Proteomic detection of a large amount of SCGFα in the stroma of GISTs after imatinib therapy
© Da Riva et al; licensee BioMed Central Ltd. 2011
Received: 11 May 2011
Accepted: 23 September 2011
Published: 23 September 2011
Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors to develop in the digestive tract. These tumors are highly resistant to conventional chemotherapy and only the introduction of imatinib mesylate has improved the prognosis of patients. However, Response Evaluation Criteria in Solid Tumors are inappropriate for assessing tumor response, and the histological/pathological response to imatinib is variable, heterogeneous, and does not associate with clinical response. The effects of imatinib on responding GISTs are still being explored, and few studies correlate the clinical response with the histological response after pharmacological treatment. Recently, apoptosis and autophagy were suggested as possible alternative mechanisms of pharmacological response.
Here, we used a proteomic approach, combined with other analyses, to identify some molecular stromal components related to the response/behavior of resected, high-risk GISTs after neoadiuvant imatinib therapy.
Our proteomic results indicate an elevated concentration of Stem Cell Growth Factor (SCGF), a hematopoietic growth factor having a role in the development of erythroid and myeloid progenitors, in imatinib-responsive tumor areas. SCGFα expression was detected by mass spectrometry, immunohistochemistry and/or western blot and attributed to acellular matrix of areas scored negative for KIT (CD117). RT-PCR results indicated that GIST samples did not express SCGF transcripts. The recently reported demonstration by Gundacker et al.  of the secretion of SCGF in mature pro-inflammatory dendritic cells would indicate a potential importance of SCGF in tissue inflammatory response. Accordingly, inflammatory infiltrates were detected in imatinib-affected areas and the CD68-positivity of the SCGF-positive and KIT-negative areas suggested previous infiltration of monocytes/macrophages into these regions. Thus, chronic inflammation subsequent to imatinib treatment may determine monocyte/macrophage recruitment in imatinib-damaged areas; these areas also feature prominent tumor-cell loss that is replaced by dense hyalinization and fibrosis.
Our studies highlight a possible role of SCGFα in imatinib-induced changes of GIST structure, consistent with a therapeutic response.
Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors to develop in the digestive tract. They typically arise from the stomach (40-70%) or small intestine (20-40%) but can also occur in the colon-rectum (10-15%) and rarely in the esophagus. At least 10-30% of tumors are discovered incidentally during laparotomy, endoscopy, or other imaging studies; 15-47% of patients present with overt metastatic disease and common sites of metastases include liver, peritoneum, and omentum [2–4]. GISTs are thought to originate from the interstitial cells of Cajal or their precursor cells . Most GISTs are characterized by gain-of-function mutations in the genes encoding KIT and platelet-derived growth factor receptor alpha (PDGFRA) , mutations that appear to be mutually exclusive. The emerging role of stem cell factor (SCF) as the ligand of the receptor tyrosine kinase KIT [7–9] suggests that an autocrine-paracrine loop serves as a possible further mechanism of action . However, the SCF/KIT system plays an important role not only in the differentiation and proliferation of interstitial cells of Cajal and the development of GISTs but also in the development of hematopoietic cells such as mast cells, erythroblasts, and melanocytes .
GISTs are highly resistant to conventional chemotherapy ; in the past decade, the introduction of imatinib mesylate (Gleevec®, Novartis Pharmaceutical Corporation, NJ, USA), a KIT receptor blocker, has significantly improved the prognosis of GIST patients. Tumor response depends on the presence/absence and type of mutations in KIT or PDGFR. Unfortunately, the major problem with imatinib treatment is resistance, mainly secondary resistance that generally evolves in most patients after a median of two years of therapy [13, 14].
Eighty to eighty-five percent of patients with advanced GISTs exhibit an initial benefit from imatinib treatment; however, the response level varies from rapid and gross reduction in tumor volume to little or no tumor shrinkage (described as stable disease) . Size-based response criteria such as the World Health Organization criteria or the current international Response Evaluation Criteria in Solid Tumors are thus thought to underestimate the response and are not appropriate tools to assess tumor response to imatinib . Consequently, the clinical management of these patients and the criteria used to assess clinical response to imatinib therapy have recently been redefined . In accordance with previously reported criteria, GISTs can be classified using the following scores: high responders, 0 to <50% residual viable tumor cells with no mitosis, and no obvious Ki-67 immunostaining; moderate responders, >50% to 90% tumor cells, no mitosis, and Ki-67 immunostaining in 0 to <10% of cells; low responders, >50% to 90% tumor cells, mitotic index > 10/50 high-power fields, Ki-67 immunostaining in 20-30% or >30% of cells; and non-responders, >90% tumor cells [17, 18].
The histological/pathological response of GIST to imatinib therapy is also variable and heterogeneous from nodule to nodule within the same resection, as well as within the same lesion, and does not correlate well with clinical response. Limited studies of the histopathological changes in imatinib-treated patients indicate a significant change in the appearance of the tumor tissue following preoperative systemic imatinib therapy in GISTs; alterations particularly occur in the stromal compartment and are visible during standard pathological assessment. The density of the tumor and the number of intratumoral vessels decrease significantly and areas of cystification and hemorrhage are clearly visible. Tumors become homogeneous and hypodense. However, even in highly responsive tumors, microscopic foci of viable cells are seen either as isolated tumor cells or as distinct micronodules embedded in an extensively hyalinized background.
The molecular effects of imatinib on responding GISTs are currently being explored. Apoptosis (programmed cell death type I) has been frequently described in GIST cell lines treated with imatinib mesylate [19, 20]; importantly, McAuliffe et al.  indicate that the action of imatinib may be both cytotoxic (by evidence of apoptosis) and cytostatic. Recently, autophagy (programmed cell death type II) has been suggested as a possible alternative mechanism of response to the imatinib mesylate treatment in clinical biopsies . Autophagy is the major self-degradative process in eukaryotic cells and has multiple physiological functions, including protein degradation, organelle turnover, and response of cancer cells to chemotherapy .
In this report, we used a proteomic approach in combination with other analyses to investigate the protein composition of highly responsive, resected GISTs after imatinib mesylated neoadjuvant therapy. Our aim was to identify several molecular components of the stroma with expression patterns possibly related to tumor response/behavior.
Patients and materials
Clinical, pathological and molecular characterists of GISTs
KIT exon 11 Del 558-563
KIT exon 11 L576P
>90% of viable cells-non responder
KIT exon 11 Del 554-558
10% of viable cells-high responder
KIT exon 11 Del 557-558
10% of viable cells-high responder
KIT exon 11 V559D
10% of viable cells-high responder
PDGRF alpha D842V
25% of viable cells-high responder
KIT exon 9 Dup 502-503
>90% of viable cells-non responder
KIT exon 11 Del 558-560 and V557C
>5% of viable cells-high responder
PDGFR alpha D842V
>90% of viable cells-non responder
>90% of viable cells-non responder
KIT Dup P577-K581
>90% of viable cells-non responder
KIT Dup A502-Y503 and N822K
>90% of viable cells-non responder
KIT K642E and N822K
>90% of viable cells-non responder
KIT Del M552-Y553 and E554K
50-90% of viable cells-moderate responder
50-90% of viable cells-moderate responder
KIT Dup 502-503
50-90% of viable cells-moderate responder
Tumor specimens were pulverized in a Mikro-Dismembrator II (B. Braun Biotech International, Melsungen, Germany). The pulverized tissue samples and the cell pellets from cell culture were recovered in ice-cold buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM Na4P2O7, 100 mM NaF, 1 mM PMSF, 1 mM sodium orthovanadate, and Complete Mini protease inhibitors cocktail (Roche, Milan, Italy) according to the manufacturer's instructions. After 30 min incubation with gentle rocking at 4°C, lysates were cleared by centrifugation for 20 min at 13,000 rpm. Supernatants were collected and protein quantification was performed with the BCA™ Protein Assay Kit (Thermo Scientific, Milan, Italy) according to the manufacturer's instructions.
Thirty micrograms of total extract from GIST samples and of plasma were loaded on one-dimensional 4-12% NuPAGE® precast gels (Invitrogen, Milan, Italy). Proteins were visualized with G250 Coomassie Blue (Bio-Rad, Milan, Italy) by standard procedures.
In-gel tryptic digestion, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), and peptide mass fingerprinting
For protein profiling, protein bands were excised from Coomassie-stained preparative gels and processed as previously described . MALDI-TOF-MS was carried out using a Voyager-DE STR (Applied Biosystems, Milan, Italy), equipped with a nitrogen laser (337 nm).
Monoisotopic peptide masses were analyzed using the Aldente software http://www.expasy.org/tools/. Input was searched according to: Aldente, UniProtKB/SwissProt; predefined taxon, Mammalia; Spectrometer internal error max, 25. Only proteins identified in at least three separate experiments were considered.
OFFGEL protein fractionation
A preparative-scale OFFGEL was used for isoelectric focusing of proteins. To perform protein fractionation according to isoelectric point, the 3100 OFFGEL Fractionator and the OFFGEL Kit 3-10 (Agilent Technologies, Milan, Italy) were used following the manufacturer's instructions . The device was set up for the 24-fraction separation using the 24 cm-long IPG gel strip with a linear pH gradient from 3 to 10. The proteins were separated in a two-phase system consisting of a liquid upper phase (focusing buffer provided by the supplier) separated in wells and a lower IPG gel strip phase. The sample was focused using the recommended method for 24-well OFFGEL fractionation with a maximum current of 50 μA. The separation method consisted of a cooling platform temperature of 15°C with electrical setting parameters of 8,000 V/h, 100,000 V, 200 W, and 50 μA/strip. The focusing was stopped after the total voltage reached 64 kVh. After focusing, samples were recovered from each well and transferred to individual microtubes. Corresponding protein fractions were purified with the 2-D Clean Up (GE Healthcare, Milan, Italy) and the protein pellets were dissolved in running sample buffer compatible with one-dimensional SDS-PAGE. For protein profiling, protein bands were excised from Coomassie-stained preparative gels and processed as previously described .
The human papillary thyroid carcinoma cell line, TPC1, was grown adherently in Dulbecco's Modified Eagle's Medium (Gibco, Milan, Italy) supplemented with 10% fetal bovine serum HyClone (Celbio, Milan, Italy) and 1 mM sodium pyruvate (Lonza, Milan, Italy). Human Embryonic Kidney (HEK) 293T (ATCC number CRL-1573) cells were grown adherently in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and L-glutamine (Lonza).
Conditioned medium concentration
An equal volume (~5 ml) of conditioned medium for each sample (mock and transfected cells) was loaded into a spin concentrator (Agilent Technologies) with a 5 kDa molecular weight cut-off and centrifuged at 4,000 rpm and 10°C until samples were concentrated to a final volume of 150-200 μl. Protein concentration was determined by BCA assay.
An equal amount of protein for each sample (30 μg) was loaded on a one-dimensional 4-12% NuPAGE® precast gel (Invitrogen). Proteins were transferred in NuPAGE® transfer buffer (Invitrogen) and 20% ethanol onto a nitrocellulose membrane (Hybond™-C Super, Amersham Biosciences, Milan, Italy) and checked for equal sample loading by Red Ponceau S (Sigma-Aldrich, Milan, Italy) staining. Blots were blocked for 1 h with TBS (10 mM Tris-HCl [pH 7.5], 150 mM NaCl) plus 0.1% Tween 20 (TBS-T buffer) containing 1% bovine serum albumin (Sigma-Aldrich) and 3% ovalbumin (Sigma-Aldrich), then hybridized in the same buffer with specific antibodies at 4°C overnight using the recommended dilutions. After incubation, the blots were washed in TBS-T buffer and incubated for 1 h at room temperature in previously described buffer using appropriate secondary antibodies (1:4,000). After incubation, the blots were washed in TBS-T buffer, and immunoreactive proteins were visualized using an enhanced ECL system (ECL® Western blotting detection reagents, GE Healthcare Life Sciences, Milan, Italy) according to the manufacturer's protocol. Monoclonal mouse anti-human SCGF/CLEC11a antibody was supplied by R&D Systems (Milan, Italy). The ECL® anti-mouse IgG, horseradish peroxidase-linked whole antibody from sheep was obtained from GE Healthcare Life Sciences.
Immunohistochemistry was performed on 2-μm formalin-fixed and paraffin-embedded sections of representative tumoral areas deparaffined in xylene and rehydrated in graded alcohols. Endogenous peroxidase activity was blocked by treatment for 10 min with 0.3% hydrogen peroxide in distilled water. Antigen retrieval was obtained by autoclaving for 15 min.
The slides were cooled under tap water, washed three times in 0.05 M phosphate-buffered saline plus 0.1% Triton, and incubated with ultra v-block (Lab Vision Corp, Newmarker, UK) for 10 min at room temperature. Then, they were incubated at room temperature for 1 h with the monoclonal mouse anti-human SCGF/CLEC11a antibody (R&D Systems) diluted 1:400 in citrate buffer (pH 6). The slides were washed again three times in 0.05 M phosphate-buffered saline plus 0.1% Triton and developed using the Ultra Vision LP Volume Detection System (Lab Vision Corp). After washes in 0.05 M phosphate-buffered saline, peroxidase activity was detected with diaminobenzidine for 10 min in the dark. The slides were counterstained with hematoxylin. Immunohistochemical analysis for KIT was performed using an antibody against CD117, as previously described . Positivity was defined as the detection of immunopositivity in >90% of cells.
Total RNA of the TPC1 cell line was prepared using a TRIZOL reagent (Life Technologies, Italy), the oligo(dT)-primed cDNA was synthesized using a RT-PCR kit (Stratagene, Milan, Italy). Oligonucleotides 5'-CCAAGCTTTCCAGCTTAATGCAG-3'(forward) and 5'-TAAAGCGGCCGCCCCGCTAGAA-3'(reverse) were used in PCR to amplify the full human SCGF- α (hSCGF-α, UniProt accession number Q9Y240) sequence. Taq Phusion® High Fidelity DNA polymerase (Finnzymes, Espoo, Finland) was used with the following thermal cycling conditions: initial denaturation at 98°C for 30 sec; 35 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 30 sec, extension at 72°C for 40 sec; and final extension at 72°C for 7 min. The hSCGF-α cDNA was subcloned into the pcDNA3.1 plasmid vector (Invitrogen). The plasmid expressing hSCGF-β was obtained by mutagenesis of the plasmid expressing hSCGF-α using the Quick-Change® II XL Site-Directed Mutagenesis Kit (Stratagene, Milan, Italy), according to the manufacturer's protocol. The DNA sequences contained in both vectors were checked by automatic sequencing.
HEK 293T cells were transiently transfected by calcium phosphate precipitation, as previously described , with the pcDNA3 expression vector (Invitrogen) alone (mock) or with the plasmid carrying the insert for SCGF-β.
Removal of O-linked oligosaccharides from SCGF
Suitable amounts (based on western blotting with anti-SCGF) of cell and tissue protein extracts and secreted proteins were incubated at 37°C for 4 h with O-glycosidase (Sigma-Aldrich) and α(2→3,6,8,9)neuraminidase (sialidase, Sigma-Aldrich) according to the manufacturer's protocol. The digested samples were analyzed by western blotting with anti-SCGF.
List of proteins identified after OFFGEL and SDS-PAGE fractionation, tryptic digest, and MALDI-TOF MS analysis
Actin, cytoplasmic 1
Actin, cytoplasmic 2
Complement factor B
Complement factor H
Alpha-2-HS-glycoprotein chain A
Fibrinogen gamma chain
Ferritin light chain
Hemoglobin subunit beta
Heat shock protein HSP 90-beta
Heat shock protein HSP 90-alpha
Ig alpha-1 chain C region
Ig gamma-1 chain C region
Ig gamma-2 chain C region
Inter-alpha-trypsin inhibitor heavy chain
Keratin, type I cytoskeletal 10
Keratin, type I cytoskeletal 9
Keratin, type II cytoskeletal 1
Vitamin D-binding protein
In the GIST 5 extract, Coomassie staining revealed bands (bands 8-10 in Figure 1A, lane E, and Figure 1B) that were not present in the plasma sample. Bands 8 and 9 corresponded to SCGF, also known as C-type lectin domain family member 11A (CLEC11A), an important hematopoietic growth factor with burst-promoting activity for human bone marrow erythroid progenitors . Band 10 corresponded to C1q, the initiator of the classical complement cascade .
SCGF is a secreted cytokine expressed in two distinct forms; SCGF-α is the full size form (323 amino acids, 35,695 Da), while SCGF-β is the shorter form (245 amino acids, 26,902 Da) characterized by a deletion within a conserved carbohydrate recognition domain . These theoretical masses only partially explain the observed molecular weights in electrophoretic separations. The spectra from our MALDI-TOF-MS of tryptic peptides from two SCGF-positive bands (band 8 and 9 in Figure 1, lane E) were nearly identical in size and were attributed to the α form (lanes G and H in Figure 1A).
To further define the nature of SCGF isoforms expressed in the GIST 5 sample, we tested the possibility that the other form corresponded to isoform β. We transfected the HEK293 cell line that scored negative for SCGF proteins (Figure 6, lanes 6 and 7) with SCGF-β cDNA. Western blotting detected a protein with the size expected for the SCGF-β backbone (Figure 6, lanes 8-10) that did not correspond to band d of the GIST 5 sample (Figure 6, lane 2). Thus, we concluded that band d was not the SCGF-β form.
Few studies have correlated clinical response with histological response in GISTs after prolonged imatinib treatment. However, it is widely recognized that clinical outcome in stable or partially responsive GIST patients does not seem to be influenced by the duration of imatinib treatment, the histological response, or the size of the tumor . In this study, we analyzed the proteomic, histological, immunohistochemical, and clinical features of a small group of GISTs resected after prolonged imatinib treatment.
SCGF, a novel cytokine, exerts its action on primitive hematopoietic progenitor cells. In combination with other hematopoietic growth factors such as granulocyte-macrophage colony-stimulating factor and erythropoietin, SCGF stimulates the formation of erythroid and granulocyte/macrophage colonies, although SCGF alone cannot induce colony formation . Using a proteomic approach, we detected a large amount of SCGF in the protein extract of one imatinib-treated GIST sample (Figure 1). This sample also had a residual cell component of <10% and was negative or very slightly positive for CD117 staining. Biochemical analyses revealed the presence of a small number of KIT receptors with very low activation (data not shown). In parallel, we found extensive SCGF positivity in the abundant stromal component, which appeared homogenous, hypodense, and eosinophilic. This observation was replicated in part of the progressive lesion of another treated GIST case in which we identified strong SCGF positivity exclusively in CD117-negative areas. Interestingly, SCGF expression occurred in the imatinib-affected areas of three GISTs that was not detected in the unaffected or scarcely affected areas of the same tumors; SCGF-positive bands were also identified in two out three responding tumors but were absent in tumors from five non-responder patients.
A study carried out on in vivo material demonstrated that SCGF is strongly expressed in bone marrow and only faintly in lymphoid organs; in bone marrow, SCGF is concentrated in the cytoplasm of immature neutrophils, but not in myeloblasts, mature neutrophils, or the extracellular bone marrow fluid . Since immature neutrophils play a role in tumor-induced immuno-inflammatory responses, SCGF may impact mechanisms regulating these responses. These observations are consistent with RNA expression patterns in mouse and human protein-encoding transcriptomes  and are attributed as follows: high expression levels to CD34+ cells, low expression levels to CD33+ (myeloid) cells, cardiomyocytes, and smooth muscle cells, and very low expression levels to all other cell lines or tissues.
Hiraoka  recently demonstrated that leukemia cell lines require self-secreted SCGF for their proliferation in tumors, indicating a putative autocrine SCGF mechanism, and that loop blockage with neutralizing antibody prevents extracellular SCGF from inducing apoptosis. Levina et al.  demonstrated that the high tumorigenic and metastatic potentials of lung cancer stem cells correlated with superior production of angiogenic factors and growth factors involved in cell proliferation and angiogenesis, describing increased levels of SCGF, stroma-derived factor 1α, and SCF in tumors from cancer stem cells in association with the stem cell phenotype. Gene-expression profiles from 35 childhood acute lymphoblastic leukemia matched diagnosis/relapse pairs, as well as 60 uniformly treated children at relapse, indicated that SCGF is significantly overexpressed at relapse .
The presence of SCGF in the CD117-negative stromal compartment of imatinib-treated GISTs suggests that its expression is associated with the histological response of GISTs to imatinib therapy. Our RT-PCR investigation revealed that SCGF is not actively transcribed in GIST samples, and thus it is difficult to determine the possible sources of SCGF in these specimens. A recent study described a subgroup of GISTs surgically resected after neoadjuvant imatinib treatment that exhibited reduced numbers of tumor cells in the hypocellular myxohyaline stroma, with small numbers of scattered atypical nuclei and occasional stromal hemorrhages . A separate investigation assessed a GIST case treated with imatinib therapy for four weeks in which most of the tumor cells were replaced by myxoid stroma and the remaining tumor cells did not appear to be actively dividing . However, to date no study has reported data on SCGF in GIST samples.
In our study, SCGF appeared as part of the stromal GIST component and, in particular, as part of the eosinophilic proteinaceous matrix described as myxoid, collagenous, or hyaline. Our proteomics experiment uncovered high plasma-protein content in treated tumors, and immunohistochemistry revealed the SCGF positivity in CD117-negative and CD68-positive areas. These observations could link SCGF positivity with the imatinib-induced inflammatory response that elicits monocyte/macrophage tissue migration, promoting scarring and removal of cell debris. CD68 positivity confirms macrophage infiltration, which may also explain the high level of C1q  in our GIST 5 proteomic analysis. Recent data support the hypothesis that induced type I maturation of dendritic cells is associated with a peak of SCGF production , supporting a pro-inflammatory role for this cytokine. It is therefore plausible that these are immunological reactions, and that liquidation of the dead tumor cells via macrophages leads to lesion shrinkage.
SCGF function may be related to the imatinib-induced inflammation response in responding GIST patients. Further studies are necessary to identify the receptor of this cytokine, to further clarify its origin, and to determine the reason for its accumulation in some imatinib-treated GISTs. These investigations may answer fundamental questions about the composition of the stromal matrix after imatinib therapy and identify proteins related to desirable tumor response/behavior.
This study was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan, Italy) and Alleanza Contro il Cancro (ACC). We thank Ms. Mazzadi Cristina for secretarial assistance.
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