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Blood First Edition Paper, prepublished online May 12, 2008; DOI 10.1182/blood-2008-02-139378
CONSTITUTIVE EXPRESSION OF IL-12RB2 ON HUMAN MULTIPLE MYELOMA
CELLS DELINEATES A NOVEL THERAPEUTIC TARGET
Irma Airoldi1∗, Claudia Cocco2∗, Nicola Giuliani3, Marina Ferrarini4, Simona Colla3, Emanuela
Ognio5, Giuseppe Taverniti5, Emma Di Carlo6, Giovanna Cutrona7, Vittorio Perfetti8, Vittorio
Rizzoli3, Domenico Ribatti9 and Vito Pistoia2
1
Department of Experimental and Laboratory Medicine, G. Gaslini Institute, Genova, Italy
2
Laboratory of Oncology, G. Gaslini Institute, Genova, Italy
3
Hematology and BMT Center, Department of Internal Medicine and Biomedical Science,
University of Parma, Italy
4
Laboratory of Tumor Immunology and Department of Oncology, Istituto Scientifico H. San
Raffaele, Milano, Italy
5
Animal Model Facility, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
6
Department of Oncology and Neurosciences, "G. d'Annunzio" University and Ce.S.I. Aging
Research Center, "G. d'Annunzio" University Foundation, Chieti, Italy
7
Oncologia Medica C, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
8
Internal Medicine and Medical Oncology, IRCCS Policlinico S. Matteo, 27100 Pavia, Italy
9
Department of Human Anatomy and Histology, University of Bari, Bari, Italy
*
Both authors equally contributed to this work
Scientific Category: Neoplasia
Corresponding Author
Irma Airoldi, Ph.D.,
Department of Experimental and Laboratory Medicine, G. Gaslini Institute, Genova, Italy, 16148
Genova, Italy; Phone: +390105636342, Fax: +390103779820
E-mail: [email protected]
Copyright © 2008 American Society of Hematology
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ABSTRACT
The IL-12 receptor(R)B2 gene acts as tumor suppressor in human acute and chronic B cell
leukemias/lymphomas
and
IL-12rb2
deficient
mice
develop
spontaneously
localized
plasmacytomas. With this background, we have investigated the role of IL-12RB2 in multiple
myeloma (MM) pathogenesis.
Here we show that i) IL12Rβ2 was expressed in primary MM cells, but downregulated in
comparison with normal polyclonal plasmablastic cells (PPC) and plasma cells (PC). IL-6
dampened IL-12Rβ2 expression on PPC and MM cells, and ii) IL-12 reduced the pro-angiogenic
activity of primary MM cells in vitro and decreased significantly (P=0.0001) the tumorigenicity of
the NCI-H929 cell line in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. The
latter phenomenon was found to depend on abolished expression of a wide panel of pro-angiogenic
genes and up-regulated expression of the antiangiogenic genes IFN-γ, IFN-α, platelet factor-4 and
TIMP-2.
Inhibition of the angiogenic potential of primary MM cells was related to down-regulated
expression of the pro-angiogenic genes CCL11, vascular endothelial-cadherin, CD13 and AKT and
to up-regulation of an IFN-γ related anti-angiogenic pathway. Thus, IL-12Rβ2 restrains directly
MM cell growth, and targeting of IL-12 to tumor cells holds promise as new therapeutic strategy.
2
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INTRODUCTION
Multiple myeloma (MM) is a monoclonal post-germinal center tumor that has phenotypic features
of plasmablasts/long lived plasma cells and usually localizes at multiple sites in the BM.1 MM is the
second most common haematological malignancy worldwide and its prognosis remains grim in
spite of advanced therapeutic protocols.2 Promising targeted therapies for MM have emerged,
including proteasome inhibitors, heat shock protein 90 inhibitors, AKT inhibitors, and antiangiogenic molecules with immuno-stimulatory properties (e.g. thalidomide), but treated patients
still relapse.2 Thus, novel therapeutic strategies are warranted to improve MM prognosis.
IL-12 is a cytokine that exerts potent anti-tumor activity through a combination of
immunostimulatory and anti-angiogenic mechanisms.3-6 The latter are related to induction of IFN-γ,
which in turn triggers the release of the anti-angiogenic chemokines CXCL9, CXCL10 and
CXCL11. In addition, IL-12 down-regulates the production of the pro-angiogenic molecules VEGF
and FGF-2.7-11
We3,12 have previously shown that the IL-12RB2 gene, encoding the IL-12R chain essential for IL12 signal transduction, functions as a tumor suppressor in human neoplastic B cells from various
chronic lymphoproliferative disorders and acute lymphoblastic leukemia. We13 have also
demonstrated that IL-12rb2 deficient mice develop spontaneously multiorgan lymphoid infiltrates,
systemic IL-6 up-regulation and CD138+ plasma-cell hyperplasia.13 Finally, aged IL-12rb2 KO
animals develop localized lymph node plasmacytoma, that is exceedingly rare in humans14, possibly
in relation to IL-6 over-expression.13
MM progression is characterized by changes in the BM microenvironment including
overexpression of IL-6 and VEGF that support tumor growth through paracrine loops, induction of
angiogenesis and suppression of cell-mediated immunity.15,16 Here we have asked whether IL12RB2 plays a role in human MM pathogenesis and investigated for the first time the expression
and function of IL-12Rβ2 in MM cells and their normal counterparts. Next, we have performed
3
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functional studies in order to assess the direct anti-tumor activity of IL-12 on MM cells and to
unravel the molecular mechanisms involved.
4
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MATERIALS AND METHODS
Patients
The study design was approved by the Ethical Committee of the University of Parma, Parma, Italy.
Nineteen MM patients were studied (Table 1). Thirteen of them were male, six female. Patient age
ranged from 49 to 92 years. Ten patients had stage IIIa, 3 stage IIIb, 2 stage IIa and 4 stage Ia
disease, according to the Durie and Salmon staging system.17 The monoclonal serum component
was IgGκ in nine cases, IgGλ in 5 cases, IgAκ in 2 cases, IgAλ in 2 cases and λ in the remaining
case. BM infiltration with malignant plasma cells at diagnosis ranged from 27% to 98%. At study,
all patients were untreated. Aliquots of BM aspirates performed for clinical evaluation were
obtained after informed consent at diagnosis in thirteen cases and at relapse in the remaining six.
BM aspirates from four healthy donors were obtained following their informed consent.
Generation of normal PPC
Normal PPC were generated in vitro from peripheral blood samples of twelve healthy volunteers
obtained after informed consent. CD19+ B cells were positively selected using MACS microbeads
(Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and subjected to two different
procedures.18-20 In some experiments, CD19+ B cells were cultured in the presence of CD40L (1
μg/ml), IL-2 (20 U/ml), IL-4 (50 ng/ml), IL-10 (50 ng/ml) and CpG 2006 oligonucleotide21 (2.5
μg/ml). After 4 day culture, B cells were washed in PBS and cultured with IL-2, IL-10 and IL-6 (5
ng/ml). On day 6 of culture, PPC were purified by depletion of CD20+ cells using immunomagnetic
bead manipulation. Alternatively, PPC were obtained after 6 day culture of CD19+ and CD3+ T cells
(ratio 0.5:1), isolated by immunomagnetic beads, in the presence of 1 μg/ml pokeweed mitogen
(Sigma Chemical Company, St. Louis, MO), as previously described.18,19 PPC were negatively
selected using CD3 and CD20 monoclonal antibodies and anti-mouse Ig beads. PPC obtained using
the two procedures were CD19+CD20-CD38++CD138+/-
20
purity ranged from 90 to 95% in the different experiments.
5
, as assessed by flow cytometry. Their
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In some experiments, tonsil PB were sorted as CD19+,CD38bright, IgDnegative/low, stained with IL12RB2 monoclonal antibody and analyzed by flow cytometry.
BM aspirates from patients with MM and from healthy donors were depleted of erythrocytes by
osmotic lysis and neoplastic cells were purified to homogeneity by positive selection using CD138
coated magnetic beads (Miltenyi Biotec).
Cell culture, antibodies, reagents and flow cytometry
LP-1, U266, Karpas 620, RPMI 8226, H-Sultan, OPM-2, JJN3, XG-1, XG-6 and NCI-H929 MM
cell lines were cultured in RPMI 1640 medium with 10% FCS (Seromed-BiochromKG, Berlin,
Germany). Human recombinant (hr) IL-12 was provided by Wyeth Inc., Cambridge, MA. Human
recombinant IL-2, IL-10, IL-4 and IL-6 were from R&D Systems, Abingdon, United Kingdom.
Trimeric CD40L was from Alexis (Axxora, LLC, San Diego, CA). Normal PPC generated in vitro,
CD138+ MM cells, NCI-H929 and XG-1 cells were cultured in the presence of 50 ng/ml IL-6 and
tested for IL-12Rβ2 surface expression by flow cytometry. The source of anti-CD3 mAb was the
supernatant of OKT3 murine hybridoma, ATCC, Manassas, VA, USA. Fluorochrome-conjugated
anti-human IL-12Rβ2, CD19, CD20, CD38, CD138, anti-IL6, anti-IFN-γ, anti-Ig and anti-Ki67
mAbs were from BD-Pharmingen (San Josè, CA). An anti-human IL-12Rβ2 goat IgG that detects
an intracellular epitope of
IL-12Rβ2 (Santa Cruz Inc, Santa Cruz, CA) was used
in some
experiments. This antibody was tested using BD Cytofix/CytopermTM fixation/permeabilization kit
(BD Biosciences). Isotype-matched mAb of irrelevant specificity or non-immune goat IgG (Caltag,
Burlingame, CA) were used as controls. Cells were scored using a FACSCalibur analyzer (BD
Bioscences, San Josè, CA) and data processed using CellQuest software (BD).
Cell proliferation and apoptosis assays
The NCI-H929 MM cell line was cultured for 48h in the presence or in the absence of 20 ng/ml
hrIL-12. Cells were then stained intracellularly with anti-Ki67 mAb and analyzed by flow
cytometry. Apoptosis was assessed using the rhAnnexin V/FITC kit from Bender MedSystems,
Burlingame, CA.
6
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Real-Time PCR
Quantitative analysis of IL-12RB2 transcript was performed as follows. One μg of total RNA was
reverse transcribed using the ReactionReady™ First Strand cDNA Synthesis kit (SuperArray
Bioscience Corporation, Frederick, MD). cDNA was subjected to real-time PCR using the RT2
Real-Time SYBR Green PCR master mix, human GAPDH and human IL-12RB2 primer sets
purchased from SuperArray (proprietary primers, sequence not disclosed). PCR was performed in
triplicate using an ABI PRISM 7700 sequence detector instrument (Applied Biosystem, Foster City,
CA) in a final volume of 25 μl. A standard two-step amplification with 60°C annealing temperature
was used, as suggested by datasheet. Relative quantification of IL-12RB2 transcript was obtained
using comparative Ct method.22 The copy number in the unknown samples was normalized to an
endogenous reference (GAPDH gene) and expressed relative to a calibrator sample (positive
control) using the 2-(ΔΔCt ± SD) method.
RT-PCR and methylation assay
RNA was extracted from freshly isolated MM cells and MM cell lines using RNeasy Mini Kit from
Qiagen (Qiagen GmbH, Hilden, Germany) and subjected to RT-PCR.23 Expression of IL-12RB2
mRNA was investigated by RT-PCR using the conditions and the primers published elsewhere.23
DNA was extracted using GenElute DNA miniprep kit from Sigma and the methylation status of
the target sequence was assessed by Methylation Specific PCR as previously described.3
Mice studies
Four- to six-week-old SCID-NOD mice (Harlan Laboratories, Udine, Italy) were housed under
specific pathogen-free conditions. All procedures involving animals were performed in the respect
of the National and International current regulations (D.l.vo 27/01/1992, n.116, European Economic
Community Council Directive 86/609, OJL 358, Dec. 1, 1987).
Two groups of 16 animals each were injected i.p. with 8x106 NCI-H929 cells. One group of mice
was treated with 3 weekly doses of hrIL-12 (1 μg/mouse/dose) starting from 8 hours after injection
of tumor cells. The other group of mice was injected with PBS following the same time schedule.
7
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Twenty-three days after tumor cell inoculation, mice were sacrificed and autopsies were carried out.
Tumor masses were measured as described.3
Morphologic and immunohistochemical analyses
For histological evaluation, tissue samples were fixed in 4% neutral buffered formalin, embedded in
paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (H&E). For
immunohistochemistry, formalin-fixed, paraffin-embedded sections were immunostained with
rabbit anti human/mouse laminin Ig (Biogenex, San Ramon, CA, USA) or mouse anti human
PCNA mAb (proliferating cell nuclear antigen) (clone PC10; Dakocytomation, Glostrup, DK).
After washing, sections were overlaid with goat anti-mouse/rabbit Ig conjugated to peroxidaselabelled dextran (EnVision+ Peroxidase, rabbit/mouse) (Dakocytomation) for 30 min. Unbound
immunoglobulin was removed by washing and slides were incubated with ABC (avidin-biotin
complex)/alkaline phosphatase (DAKO) for 30 min, then sections were counterstained with H&E.
CAM assay
Fertilized White Leghorn chicken eggs (20/group) were incubated at 37°C at constant humidity. On
day 3, a square window was opened in the shell, and 2 to 3 ml of albumen was removed to allow
detachment of the developing chorioallantoic membrane (CAM). The window was sealed with a
glass, and the eggs were returned to the incubator. On day 8, eggs were treated with: 1 mm3
sterilized gelatin sponges (Gelfoam Upjohn, Kalamazoo, MI) placed on top of the growing CAM,
as reported24, and loaded with: 1 μl of PBS (negative control); 1μl of PBS with 250 ng VEGF
(R&D Systems) as positive control; 1 μl of medium from NCI-H929, XG-1, U266 cell lines or
purified MM cells from patients cultured 48h with or without hrIL12; 1 μl of medium containing
hrIL-12. The viability of cells from primary MM samples and MM cell lines was checked before
supernatant harvest by cell count using Trypan Blue dye exclusion test. All supernatants were tested
in triplicate and means ± SD were calculated. CAM were examined daily until day 12 and
photographed in ovo with a stereomicroscope equipped with a camera and image analyzer system
8
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(Olympus Italia, Italy). On day 12, the angiogenic response was evaluated by the image analyzer
system as the number of vessels converging toward the sponges.
Angiogenesis PCR-Array
RNA was extracted from tumors removed from SCID-NOD mice 23 days after injection of NCIH929 cells and treatment with hrIL-12 or PBS, using TRIZOL® from Invitrogen (Carlsbad, CA,
USA) and retro-transcribed by the ReactionReady™ First Strand cDNA Synthesis kit (SuperArray
Bioscience Corporation). In some experiments, CD138+ myeloma cells purified from BM of MM
patients, were cultured for 48 hours with medium in the presence or absence of hrIL-12 and RNA
was extracted using Trizol. Contaminant genomic DNA was removed by Dnase treatment using
Rneasy Micro Kit (Qiagen GmbH) and IL-12RB2 expression was tested by PCR before starting
PCR-Array procedure.
Human Angiogenesis RT2 ProfilerTM PCR Array and RT2 Real-TimerTM SyBR Green/ROX PCR
Mix were purchased from SuperArray Bioscience Corporation. PCR was performed on ABI
PrismTM 7700 Sequence Detector (Applied Biosystems).
For data analysis the ΔΔCt method was used; for each gene fold-changes were calculated as
difference in gene expression between tumors formed by NCI-H929 cells in hrIL-12 or PBS treated
animals or MM primary cells treated or not with IL-12 in vitro. According to the instructions of the
manufacturer, a significant threshold is defined as a four-fold change in gene expression. To render
the assay more stringent, we elevated such threshold to five.
Statistical methods.
Results were calculated with 99% confidence interval. Data were analyzed using Student’s t test for
the analysis of the Results of the CAM assay or Mann Whitney test for the analysis of the results of
the remaining experiments. A P value lower than 0.05 was considered statistically significant.
9
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RESULTS
Expression and function of IL-12RB2 in normal polyclonal plasmablastic cells (PPC), plasma
cells (PC) and primary MM cells
In the following experiments, we first investigated expression and function of IL-12RB2 in normal
PPC generated in vitro from normal peripheral blood or isolated from tonsil, as well as in normal
PC purified from BM or tonsil. Both PPC and PC represent potential counterparts of MM cells.
Figure 1A, panels a and b, shows two experiments, representative of the 12 performed with similar
results, in which surface IL-12Rβ2 expression was consistently detected by flow cytometry using
the mAb from BD Biosciences in PPC generated in vitro from normal peripheral blood (mean
percentage of IL-12Rβ2+ cells=58.6%, range from 43% to 75%). In four different experiments, IL12Rβ2 was found to be expressed also by PPC sorted from tonsil (mean percentage of IL-12Rβ2+
cells=97%, range from 95% to 99%). Figure 1A, panels c and d, shows two representative
experiments.
Figure 1B shows that CD138+ PC from normal BM (panels a and b) or tonsil (panels c and d)
expressed IL-12Rβ2 on the cell surface using the mAb from BD Biosciences (BM CD138+ cells:
mean percentage of IL-12Rβ2+ cells from four different experiments=76.5%, range from 62% to
84%; tonsil CD138+ cells: mean percentage of IL-12Rβ2+ cells from four different
experiments=80.3%, range from 71% to 87%).
The ubiquitous IL-12Rβ1 chain was expressed in all PPC suspensions in vitro or in PPC and PC ex
vivo (not shown).
In order to investigate whether IL-12R was functional in normal PPC, four PPC suspensions
generated in vitro were incubated with hrIL-12 or medium for 48h and subsequently tested for IFNγ production by intracellular staining. As apparent from Figure 1C, IL-12 up-regulated significantly
(P=0.0286) IFN-γ production, thus indicating that the IL-12R was functional.
Next, we investigated the expression of the IL-12RB1 and B2 chains in primary neoplastic cells
isolated from the BM of nineteen MM patients. Primary MM cells from all patients expressed IL10
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12RB1 (upper panel) and IL-12RB2 (lower panel) mRNA, as assessed by RT-PCR (Fig. 2A, 11
patients shown). Nonetheless, primary MM cells from the same eleven patients tested negative for
IL-12Rβ2 surface expression, as assessed by flow cytometry using the mAb from BD Biosciences,
indicating that IL-12Rβ2 was strongly down-regulated in comparison with normal PPC or PC. Fig.
2B shows two representative profiles from Pt #1 and #2 (BD mAb). However, the same MM cell
suspensions still displayed evident expression of IL-12Rβ2 upon staining with the anti IL-12Rβ2
antibody from Santa Cruz (Fig. 2B, SC Ab). Fig. 2C shows comparative staining of one
representative normal PPC sample (upper panels), of IL-12RB2 transfected RAJI Burkitt lymphoma
cell line3 (middle panels), and of a normal T helper 1 clone (lower panels) with the two anti-IL12Rβ2 antibodies, clearly highlighting the superior performance of the Santa Cruz reagent.
By comparing the results of PPC (Fig. 2C, upper panels) and primary MM cell (Fig. 2B, SC Ab)
staining with the Santa Cruz antibody, downregulation of IL-12Rβ2 protein in tumor cells vs PPC is
confirmed (mean percentage of IL-12Rβ2+ PPC from three different experiments=92%, range from
89% to 95%; mean percentage of
IL-12Rβ2+ primary MM cells from three different
experiments=61.5%, range from 55% to 68%).
Quantitative PCR performed with cDNA from four normal PPC preparations and ten CD138+ MM
cell suspensions revealed that IL-12RB2 mRNA was expressed at similar levels in all samples
tested (data not shown), thus suggesting that down-regulation of IL-12Rβ2 protein on the cell
surface of MM cells occurred through post-transcriptional mechanisms.
To gain more insight into this issue, we hypothesized that IL-12Rβ2 chain down-regulation on
primary MM cells, as compared to normal PPC or PC, was related to IL-6 over-produced in the BM
microenvironment. IL-6 is a major paracrine myeloma growth factor both in vitro and in vivo and
high serum IL-6 levels were detected in MM patients with active disease.1 Thus, we cultured four
different CD138+ MM cell suspensions for 48h in the presence or absence of hrIL-6 and
subsequently tested them for IL-12Rβ2 expression by flow cytometry using the Santa Cruz
11
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antibody. Figure 3A shows that the IL-12Rβ2 chain was expressed on CD138+ MM cells that had
been cultured for 48h with medium alone and downregulated significantly (P=0.0022) upon 48h
incubation with hrIL6. The same MM cell suspensions cultured with or without IL-6 as above
displayed similar levels of IL-12RB2 mRNA, indicating that protein downregulation induced by the
latter cytokine occurred through post-transcriptional mechanisms (Fig. 3B).
Figure 3C shows two representative experiments out of the four performed in which IL-6 48 h
treatment strongly down-regulated surface expression of IL-12RB2 also in PPC (P=0.019).
hrIL-12 inhibits the pro-angiogenic activity of primary MM cells
MM cells are known to release several pro-angiogenic factors.25 We then asked whether this feature
was affected by IL-12, which we have found to exert direct anti-tumor activity through inhibition of
angiogenesis in other tumor models.3,7 We therefore incubated CD138+ neoplastic cells from four
MM patients (Patients #1, 5, 14 and 18) with hrIL-12 or medium alone and tested the angiogenic
activity of culture supernatants in the chorio-allantoid membrane (CAM) assay. We also checked
tha viability of primary tumor cells incubated with or without IL-12 before harvesting supernatants
by Trypan blue staining. There were no statistically significant differences in the proportions of
viable cells between cultures performed in the presence or absence of IL-12.
CAM treated with sponges loaded with VEGF (positive control) or with supernatants from primary
MM cells were surrounded by allantoic vessels developing radially towards the implant in a
‘spoked-wheel’ pattern. In the representative experiment shown in Fig. 4A, left panel, the mean
number of vessels formed in the presence of supernatant from CD138+ MM cells (Pt #1) was 30±3,
while that formed in the presence of VEGF was 30±5 (not shown). No vascular reaction was
detected around the sponges upon exposure to hrIL12 diluted in medium at the same final
concentration used to treat tumor cells (mean number of vessels = 7±3 in the presence or absence of
hrIL-12, not shown). When the supernatants from hrIL-12 treated CD138+ cells from the same MM
12
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patient was tested in the CAM assay, a significant (P<0.001) reduction of the angiogenic response
was appreciable (mean number of vessels = 12±2) (Figure 4A, right panel), as compared to positive
control. Similar results with the same statistical significance were obtained when supernatants from
the three remaining CD138+ primary MM cell suspensions were tested (Patient #5: mean number of
vessels formed in the presence of untreated supernatant 32±3, of IL-12 treated supernatant 14±3.
Patient 14: mean number of vessels formed in the presence of untreated supernatant 35±4, of IL-12
treated supernatant 16±3. Patient 18: mean number of vessels formed in the presence of untreated
supernatant 28±2, of IL-12 treated supernatant 12±2). Taken together, these findings demonstrated
that IL-12R was functional in primary MM cells and that IL-12 treatment damped their proangiogenic activity.
Next, we investigated expression of pro-angiogenic and anti-angiogenic genes in primary MM cells
incubated with IL-12 or medium. Purified CD138+ cells from three different MM patients (Patients
#4, 13 and 19) were cultured for 48h in the presence or absence of hrIL-12. RNA was extracted
from cultured cells, reverse transcribed and tested by PCR Array.
Fig. 4B shows the pooled results from the 3 samples analyzed. IL-12 treatment downregulated
significantly mRNA of the pro-angiogenic factors CCL11 (p=0.02), VE-cadherin (p=0.013), AKT
(p=0.021) and CD13 (p=0.05), whereas up-regulated mRNA of the angiogenesis inhibitors IFN-γ
(p=0.012), CXCL9 (p=0.02) and CXCL10 (p=0.015). Notably, also the transcripts of the proangiogenic CCL2 (p=0.013), angiopoietin (ANGPT)-1 (p=0.032) and ANGPT-5 (p=0.029) genes
were up-regulated in these cells (Fig. 4B).
Since PCR array studies pointed to a major role of an IFN-γ driven pathway in angiogenesis
inhibition, we investigated by flow cytometry whether purified CD138+ primary MM cells
incubated with IL-12 up-regulated expression of the IFN-γ protein. Indeed, as shown in Fig. 4C,
constitutive production of IFN-γ by primary MM cells was significantly increased following culture
with IL-12.
13
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Expression and function of IL-12RB2 in MM cell lines
Figure 5A, upper panel, shows that LP1, U266, JJN3, Karpas 620, RPMI 8226, H-Sultan, and
OPM-2 MM cell lines did not express the IL-12RB2 chain, as assessed by RT-PCR. Lack of
expression of IL-12RB2 gene in these cells was found to depend on methylation of the CpG island
within exon 1 (Fig. 5A, lower panel), as previously described for other human B cell malignancies.3
In contrast, XG-1, XG-6 and NCI-H929 MM cell lines expressed IL-12RB2 mRNA (Figure 5A,
upper panel) as well as the corresponding protein (Figure 5B, and data not shown for XG-6 cell
line), as assessed by staining with the Santa Cruz antibody.
Fig. 5C, left panel, shows that, similarly to primary MM cells, incubation of the IL-6 independent
NCI-H929 MM cell line with IL-6 down-regulated IL-12Rβ2 protein expression. Fig. 5C, right
panel, demonstrates that such down-regulation was not attributable to reduced gene expression
since similar levels of IL-12RB2 mRNA were detected in cells that had been cultured with or
without IL-6.
NCI-H929 cells were next cultured in the presence or absence of hrIL-12 for 48h and subsequently
tested for proliferation or apoptosis by Ki-67 mAb or propidium iodide/Annexin V staining,
respectively. In three different experiments, IL-12 inhibited the proliferation and induced apoptosis
of NCI-H929 cells by 5-10%. Likewise, IL-12 had minimal effects on apoptosis of two primary
MM cell suspensions in 48h cultures (not shown).
The angiogenic activity of IL-12Rβ2+ NCI-H929 and XG-1 cells, and of IL-12Rβ2- U266 cells was
subsequently investigated in the CAM assay using supernatants from cells cultured in the presence
or absence of hrIL12. Again, the proportions of viable cells did not differ significantly in cultures
performed with or without IL-12 for all the cell lines tested.
In these experiments, the XG-1 cell line was cultured without IL-6 for 48h before treatment with
hrIL-12.
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In the representative experiment shown in Fig. 5D, left upper panel, the mean number of vessels
formed in the presence of supernatant from the NCI-H929 cells was 28±4. When the supernatant
from the NCI-H929 cell line incubated with hrIL12 was tested, a significant (P<0.001) reduction of
the angiogenic response was appreciable (mean number of vessels=14±5), as compared to positive
control (VEGF, mean number of vessels 31±5). The mean number of vessels formed in the presence
of supernatants from XG-1 or U266 cells was 27±4 and 28±3, respectively (Fig. 5D, left, middle
and lower panels, respectively). When supernatant from the XG-1 cells incubated with hrIL-12 was
tested, a significant inhibition of their angiogenic potential was observed (mean number of vessel
13±4, P=0.001, Fig. 5D, middle right panel). In contrast, supernatant from U266 cell line tested as
negative control did not shown any significant reduction in vessel formation irrespective of
incubation with hrIL-12 (mean number of vessel 25±2, Fig. 5D, lower right panel). Taken together,
these results demonstrated unambiguously that the anti-angiogenic activity of IL-12 on MM cell
lines was absolutely dependent on IL-12R.
hrIL-12 strongly inhibits tumorigenicity of NCI-H929 cells in SCID-NOD mice
In subsequent experiments, the tumorigenicity of NCI-H929 cells was investigated in SCID-NOD
mice. Two groups of sixteen animals each were injected i.p. with 8 x 106 cells, treated with hrIL-12
or PBS i.p., and sacrificed after 23 days. By the end of the follow-up period, all mice developed
tumors that grew in the peritoneal cavity in the absence of metastases at distant sites.
Mice injected with NCI-H929 cells and treated with hrIL-12 developed tumors significantly smaller
(P < 0.0001) than mice inoculated with the same cells and treated with PBS (n=10 for both groups;
IL-12 treated, median volume 24 mm3; range 18.5-65 mm3. PBS treated, median volume 268 mm3;
range 88-1668 mm3) (Figure 6A).
The angiogenic phenotype of tumors formed in IL-12 vs PBS treated animals was next investigated
using PCR Array. In two different experiments performed with superimposable results, expression
of the following angiogenesis activators was virtually abolished by IL-12 (Fig. 6B, left panel):
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ANGPT-2, ANGPT-5, angiopoietin like (ANGPTL)-4, CD13, endothelial differentiation gene
(EDG)1, endoglin (END), ephrin (Eph) receptor B4, FGFb, FGF receptor 3 (ACH), VEGF-A, –C
and -D, heart and neural crest derivates expressed (HAND)2, HGFb, hypoxia inducible factor
(HIF)1α, inhibitor of DNA binding (ID)3, IL-6, IL-1β, CD51, neuropilin (NP)2, PDGFα, CD31,
COX1, stabilin (Clever)1, TGFα, TGFβ1, TGFβR2, thrombospondin (TSP)1 and TNF. All these
molecules are expressed during tumor neo-vascularization and promote organization, survival or
migration of endothelial cells26-51. Conversely, IFN-α and γ, platelet factor (PF)4 and tissue inhibitor
of metalloproteinase (TIMP)-2, which are inhibitors of angiogenesis10,11,52-55, were found to be upregulated in tumors grown in IL-12 vs PBS treated animals. (Figure 6B, right panel). Finally,
expression of the pro-angiogenic ID3, IGF-1 and EGF was up-regulated (Figure 6B, right panel)
We next investigated the histological and immunohistochemical features of tumors formed by the
NCI-H929 MM cell line in IL-12 vs PBS treated mice (Fig. 6C). Tumors from IL-12 treated animals
displayed a wide focus of ischemic-coagulative necrosis (Fig. 6C, panel d) as compared to control
tumors (Fig. 6C, panel a). In the former tumors, microvessel density, as assessed by laminin
staining, and proliferation index, as assessed by anti-PCNA staining (Fig. 6C, panels e and f,
respectively), were strongly reduced in comparison to control tumors (Fig. 6C, panels b and c,
respectively).
16
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DISCUSSION
Pathogenesis of MM is complex and dependent on the interactions between tumor cells and their
microenvironment in the BM, the primary site of MM development.1,15,56 Different cytokines,
chemokines and pro-angiogenic factors released in the tumor microenvironment are known to
promote MM cell growth and metastatic dissemination, especially to the skeleton.15,25,57,58 The most
prominent of these molecules is IL-6 that is produced by stromal cells from tumor infiltrated BM
and supports survival and proliferation of MM cells.1,15 Other molecules that exert similar effects
are VEGF and the chemokine CCL2.59,60
No information was so far available on the involvement of the IL-12/IL-12R system in MM
pathogenesis, with the exception of the reported increase of serum IL-12 levels in a cohort of MM
patients.61
We addressed the role of the IL-12/IL-12R system in MM based upon our previous finding that
aged IL12rb2 mice, who produce but cannot utilize IL-12, develop spontaneously localized
monoclonal plasmacytomas in the setting of a systemic autoimmune lymproliferative disorder
characterized by IL-6 overproduction.13 Other studies from our group lend cogent support to the
notion that IL-12 acts a negative regulator of the growth of both hematopoietic and nonhematopoietic tumors.3,7,12,13
In this study we demonstrate that IL-12Rβ2 is expressed on the surface of normal PPC and PC but
down-regulated in primary MM cells. Since quantitative PCR experiments disclosed similar levels
of IL-12RB2 mRNA in normal PPC and primary MM cells, downregulation of IL-12Rβ2 protein
expression in the latter cells must depend on post-transcriptional events. Consistent with the above
findings, the CpG island in exon 1 of the IL-12RB2 gene3 was never found to be methylated in
primary MM cells (not shown), at variance with that reported from our group in a large number of
chronic B cell malignancies including B-CLL, follicular lymphoma, mantle cell lymphoma and
marginal zone lymphoma3, as well as in B cell acute lymphoblastic leukaemia.12
17
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Many human MM cell lines did not express IL-12RB2 either at the protein or the mRNA level and
most of them displayed methylation of the CpG island in exon 1 of the gene. Differences in IL12RB2 gene expression between primary MM cells and the MM cell lines here investigated may be
related to the following, i) these cell lines, as most MM cell lines, were derived from
extramedullary sites where more aggressive tumors develop in comparison with MM confined to
BM, and/or ii) long-term in vitro culture of MM cell lines may have caused epigenetic changes
including IL-12RB2 gene methylation.
Nonetheless, three MM cell lines (XG-1, XG-6 and NCI-H929) were found to express IL-12RB2
mRNA and protein. We focused on the NCI-H929 cell line since it grows independently of
exogenous growth factors whereas the two remaining cell lines are IL-6 dependent. NCI-H929 cells
showed various analogies with primary MM cells that made them attractive candidates for in vivo
studies, and namely i) expression of similar patterns of IL-12RB2 mRNA and protein, and ii)
comparable inhibition of the angiogenic potential of tumor cells in the CAM assay induced by IL12.
We next investigated the in vivo effects of human IL-12 on tumorigenicity of the NCI-H929 cell
line in SCID/NOD mice. This model allows to assess the direct effects of human IL-12, that is
species-specific and inactive in the mouse, on human tumor cells injected in severely
immunodeficient animals. We injected NCI-H929 cells i.p. in order to generate a tumor mass
suitable for the investigation of IL-12 mediated anti-angiogenic effects. Although this model does
not mimick closely human disease that develops in the BM, the results obtained provide useful
translational information.
These experiments demonstrated that MM growth was virtually abrogated by IL-12 treatment
primarily through impaired formation of laminin lined mature blood vessels. The reduced MM cell
proliferation detected in tumors was consequent to angiogenesis inhibition since IL-12 had minimal
effects on the in vitro proliferation of NCI-H929 cells. This latter finding may depend on the
intrinsic properties of NCI-H929 cells rather than on those of IL-12, since the cytokine was
18
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previously found to inhibit the in vitro proliferation of IL-12Rβ2 expressing RAJI B lymphoma
cells.
Ex vivo PCR Array analysis of tumor masses showed that IL-12 damped the expression of a wide
set of pro-angiogenic genes including VEGF A, C and D, FGFb, ANGPT-2 and -5, COX-1,
PDGFα, and HIF-1α. A concomitant up-regulation of a limited number of anti-angiogenic genes
including IFN-γ, IFN-α, PF-4 and TIMP-2 was also detected.
Also primary MM cells exposed to IL-12 in vitro showed down-regulated expression of proangiogenic genes and up-regulated expression of anti-angiogenic genes. The former included AKT,
a key component of the phosphatydil inositol 3 phosphate kinase pathway that is now being targeted
for therapeutic purposes62, VE-cadherin, an endothelial cell specific adhesion molecule whose
soluble form correlated with tumor burden63, and CCL11, a chemokine binding to CCR3 expressed
by MM cells and driving their chemotaxis.64 IL-12 up-regulated anti-angiogenic factors included
IFN-γ and the IFN-γ inducible chemokines CXCL9 and CXCL10, pointing to the involvement of
this pathway in angiogenesis inhibition. Expression of four pro-angiogenic genes, i.e. ANGPT1,
ANGPT5, CXCL1 and CCL2, was also increased by IL-12, but the functional significance of this
finding remains to be established.
Differences in IL-12 induced expression profiles of angiogenesis related genes between NCI-H929
cell tumors and primary MM cells may be related to intrinsic differences between the two cell types
and/or to the different microenvironments to which they have been exposed in vivo.
We finally attempted to identify potential mechanisms involved in IL-12Rβ2 down-regulation on
MM cells as compared to normal PPC and PC. We pointed to IL-6 since it is overproduced in MM
microenvironment and strongly up-regulated in IL-12rb2 knock out mice. Indeed, incubation of
primary MM cells and the NCI-H929 cell line, as well as of normal PPC, with IL-6 downregulated
significantly surface expression of IL-12Rβ2. These results candidate IL-6 as a major regulator of
IL-12Rβ2 expression on MM cells in vivo.
19
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The present findings have translational relevance since the prognosis of MM patients remains grim
in spite of recent therapeutic improvements. IL-12 has been already tested as investigational drug in
patients with different malignancies65-67 and its safety and pharmacokinetics profiles are well
known. Thus, a clinical trial in MM patients appears to be feasible. IL-12 may be targeted directly
to tumor cells and/or administered systemically in order to take advantage also of the well known
ability of this cytokine to activate anti-tumor CTL and NK cell mediated responses.5
20
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ACKNOWLEDGMENTS
This work was supported by grants from A.I.R.C., Milano, Italy (#1429 to V.P. and #4014 to I.A.),
Ministero della Salute, Ricerca Finalizzata 2006 (to V.P.), Fondazione CARIGE Genova (to V.P.),
Fondazione Querci (to V.P.), and
Fondazione Cassa di Risparmio della Provincia di Chieti
(CariChieti), Italy to E.D.C. C.C. is the recipient of a fellowship from F.I.R.C., Milano, Italy. The
excellent secretarial assistance of Mrs. Chiara Bernardini is acknowledged.
The authors declare no competing financial interests.
AUTHOR CONTRIBUTION
I.A. designed research, performed research, collected data, analyzed and interpreted data, performed
statistical analysis, drafted the manuscript. C.C. performed research, collected data, performed
statistical analysis, analyzed and interpreted data. N.G. and S.C. performed research and provided
reagents. M.F., V.Perfetti and V.R. provided reagents. E.O., G.T. and G.C. performed research.
D.R. performed research, analyzed and interpreted data. V. Pistoia designed research, analyzed and
interpreted data, drafted the manuscript.
21
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27
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Table 1. CLINICAL AND LABORATORY FEATURES OF MM PATIENTS
PATIENTS
#1
#2
#3
#4
#5
#6
#7
#8
#9
# 10
# 11
# 12
# 13
# 14
# 15
# 16
# 17
# 18
# 19
SEX
AGE
STATE
STAGE
TYPE
F
F
M
F
M
M
F
M
M
M
M
M
M
F
F
M
M
M
M
92
85
49
82
56
62
66
70
66
74
62
75
73
78
89
76
68
55
74
Diagnosis
Relapsed
Diagnosis
Diagnosis
Diagnosis
Relapsed
Relapsed
Relapsed
Relapsed
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Relapsed
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIb
IIIb
IIIb
IIIa
Ia
Ia
Ia
IIa
Ia
IIIa
IIIa
IIa
IgGλ
IgGλ
IgGk
IgGk
IgGk
IgGk
IgGk
IgGk
Igλ
IgGk
IgGλ
IgAk
IgGλ
IgAκ
IgGκ
IgG λ
IgA λ
IgGκ
IgA λ
28
BM
Plasmacytosis
40%
38%
30%
75%
38%
60%
70%
35%
48%
70%
98%
50%
36%
52%
64%
27%
56%
52%
52%
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FIGURE LEGENDS
Figure 1.
1A. IL-12Rβ2 surface expression in PPC generated in vitro (panels a and b) or sorted from tonsils
(panel c and d), as assessed by flow cytometry using the BD mAb. Open profile: IL-12Rβ2 staining;
dark profile: isotype matched mAb staining.
1B. IL-12RBβ2 surface expression in CD138+ PC purified from normal BM (panels a and b) or
tonsil (panels c and d), as assessed by flow cytometry using the BD mAb. Open profile: IL-12Rβ2
staining; dark profile: isotype matched mAb staining.
1C. Up-regulation of IFN-γ production in PPC generated in vitro upon incubation with medium
(white column) or hrIL-12 (black column) for 48 h, as assessed by flow cytometry. Results
represent median IFN-γ+ cells ± SE from four different experiments.
Figure 2.
2A. IL-12RB1 and B2 expression in primary CD138+ MM cells, as assessed by RT-PCR. From left
to right: MW=molecular weight; NC=negative control (water in the place of cDNA); PC=positive
control (total tonsil B cells); eleven MM cases (Pt 1 to Pt 11) are shown.
2B. IL-12Rβ2 surface expression in primary CD138+MM cells (Pt #1 and 2), as assessed by flow
cytometry using the mAb from BD Biosciences (BD mAb) or the Santa Cruz antibody (SC Ab).
Open profile: IL-12Rβ2 staining; dark profile: isotype matched Ab staining.
2C. Flow cytometric analysis of IL-12Rβ2 expression in PPC generated in vitro (upper panels), IL12RB2 transfected Raji Burkitt lymphoma cells (middle panels) and a normal T helper 1 clone
using the BD Bioscences mAb (left panels) and the Santa Cruz antibody (right panels). Open
profile: IL-12Rβ2 staining; dark profile: isotype matched antibody staining.
29
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Figure 3.
3A. Down-regulation of IL-12Rβ2 expression in primary MM cells upon incubation with medium
(white column) or IL-6 (black column) for 48 h, as assessed by flow cytometry using the Santa Cruz
antibody. Results represent median IL-12Rβ2+ cells ± SE from four different experiments.
3B. Quantitative analysis of IL-12RB2 vs GAPDH transcript in two MM cell suspensions (Pt#1 and
Pt#2) cultured with (white columns) or without (black columns) IL-6 as above.
3C. IL-12Rβ2 surface expression in normal PPC before (open profile) and after (dashed line)
treatment for 48h with hrIL-6, as assessed by flow cytometry using the BD Bioscience mAb. Dark
profile indicates staining with isotype matched mAb. Two different experiments out of the four
performed with superimposable results are shown.
Figure 4.
4A. Angiogenic activity of supernatants from one representative CD138+ MM sample cultured in
the presence or absence of hrIL12. CAMs treated with sponges loaded with the conditioned medium
from the untreated cells were surrounded by allantoic vessels developing radially towards the
implant in a ‘spoked-wheel’ pattern (left panel). When medium from the same MM sample cultured
with hrIL12 was tested, a significant reduction of the angiogenic response was evident (right panel).
Original magnification: x 50.
4B. Results from human angiogenesis PCR array performed in one representative CD138+ MM
sample cultured in the presence or absence of hrIL-12 are shown.
4C. Purified CD138+ primary MM cells incubated with IL-12 for 48h up-regulated significantly
expression of the IFN-γ protein. Results are median mean fluorescence intensity (MFI) values ± SE.
Figure 5.
5A. Upper panel. IL-12RB2 expression in MM cell lines, as assessed by RT-PCR. From left to
right: MW=molecular weight; NC=negative control (water in the place of cDNA); PC=positive
30
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control (total tonsil B cells); different MM cell lines (LP-1, U266, JJN3, Karpas 620, RPMI 8226,
H-Sultan, OPM-2, XG-1, XG-6 and HCI-H929) are shown. Lower panel. Methylation Specific
PCR analysis of MM cell lines. MM cell lines that do not express the IL-12RB2 mRNA (LP-1,
U266, Karpas 620, RPMI8226, H-Sultan and OPM-2) show the amplification band corresponding
to the methylated target sequence, whereas MM cell lines that express IL-12RB2 mRNA (XG-1,
XG-6 and NCI-H929) failed to amplify the methylated sequence.
5B. IL-12Rβ2 expression in NCI-H929 and XG-1 MM cell lines, as assessed by flow cytometry
using the anti IL-12Rβ2 antibody from Santa Cruz. Open profile: IL-12Rβ2 staining; dark profile:
isotype matched antibody staining.
5C. Left panel. IL-12Rβ2 protein expression in NCI-H929 cells cultured with medium alone or
with hrIL-6 for 48h, as assessed by flow cytometry using the Santa Cruz antibody. Right panel.
Quantitative analysis of IL-12RB2 vs GAPDH transcript in the same NCI-H929 cell suspensions
analyzed in the left panel, cultured with (white columns) or without IL-6 (black columns).
5D. Angiogenic activity of supernatants from NCI-H929 (upper panels), XG-1 (middle panels) and
U266 (lower panels) cells cultured in the presence or absence of hrIL-12, as assessed by CAM
assay.
Figure 6.
6A. Volume of tumors grown intra peritoneum in PBS and IL-12 treated animals twenty-three days
after NCI-H929 cell inoculation. The differences in size between tumors removed from PBS and
IL-12 treated mice were evaluated by Mann-Whitney U test. Boxes indicate values between the 25th
and 75th percentiles, whisker lines represent highest and lowest values for each group. Horizontal
lines represent median values.
6B. Human Angiogenesis PCR Array on tumors explanted from IL-12 vs PBS treated animals 23
days after NCI-H929 cell inoculation. Left panel enlists the gene whose expression has been
31
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
abolished in tumors from IL-12 vs PBC treated mice. Histogram in right panel shows fold
expression changes of genes upregulated in tumors from IL-12 vs PBC treated mice.
6C. Histological and immunohistochemical features of tumors developed in PBS treated (a, b, c)
and hrIL-12 treated (d, e, f) SCID/NOD mice 23 days after NCI-H929 tumor cell injection.
NCI-H929 tumors are mostly formed by undifferentiated, proliferating (mitotic features indicated
by arrows) blast cells that are large and pleomorphic and sometimes binucleated or endowed with
very prominent nucleoli (a). These tumors are supplied by a distinct network of mature microvessels, as assessed by laminin staining (b), and show frequent PCNA expression (c). In hrIL-12
treated mice these morphologic features are frequently altered by the appearance of ischemichemorrhagic foci of necrosis (N)(d) associated with defective microvascularization (e) and
decreased tumor cell proliferation (f). (Magnification at x400).
32
Figure 1
A
a
b
c
PPC generated in vitro
d
PPC Sorted from tonsil
B
a
b
c
BM CD138+ PC
d
Tonsil CD138+ PC
C
% producing cells IFN-γ
70
∗
60
50
P=0.0286
40
30
20
10
0
medium
IL-12
Pt #11
Pt #10
Pt #9
Pt #8
Pt #7
Pt #6
Pt #5
Pt #4
Pt #3
Pt #2
PC
NC
MW
A
Pt #1
Figure 2
IL-12RB1
IL-12RB2
B
BD mAb
SC Ab
BD mAb
Pt #1
SC Ab
Pt #2
C
BD mAb
SC Ab
PPC
generated in vitro
BD mAb
SC Ab
IL-12RB2 RAJI
transfected cells
BD mAb
SC Ab
TH1 clone
Figure 3
A
∗
IL-12Rβ2+ MM cells
100
80
60
40
20
0
medium
IL-6
B
IL-12RB2/GAPDH
ratio
1,2
medium
IL-6
0,9
0,6
0,3
0
Pt#1
Pt#2
C
isotype
isotype
IL-6
medium
IL-12Rβ2
IL-6
medium
Figure 4
A
medium
IL-12
B
Up-regulated genes
Down-regulated genes
50
0
-6 8 0
C
VE-cadherin
CD13
CCL11
AKT
CXCL1
CCL2
ANGPT5
ANGPT1
CXCL10
CXCL9
IFN-γ
-7 8 0
∗
500
P=0.02
400
MFI
Fold change
100
300
200
100
0
medium
IL-12
NCI-H929
XG-6
XG-1
OPM-2
H-Sultan
RPMI 8226
JJN3
U266
LP-1
NC
PC
MW
A
Karpas 620
Figure 5
IL-12RB2
Methylated
IL-12RB2
B
C
Isotype
(med)
Isotype
(IL6)
IL-12Rβ2
(IL6)
IL-12Rβ2
(med)
IL-12RB2/GAPDH
ratio
NCI-H929
XG-1
1,2
medium
IL-6
0,8
0,4
0
IL-12Rβ2
D
medium
IL-12
NCI-H929
XG-1
U266
A
Tumor volume mm3
Figure 6
2000
P<0.0001
1000
0
PBS
IL-12
B
C
H&E
Laminin
PCNA
PBS
2800
1800
60
hrIL-12
IGF1
ID3
EGF
TIMP2
PF-4
0
IFN-γ
30
IFN-α
ANGPT2, TNF, ANGPT5,
ANGPTL4, CD13, EDG1,
END,
EphRB4,
FGFb,
FGFR3, VEGF-A, VEGF-C,
TSP, VEGF-D, HAND2,
HGFb, HIF1α, HPA, ID3,
IL1β, IL6, CD51, NP2,
PDGFα,
CD31,
COX1,
CLEVER1, TGFα, TGFβ1,
TGFβ2, TGFβR1
3800
Fold change
Genes whose expression is
abolished in tumors from
IL-12 treated mice
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Prepublished online May 12, 2008;
doi:10.1182/blood-2008-02-139378
Constitutive expression of IL-12RB2 on human multiple myeloma cells
delineates a novel therapeutic target
Irma Airoldi, Claudia Cocco, Nicola Giuliani, Marina Ferrarini, Simona Colla, Emanuela Ognio,
Giuseppe Taverniti, Emma Di Carlo, Giovanna Cutrona, Vittorio Perfetti, Vittorio Rizzoli, Domenico
Ribatti and Vito Pistoia
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