Tumorigenicity of IL-1α– and IL-1β–Deficient Fibrosarcoma Cells

Analyzing the growth of fibrosarcoma lines derived from IL-1α–, IL-1β–, or IL-1αβ–knockout () mice in the immunocompetent host revealed that tumor-derived IL-1α and IL-1β exert strong and opposing effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1–deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1–deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1αβ–competent () lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1β lines, IL-1α strengthens anchorage-independent growth, but both IL-1α and IL-1β support drug resistance. Corresponding to the aggressive growth, IL-1β cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated αv/β3 and matrix metalloproteinase expression. Recruitment of myeloid cells requires IL-1β but is regulated by IL-1α, because inflammatory chemokine and cytokine expression is stronger in IL-1αβ than in IL-1 lines. This regulatory effect of tumorderived IL-1α is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1β. Both sarcoma cell–derived IL-1α and IL-1β promote tumor growth. However, IL-1α exerts regulatory activity on the tumor cell–matrix cross-talk, and only IL-1β initiates systemic inflammation. Neoplasia (2008) 10, 549–562 ors derived from mice with a targeted deletion of the corresponding genes; IL-1, derived suppressor cells; MMP, matrix metalloproteinase; RPMI-s, RPMI 1640, supcutaneously; wt, wild type une Defense, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 an Cancer Research Center, Heidelberg, Germany (M.Z., R.N.A.), the Tumorzentrum , the Israel Science Foundation founded by the Israel Academy of Sciences and Hu), Association for International Cancer Research (R.N.A.), the German–Israeli DIP dation (E.V.), and the Israel Cancer Association (E.V.). R.N.A. is an incumbent of 550 Fibrosarcoma-derived IL-1 Nazarenko et al. Neoplasia Vol. 10, No. 6, 2008 Introduction The IL-1 family consists of the agonistic proteins IL-1α and IL-1β and the IL-1 receptor antagonist (IL-1Ra) [1,2], which by binding to IL-1 receptors without transmitting an activation signal acts as a physiological inhibitor [3]. IL-1α and IL-1β are synthesized as 31-kDa precursors and are processed by proteases to their mature 17-kDa forms. IL-1β–converting enzyme cleaves the inactive IL-1β precursor; ProIL-1α is processed by calpain [4,5]. Many cell types produce and secrete IL-1α, IL-1β, and IL-1Ra on activation with environmental stimuli [6,7]. Mononuclear cells secrete the highest levels, mainly of IL-1β [1,7]. Secreted IL-1α and IL-1β bind to the same receptors and exert similar biologic activities. However, IL-1α and IL-1β differ inasmuch as IL-1β is solely active as a secreted product; IL-1α is mostly active as an intracellular precursor and in its membrane-associated form. The active membrane form of IL-1α is derived from myristoylation of proIL1α and is anchored to the membrane through a mannose-like receptor [1,2,7–9]. IL-1 is a pleiotrophic cytokine that primarily affects inflammatory responses, immune reactivity, and hematopoiesis [1,2,7,9,10]. Its potency stems from inducing cytokine, chemokine, proinflammatory molecule secretion, and adhesion molecule expression in diverse cells, thereby amplifying and sustaining the response. Both the localization of the IL-1 molecules in the producing cell and the microenvironment dictate their biologic functions [8]. Membrane-associated IL-1α is supposed to be immunostimulatory. Cytosolic proIL-1α may control gene expression, proliferation, and differentiation [5, 11–15]. Low-level secreted IL-1β induces limited inflammatory responses followed by T cell activation. High doses of IL-1β are accompanied by broad inflammation with tissue damage [1,2,7,16]. IL-1 is abundant at tumor sites, being secreted by the malignant cells and/or cells in the tumor microenvironment in response to local inflammatory signals. It can promote invasiveness and metastasis formation or induce an antitumor immune response and inhibit tumor growth [6,7,17]. Over-expression of the precursor of IL-1α by fibrosarcoma cells can initiate a strong immune response [11– 13,15,18]. In contrast, IL-1β–transfected tumor cells are more invasive than wild type cells [19,20]. Increased invasiveness correlates with enhanced angiogenesis and activation of immunosuppression [18–23], which may be a consequence of tumor-derived IL-1β supporting extramedullary myelopoiesis [18,23,24]. To explore whether these effects are exclusively or at least predominantly due to tumor-derived IL-1, fibrosarcoma were induced in IL-1α–, IL-1β–, or IL-1αβ–deficient mice [19,25]. Because IL-1 expression and secretion was unimpaired in the syngeneic host, this system allowed to selectively elaborate the contribution of tumorderived IL-1 on tumor growth. The analysis of in vivo growth of these IL-1α–, IL-1β–, or IL-1αβ–deficient lines in the immunocompetent, IL-1–competent host confirmed that tumor-derived IL-1 induces an utmost strong T cell response (IL-1α) or immunosuppression (IL-1β) [26]. Thus, it became important to explore whether tumor-derived IL-1 affects exclusively the host’s immune system or also the tumor cell itself. A detailed in vitro analysis of the tumor lines and their growth in nu/nu mice revealed that IL-1β comp tumors are more aggressive, which is a consequence of tumor-derived IL-1β locally and systemically initiating an inflammatory milieu. Tumor-derived IL1α exerts no systemic effects, but promotes transcription of genes, which support tumor cell survival and the cross-talk of IL-1β with the tumor environment. Material and Methods Mice and Tumors nu/nu mice (WIGA, Sulzfeld, Germany), kept under specific pathogen-free conditions, fed sterilized food, and water ad libitum, were used for experiments at the age of 8 to 10 weeks. IL-1RI mice were purchased from Jackson Laboratories (Bar Harbor, ME). Fibrosarcoma were induced by 3-methylcholanthrene treatment of wt, IL-1α, IL-1β, IL-1αβ, and IL-1RI C57BL6 mice [19]. The IL-1–deficient mice were generously provided to R.N. A. by Prof. Yoichiro Iwakura, University of Tokyo, Tokyo, Japan [25]. 3Methylcholanthrene (Sigma Israel, Rehovot, Israel) was dissolved in olive oil (200 μg/mouse) and was subcutaneously (s.c.) injected into the right thigh [27]. Local fibrosarcomas developed within 3 to 5 months. When tumors reached a diameter of 10 mm, mice were killed and the tumor tissue was aseptically removed. Part of the tissue was processed for the establishment of cell lines by enzymatic digestion in trypsin. The following fibrosarcoma lines were used: IL-1wt lines 2, 19, 21 (wt2, wt19, wt21), IL-1α lines 3, 15, 16 (α3, α15, α16), IL-1β lines 3, 4, 17 (β3, β4, β17), IL-1αβ lines 6, 11, 13 (αβ6, αβ11, αβ13), and IL-1RI −/− lines PV and R1. Each of these 14 tumor lines was derived from a different 3-methylcholanthrene–treated C57BL6 mouse. The lines were maintained in RPMI 1640, 10% fetal calf serum. Antibodies Anti-CD11b, -CD54 (European Collection of Animal Cell Cultures, Salisbury, UK), -panCD44 (American Type Culture Collection, Manassas, VA), CD49d [28], -CD44v6, -CD51, -CD49c, -CD49f, -CD29, -CD61, -CD154, -CD31 (PECAM), -CD62E (E-selectin), CD105 (endoglin), -Gr-1, -CD95, -CD178 (CD95L), -CD120a (TNFRI), -CD120b (TNFRII), -CD121a (IL-1RI), -CD121b (IL-1RII), -CD87 (uPAR), -MMP2, -CCL1, -CCL2, -CCL3, -CCL5, -CCL15, -CCL17, -CCL19, -CCL20, -CXCL10, -OPN, -CCR3, -CCR4, -CCR6, -CCR7, -CCR8, native and biotinylated anti–IL-1α, –IL-1β, –IL-4, –IL-6, –IL-10, –IL-12, -IFNγ, -TNFα, -TGFβ, -ERK1,2, -pERK1,2, -pJNK, -pJun, -PTEN, -pIκBα, -pAkt, -pBAD, -Bid, -BIM, -Bcl-2, –Bcl-Xl, -BAX, -PARP, -actin, and secondary labeled [biotin, fluorescein isothiocyanate (FITC), phycoerythrin, allophycocyanin, and HRP] antibodies, were obtained commercially. Flow Cytometry Approximately 2.5 to 5 × 10 cells were stained according to standard protocols. For intracellular staining, cells were fixed (formaldehyde) and permeabilized (PBS, 1% Tween 20). Apoptosis was determined by Annexin V–FITC/PI staining. Fluorescence was determined using a FACStar and the CellQuest program (BD, Heidelberg, Germany). Cytokine ELISA Standard sandwich ELISA procedures were used to measure IL-1α, IL-1β, and IL-1Ra secretion. Immunohistology Sections (5 μm) of snap-frozen tumor were fixed (chloroform/ acetone, 1:1, 4 minutes) and treated with levamisole solution to ablate tissue alkaline phosphatase activity. Nonspecific binding was Neoplasia Vol. 10, No. 6, 2008 Fibrosarcoma-derived IL-1 Nazarenko et al. 551 blocked using an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA) and 2% normal serum from the same species as the secondary antibodies. For intracellular staining, tissues were fixed and permeabilized (4% paraformaldehyde, 0.1% Triton X-100). Tissues were incubated with the primary antibody (1 hour), the biotinylated secondary antibodies (30 minutes), and alkaline phosphatase– conjugated avidin-biotin complex solutions (5–20 minutes). Sections were counterstained with Mayer’s hematoxylin. Primary antibodies were replaced by rat or rabbit IgG for negative controls. Western Blot Analysis Cells (5 × 10) were lysed in 1% Triton X-100. Where indicated cells were cultured for 24 hours in the presence of recombinant IL-1α and/or IL-1β (10 ng/ml) (CyoLAB/Peprotech, Rocky Hill, NJ). Lysates were resolved on 10% SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose membranes (30 V, overnight). After blocking (5% fat-free milk powder, PBS, 0.1% Tween 20), immunoblot analysis was performed with the indicated antibodies, followed by HRP-labeled secondary antibodies. Blots were developed with the enhanced chemiluminescence detection system. Tumor Cell Proliferation and Apoptosis Tumor cells (1 × 10) were seeded in F-bottom 96-well plates, adding 10 μCi/ml H-thymidine and harvesting cultures after 16 hours. Apoptosis resistance was evaluated by the MTT assay and by Annexin V–FITC/PI staining. Cells (1 × 10), cultured overnight in F-bottom 96-well plates, were grown for 3 days in RPMI-s containing serial dilutions of cisplatin [cis-diamineplatinum(II) dichloride; Sigma, Munich, Germany], starting with 35 μg/ml. Adhesion Assay Cells (1 × 10) were seeded on F-bottom 96-well plates coated with collagens I and IV (10 μg/ml) or vitronectin (2 μg/ml). After 2 hours (37°C, 5% CO2), nonadherent cells were removed by washing, and adherent cells were stained with crystal violet. Absorbance was measured at 595 nm. Soft Agar Assay Tumor cells, suspended in 0.3% agar, were seeded on a preformed 1% agar layer. Where indicated, the agar contained 10 μg/ ml anti–IL-1RI. Colonies was counted at an inverted microscope after 3 weeks. Migration Assays Tumor cells were seeded in 2-cm diameter, fibronectin-coated Petri dishes. Subconfluent cultures were wounded by scratching with a micropipette tip, washed, and incubated for 48 hours. Wound healing was evaluated microscopically after staining with hematoxylineosin. Alternatively, tumor cells (1 × 10) were seeded in the upper part of a Boyden chamber in 30 μl of RPMI/0.1% BSA. The lower part of the chamber, separated by an 8-μm–pore size polycarbonate membrane (Neuroprobe, Gaithersburg, MD), contained 30 μl of RPMI/10% fetal calf serum. After 4 hours, cells on the lower side of the membrane were stained with crystal violet. Absorbance was measured at 595 nm. Zymography Tumor cells (5 × 10) were starved for 24 hours, the conditioned medium was centrifuged (15 minutes, 15,000g), and aliquots of the supernatants were incubated with Lämmli buffer (15 minutes, 37°C) and separated in a 10% acrylamide gel containing 1 mg/ml gelatin. After washing, gels were stained with Coomassie blue. Matrigel Assay Tumor cells (2 × 10) were seeded on matrigel-coated 24-well plates. Cable formation was evaluated after 24 hours by light microscopy. Tumor Growth nu/nu mice received 5 × 10 tumor cells, s.c. Tumor growth was controlled (mean diameter) twice per week. Mice were killed at the indicated time points after tumor cell application, but latest when the s.c. tumor mass reached a mean diameter of 3 cm (survival time). Mice were bled by eye puncture, and serum was collected. Tumor, spleen, femura, and tibiae were excised. The tumor was shock frozen. The spleen was teased through fine gauze. The bones were flushed with PBS. Single spleen cell (SC) and bone marrow cell (BMC) suspensions were washed and used for flow cytometry. Animal experiments were approved by the governmental authorities for animal health care. Statistical Analysis Significance of differences was calculated by the Student’s t-test (in vitro assays) or the Wilcoxon rank sum test (in vivo assays). P values <.05 were considered significant. Results Using fibrosarcoma lines derived from IL-1α, IL-1β, and IL1αβ mice, we recently demonstrated that tumor-derived IL-1α and IL-1β have very strong bearing on immune response induction in the immunocompetent, IL-1–competent host, such that IL-1α tumor lines induce a T cell–mediated rejection response, whereas IL-1β comp tumors induce strong immunosuppression that suffices to counterregulate immune response induction by IL-1α with the consequence that IL-1αβ comp tumors grow in the immunocompetent host [26]. These unexpectedly strong effects exclusively of tumorderived IL-1 on the host immune system prohibited a judgment on the impact of tumor-derived IL-1 on tumorigenicity. However, an answer to this question is essential for estimating potential therapeutic efficacy of IL-1α in tumor rejection. Tumorigenicity of IL-1α– and/or IL-1β–Deficient Tumor Cells IL-1αIL-1β tumor lines do not grow in the immunocompetent host [26]. To elaborate whether this failure to grow is exclusively a consequence of induction of a potent T cell–mediated response or whether IL-1αIL-1β tumor lines are less tumorigenic, the experiment was repeated in nu/nu mice. All tumor lines grew in nu/nu mice. However, the growth rate of IL-1β and IL-1αβ tumors was reduced, and accordingly, the mean survival time was prolonged (Figure 1, A and B). In addition, IL-1β comp tumors grew more aggressively, infiltrated the surrounding tissue, and showed massive infiltration by myeloid cells, which were not seen in IL-1β tumors (Figure 1C ) but have also been seen in IL-1β comp tumors in the immunocompetent host [26]. Importantly, too, IL-1β comp tumors strongly supported angiogenesis, whereas only few capillaries were detected in IL-1β tumors (Figure 1D). 552 Fibrosarcoma-derived IL-1 Nazarenko et al. Neoplasia Vol. 10, No. 6, 2008 Figure 1. Growth of wt, IL-1α, IL-1β, and IL-1αβ fibrosarcoma lines in nu/numice. (A and B) nu/numice (10 mice/line) received a s.c. injection of 5 × 10 fibrosarcoma cells from 3 wt, IL-1α, IL-1β, and IL-1αβ fibrosarcoma lines. (A) Starting 7 days after tumor cell inoculation, tumor growth was monitored twice per week by measuring the tumor diameter. The mean tumor diameter ± SD of 30 mice/group (10 mice/line) is shown. (B) Mice were sacrificed when the mean tumor diameter reached 3 cm (survival time). The mean survival time ± SD (30 mice/group; 10 mice/line) is shown. Significant differences in the mean tumor diameter and the survival time as compared to mice receiving wt clones are indicated by an asterisk. (C and D) Wt, IL-1α, IL-1β, and IL-1αβ fibrosarcoma cells (5 × 10) were s.c. implanted into nu/numice. Tumors of a mean diameter of 0.5 cm were excised, shock frozen and 5 μm sections were stained with anti–Gr-1 (C) and anti-CD31 (D). Representative examples of one wt, IL-1α, IL-1β, and IL-1αβ fibrosarcoma lines are shown. (E) IL-1α, IL-1β, and IL-1Ra expression and secretion was evaluated in lysates (corresponding to 1 × 10 cells) and culture supernatants (fresh medium being added to subconfluent cultures and being collected after 24 hours) of wt, IL-1α, IL-1β, IL-1αβ, and IL-1RI fibrosarcoma lines by ELISA. Secreted IL-1α hardly could be detected in any of the tested lines. Yet, even in supernatants and lysates of IL-1 and IL-1RI cells, IL-1α and IL-1β was too low for a reliable quantification. Therefore, extinction values (mean ± SD of triplicates) are shown. Background values have been subtracted. Neoplasia Vol. 10, No. 6, 2008 Fibrosarcoma-derived IL-1 Nazarenko et al. 553 Taken together, irrespective of IL-1 expression, 3-methylcholanthrene– induced fibrosarcoma are tumorigenic in the immunocompromised host. However, tumor-derived IL-1β appears to promote tumor growth more efficiently than tumor-derived IL-1α. Notably, the tumors express/secrete a very low level of IL-1α and IL-1β, and there is no evidence that the absence of IL-1α or IL-1β would be compensated by considerably up-regulated expression/secretion of the remaining IL-1 gene. IL-1 expression was also low in an IL-1RI line. IL-1Ra expression and, particularly, secretion were slightly increased in IL-1αβ comp lines and slightly reduced in IL-1β lines. It was not influenced by an IL-1RI deficiency (Figure 1E ). These findings suggested that low-level tumor-derived IL-1 or the host environment during carcinogenesis may have strong bearing on the expression of distinct genes. To control for these hypotheses, we evaluated several central features of tumorigenicity and tumor aggressiveness in the wt, IL-1α, IL-1β, and IL-1α/β tumor lines. To avoid, as far as possible, in vitro selection processes, the experiments were performed with uncloned tumor lines that were cultured for less than 10 passages in vitro. Furthermore, each line was derived from a different 3-methylcholanthrene–treated mouse. IL-1α Competence Promotes Anchorage-Independent Growth and Both Tumor-Derived IL-1α and IL-1β Support Apoptosis Resistance The accelerated growth of IL-1β comp tumors could have been a consequence of accelerated cell cycle progression or pronounced apoptosis resistance. Therefore, we explored the impact of IL-1 competence on these parameters in vitro. We expected accelerated proliferation and/or high-efficacy anchorage-independent growth possibly combined with increased apoptosis resistance of IL-1β comp lines. This has not been the case. Neither an IL-1β nor an IL-1α deficiency influenced tumor cell proliferation (Figure 2A). In addition, IL-1α, but not IL-1β competence, supported anchorage-independent growth in soft agar, such that colony formation of IL-1α lines was strongly reduced. Anchorage-independent growth of IL-1αβ lines also was impaired, but less efficiently. The high cloning efficacy of IL-1α lines was maintained in the presence of anti–IL-1RI. In addition, an IL-1RI line also revealed high cloning efficacy. However, the low cloning efficacy of IL-1αβ comp lines was further reduced in the presence of anti–IL-1RI (Figure 2B). Thus, IL-1α supports anchorage independent growth likely independent of IL-1RI expression, but IL-1β (minor contribution, if at all) might act through the IL-1RI. Apoptosis/drug resistance of the IL-1–deficient lines also did not meet our expectation. When cultured in the presence of an increasing dose of cisplatin, apoptosis resistance was strongly reduced in IL-1α and IL-1β cells, whereas IL-1αβ cells displayed unaltered or slightly increased cisplatin resistance compared with wt cells. This has been evaluated by MTT (Figure 2C ) and Annexin V/PI staining (Figure 2D) and has been confirmed by reduced Hthymidine uptake of cisplatin-treated IL-1α and IL-1β, but not IL-1αβ −/− compared with IL-1 cells (data not shown). Trying to explain these unexpected findings, expression of several proand antiapoptotic proteins was analyzed by flow cytometry and Western blot analysis. CD95 expression was reduced in IL-1α, IL-1β, and IL-1αβ sarcoma lines (Figure 2E ). Expression of CD95L and TNFRI was largely independent of tumor cell–associated IL-1. Instead, TNFRII expression was particularly low in IL-1α and IL-1αβ lines (Table 1). Expression of some proapoptotic proteins, such as BIM, PARP (data not shown), and Bid, varied between the individual lines and thus could not account for the uniformly decreased apoptosis resistance of IL-1α and IL-1β lines. However, in line with unimpaired drug resistance, both Bcl2 and proapoptotic Bax expression was significantly increased in IL-1αβ lines. Decreased apoptosis resistance of IL-1β lines was accompanied by reduced Bcl2 expression. In IL-1α lines, a reduction in Bcl2 expression was not consistently observed (Figure 2F ). These findings suggest that IL-1α and IL-1β might be distinctly involved in the regulation of both proand antiapoptotic signaling cascades. To support this hypothesis, Bcl2 and Bax expression was evaluated after culturing cells in the presence of recombinant IL-1α and/or IL-1β. Recombinant IL-1α and IL-1β did not influence Bax expression. This also accounted for an IL-1RI line. Surprisingly, Bcl2 expression became up-regulated in the presence of exogeneous IL-1α plus IL-1β in the IL-1RI line and down-regulated in an IL-1αβ line. Instead, exogeneous IL-1α or IL-1β had no impact on Bcl2 expression in IL-1, IL-1α, and IL-1β cells (Table 2). Thus, one could hypothesize that possibly in the absence of IL-1α and IL-1β the Bcl2 protein may become stabilized or, at least, become less efficiently degraded. So far, we failed to identify the relevant signaling pathways. However, several pathways could be excluded. Akt phosphorylation and Pten expression were not or not consistently affected by an IL-1α or IL-1β deficiency. In addition, extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation, which could have been indicative for apoptosis resistance and anchorage independence [29], was not affected by an IL-1α and/or IL-1β deficiency (Figure 2G ). As a possible alternative explanation for the distinct effect of tumor-derived IL-1α and IL-1β on tumor cell survival, we hypothesized that IL-1β, possibly together with TNFα, might sustain an inflammatory milieu that supports the activation of an apoptotic signaling cascade. To control the latter, inflammatory cytokine expression was evaluated in the IL-1 and IL-1 tumor lines. Unexpectedly, IL-10 and TNFα expression was increased in IL-1α β , but also, although less pronounced, in IL-1αβ lines. This suggests that within the tumor cell inflammatory cytokines expression may be actively controlled and down-regulated by IL-1α. In line with this interpretation, inflammatory cytokine expression was comparably low in IL-1α, IL-1RI lines (Figure 2H ). It should be mentioned that IL-1RI expression was unaltered in IL-1 cells and IL-1RII expression was low and not significantly influenced by tumor-associated IL-1 (data not shown). Although these experiments confirm opposing effects of IL-1α and IL-1β on proand antiapoptotic gene expression by the IL-1– producing tumor cell and an involvement of IL-1α in proinflammatory cytokine expression, we have no unequivocal answer on the impact of tumor-derived IL-1α and IL-1β on the tumor cell’s apoptosis resistance. In addition, only IL-1α promotes anchorageindependent growth, possibly through sustained TNFRII expression [30]. Both apoptosis resistance, distinctly regulated by tumor-derived IL-1α and IL-1β and anchorage-independent growth promotion by IL-1α, do not explain accelerated tumor growth of IL-1β comp tumor lines in vivo. Tumor Cell–Derived IL-1α and IL-1β: Cell Adhesion, Migration, and Angiogenesis IL-1β fibrosarcoma lines grow less invasively and are poorly vascularized. We interpreted the finding in the sense that tumorderived IL-1β might modulate tumor cell adhesion, migration, 554 Fibrosarcoma-derived IL-1 Nazarenko et al. Neoplasia Vol. 10, No. 6, 2008 Neoplasia Vol. 10, No. 6, 2008 Fibrosarcoma-derived IL-1 Nazarenko et al. 555 matrix degradation, and angiogenesis. To support the assumption, adhesion molecule, matrix metalloproteinase (MMP), and chemokine expression was analyzed. From the adhesion molecules tested, that comprised panCD44, CD44v6, the integrins CD49c (α3), CD49d (α4), CD49f (α6), CD51 (αv), CD29 (β1), CD61 (β3), CD104 (β4), CD54 (ICAM1), and CD62E (E-selectin), reduced CD51 and CD61 expression in IL-1β lines was the most prominent change. CD44v6 and CD54 were up-regulated in IL-1αβ lines. CD62E was up-regulated in IL-1αβ and down-regulated in IL-1αβ −/− lines (Figure 3A). Additional changes in adhesion molecule expression were not significant (data not shown). Reduced CD51/CD61 expression of IL-1β −/− and IL-1αβ −/− cells correlates with reduced adhesion to collagens I and IV and vitronectin (Figure 3B). Adhesion to hyaluronan, collagen III, fibronectin, and laminins 1 and 5 was unaltered (data not shown). IL-1β −/− and IL-1αβ tumor cells also revealed reduced transwell migration and in vitro wound healing (Figure 3, C and D). In line with the reduced CD51/CD61 expression, IL-1β and IL-1αβ tumor cells formed small clusters but not cablelike structures on matrigel. Instead, IL1β comp lines formed a net of cablelike structures (Figure 3E ). Cable formation is suggested to be an angiogenesis-related morphogenic feature. Thus, cable formation by IL-1β comp tumor lines corresponds well to the stronger in vivo angiogenesis by these tumors, and it became interesting to see whether IL-1β would have bearing on additional angiogenesis-related factors. Vascular endothelial growth factor and basic fibroblast growth factor expression was low and was not reduced in IL-1β or IL-1αβ lines (data not shown). Instead, in the absence of IL-1β, low uPAR expression was further reduced (Figure 3F ), and MMP9 secretion was strongly diminished in IL1αβ and distinctly in IL-1αβ lines (Figure 3G ). Taken together, IL-1β comp tumor lines are more adhesive and motile and display angiogenesis-related features. These findings are in line with high CD51/CD61, up-regulated CD62, up-regulated uPAR expression, and increased MMP provision. Neither was there evidence for an active contribution of tumor-derived IL-1α to CD51/ CD61, uPAR, and MMP expression/secretion nor has there been evidence that IL-1α may counterregulate expression of these molecules. As outlined previously, this was distinct for cytokines but is also distinct for chemokine expression. A deficiency in IL-1α or IL-1β had no negative impact on chemokine secretion by the tumor cells. On the contrary, the expression of CCL2, CCL3, CCL5, CCL15, CCL19, and OPN, chemokines preferentially attracting leukocytes [31], was increased in IL-1α tumor lines, indicating that IL-1α Figure 2. Tumor cell–associated IL-1, proliferation, anoikis, and apoptosis resistance. (A) Three wt, IL-1α, IL-1β, and IL-1αβ fibrosarcoma lines (2 × 10 cells) were grown for 16 hours in RPMI in the presence of 10 μCi of H-thymidine and incorporation was determined in a β-counter. (B1) Three wt, IL-1α , IL-1β, IL-1αβ and 2 IL-1RI fibrosarcoma lines (10 cells) were suspended in 0.3% agar and seeded on a 1% agar layer. Where indicated, cultures contained 10 μg/ml anti–IL-1RI. After 3 weeks of culture, colonies were counted. (B2) Representative examples for one wt, IL-1α , IL-1β, and IL-1αβ fibrosarcoma lines are shown. (C and D) Three wt, IL-1α, IL-1β and IL-1αβ fibrosarcoma lines (1 × 10 cells) were cultured for 48 hours in the presence of 1.56 to 25 μg cisplatin. Survival was evaluated by MTT staining (C) and Annexin V–FITC/PI staining (D). The percentage of live cells (C) and apoptotic cells (D) as compared to cells grown in the absence of cisplatin (100%) is shown (mean values of triplicates). The amount of cisplatin required for a 50% reduction in cell survival is indicated. (E) CD95 expression was evaluated on three wt, IL-1α, IL-1β, and IL1αβ fibrosarcoma lines by flow cytometry. The percentage of stained cells and the mean intensity of staining is shown. (F and G) Wt, IL-1α, IL-1β, and IL-1αβ fibrosarcoma cells were lysed. Lysates were separated by SDS-PAGE. After transfer, membranes were blotted with the indicated antibodies specific for proand antiapoptotic proteins and for proand antiapoptotic signal transducing molecules. For Bcl2 and Bax, the intensity ratio in comparison to the actin loading control is indicated. One of 3 representative experiments are shown. (H) Three wt, IL-1α, IL-1β, IL-1αβ, and 2 IL-1RI fibrosarcoma lines were tested for cytokine expression by flow cytometry. The mean percentage of stained cells is shown: (A, B1, C, D, E, and H) mean ± SD of the distinct lines are presented and significant differences between wt, IL-1α, IL-1β, IL-1αβ, and IL-1RI lines are indicated by an asterisk, a reduction in colony formation (B1) by anti–IL-1RI is indicated by a boldface “s.” Experiments were repeated two to three times and revealed comparable results. Table 1. Flow Cytometry Analysis of Apoptosis Marker Expression in wt, IL-1α, IL-1β , and IL-1αβ −/− Fibrosarcoma Lines. Marker Tumor Lines (P )