Laminin-332 (Laminin-5) is the major motility ligand for B cell chronic lymphocytic leukemia

Matrix Biology 26 (2007) 473 – 484 www.elsevier.com/locate/matbio

Laminin-332 (Laminin-5) is the major motility ligand for B cell chronic lymphocytic leukemia Paola Spessotto a , Antonella Zucchetto b , Massimo Degan b , Bruna Wasserman a , Carla Danussi a , Riccardo Bomben b , Roberto Perris c , Vincenzo Canzonieri d , Oriano Radillo e , Alfonso Colombatti a,f,⁎, Valter Gattei b a

Experimental Oncology 2, Centro di Riferimento Oncologico, IRCCS, Aviano (PN), Italy b Clinical and Experimental Hematology Research Unit, Italy c Dipartimento di Biologia Evolutiva e dello Sviluppo, University of Parma, Italy d Anatomy, Centro di Riferimento Oncologico, IRCCS, Aviano (PN), Italy e Laboratorio di Analisi, IRCCS, Burlo Garofolo, Trieste, Italy f Immunology Section, MATI Excellence Center and DIBI, University of Udine, Italy

Received 2 January 2007; received in revised form 29 March 2007; accepted 2 April 2007

Abstract Cell adhesion and motility are central aspects in the pathophysiology of B cell chronic lymphocytic leukemia (B-CLL), but the role of specific extracellular matrix proteins is still to be completely unveiled. Purified peripheral blood neoplastic cells of B-CLL patients migrated poorly on laminins-111,-411,-511, but showed pronounced motility on laminin (LM)-332 in a high percentage of cases. B-CLL cell motility on LM-332 was mediated by the α3β1 integrin and was preferentially observed in cells carrying a mutated IgVH gene profile. Within normal lymph nodes, LM-332 was circumscribed around blood vessels and to areas corresponding to marginal zones, where it was deposited in a pattern reminiscent of reticular fibers. Conversely, in B-CLL involved lymph nodes, a positive LM-332 reticular mesh was diffusely evident, throughout the disrupted nodal architecture. In the present study we identified LM-332 as a crucial motility-promoting factor for B-CLL lymphocytes and as a potential constituent favoring the dissemination of B-CLL lymphocytes through vascular basement membranes and possibly lymph node compartments. © 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Laminins; B-CLL; Cell migration; IgVH mutational status

1. Introduction Interaction of neoplastic B cells with ECM components is a crucial event for the regulation of their dissemination pathways and is particularly relevant for their trafficking and retention within lymph nodes, accumulation of cells into lymphatic channels, and infiltration of the bone marrow microenvironment (Wagstaff et al., 1981). The patterns of ECM interaction of leukemia/lymphoma B cells may differ from those of normal B

⁎ Corresponding author. Experimental Oncology 2, CRO-IRCCS, National Cancer Institute, Aviano, Via Pedemontana Occidentale, 12, 33081 Aviano, Italy. Tel.: +39 0434 659301; fax: +39 0434 659428. E-mail address: [email protected] (A. Colombatti).

cells and diverge with the phenotype and maturation status of the cells (Segat et al., 1994; Baldini and Cro, 1994; De Rossi et al., 1994; Vincent et al., 1996; Behr et al., 1998; Hewson et al., 1996; Till et al., 1999; Sbaa-Ketata et al., 2001). In addition, in the case of B cell chronic lymphocytic leukemia (B-CLL), diversified expression of certain integrins may correlate with the disease stage and aggressiveness and may modulate the cells' interplay with interstitial matrices, basement membranes, and endothelial cell surfaces (Eksioglu-Demiralp et al., 1996; Zucchetto et al., 2006a,b). Although a clear importance has been assigned to certain growth factor-, cytokine- and chemokine receptor systems for the entry of B-CLL cells into secondary lymphoid organs and bone marrow (Sbaa-Ketata et al., 2001; Till et al., 2005; Burger and Kipps, 2002; Gretz et al., 2000), virtually nothing is known about the ECM components that are

0945-053X/$ - see front matter © 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2007.04.003

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Fig. 1. Intrinsic versus LM-promoted migration capabilities of B-CLL lymphocytes. (A) Averaged LM isoform-induced migration of B-CLL cells derived from 27 randomly selected patients after 24 h assayed in triplicates. (B) Representative fields of B-CLL cells allowed to migrate in response to FN and LM-332 substrates at 24 h in our FATIMA system and which were derived from samples displaying high inter-patient variability in their intrinsic motile properties. Left, spontaneous motility.

implicated in the invasion and movement of these cells through these preferential target tissues. Within the lymph node environment, the areas primarily involved by the outgrowing B-CLL process have been iden-

tified among those localized outside the secondary follicles, in close proximity of the outer space of mantle zone, i.e. the socalled marginal zone, and/or within the interfollicular spaces (Chiorazzi et al., 2005; Kuppers, 2005). In this compartment,

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Fig. 2. Relative levels of migration on LM-332. Lymphocytes derived from 46 randomly selected B-CLL patients reported as the ratio of LM-332/FN migration. Dotted line indicates the cut-off value of 3 arbitrarily adopted to define highly specific movement in response to LM-332.

in fact, subsets of memory B cells expressing either heavily mutated IgVH genes are localized, as the result of a T celldependent germinal center (GC) differentiation, or few or no IgVH mutations, as in the case of B cell maturation taking place entirely outside the GC in the absence of T-B cell interactions (Chiorazzi et al., 2005; Kuppers, 2005). These B cell subsets have been recognized as the bona fide normal counterparts of B-CLL cells bearing either mutated (M) or unmutated (UM) IgVH gene products, respectively (Chiorazzi et al., 2005; Kuppers, 2005; Oscier et al., 1997; Fais et al., 1998; Naylor and Capra, 1999; Rosenwald et al., 2001; Klein et al., 2001). LMs are a family of trimeric ECM proteins characteristically present in basement membranes which can assemble in many different combinations, for instance, α1β1γ1 (LM-111), α3β3γ2 (LM-332), α5β1γ1 (LM-511). They are expressed in specific locations (Aumailley et al., 2005) including hematopoietic and lymphoid tissues (van den Berg et al., 1993; Jaspars et al., 1996; Gu et al., 1999, Sorokin et al., 1997; DrumeaMirancea et al., 2006); in particular, α3 chain containing LMs

have been described in the lymph node reticular network in a hollow or ring staining pattern (Jaspars et al., 1996; Kaldjian et al., 2001). It has been suggested that the function of these LMs may be to anchor the follicular reticular cells to the reticular fibers, where the spaces act as a conduit system for soluble molecules as well as for lymphocytes, to promote cell adhesion and migration within the node environment (van den Berg et al., 1993) and affect immune cell activation or differentiation. In this regard, we and others have previously shown that leukemia/lymphoma cell lines can interact with different LM isoforms (Segat et al., 1994; Gretz et al., 1997), some of them being particularly effective in promoting movement of lymphoid tumor cell lines (Spessotto et al., 2003). Given these cumulative observations, we have investigated here the potential involvement of LM isoforms in regulating the transmigration of B-CLL cells in the context of the lymph node environment. To this end, a number of purified native laminins (LM-111, LM-322, LM-411, LM-511) and one native multilaminin complex (LM-111/511/521) were assayed for their

Fig. 3. Integrin and metalloproteinase expression. B-CLL samples were divided into two groups according to their LM-332/FN migration ratio, using the cut-off value of 3 as a threshold and the individual cases and ordering the individual cases according to their α3β1 integrin levels of expression reported as percentage of positive cells. Bar color coding refers to the presence/absence of MMP-2 or MMP-9 transcripts evaluated following RT-PCR: white bars, cases lacking both MMP-2 and MMP-9 transcripts; grey bars, cases lacking MMP-2 transcript; black bars, cases transcribing both MMP-2 and MMP-9. Inset shows representative PCR amplifications in MMP-9 positive (case B157), MMP-2 and MMP-9 double-negative (case B159) and double-positive (case B158) cases.

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Fig. 4. Role of α3β1 integrin in B-CLL migration on LM-332. Function blocking mAbs against α1, α3 and α4 integrin subunits were added at 5 μg/ml to cells just before cell plating in the upper side of the insert membrane. The results are the mean ± SD of three independent experiments. Fields of migrated cells at 24 h were photographed under phase contrast from representative samples.

capability to promote migration of neoplastic B cells derived from B-CLL patients. Motility responses to laminins were normalized to FN and compared with integrin subunit and metalloprotease (MMP) expressions, as well as with the IgVH gene mutational status of the cells. We identified isoform LM332 as the candidate LM responsible for the locomotion of BCLL cells. We propose that LM-332 may represent a key molecule involved in promoting the dissemination of neoplastic B-CLL lymphocytes through the basal endothelial matrix and within the lymph node microenvironment. 2. Results 2.1. Differential B-CLL cell migration towards LM isoforms In exploratory experiments, purified neoplastic cells from 27 B-CLL cases were tested for migration on several LM substrates by a specific migration assay set up in our laboratory (Spessotto et al., 2002). Fibronectin (FN) was used as standard ECM substrate, while spontaneous motility of B-CLL cells was determined in uncoated inserts. Of the 20 B-CLL cases whose cells showed specific migration (i.e. more than 3% migrating cells

over controls) on one or more of the investigated substrates, 13 (65%) migrated on LM-332, 7 (35%) on LM-411 and LM-511, 4 (20%) on FN, and only 3 (14%) on LM-111 (Fig. 1A). In addition, the average specific migration of B-CLL cells on the test substrates was 3–5-fold more efficient in response to LM332 than FN, or other LM isoforms, including LM-411, -511 and the complex composed of LN-111/511/521. Moreover, specific locomotion on LM-111 was negligible for virtually each of the patient samples, since it rarely exceeded migration on uncoated inserts. We also asserted that the poor motility detected for cells of certain B-CLL patients was not due to an inactive/nonfunctional nature of the substrate molecules, as determined by parallel control tests with other solid tumor and hematopoietic cell lines (data not shown). The range of variability in term of specific migration among the various B-CLL cases was rather high, with a number of samples displaying a high spontaneous motility towards uncoated inserts. In particular, we observed cases (Fig. 1B) in which specific migration could be documented only as an increased locomotion towards a given ECM substrate, along with other samples which displayed low or no motility on uncoated inserts and exclusively migrated onto specific ECM substrates. Thus, by subtracting the variable spontaneous

Fig. 5. Expression of LM-332 in reactive and B-CLL affected lymph nodes. (A) Reactive lymph node. Distribution of LM-332 is evident in capillary basement membranes through the medulla and cortex including the germinal center (GC). At high magnification a fibrillar positivity of LM-332 is particularly concentrated in the outskirts of the mantle zone (MaZ) at the level of the marginal zone (MgZ). Bar = 200 μm. (B) B-CLL lymph node. The uniform pattern of neoplastic B-CLL lymphocytes is interspersed with a LM-332 positive and homogeneous reticular mesh, visualized with R13 polyclonal antibody, and more clearly evident at higher magnification (right panel). Black asterisks indicate reticular mesh. In the middle image B-CLL nuclei are visualized by propidium iodide. Bar = 75 μm. (C) Sections of B-CLL lymph nodes were treated with monoclonal antibodies against either LM-332 chains. The staining with anti α3, β3 and γ2 chains revealed the presence of a reticular mesh (white asterisks). A section treated with control mAbs was counterstained with propidium iodide to visualize nuclei (upper panel, left). Bar = 75 μm. (D) Western blotting analyses of B-CLL lymph node extracts from three patients (p1, p2, p3). Anti β3 chain and anti γ2 chain antibodies were used. LM-332, LM-111 and fibronectin (FN) were loaded as controls.

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motility of B-CLL cells towards uncoated membranes, a preferential migration of B-CLL cells towards LM-332 rather than FN or any of the other LM substrates could be clearly documented (Fig. 1A). The specific capability of isolated B-CLL cells to migrate towards LM-332 was then investigated in a larger cohort of randomly selected B-CLL patient samples, i.e. a total of 46 cases. These experiments confirmed the pronounced migration of B-CLL cells when confronted with LM-332, as compared to FN substrates. To more precisely identify B-CLL patients whose cells specifically responded to LM-332, we adopted the ratio of cell movement on LM-332 versus that on FN, that is, a ratio between the specific migration towards LM-332 and the specific migration on FN. Given the fact that FN is widely expressed in the lymph node environment, but is absent from basement membranes, this parameter provides a strong indication that B-CLL cells may have acquired a higher propensity for trans-basement membrane migration rather than simply a more generalized locomotory propensity that could be supported by any cell-adhesive ECM ligand including FN. We then arbitrarily set the threshold level for the assignment of LM332-responding and non-responding B-CLL cases at ≥ 3-fold greater movement on this LM isoform when compared to FN (Fig. 2). By this means, we could compensate for intra-and inter-sample variability, the use of fresh samples along with samples stored under liquid nitrogen, and the highly variable degree of cell migration observed with cells of some patients. When applying such a highly stringent cut-off level, lymphocytes from 21 out of 46 B-CLL patients examined revealed a LM-332/FN migration ratio above or equal to 3 and, amongst them, 10 patients had neoplastic cells displaying a migration ratio exceeding the value of 5. 2.2. Integrin and MMP involvement in B-CLL cell migration towards LM-332 We next investigated the molecules responsible for the diverse B-CLL cell migratory responses to LMs by examining the putative cell surface receptors involved in the process and considering that several integrins, including α3β1, α6β1 and α6β4 (Hintermann and Quaranta, 2004), may recognize LM332. Flow cytometry analyses were performed on 40 B-CLL patient samples, comprising 23 cases with a migration ratio below the cut-off of 3 and 17 cases with a LM-332/FN ratio higher than the established cut-off. Integrin subunits α1, α2 and α6 were mostly absent (mean expression level of about 3.0% with 1–25% of the cells showing this weak positivity; data not shown), whereas the α3 integrin subunit was detected at variable levels in the majority of B-CLL cells (31 out of the 40 cases analyzed with N10% cells showing significant expression), with no differences between cells capable of migrating on LM-332 versus those that were unable to do so (Fig. 3). The lack of an apparent correlation between migration towards LM-332 and α3 integrin expression levels raised the question regarding the effective and necessary involvement of α3 integrins in B-CLL cell migration towards LM-332. To further assert the importance of this integrin, we selected cells

from a set of 5 B-CLL patients that had been found to exhibit a LM-332/FN migration ratio above 3 and which had low (between 10 and 20% of positive cells in B114 and B164 cases) or high (N 20% of positive cells in B125, B173 and B174) α3 integrin subunit expression (Fig. 4). These cells were then comparatively assayed on FN and LM-332 substrates in the presence of function-blocking antibodies. A strong inhibition of B-CLL cell migration on LM-332 was obtained with the anti-α3 mAb P1B5 in all cases, whereas no significant inhibition of cell migration was detected using anti-α1 or anti-α4 blocking antibodies. Conversely, as expected, anti-α3 antibodies failed to block migration of B-CLL cells towards FN substrates (not shown). We also verified morphologically by direct microscopic analysis that the reduced B-CLL cell migration following blockade of the α3β1 integrin was not due to enhanced intercellular adhesion and aggregation caused by the antibody treatment itself. That α3 integrin might be involved in B-CLL cell migration towards LM-332 even in the absence of high expression levels of the receptor molecule (B114 or B164 cases), underscores the importance of integrin activation levels instead of expression levels for integrin functions also in this cell system. The involvement of α3 integrins in B-CLL cell migration onto LM-332 substrates in the absence of a clear correlation with α3 integrin expression levels suggests that other mechanisms, in addition to α3 integrins, might be implicated in the process of B-CLL cells migration towards LM-332 substrates. At least two metalloproteinases, MMP-2 (secreted) and MTI-MMP (MMP14; surface-bound) have been proposed to act proteolytically on LM-332 to enhance its motility-promoting activity in neutrophils and epithelial tumours (Giannelli et al., 1997; Koshikawa et al., 2000), while MMP-9 has been described as the major metalloproteinase produced by B-CLL cells (Bauvois et al., 2002; Redondo-Munoz et al., 2006). We therefore examined the relative expression of MMP-2 and MMP-9 in our BCLL cells and matched this analysis with the migratory data and expression pattern of the α3 integrin subunit (Fig. 3). Cells derived from the great majority of B-CLL patients expressed MMP-2 and/or MMP-9 transcripts (35/40 cases), whereas no cases were found to solely express the MMP-2 mRNAs. Expression of MMP-9 alone, or MMP-9 plus MMP-2, was found, respectively, in 14 and 21 of the 40 cases analyzed (Fig. 3). The rather ubiquitous expression of MMP transcripts by B-CLL cells did not highlight any consistent and/or significant correlation with the migration capability of the cells. However, the 5 B-CLL cases noted to lack both MMP-2 and MMP-9 expression clustered within the group of lymphocytes showing a LM-332/FN migration ratio b 3 (Fig. 3). These cells were characterized by a generalized poor migration onto other ECM substrates as well (not shown). 2.3. Immunolocalization of LM-332 in lymph node microenvironment The suggestion that LM-332 could be an appropriate substrate for B-CLL cell migration prompted us to investigate the in situ tissue distribution of LM-332 components in cryostat

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sections of normal reactive and B-CLL-involved lymph nodes. As clearly shown in Fig. 5A, there was a paucity of staining for LM-332 components within B cell follicles germinal centers and mantle zones, specific staining in these areas being confined to vessel walls and basal membranes. Interestingly, the areas with a specific and strong LM-332 staining were located at the far periphery of mantle zones, corresponding to marginal zones, as well as in the interfollicular spaces (Fig. 5A); in these areas LM332 staining acquired a pattern reminiscent that of reticular fibers with no expression detected in the spaces between fibers (Fig. 5A right, enlargement). Conversely, expression of LM-332 in B-CLL affected lymph nodes displayed a uniform distribution according to the disruption of the normal tissue architecture upon the outgrowth of neoplastic lymphocytes (Fig. 5B). A LM332 positive and homogeneous reticular mesh was clearly evident at higher magnification (Fig. 5B, middle and right). The specificity of staining was confirmed by using monoclonal antibodies against either subunits of LM-332: all three chains were well detected in B-CLL lymph nodes, showing a characteristic reticular pattern of distribution (Fig. 5C). Lymph nodes extracts from the immunostained tissue of B-CLL patients were subjected to Western blotting analyses. In all three patients a marked band corresponding to β3 subunit of LM-332 was present, whereas anti γ2 antibody revealed three faint bands migrating at 105, 75 and 50 kDa indicating a possible proteolytic processing of this chain as suggested in some recent studies (Koshikawa et al., 2004). 2.4. B-CLL migration in response to LM-332 correlates with the mutational status at the IgVH locus The distribution of LM-332 at the periphery of the lymph node follicles, where normal B cells usually terminate their GC-dependent differentiation process, as well as the diffuse reticular mesh of LM-332-containing fibers found in B-CLLinvolved lymph nodes, prompted us to verify whether enhanced motility of B-CLL cells on LM-332 correlated with the mutational status of IgVH genes, or with antigen-driven selection in IgVH gene sequences (Degan et al., 2004a). Therefore, the LM-332/FN migration ratios were compared between 27 unmutated and 19 mutated B-CLL cases; thus, only cells of 6 out of 27 (22%) unmutated B-CLL cases displayed specific migration on LM-332 substrates (Fig. 6A, upper). Conversely, in the mutated B-CLL subset, the cases having cells that preferentially migrated towards LM-332 amounted to 58% (11 out of 19; Fig. 6A, lower) and, hence, significantly different from unmutated cases ( p = 0.03). Comparison of the mean values of LM-332/FN ratios between the two groups showed a significantly higher propensity of the mutated B-CLL group, as compared to the unmutated B-CLL cells, to migrate in response to LM-332 (mean migration ratios of 5.3 ± SD and 2.5 ± SD, respectively; p = 0.03; Fig 6B, upper). Conversely, no differences in terms of biased migration towards LM-332 substrates were found when comparing the 10 B-CLL cases expressing a mutated IgVH gene configuration with evidence for affinity maturation (reported elsewhere as “significantly mutated”, Degan et al., 2004a) with the residual mutated

Fig. 6. IgVH gene mutational status-dependent B-CLL migration on LM-332. Migration ratio (LM-332/FN) values of 27 unmutated (UM) B-CLL cases (A, upper panel) and 19 mutated (M) cases (A, lower panel). The 9 grey bars in the left panel represent those M B-CLL cases with evidence of antigen-driven selection (“significantly mutated”, sM). Dotted lines refer to the migration ratio cut-off of 3. Chi-square (p = 0.03) between IgVH mutational status (cut-off: 2% mutations) and migration ratio (cut-off = 3) calculated on the UM versus M cases. (B) Migration ratio (LM-332/FN) values between UM and M, and (C) between UM, non-significantly mutated (nsM), and sM cases. Values for statistical significance were obtained by a Student's t-test.

B-CLL lacking such an evidence (Fig. 6B, lower). However, a stronger statistically significant difference was found when the migratory ratios between significantly mutated and

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unmutated B-CLL subgroups were compared (Fig. 6B, lower, p = 0.01). 3. Discussion While a wealth of data has been gathered on the effects of LM-332 on epithelial cells adhesion and migration phenomena, much less, if not anything, is known on the impact of LM-332 interaction on lymphoid cell adhesion and migration. In particular, by excluding recent data indicating that LM-332 can be a selective ligand and promotes migration of mature medullary thymocytes (Sorokin et al., 1997; Drumea-Mirancea et al., 2006; Wayner et al., 1993; Kutlesa et al., 2002) no information is currently available on the importance of this specific LM in the context of the dissemination and local growth of B-CLL. This despite the fact that LM-332 has been described as a primary component of basement membranes of both bone marrow and lymph nodal vessels (van den Berg et al., 1993; Jaspars et al., 1996; Kaldjian et al., 2001; Gretz et al., 1997). In the present study, we were able to demonstrate that neoplastic lymphocytes isolated from patients afflicted by BCLL displayed diverse migratory capabilities, with the striking finding that, in the vast majority of cases, cells locomoted considerably more effectively on LM-332 than on FN and any other LM isoforms. Since viability rate of the ex-vivo B-CLL cells assayed in our migration system exceeded 90%, with no overt differences observed among the various ECM migratory substrates, we could rule out that the different extent of migration of B-CLL cells on LM-332 could be due to an enhanced survival of cells interacting with this specific molecule. Another intriguing finding of our study is the correlation between preferential B-CLL cell movement on LM-332 and extent of IgVH locus mutations. In this regard, specific migration towards LM332 substrates was observed in a much higher percentage of cases with a mutated IgVH gene configuration, as compared to unmutated B-CLL cases (58% versus 22%). In addition, B-CLL lymphocytes harboring mutated IgVH genes migrated more efficiently on LM-332 than their unmutated counterparts displaying a mean migration ratio of 5.3 compared to 2.5. This latter divergent LM-332-interactive capability was even more significant when we compared the migratory response of unmutated B-CLL cells with those of cells from “significantly mutated” BCLLs, i.e. the mutated B-CLL subset in which a qualitative analysis of IgVH mutations revealed evidence of antigen-driven selection (Degan et al., 2004a; Bomben et al., 2005). In epithelial cells, integrins α3β1 and α6β1 are known to mediate cell motility through LM-332 substrates (Watt, 2002). In agreement with literature data (Baldini and Cro, 1994; Zucchetto et al., 2005), α3β1 was the most expressed integrin also in the present B-CLL series, while levels of α6/β1 were in turn almost undetectable. In accordance, the motility of B-CLL onto LM332 specific substrates was exclusively dependent upon engagement of α3β1 integrins, as demonstrated by a nearly complete inhibition of B-CLL motility onto LM-332 in the presence of anti-α3 blocking mAbs. α3β1 integrin is also a receptor for LM-511 (Kikkawa et al., 1998). We have previously described that lymphoma cells

(HUT-78 cell line) migrate on LM-332 as well as on LM-511/ 521 using α3β1 receptor (Spessotto et al., 2003). B-CLLs adhered to LM-511 (data not shown), but migrated poorly on this specific LM isoform (present data). Goldfinger and colleagues (1999) demonstrated that functional LM-332 was required for maximal epithelial cell migration in the skin. Most probably a functional form of LM-511 would be necessary also for B-CLLs in order to migrate. The observation that LM-332-dependent migration was more effective in B-CLL cells with a mutated IgVH locus emphasized a correlation between expression of the α3 integrin subunit and a specific IgVH mutational status of B-CLL cells. Since migration on LM-332 was assayed directly on ex-vivo BCLL cells without any additional activation of neoplastic cells, it underscored the higher constitutive activation state of α3β1 in cells with mutated (M) and/or significantly mutated (sM) IgVH genes when compared with the receptor status in B-CLL cells with unmutated loci. Alternatively, ancillary cell surface components assisting/influencing the activity of this specific LM-binding integrin may be diversely modulated and/or expressed in mutated versus unmutated B-CLL cells. These findings seem in contrast with previous observations suggesting that binding of B-CLL cells to basement membranes required a putative inside-out integrin activation elicited by triggering of intracellular signaling cascades (Vincent et al., 1996). However, these studies focused on B-CLL cell adhesion to murine EHS LM-111, which we found here to be a poor substrate for B-CLL locomotion. Studies of extensive immunophenotypic analysis of B-CLL cells performed by some of us (Zucchetto et al., 2005, 2006a,b), allowed the identification of a specific immunophenotypic profile of α3-expressing B-CLL cells, as being basically characterized by overexpression of CD62L, CD54, and CD25, usually in association with higher levels of CD40. Of note, α3expressing B-CLL lymphocytes with the CD62L+ CD54+ CD25+ CD40+ phenotype harbored at higher frequency a larger number of IgVH mutations (Degan et al., 2004a; Hamblin et al., 1999; Damle et al., 1999), as well as an IgVH mutational status consistent with antigen-driven selection (Bomben et al., 2005; Chang and Casali, 1994; Chen et al., 1999; Degan et al., 2004a; Lossos et al., 2000; Zucchetto et al., 2005). Noteworthy, CD62L, CD54, CD25 and CD40 correspond to surface components that are involved in the cross-talk between B-CLL lymphocytes with neighboring endothelial and/or T cells within the lymph node microenvironment (Chen et al., 1999; Lane et al., 1992; Poudrier and Owens, 1994; Gu et al., 2001; Mills and Cambier, 2003) and their expression is consistent with the fact that IgVH mutations usually occur as the result of T celldependent interactions during GC-maturation of B cells (Dorner et al., 1998). Findings reported in the present study, indicating the presence of a specific mesh of LM-332 fibrils in the context of marginal zones and/or follicle interspaces of normal reactive lymph nodes, corroborate this hypothesis. In these areas, in fact, are localized subsets of memory B cells expressing a heavily mutated IgVH genes, as the result of a T cell-dependent GC differentiation (Chiorazzi et al., 2005; Kuppers, 2005) and such cells are presumed to correspond to the normal counterparts

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of B-CLL cells bearing mutated IgVH gene products (Chiorazzi et al., 2005; Kuppers, 2005; Rosenwald et al., 2001; Aumailley et al., 2005). This also suggests that an origin of the malignant B-CLL clone outside the follicles, in close vicinity of lymph node marginal zones, may be favoured by interactions of B cells with specific ECM component, including LM-332. Although the cellular source of the LMs assembled in these basement membranes is not known, the epithelial-like traits of extra-follicular reticular cells (Franke and Moll, 1987) suggest that these may be the cells depositing LMs in these lymph nodal compartments. In B-CLL, the normal architecture of lymph nodes is disrupted by infiltrating malignant lymphocytes, although specific microenvironmental cell–cell and cell–matrix interactions continue to take place, thus facilitating the growth, survival and motility of neoplastic cells (Caligaris-Cappio and Hamblin, 1999). A further finding of the present study is that B-CLL involved lymph nodes displayed a mesh of LM-332 no longer circumscribed to specific areas, as in normal tissues, but diffuse throughout the lymph node architecture. Although little is known about the mechanisms determining lymph node enlargement in B-CLL, this process clearly involves a complex set of steps, including malignant cell invasion into and within all lymph node areas and either their enhanced accumulation or reduced exit through lymphatic channels and/or high endothelial venules (Jung and Littman, 1999). High endothelial venules are an important route of entry of lymphocytes into lymph nodes and malignant B-CLL cells can be observed migrating through these vessels (Csanaky, 1994). Since both lymphatic and vascular basal membranes, including those of high endothelial venules, contain high amounts of LM-332, it is tempting to speculate that this de novo LM-332 network may represent an additional migration route for B-CLL lymphocytes mobility and migration, playing a role in the malignant cell movement into as well as within lymphoid tissues. How the distribution of LM-332 in B-CLL affected lymph nodes relates to the LM-411 and LM-511 rich conduit system recently described (Sixt et al., 2005) remains to be determined. In conclusion, LM-332 may be considered an important microenvironmental mediator not only for B-CLL cell locomotion but also as part of a more complex picture in which the convergent signals delivered by soluble factors and by direct cell-cell contacts to rescue B-CLL cells from undergoing apoptosis interplay with this LM to promote cell growth and motility within the nodes. 4. Experimental procedures 4.1. B-CLL cell samples and immunophenotyping The study includes peripheral blood samples, collected after informed consent for routine diagnostic and follow-up procedures, from 46 non-randomized patients (28 males, 18 females; median age 65 years, range 34–97) affected by typical B-CLL (Matutes et al., 1994). Expression of diagnostic phenotypic markers was investigated by flow cytometry by combining phycoerithrin (PE)-,

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FITC- and allophycocyanin (APC)-conjugated monoclonal antibodies (mAbs) as reported elsewhere (Gattei et al., 1997; Zucchetto et al., 2005). For integrin subunits expression, the following non-conjugated mAbs obtained by Immunotech (Marseille, France) and utilized in conjunction with PE-labeled secondary antibody (BD Biosciences Inc., Palo Alto, CA), were employed: HP2B6 (α1), Gi9 (α2), C3VLA3 (α3), HP2/1 (α4), GoH3 (α6) and 4B4 (β1). Unrelated isotype-matched antibodies (BD Biosciences, Inc) were used to determine background fluorescence. Viable, antibody-labeled B-CLL cells were identified according to CD19 expression/side-scattering, electronically gated and analyzed on a FACScalibur flow cytometer by means of the CellQuest software (BD Biosciences, Inc). 4.2. Amplification, cloning and sequencing of VHDJH transcripts The IgVH mutational status analysis of B-CLL samples was performed as previously described (Degan et al., 2004a). Briefly, total RNA, extracted, reverse–transcribed and checked for firststrand synthesis (Gattei et al., 1997; Degan et al., 2000), was amplified using a mixture of sense primers annealing either to the VH1 through VH6 leader sequences or to the 5′ end of VH1– VH6 FR1 utilized in conjunction with a mixture of antisense primers complementary to the germ line JH region (Fais et al., 1998; Hamblin et al., 1999; Gurrieri et al., 2002; Degan et al., 2004b). The purified amplified products were cloned into plasmids and sequenced. Sequences were aligned to the IgBLAST, VBASE or ImMunoGeneTics directories. Only when the same rearrangement was identified in at least 5–10 clones, a given IgVH sequence was assigned and the number of IgVH mutations computed. 4.3. Immunohistochemistry and immunofluorescence Fresh tissue samples were embedded in OCT compound (Miles Laboratories, Naperville, IL), snap-frozen in liquid nitrogen-cooled isopentane, and stored at − 70 °C until time of processing or directly cryosectioned. We processed lymph nodes obtained from three B-CLL patients. The number can be considered low but for ethical reasons the lymph node biopsy in B-CLL patients is not a suggested procedure. Sections (5 μm thickness) were stained with rabbit polyclonal anti LM-332 (R13), kindly provided by Dr. Manuel Koch followed by antirabbit IgG and peroxidase ABC-Elite complex (Vector Laboratories, Burlingame, CA) or by goat anti-rabbit Alexa Fuor® 488labelled secondary antibodies (Molecular Probes, Eugene, OR). Images were taken with a BX51 Olympus microscope equipped with a DP50 camera (Olympus Italia S.r.l., Segrate Milano, Italy) or acquired with LEICA TCS SP2 confocal system (Leica Microsystems Heidelberg GmbH, Mannheim, Germany), using the Leica Confocal Software (LCS) and a 40× fluorescence objective on a Leica DM IRE2 microscope. Other sections were treated with monoclonal antibodies specific for LM-332 chains: anti α3 chain (BM2) and anti β3 chain (6F12) were kindly provided by Dr. Manuel Koch and anti γ2 chain (D4B5) was purchased from Chemicon International, Temecula, CA.

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These sections were treated for immunofluorescence using chicken anti-mouse Alexa Fuor® 488-labelled secondary antibodies (Molecular Probes). Nuclei were in some cases visualized with propidium iodide (Molecular Probes) and images were acquired with LEICA TCS SP2 confocal system (Leica Microsystems). 4.4. Purification and characterization of LM isoforms Native LM-111-nidogen complex from EHS mouse tumor were purified by a previously described EDTA-extraction procedure (Spessotto et al., 2003). LM-332, isolated from cultured human keratinocytes through immunoaffinity chromatography on mAb BM165 (Rousselle and Aumailley, 1994) was kindly provided by Dr. Patricia Rousselle. Native LM-411 isolated from primary bovine aortic endothelial cells as previously described (Spessotto et al., 2001, 2003) was kindly provided by Dr. Lydia Sorokin. ELISA, SDS-PAGE and Western blotting with anti-α5 and anti-α2 laminin chain antibodies confirmed the previously reported (Sorokin et al., 1997) absence of significant levels of LM-421,-511 and -521 (not shown). A pepsin-extracted LM-511 preparation from human placenta, isolated by immunoaffinity chromatography on mAb 4C7, was purchased from Chemicon. The LM-111/511/521 complex from adult bovine kidney was purified under native conditions and has been extensively characterized in our previous publications (Spessotto et al., 2001, 2003). Human plasma FN was purchased from Calbiochem-Novabiochem Corporation (San Diego, CA). 4.5. Western blotting

were washed, re-suspended in RPMI with 0.1% BSA and then added to the upper side of the inserts (2 × 105 cells/insert). Migratory behaviour of the cells was then monitored at different time intervals by independent fluorescence detection from the top (corresponding to non-transmigrated cells) and bottom (corresponding to transmigrated cells) side of the membrane using a computer-interfaced GENios microplate reader (Tecan Italia). In some instances, cells were pre-incubated with blocking antibodies or non-blocking control antibodies for 30 min and then added to the upper surface of the inserts. For these experiments, blocking mAbs anti-α1 (clone FB12), anti- α3 (clone P1B5), anti- α4 (clone P1H4), anti-α6 (GoH3; Chemicon International) were used at previously established optimal concentrations. A specifically designed FATIMA software was used for the elaboration of fluorescence values and to determine the exact percentage of transmigrated cells out of the total amount introduced into the system. The integrated mean values from 25 distinct acquisition points/well are reported. 4.7. Detection of MMP-2 and MMP-9 mRNA expression A volume of 2 μl of the cDNA preparations, obtained as described above, was amplified in a 50 μl final volume containing 25 pmoles of primers specific for MMP-2 (sense: 5′GTTTCCATTCGCTTCCAGGGCAC-3′; antisense: 5′GGTCGCACAC CACATCTTTCCGTC-3′) or MMP-9 (sense: 5′-CACCATGAGCCT CTGGCAGC-3′; antisense: 5′-GCCCAGGGACCACAACTCGT-3′). For all primer pairs, the PCR amplification protocol was a two-step procedure consisting of 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C and 75 s at 68 °C.

Tissue extracts from 3 B-CLL lymph nodes or purified proteins (LM-332, LM-111 and fibronectin) were loaded onto a SDS-acrylamide gel (4–15% polyacrylamide), subjected to electrophoresis under reducing conditions and transferred to a nitrocellulose membrane. The membranes were saturated with TBS buffer (containing 20 mM TRIS and 0.15 M NaCl) supplemented with 0.1% Tween-20 (TBST) and 5% not fat dry milk or BSA for 2 h at room temperature and then incubated with primary antibodies at 4 °C overnight. After extensive washing in TBST, the membranes were incubated with HRP-conjugated appropriate secondary antibodies (Amersham Biosciences Europe, Orsay, France) and then revealed with the ECL Plus chemiluminescence kit (Amersham).

Correlations between IgVH mutational status and B-CLL cell migration were performed by means of Chi-square test (Armitage) and for IgVH mutation, analyses were performed by using the standard cut-off of 2% to discriminate between unmutated and mutated B-CLLs (Degan et al., 2004a; Hamblin et al., 1999). The B-CLL subsets, with or without evidence of antigen-driven selection (Chang and Casali, 1994; Dorner et al., 1998; Lossos et al., 2000) were defined by applying the available binomial and multinomial distribution models (Chang and Casali, 1994; Lossos et al., 2000), as described in other publications (Degan et al., 2004a,b). Statistical significance was set at p values lower than 0.05.

4.6. Cell motility assay

Acknowledgments

Migration of B-CLL cells in response to ECM substrates was assessed by FATIMA system (Spessotto et al., 2002; Spessotto et al., 2003). The insert membranes were coated with the various purified ECM molecules, in 0.05 M bicarbonate buffer, pH 9.6, at 4 °C overnight and subsequently blocked with 1% BSA for 1 hour at room temperature. B-CLL cells were fluorescently tagged with the lipophylic dye Fast DiI (Molecular Probes) used at a final concentration of 5 μg/ml for 10–15 min at 37 °C. The cells

We are particularly grateful to Patricia Rousselle and Giancarlo Giannelli for providing us with purified LM-332, and to Manuel Koch for the gift of R13 polyclonal antibody, α3 chain (BM2) and anti β3 chain (6F12) monoclonal antibodies. The work was supported by grants from the Italian Ministry of Health (Ricerca Finalizzata IRCCS and “Alleanza Concro il Cancro, FSN” to VGRP and A.C.), Associazioni Italiana per la ricerca sul Cancro (AIRC to RP) and institutional

4.8. Statistical procedures

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