Increased hyaluronan levels and decreased dendritic cell activation are associated with tumor invasion in murine lymphoma cell lines

Immunobiology 217 (2012) 842–850

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Increased hyaluronan levels and decreased dendritic cell activation are associated with tumor invasion in murine lymphoma cell lines Rosalia I. Cordo Russo a,∗ , Glenda Ernst a , Silvina Lompardía a , Guillermo Blanco a , Élida Álvarez a , Mariana G. Garcia a,b , Silvia Hajos a,∗ a b

Department of Immunology, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), IDEHU-CONICET, Buenos Aires, Argentina Gene Therapy Laboratory, School of Medicine, Austral University, Buenos Aires, Argentina

a r t i c l e

i n f o

Article history: Received 20 June 2011 Accepted 15 December 2011 Keywords: Dendritic cells Hyaluronan Hyaluronan synthases Hyaluronidases Lymphoma

a b s t r a c t Hyaluronan (HA), a component of the extracellular matrix surrounding tumors, modulates tumor progression and the immune response. Dendritic cells (DC) may tolerize or stimulate immunity against cancer. In this report, we study the association between tumor progression, HA levels and DC activation in a lymphoma model. Mice injected with the cells with highest invasive capacity (LBR-) presented increased HA in serum and lymph nodes, and decreased DC activation in infiltrated lymph nodes and liver. These findings could be related to lack of an effective antitumor immune response and suggest that serum HA levels could have a prognostic value in hematological malignancies. © 2011 Elsevier GmbH. All rights reserved.

Introduction Tumor progression occurs because of the existence of tumor cells and variations in the microenvironment surrounding these cells. The tumor microenvironment is composed of cells (such as stromal and immune cells), extracellular matrix components (such as hyaluronan), growth factors and cytokines (Joyce and Pollard 2009). In cancer, the remodeling of this microenvironment is closely associated with tumor progression (Christofori 2006). Metastasis constitutes the main cause of death in cancer patients and especially in those which become resistant to conventional therapies. Hyaluronan (HA) is a linear glycosaminoglycan (GAG), composed of repeated disaccharide units of d-glucuronic acid and

Abbreviations: bHABP, biotinylated hyaluronic acid binding protein; Da, Dalton; DC, dendritic cells; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; H–E, hematoxylin–eosin; HAS, HA synthase; Hyal, hyaluronidase; HMW-HA, high molecular weight-HA; i.p., intraperitoneal; LBR-, sensitive cell line; LBR-V160, vincristine resistant cell line; LMW-HA, low molecular weight-HA; MHCII, major histocompatibility complex class II; PBS, phosphate-buffered saline; Pgp, P-glycoprotein; RT, room temperature; RT-PCR, reverse transcription-polymerase chain reaction; SEM, standard error of the mean; VCR, vincristine. ∗ Corresponding authors at: Department of Immunology, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), IDEHU-CONICET, Junin 956 4th Floor, Buenos Aires, CP 1113, Argentina. Tel.: +54 11 49648259; fax: +54 11 49640024. E-mail addresses: [email protected] (R.I. Cordo Russo), [email protected] (S. Hajos). 0171-2985/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2011.12.006

N-acetyl-d-glucosamine, with a molecular weight ranging from 105 to 107 Da. HA is biosynthesized at the plasma membrane by hyaluronan synthases (HAS) and the growing polymer is extruded through the membrane into the extracellular space. In mammals, there are three HAS isozymes: HAS-1, HAS-2 and HAS-3 (Weigel and DeAngelis 2007; Heldin et al. 2009). The main control for HA deposition occurs at catabolism. Degradation of HA in peripheral tissues takes place both in the tissue and by release into the lymph and vascular systems. The HA entering the lymph system is removed by lymph nodes, whereas the HA in the blood stream is removed mainly by the liver (Laurent and Fraser 1992). Once inside the cells, HA is degraded by the concerted action of three enzymes: a hyaluronidase (Hyal) and two exoglycosidases. Among Hyal, HYAL-1 and HYAL-2 are of great importance in the degradation of high molecular weight HA (HMW-HA > 1000 kDa) and HYAL-3 is expressed in many tissues (Stern 2009). HA is a component of the mammalian extracellular matrix (ECM), where it possesses functions in tissue injury and repair, inflammation, and tumorigenesis (Toole 2004; Jiang et al. 2007; Johnson and Ruffell 2009). Besides, HA is increased in the tumor microenvironment and is related to tumor progression. Differential expression of HAS and Hyals is associated with increased HA levels (Itano and Kimata 2008). Indeed, HAS overexpression is correlated with tumor aggressiveness (Bourguignon et al. 2007). On the other hand, increased expression of Hyal generates HA fragments (o-HA), which inhibit signaling pathways induced by HMW-HA and decrease tumor growth in vivo (Ghatak et al. 2002). However, in some tumors overexpression of Hyal would favor tumor progression (Lokeshwar and

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Selzer 2009). The role of HA in tumor progression is complex and depends mainly on its molecular weight. Upon interaction with cell surface receptors like CD44, RHAMM, TLRs (toll-like receptors) or different HA-binding proteins (TSG-6, SHAP), HA modulates processes such as proliferation, adhesion, migration, differentiation and multidrug resistance (Toole and Slomiany 2008; Cordo-Russo et al. 2010). The immune system generates an antitumor immune response to control and eradicate cancer. However, tumors use mechanisms that circumvent these immune reactions to enhance their growth. Dendritic cells (DC), the most powerful antigen-presenting cells, are key regulators of the antitumor immune response. DC modulate the immune system by distinct activation states, different DC subtypes and environmental conditions (Coquerelle and Moser 2010). In fact, an inflammatory microenvironment favors DC activation and induction of an effective immune response, while an anti-inflammatory microenvironment induces tolerance (Chaput et al. 2008). Activation of DC is associated with increases in MHC class II molecules, enhanced expression of costimulatory molecules, such as CD80 and CD40, and secretion of Th1 cytokines (Steinman and Idoyaga 2010). Several reports have demonstrated that DC functionality is altered in animals and patients with different tumors, favoring evasion of the immune system and tumor growth (Chaput et al. 2008). HA modulates the immune system (Mummert 2005). During inflammation, HA undergoes degradation into small fragments (o-HA), which are potent activators of DC (Termeer et al. 2002). Recently, it has been demonstrated that LMW-HA (∼500 kDa) inhibits colorectal carcinoma growth in vivo by decreasing tumor cell proliferation and by stimulating DC activation and lymphocyte recruitment (Alaniz et al. 2009). Since HA is a component of the ECM surrounding tumors – which modulates DC function – and tumors generate factors that modulate DC functionality, we decided to study the correlation between HA presence and DC activation in lymphoma. To this end, we analyzed the ability of lymphoma cell lines with different invasive capacity to produce HA. Besides, we evaluated HA levels and the activation state of DC within the tumors induced by these cells. Our results showed that mice injected with the cells with highest invasive capacity presented increased HA in serum and lymph nodes, and decreased DC activation in infiltrated lymph nodes and liver. Such findings could be related to the lack of an effective antitumor immune response and suggest that serum HA levels could have a prognostic value in hematological malignancies.

Materials and methods Cell culture and animals The vincristine-resistant (LBR-V160) and sensitive (LBR-) murine lymphoma cell lines were obtained in our laboratory and were grown as previously described (Lopes et al. 2003). Mice were purchased from School of Science’s animal facility (University of Buenos Aires) and maintained with a 12 h light/dark cycle. Animal experiments were conducted according to the NIH “Guide for the care and use of Laboratory Animals”. BALB/c mice (6- to 8-week old) were injected i.p. with tumor cells (1 × 106 cells/mouse, n = 5 per group) or vehicle. At day 10, mice were sacrificed; spleen, lymph nodes, liver, and lung were removed and divided into pieces. Afterwards, cell suspensions were prepared from one section for DC analysis while other sections were frozen for RT-PCR and GAGs isolation. Other section was fixed for histological analysis. Before sacrifice, blood samples were obtained by retro-orbital bleeding under anesthesia, centrifuged and the decanted serum was stored.

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Table 1 Primer sequences for mouse HAS-1, HAS-2, HAS-3, Hyal-1, Hyal-2, Hyal-3 and ˇ-actin. Ta: anneling temperature. bp: base pair. Gene HAS-1n Sense Antisense HAS-2 Sense Antisense HAS-3n Sense Antisense Hyal-1n Sense Antisense Hyal-2n Sense Antisense Hyal-3 Sense Antisense ˇ-actin Sense Antisense

Primer sequence

Ta (◦ C)

bp

5 -CATTCCTCAGCGCACACCTA-3 5 -TGATGCAGGACACACAGTGG-3

56

752

5 -TGGAACACCGGAAAATGAAGAAG-3 5 -GGACCGAGCCGTGTATTTAGTTGC-3

57

805

5 -AGGTGGTCATGGTAGTGGAT-3 5 -CACTGTTAGCAGGAAGAGGA-3

58

898

5 -TGGGCACAGAGAAGTCACAG-3 5 -GTCTCTTCTTGCTGGGCAAC-3

62

992

5 -GATGTGAAGGCTACACCGAA-3 5 -TAAGGTCCACCTGGCTCAGT-3

60

750

5 -CCTAGGCCTAATGATGGTG-3 5 -GCTAGTATGGGCTTTGTGG-3

53

507

5 -ATGGATGACGATATCGCT-3 5 -ATGAGGTAGTCTGTCAGGT-3

52

569

Measurement of HA levels by enzyme linked immunosorbent assay (ELISA) HA levels in cell supernatant, serum and GAGs were measured with a competitive ELISA (Cordo-Russo et al. 2009). Ninety-six well plates were coated with 100 ␮g/ml HMW-HA (CPN spol.s.r.o Czech Republic, Farmatrade Argentina) at 4 ◦ C overnight. The samples or standard HMW-HA were incubated with 0.75 ␮g/ml biotinylated HA binding protein (bHABP, #385911, Calbiochem) at 37 ◦ C. Afterwards, the plate was blocked and incubated with the samples at 37 ◦ C for 4 h. The bHABP bound was determined using an avidin–biotin detection system. Sample concentrations were calculated from a standard curve. In this assay, HA of different molecular sizes are determined. GAGs were isolated as previously described (Cordo-Russo et al. 2009). Histological and histochemistry analyses Tissue specimens were fixed and 5 ␮m sections were stained with hematoxylin–eosin. For histochemical analysis, sections were buffered and washed in PBS, de-paraffined and rehydrated. Then, the specimens were incubated with 3% H2 O2 in methanol, followed by avidin–biotin blocking solution (Vector) and by 5 ␮g/ml BSA. Then, 5 ␮g/ml bHABP was applied overnight. Control sections were pretreated with 100 U/ml hyaluronidase (#385931, Calbiochem). As revealing system, avidin peroxidase complex (Vector) was used. The signal was detected by incubating sections with 0.5 mg/ml diaminobenzidine, 0.05% H2 O2 , and 0.05 g/ml ammonium nickel II sulfate hexahydrate. Slides were examined using a Nikon microscope (Eclipse E800). Total RNA extraction and semi-quantitative RT-PCR Total RNA from tumor cell lines (5 × 106 ) and samples of tissues (∼50 mg) was isolated employing TRIZOL (Invitrogen) and following manufacturer’s instructions (Cordo-Russo et al. 2009). Isolated RNA was reverse transcripted and cDNA was then amplified with specific primers for HAS-1, HAS-2, HAS-3, Hyal-1, Hyal-2 and Hyal-3 (Table 1) by 30 cycles (30 s at 94 ◦ C, 1 min at annealing temperature – Table 1 – and 1 min at 74 ◦ C), followed by an extension at 72 ◦ C for 10 min. As reference gene, ˇ-actin was amplified. Serial dilutions were used for each cDNA to ensure that amplification occurred

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within the exponential range. PCR products were separated in 2% agarose gel, stained with ethidium bromide and visualized under UV light (Cole Palmer Instrumental, IL).

Preparation of cell suspensions Tissues were removed and enzymatically digested with 0.5 mg/ml collagenase (#C2139, Sigma) diluted in HBSS (Hanks’ buffer saline solution) at 37 ◦ C for 45 min (or 15 min for spleen). Thereafter, the isolated cells were collected in a tube through a 40 ␮m net and washed. The procedure was repeated and the cells were resuspended in flow cytometry buffer. Trypan blue exclusion test revealed that cell viability was 95%.

Flow cytometry analysis Cell suspensions were washed with flow cytometry buffer (FC buffer, PBS supplemented with 1% BSA and 0.1% sodium azide). Human ␥ globulins were added to block Fc receptors. Cells were then incubated at 4 ◦ C for 45 min with mAb anti-CD11c biotinylated, PECy7-conjugated streptavidin, anti-CD80 conjugated with FITC, and anti-MHCII conjugated with PE (#553800, #557598, #553769, and #553544, respectively, Beckton Dickinson). Afterwards, cells were washed and acquired with a PAS III flow cytometer (Partec). As controls, cells were stained with the isotypematched mAb. Instrument compensation was set using single-color stained samples. For examining the surface expression of HA, cell lines were incubated with bHABP and PECy7-conjugated streptavidin and the control of immunoreactive specificity was performed by incubation without bHABP. Data were analyzed by WinMDI software (Scripps Institute, USA).

Statistical analysis Statistical significance was evaluated by one way-ANOVA and means were compared by the Tukey’s test. When only two groups were compared a T test (Mann–Whitney test) was used. Differences were considered significant at the level of p < 0.05. Analysis was performed using Prism 4 software (Graph Pad).

Results Lymphoma cells produce HA in vitro First, we analyzed the ability of different lymphoma cell lines to produce HA in vitro by evaluating HA expression on the cell surface. For this purpose, the sensitive (LBR-) and the vincristineresistant (LBR-V160) lymphoma cells were incubated with bHABP and PECy7-conjugated streptavidin, and then flow cytometry was performed. LBR- presented a higher percentage of cells expressing HA than LBR-V160 (P < 0.01). A tumor pancreatic cell line (PANC-1) was used as positive control (Fig. 1A). Besides, we evaluated HA concentration in the supernatant of the cells by ELISA. We found that LBR- produced higher HA levels than LBR-V160 (41.3 ± 4.1 vs 28.2 ± 3.4 ng/ml, P < 0.05, Fig. 1B). To identify the enzymes involved in HA metabolism, we evaluated mRNA expression for HAS (HAS-1, HAS-2 and HAS-3) and Hyal (Hyal-1, Hyal-2 and Hyal-3) by RT-PCR. mRNA of HAS-1 and HAS-2 could not be detected in the cell lines, while HAS-3 mRNA was found in both LBR- and LBR-V160. Besides, we detected Hyal-1, Hyal-2 and Hyal-3. Interestingly, both Hyal-2 and Hyal-3 mRNA was increased in LBR-V160 as compared with LBR- (Fig. 1C).

Serum HA levels in mice injected with lymphoma cells Our next step was to evaluate HA levels in sera from mice injected with tumor cells. We have previously reported that LBRis more aggressive in vivo than LBR-V160, being the survival times 23 and 41 days, respectively (Lopes et al. 2002). These data were considered to perform this study on day 10 post-tumor injection, in a non-terminal state. Serum HA levels were assessed by ELISA and showed that HA concentration was two-fold increased in mice injected with LBR- compared to control and LBR-V160-injected animals (P < 0.001, Fig. 2A). HA levels, distribution and expression of HAS and Hyal in infiltrated tissues Since LBR- infiltrates the spleen, lymph nodes, liver and lungs (Lopes et al. 2002), we analyzed HA levels, localization and HAS and Hyal expression in those organs on day 10 post-injection. In spleen, we found no differences in HA levels between mice injected or not with tumor cells (Fig. 2B). Histochemical analysis showed conservation of the spleen architecture in all the groups and normal presence of HA in the perivascular and follicular zone (Fig. 2C and D). In lymph nodes, we found that HA levels were increased in mice injected with LBR- compared with control and LBR-V160-injected ones (Fig. 2B). Histochemical analysis showed conservation of the lymph node architecture in control and LBR-V160-injected mice but not in mice injected with LBR-. Indeed, mice injected with LBR- showed absence of defined lymph nodes, presence of cells 2–3 times larger than normal lymphocytes with nucleolus, thus indicating incipient tumor infiltration (Fig. 2C). Interestingly, HA histochemistry showed no differences in HA localization between tumor-bearing and control mice. All groups presented HA in subcapsular sinus (Fig. 2D). In the liver, we found no differences in HA levels between control and tumor-injected mice (Fig. 2B). Hematoxylin–eosin showed conservation of the hepatic architecture in control and LBR-V160injected mice; however, animals injected with LBR- presented tumor infiltrates (Fig. 2C). HA histochemistry showed positive staining in the portal triad both in control and tumor-bearing mice (data not shown) and mice injected with LBR- also presented HA in the tumor infiltrate (Fig. 2D). Finally, no changes in HA levels were observed in the lungs of mice injected with LBR- and LBR-V160 compared to controls (Fig. 2B). Besides, histochemical analysis showed conservation of the lung architecture and HA presence in peribronchiolar and perivascular regions in all the groups (Fig. 2C and D). Since alterations in HA levels and/or tumor infiltration were observed in lymph nodes and liver, we analyzed mRNA expression of HAS and Hyal in those tissues by RT-PCR. In the lymph nodes, we detected only Hyal-3 mRNA. Besides, Hyal-3 mRNA was decreased in mice injected with LBR- and LBR-V160 compared to control (Fig. 3A). In the liver, we found mRNA for HAS-2, HAS-3, Hyal-1, Hyal-2 and Hyal-3, with no differences between the groups (Fig. 3B). DC in mice injected with lymphoma cells Since murine DC can be identified by their expression of CD11c, we first evaluated CD11c expression by flow cytometry on cell suspensions obtained from the spleen, lymph nodes, liver and lung. The percentage of CD11c+ cells in the spleen from mice injected with LBR- was decreased compared to control mice (1.8 ± 0.8% vs 3.6 ± 0.2%, P < 0.05), while no differences were observed in LBRV160-injected ones (3.6 ± 0.2%). Besides, the percentage of CD11c+ cells was also decreased in the liver from mice injected with

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Fig. 1. Production of HA by lymphoma cell lines in vitro. (A) Histograms indicating cell surface expression of HA in LBR- and LBR-V160. Cells were incubated with bHABP and PECy7-conjugated streptavidin. The full line corresponds to the sample and the black line is the specificity control. The pancreatic tumor cell line PANC-1 was used as positive control. One representative from three independent experiments is shown. In the graphic, the results are shown as % of HABP+ cells, which correlates with the % of cells expressing HA. (B) Measurement of HA levels in the supernatant of LBR- and LBR-V160 by ELISA as described in “Materials and methods”. (C) mRNA expression of HA synthesis and degradation enzymes in LBR- and LBR-V160. HAS (HAS-1, HAS-2 and HAS-3) and Hyal (Hyal-1, Hyal-2 and Hyal-3) was evaluated by RT-PCR. ˇ-actin was used as control of near equal amplification. Decidua samples were used as positive control. One representative from three independent experiments is shown. Densitometric analysis of the bands was performed and the results are expressed as the index (HAS or Hyal mRNA expression/ˇ-actin mRNA expression). Bars represent the mean ± SEM. *P < 0.05 vs LBR-.

LBR- compared to control ones (5.3 ± 0.4% vs 9.0 ± 0.9%, P < 0.05). However, in lymph nodes, the percentage of CD11c+ cells was increased in mice injected with LBR- as compared with control and LBR-V160-injected mice (2.5 ± 0.4% vs 1.1 ± 0.1% and 1.7 ± 0.5, P < 0.01). In the lung, no differences in CD11c levels between the groups (Fig. 4A). Then, we analyzed the activation of DC through the expression of the costimulatory molecules CD80 and MHC-II in CD11c+ cells. Analysis showed no differences between the groups in the spleen and lung. However, in the lymph nodes, a decrease in CD80, MHC-II and CD11c+ cells was observed in mice injected with LBRcompared with control and LBR-V160-injected ones (15.4 ± 2.4% vs 31.8 ± 5.0% and 29.2 ± 3.0, P < 0.05). Similar results were observed

in the liver (10.2 ± 2.0% vs 37.1 ± 4.5% and 35.8 ± 6.7, P < 0.01) (Fig. 4B). Discussion HA levels are related to tumor progression. However, HA synthesis by lymphoma cells and its relationship with DC activation has not been elucidated yet. In this work, we observed that the sensitive LBR- cell line presented increased HA levels in comparison with the resistant cell line LBR-V160. Different tumor cells, including lymphoma cells, produce HA (Kuwabara et al. 2003; Tammi et al. 2009). Recently, it has been observed that 4-methylumbeliferone (4-MU), an inhibitor

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Fig. 2. HA levels and localization in mice injected with lymphoma cell lines. (A) Measurement of serum HA levels in mice injected with LBR-, LBR-V160 or vehicle (control) by ELISA as described in “Materials and methods”. Data in the diagram is depicted as the mean ± SEM (n = 5 per group). (B) HA levels in GAGs isolated from spleen, lymph nodes, liver and lung determined by ELISA. Bars represent the mean ± SEM (n = 5 per group). (C) Hematoxylin–eosin (H&E) staining of spleen, lymph nodes, liver and lung of mice injected with LBR-. A representative image is shown. The presence of tumor cells in lymph nodes and liver is marked with a circle. (D) Histochemical localization of HA was performed by staining with bHABP. Representative examples of HA histochemistry from different infiltrated tissues of mice injected with LBR- are shown. Arrows depict HA positive staining. Bar = 50 ␮m. *P < 0.05 and ***P < 0.001.

of HA synthesis, displayed an anti-tumoral effect on melanoma, breast, ovarian and prostate cancer cells, thus suggesting 4-MU as a therapeutic candidate (Kultti et al. 2009; Lokeshwar et al. 2010). Besides, biopsies of patients have shown that high-grade

lymphomas correlate with increased HA production (Bertrand et al. 2005), thus indicating that different lymphoma cells produce dissimilar HA levels, a fact related to their malignancy. In this work, LBR- expressed higher HA levels than LBR-V160 and was also the

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Fig. 3. Expression of HAS and Hyal by RT-PCR. Analysis of mRNA of HAS (HAS-1, HAS-2 and HAS-3) and Hyals (Hyal-1, Hyal-2 and Hyal-3) in lymph nodes (A) and liver (B) of mice injected with LBR-, LBR-V160 or vehicle (control). ˇ-actin was used as control of near equal amplification. One representative from three independent experiments is shown. Densitometric analysis of the bands was performed and the results are expressed as the index (HAS or Hyal mRNA expression/ˇ-actin mRNA expression). Bars represent the mean ± SEM (n = 5 per group). *P < 0.05 and **P < 0.01.

most aggressive line in vivo (Lopes et al. 2002). It is noteworthy that cell surface expression of HA in lymphoma cells was significantly lower than in tumor pancreatic cells (PANC-1). These differences may be due to the origin of the cells. In fact, enhanced accumulation of HA has been described in epithelial tumors – such as breast, colon and pancreatic cancer – and their healthy tissues compared with hematological tumors (Tammi et al. 2009). Besides, we evaluated HAS and Hyal expression and observed mRNA of HAS-3, Hyal-1, Hyal-2 and Hyal-3. HAS-3 is overexpressed in colon and prostate cancer and is associated with HA synthesis and tumor growth (Simpson et al. 2001; Bullard et al. 2003). Moreover, inhibition of HAS-3 diminishes colon cancer growth in vivo (Lai et al. 2010) and mRNA of HAS-2 and HAS-3 has been found in OHK lymphoma cells (Kuwabara et al. 2003). Our findings show that only HAS-3 is involved in HA synthesis in LBR- and LBR-V160. The role of Hyal-1 and Hyal-2 in cancer is complex since they could act as a tumor promoter or suppressor depending on its concentration, location and tumor cell context (Lokeshwar and Selzer 2009). Hyal-1 overexpression has been shown to suppress tumorigenicity of colon carcinoma cells (Jacobson et al. 2002). However, Hyal-1 has been found increased in bladder, in prostate, and in head and neck tumors and was associated with tumor progression (Lokeshwar and Selzer 2009). Hyal-2 has been found overexpressed in breast cancer (Udabage et al. 2005) and decreased in ovarian and lung tumors as well as in lymphomas (Bertrand et al. 2005; Hiltunen et al. 2002).

Biopsies of high-grade lymphoma patients have shown a decrease in Hyal-2 with increased HA levels, thus suggesting that Hyal-2 may have a prognostic significance (Bertrand et al. 2005). Our results are in agreement with the latter and show that LBR-, which presented increased HA expression, also presented decreased Hyal-2 mRNA compared with LBR-V160. Finally, we also found a decrease in Hyal3 mRNA in LBR-. Although Hyal-3 expression has been found in endometrial and breast cancer, its role has not been assessed yet (Udabage et al. 2005; Paiva et al. 2005). To our knowledge, this is the first work that demonstrates Hyal-3 mRNA expression in lymphoma cells. Thus, we suggest that the increase in HA production observed in LBR- would be related to abnormal HA degradation due to a decrease in Hyal-2 and Hyal-3 mRNA. Since increased HA levels in serum have been reported in breast, ovarian, endometrial, bladder cancer and also in leukemia and lymphoma (Hiltunen et al. 2002; Hasselbalch et al. 1995; Hautmann et al. 2001; Burchardt et al. 2003; Calabro et al. 1998), we analyzed HA levels in mice injected with different lymphoma cells. It has been reported that serum HA levels are higher in patients with metastasis of breast cancer than in nonmetastatic patients (Delpech et al. 1990). Moreover, it has even been suggested that serum HA measurement would be a useful cancer prognostic marker (Xing et al. 2008; Dahl et al. 1999). In agreement, our results show that mice inoculated with LBR- present higher serum HA levels than controls and LBR-V160-inoculated

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Fig. 4. Flow cytometry analysis of DC in mice injected with lymphoma cell lines. (A) Analysis of CD11c+ cells in spleen, lymph nodes, liver and lung of mice injected with LBR-, LBR-V160 or vehicle (control). The results are plotted as the percentage of CD11c+ cells in cell suspensions of the different tissues. (B) Analysis of DC activation markers. Results are expressed as the percentage of CD11c+ cells that co-express MHC-II and CD80. Representative histograms of the experimental groups and tissues are shown. Bars represent the mean ± SEM (n = 5 per group). *P < 0.05 and **P < 0.01.

ones. Since LBR- presents a lower survival time than LBR-V160, our findings suggest that serum HA levels could have a prognostic value in lymphoma. Different mechanisms seem to be involved in serum HA increase: (i) increased synthesis by tumor cells (Tammi et al. 2009), and (ii) abnormal degradation by malignant transformation of tissues involved in HA clearance such as lymph nodes and liver (Hasselbalch et al. 1995; Miele et al. 2009). Our results suggest that enhanced serum HA levels in mice injected with LBR- would be related to HA synthesis by LBR-, together with a decrease in HA clearance both in lymph nodes and liver, which were infiltrated by tumor cells. Infiltration of lymph nodes by LBR- could decrease the ability of this tissue to degrade HA, a fact inferred by the decreased Hyal-3 expression and the consequent increased HA levels in lymph nodes. Interestingly, HA histochemistry did not show increased HA deposition in LBR- infiltrated lymph nodes. Besides, we found HA in liver areas invaded by LBR-, and this would also contribute to serum HA levels due to increased synthesis and decreased metabolism.

Serum HA levels in LBR-V160-injected mice were not increased compared with control mice, and although this cell line also synthesizes HA, no infiltration of the lymph nodes or liver was observed, with consequently no changes in HA metabolism. Several reports have demonstrated that DC functionality is altered in animals and patients bearing tumors, and that this would favor evasion of immune system and tumor growth (Chaput et al. 2008). In this work, we found that mice inoculated with LBR- presented a decrease in the percentage of DC in spleen and liver and a decrease in DC activation markers (MHC-II and CD80) in lymph nodes and liver. However, LBR-V160-injected mice presented no changes in activation of DC in any of the organs evaluated. Many studies have demonstrated a decrease in DC number and/or maturation in colorectal, ovarian and breast tumors (Ambe et al. 1989; Eisenthal et al. 2001; Iwamoto et al. 2003). In hematological malignancies, patients with chronic myeloid leukemia present a decrease in DC precursors in blood, whereas patients with chronic

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lymphocytic leukemia show immature DC (Mohty et al. 2002; Orsini et al. 2003). Besides, DC are significantly reduced in infiltrated lymph nodes of patients with non-Hodgkin’s lymphoma (Fiore et al. 2006). Recently, lymphoma patients have shown immature DC in tumor tissues and mature DC in peri-tumoral areas, suggesting that factors released by tumor cells would alter DC maturation (Hussin et al. 2009). Our results are in accordance with the latter and show that the more aggressive cell line LBR- presented a decrease in the percentage and activation of DC compared with control animals and with those injected with the less aggressive cell line LBR-V160. We suggest that this would be related to the lack of an effective antitumor immune response and would favor tumor infiltration and invasion. It is noteworthy that lymph nodes of mice injected with LBR- presented an increase in the percentage of DC along with decreased DC activation. This finding is in agreement with a report of enhanced immature DC infiltration in lesional skin of cutaneous T-cell lymphoma (Schlapbach et al. 2010) and suggests that non-activated DC would be useless to generate antitumor immune responses. Several studies have demonstrated that tumors release factors that alter the number or activation of DC so they can evade the immune system (Stewart and Abrams 2008). Indeed, TGF-␤ levels are inversely correlated with mature DC in breast cancer (Iwamoto et al. 2003). Besides, tumor cells produce HA, and HA fragments induce semimature DC, which aids tumor immune escape (Kuang et al. 2008). Our findings suggest an inverse correlation between HA levels and DC activation, since we showed that mice injected with LBR- presented enhanced HA levels in sera and lymph nodes as well as decreased DC activation in this organ. We hypothesize that LBRmay synthesize HA or favor its accumulation, leading to DC modulation, failure of antitumor immune response and consequently tumor progression. In summary, our results demonstrate that mice injected with the cell line with higher invasive capacity (LBR-) presents increased HA in serum and lymph nodes. This may be explained by the ability of LBR- to synthesize HA and by abnormal HA degradation due to tumor infiltration of the lymph nodes and liver. Besides, mice injected with LBR- also presented a decrease in DC activation in lymph nodes and liver, thus suggesting that a lack of antitumor immune response would favor tumor invasion. Our findings suggest an association between tumor progression, HA levels and DC activation in this lymphoma model and that serum HA levels could have a prognostic value in hematological malignancies. Authors’ contributions RCR performed the experiments, contributed to the design of the study, analyzed the data and wrote the manuscript. GE and SL contributed to acquisition of data. GB supplied flow cytometry data and interpretation. EA helped to the design of the study. MGG contributed to the design of the study, analysis of the data, editing and supervision of the manuscript. SH is the director of the group and supervised the work. All authors read and approved the final manuscript. Conflict of interest All authors have no conflict of interest to report. Acknowledgements We thank Ms. Romina De León, Dr. Susana Constantino and Dr. Daniela Uretra for excellent technical assistance. We are grateful to Dr. S.M. Blois (Charité, University Medicine of Berlin, Germany) for kindly providing DC antibodies and for her invaluable advice.

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