30 ~g/ml).
Immunohistochemistry
Antibodies The monoclonal antibodies (mAb) employed were MNF116 and LP34 (Dakopatts) against epithelial cytokeratins; clone V9 (Dakopatts) against vimentin; clone IA4 (Bio-Makor) against smooth-muscle-specific alpha-actin ; and clone F8/86 and a polyclonal rabbit antiserum (Dakopatts) against vimentin. Antibodies against yon Willebrand factor VIII (factor VIII) for endothelial cells, clone TE7 (Seralab Ltd) recognizing reticular fibroblasts, clone 9.4 (New England Nuclear) for the CD45 panleukocytic antigen and "QB END 10" (Melgriff) for the CD34 haematopoietic stem cell antigen were also used for cell type identification.
Cells Cells were seeded in "Lab-tek" chamber slides and grown for 24-48 hours. They were then rinsed twice in PBS and air-dried for 2 hours. Glass slides were timed in cold acetone and dried. Fixed cells were washed in TBS (Trisbuffered saline pH 7.4) 0.05 M and preincubated for 10 rain in TBS containing 0.5 07oBSA; an overnight incubation (4°C) with the various mAb appropriately diluted was followed by multiple washings in TBS. The primary antibodies were revealed with anti-m"ouse peroxidaseconjugated rabbit antiserum (Dakopatts) diluted 1:100 in TBS-BSA (1 hour at room temperature). Cells were then washed in Tris and stained using DAB (Sigma) as chromogen. Growth on Matrigel
Matrigel, a mixture of basement membrane components extracted from the EHS tumour, was prepared according to Kleinman et al. (1986) and stored at -20°C. For morphological assays, Matrigel was thawed in a 0°C ice-water bath, diluted to a final concentration of 10 mg/ml, and 0.4 ml was gently pipetted into each 13-mm diameter tissue culture well. After 1 h at 37°C, Matrigel was completely polymerized, and 5 x 1 0 4 cells in 1 ml of normal medium were carefully seeded on top of the Matrigel layer. The seeded plates were incubated at 37°C in a 5 % CO2 humidified atmosphere and photographed at various intervals. Chemotaxis assay
Chemotaxis assays were performed in Boyden's chambers according to Postlethwaite et al. (1976) as modified (Albini et al., 1987a). Briefly, the lower compartment was filled (200 ~l of total volume) with the KS-conditioned
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medium (KSCM) as chemoattractant (or with serum-free medium as controls) and covered with a polycarbonate filter (12-1an pore size; Nucleopore) coated with type IV collagen (5 ~g/filter; Sigma) to support cell adhesion. KSCM was collected after incubating KS cells in serum-free medium (SFM) for 24 hours. The medium was then centrifuged and stored frozen at -20°C. HUVEC were harvested with trypsin, then centrifuged, washed and resuspended in SFM. The cells were brought to a final concentration of 250 x 10a cells/mis~, and 400 ~l of the suspension were placed in the upper compartment. After a 6-h incubation (37°C, 5 % CO2), filters were taken, and the cells present on the upper side were removed. The migrated cells were fixed in absolute ethanol, stained with toluidine blue and counted (5 unit fields per filter) at a 160x magnification with a "Zeiss" microscope. One field represents 1/160 of the entire area of the migrated cells.
normal culture medium. After the 8th passage they began a slow process o f regression. The growth of KSIsTVIII cultures could be supported by the addition of Ultroser-G (similarly to the previously established AIDS-KSIsTIII), while AIDS-KSIsTIV cells did not respond to Ultroser-G. Ultroser-G was able to p r o m o t e AIDS-KSIsTIII and KSIsTVIII cell proliferation until 15-16 passages in culture. We phenotyped our cultures using a large panel o f antibodies (table I). Like AIDS-KSIsTIII cells, both AIDS-KSIsTIV and KSIsTVIII cell showed a smooth-muscle/fibroblast-like phenotype, being positive for smooth-muscle-specific alpha-actin, desmin (not shown) and TE7. A mesenchymal origin o f these cells was confirmed by strong positivity for vimentin (fig. 1). In contrast, both AIDS-KSIsTIV and KSIsTVIII were negative for the endothelial marker factor VIII, the pan-leukocytic marker CD45 and for epithelial cytokeratins. When these cells were cultured in Ultroser-G enriched medium, the pattern o f ch~acterization changed; in fact, after a few passages in Ultroser-G the smooth muscle actin staining almost disappeared in the KSls-rVIII cultures. However, this was reversible: the immunochemical staining for a-actin returned to the previous state after withdrawal o f the supplement. This change occurred without apparent alterations in the morphology and the staining for other markers, such as TE7 and vimentin.
Chemoinvasion assay The chemoinvasion assay (Albini et al., 1987b) was performed as for chemotaxis, except that the collagen-IVcoated polycarbonate filter was overcoated with 25 ~g of Matdgel, dried and reconstituted with SFM prior to use.
RESULTS Establishment histochemistry
of
KS
cultures
and
immuno-
Both KS primary cultures, AIDS-KSIsTIV and KSlsrVIII had the same spindle-shaped m o r p h o l o gy previously described for other KS cultures (Albini et al., 1992; Corbeil et al., 1991 ; N a k a m u r a et ai., 1988; Salahuddin et al., 1988; Wittek et al., 1991). Similarly to previous cell cultures that we have obtained f r o m KS biopsies, AIDS-KSxsTIV and KSIsTVIII cells proliferated with a doubling time o f 72 h until the 8th passage, without the addition o f supernatants o f activated P B L or N u t r i d o m a to the
Growth o f KS-derived cultures on Matrigel When grown on a three-dimensional Matrigel substrate, 24 hours after seeding, the new cell lines established showed the typical invasive branching morphology of neoplastic mesenchymal cells (fig. 2). This m o r p h o l o g y has been described previously for other KS cell lines (Albini et al., 1992) and for primary malignant sarcoma cells (Nicolb et ai., 1991).
Table I. Immunohistochemistry of KSIsT cells and relative controls. Marker Keratin Vimentin a-Actin VW-Fact. TE7 CD34 CD45
KSIsTIV
KSIsTVIII
SMC
Fb
HUVEC
+ + + .
+ + + -
nd + + + / _ (a) nd .
+ + nd
+ + _ nd
.
.
.
Cells grown on chamber slides were fixed and stained as described in "Materials and Methods". nd= not done; (a)=TE7 positive for SMC in vivo and detected in vitro in cultures of SMC derived from human carotide artery. SMC = smooth musclecell. Fb = fibroblast.
.!.J
a
¢
b
d
Fig. 1. Immunohistochemistry of KS-derived cells. a = expression of TE7 (KS~sTVIII), b = expression o f smooth muscle isoform of ~-actin (KSIsTIV), ¢ = lack of CD34 expression (KSIsTVIII) and d = expression of vimentin (KSIsrVIII); detected with immunoperoxidase method. ( × 685).
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R. B E N E L L I E T A L .
y
a
e
b
d
Fig. 2. Growth on Matrigel. a = HUVEC form tube-like structures as previously described, b = fibroblasts form small colonies, c = AIDS-KSIsTIVand d = KSIsTVIIIshowing the typical invasive branching colonies exhibited by tumours of mesenchymal origin (photograhs taken 48 hours after plating; a and b x 270; c and d x 274.
Induction of endothelial cell chemotaxis and invasiveness by KS cultures
We tested the in vitro angiogenic properties of these KS-derived cells by utilizing KSCM as an endothelial cell activator in the Boyden's chamber chemotaxis and chemoinvasion assays. The HUVEC chemotactic response to conditioned medium of both AIDS-KStsrlV and KS~-rVIII was much higher than that to SFM controls (fig. 3). KSCM also induced a three-fold increase in endothelial cell invasion through a thin layer of Matrigel. DISCUSSION The newly established AIDS-KSIsTIV and KSxsT_ VIII Kaposi's sarcoma-derived ceils show morphology, immunohistological staining, invasive behaviour and in vitro angiogenic properties similar to those seen for the previously obtained KStsTII and AIDSKSmTIII lines (Albini et al., 1992). Their morphological appearance is also similar to that described
for most established KS-cell cultures. The culture procedure appears to select for the "neoplastic" cells found in KS. The histopathological analysis of KS biopsies has always demonstrated abundant expression of endothelial markers in the lesions (Armes, 1989); however, very rarely have cells with endothelial markers been obtained from these biopsies. This is probably due to the fact that most of the endothelial cells are " n o r m a l " cells recruited from the host, and these do not survive well in culture. Immunohistochemistry also shows conflicting information between the in vivo lesion and the cultured cells. Only a few of the poorly differentiated spindle cells proliferating inside the KS lesion express smooth muscle actin; however, cultured cells have a much higher frequency of positively stained cells (20-50 %). We hypothesize that the KS spindle cell can assume more smooth-muscle-like or more fibroblasticlike (or perhaps even more endothelial-like) characteristics under different conditions or stimulation. This hypothesis is supported by the different expression of smooth muscle a-actin demonstrated by
N E W K A P O S I ' S S A R C O M A CELL CU LTU R E S F R O M A I D S A N D N O N - A I D S 100'~1
Chemotaxis and chemolnvasion of AIDS KS IST IV-stimulated HUVE cells
80 • [] _=
chemotaxis invasion
6O
257
polyclonal KS lesions. Under appropriate conditions, it is possible that some undifferentiated mesenchymal/vascular precursor cells normally present in the organism begin to proliferate but do not differentiate, giving rise to KS lesions. This hypothesis could explain the evident discrepancies in the immunohistochemical distribution of markers in vivo and in vitro. In KS lesions, the presence of activated lymphocytes, macrophages, "spindle-shaped" cells and endothelial cells indicates the presence of a very complex network of autocrine and paracrine stimulation. It would be of interest, although likely very difficult, to detect the true plasticity of spindleshaped KS cells inside the lesion. It would be of even greater interest to discover the cytokine(s) able to induce a definitive differentiation of KS spindle cells that could end the progression of the lesion.
o}
0
40,
=E z 20,
KS IV CM
SFM
Acknowledgements
I O0
We would like to thank Dr. Douglas Noonan (IST, Genova) for critical reading and for revision of the English. This work was financed by grants from the Italian Ministero della Sanit/l, VI progetto AIDS. R. BeneUi is an AIRC fellow.
80 tcJ ~t o "ID
60
Caract~risation de deux nouveHes cultures cellulaires de sarcome de Kaposi ~tablies k partir d'un cas de S I D A et d'un cas de sarcome sporadique
E 0
40 E
20
KS VIII CM
SFM
Fig. 3. Chemotaxis (black) and invasion (grey) of HUVEC in response to AIDS-KSIsTIV (top) and KSIsTVIII (bottom) conditioned media. SFM = serum-free medium controls.
KSmTVIII cells cultured with or without Ultroser G, It is probable, as has been shown for other systems, that cytokines or components of the extracellular matrix are able to induce pluripotential differentiation of vascular precursor cells. Another phenomenon which supports the emergence of an undifferentiated mesenchymal cell in the KS pathology is the frequent observation of multifocal and
A partir de biopsies cutan6es, deux nouvelles ligndes cellulaires de sarcome de Kaposi (KS= Kaposi's sarcoma) ont ~t6 &ablies: AIDS-KSxsTIV darts un cas de KS associ6 au SIDA et KS[sTVIII dans un cas de KS sporadique. Ces deux lign6es sont principalement compos6es de cellules fusiformes, elles ont des diagrammes de coloration immunohistochimique semblables et elles sont positives pour les marqueurs des muscles lisses (actine-~ de muscle lisse) et les marqueurs fibroblastoides (TE7). Aucune de ces lign6es n'exprime le marqueur endoth61ial du facteur VIII de yon WiIlebrand. Ces profils immunohistochimiques sont semblables A ceux d'autrcs ligndes cellulaires KS d6j/t &ablies. Quand elles sont ensemenc6es sur une membrane basale reconstitu6e (
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R. B E N E L L I E T A L .
Les 16sions du KS sont caract6ris~es par une n6ovascularisation tr~s 6vidente, d6riv~e des cellules h6tes. Nous avons test6 la capacit6 des surnageants des cellules KS d'induire une r6ponse angiog~nique in vitro. Les nouvelles lign6es sont capables de stimuler le chimiotactisme des cellules endoth61iales humaines et l'invasion ~ travers les filtres couverts de Matrigel. Aucune diff6rence darts le potentiel angiog6nique in vitro n'a 6t6 observ6 entre les cellules du sujet VIH + et celles du sujet V I H - comme nous l'avions d6j/l not~ pour d'autres cultures. Les nouvelles lign~es cenulaires 6tudi6es ici ont les propd6t6s des cellules du sarcome de Kaposi et elles confirment que les cellules fusiformes du KS de patients VIH + ou V I H - ont des ph6notypes et des caract6dstiques de comportement in vitro identiques. Mots-clds: Sarcome de Kaposi, SIDA; Cultures cellulaires.
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NEW KAPOSI'S SARCOMA CELL CULTURES FROM AIDS AND NON-AIDS
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