J Cancer Res Clin Oncol (2014) 140:937–947 DOI 10.1007/s00432-014-1648-9
Original Article – Cancer Research
Involvement of CD74 in head and neck squamous cell carcinomas Nadège Kindt · Jérôme R. Lechien · Denis Nonclercq · Guy Laurent · Sven Saussez
Received: 9 December 2013 / Accepted: 11 March 2014 / Published online: 25 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose While macrophage migration inhibitory factor (MIF) has been extensively studied in the context of inflammation and inflammatory disorders, less work has been devoted to its involvement in cancer, notably in neoplastic progression. In a previous study, we have found evidence that MIF plays a role in head and neck squamous cell carcinomas (HNSCC). The current investigations were undertaken in order to estimate the importance of the MIF receptor, CD74 in the progression of HNSCC. Methods and results In a cohort of 46 cases of oral cavity carcinomas, immunohistochemical staining revealed an increase in CD74 expression during progression from benign lesions to carcinoma. As shown by cell culture experiments using squamous carcinoma cell line (SCCVII) transduced with anti-CD74 shRNA, the amount of cellproduced VEGF was lower in SCCVII CD74KD cell line Guy Laurent and Sven Saussez have contributed equally to this work and should be regarded as joint last authors. N. Kindt · J. R. Lechien · S. Saussez (*) Laboratory of Anatomy and Cellular Biology, Faculty of Medicine and Pharmacy, Research Institute for Health Sciences and Technology, University of Mons (UMons), Pentagone 2A, 6 Ave du Champ de Mars, 7000 Mons, Belgium e-mail: [email protected] D. Nonclercq · G. Laurent Laboratory of Histology, Faculty of Medicine and Pharmacy, Research Institute for Health Sciences and Technology, University of Mons, 6 Ave du Champ de Mars, 7000 Mons, Belgium S. Saussez Department of Oto‑Rhino‑Laryngology, Faculty of Medicine, Free University of Brussels (ULB), CHU St‑Pierre, Rue Haute 322, 1000 Brussels, Belgium
compared with control SCCVII CD74sc cell line, suggesting that CD74 could be implicated in angiogenesis in vivo. Furthermore, knockdown of CD74 decreased proliferation of SCCVII cells in vitro. The migration of SCCVII cells, as well as the cell secretion of matrix metallopeptidase 9, was also negatively affected by CD74 knockdown. These observations in vitro were confirmed in an orthotopic mouse model of SCC where tumors produced by SCCVII CD74KD cell inoculation were found to grow more slowly than tumors generated by SCCVII CD74sc cells. Conclusion The clinical observations and experimental data reported here suggest that CD74, as well as MIF, plays a pivotal role in HNSCC progression. Keywords CD74 · MIF · Oral carcinoma · HNSCC · SCCVII
Introduction Head and neck squamous cell carcinomas (HNSCC), which account for 90 % of head and neck cancers, have been reported as the sixth leading cause of cancer-related deaths worldwide. Primary risk factors associated with HNSCC include tobacco and alcohol consumption in the western world, as well as betel quid chewing in Southeast Asia and the Indian subcontinent. Beside, one has identified populations of HNSCC patients without these typical risk factors. In these cases, the major etiological factor seems to be human papilloma virus infection (in particular HPV-16 and 18). Of note, HPV infection might be responsible for the increase in the global incidence of HNSCC observed during the last decade. Despite the fact than HNSCCs are particularly aggressive, they are often diagnosed at advanced stages,
13
938
accounting for high mortality rates in patients. Even in cases of successful curative treatments, patients generally suffer from severe and debilitating adverse effects resulting from surgery and/or chemoradiotherapy. Thus, there is an obvious need for a better knowledge of the disease, as well as for new biomarkers and alternative targeted therapeutic strategies. A number of growth factors and cytokines have been shown to contribute to the pathogenesis of HNSCC (Pries and Wollenberg 2006; Molinolo et al. 2009; Wang et al. 2009). In particular, recent work of our group has enlightened the importance of macrophage migration inhibitory factor (MIF) in the progression of this disease. Thus, MIF expression in tumor tissue, as assessed by immunohistochemistry, increases during neoplastic progression in hypopharyngeal, laryngeal and oral cavity carcinomas (Cludts et al. 2010; Kindt et al. 2012, 2013a). Moreover, an increase in MIF serum levels was also noted in patients with HNSCC as compared to non-cancer individuals (Kindt et al. 2012). Finally, we found that increased MIF expression in laryngeal carcinoma was associated with a worse prognosis in terms of local recurrence and cancer metastasis (Kindt et al. 2012). These clinical observations were complemented by experimental studies showing that MIF is somehow involved in cell proliferation and invasiveness in a squamous carcinoma cell line (SCCVII) (Kindt et al. 2012). MIF is thought to modulate target cell activity by interacting with the cell surface receptor CD74, triggering thereby intracellular cascades such as ERK 1/2 MAPK and PI3K/Akt cascades (Leng et al. 2003; Lue et al. 2006, 2007). It is noteworthy that both MIF and CD74 can be expressed on the same cells, providing the opportunity for autocrine stimulation (Kindt et al. 2013b). On the other hand, some effects of MIF might result from intracrine activity. While the importance of MIF in HNSCC pathogenesis seems well established, there is a lack of information concerning the expression of CD74 in these neoplasms. The current study was undertaken to examine clinical tumor specimens for CD74 expression and disclose possible relationship with prognosis. Furthermore, a SCCVII was used to evaluate the contribution of CD74 to cell proliferation and invasiveness, and tumor cell-mediated angiogenesis.
J Cancer Res Clin Oncol (2014) 140:937–947 Table 1 Patient population characteristics Mean age: 58 years (range between 36 and 81 years) Number of cases Gender Male Female
37 9
Under localization Tongue Mouth floor Others
5 17 24
Histological grade Well differentiated Moderately differentiated Poorly differentiated Unknown
14 23 6 3
TNM stage T1–T2 T3–T4 N0 N+ M0
27 19 25 21 46
Recurrence Yes No
21 25
Clinical follow-up (month) Range
1–120
Average
23
p value
N.S.
N.S. N.S.
N.S.
(December 1996 to February 2008) from the records of CHU Erasme (Brussels, Belgium), RHMS Baudour (Baudour, Belgium) and CHU Saint-Pierre (Brussels, Belgium). The description of the tumor status was based on the histopathological grade of tumor differentiation and the TNM staging classification (Hyams et al. 1988; Wittekind et al. 2004). Detailed information on the patients’ age, gender, tumor histopathology and follow-up data up to the last contact with the patient together with the status of the disease at the last examination was available for all patients (see Table 1). Twenty cases of tonsillectomy were used for the normal epithelium group. This study was approved by the local Institutional Review Board. Immunohistochemistry
Materials and methods Patients’ characteristics A total of 46 patients with oral cavity carcinoma who underwent surgery aimed at curative tumor resection were studied. The patient files were compiled retrospectively
13
CD74 immunohistochemistry was performed using a procedure similar to that described previously for MIF (Kindt et al. 2012). After antigen retrieval by microwave treatment, sections were pretreated with hydrogen peroxide to block endogenous peroxidase activity. Thereafter, they were successively exposed to avidin and biotin in order to avoid
J Cancer Res Clin Oncol (2014) 140:937–947
false-positive staining due to the presence of endogenous biotin. These steps were followed by sequential incubations with (1) primary antibody (rabbit polyclonal anti-human CD74 FL-296 from Santa Cruz Biotechnology, Santa Cruz, CA), (2) biotinylated secondary antibody (polyclonal goat anti-rabbit IgG), (3) avidin–biotin–peroxidase (ABC) complex. Immunocomplexes were finally visualized by exposure to the chromogen diaminobenzidine in the presence of H2O2. Sections were counterstained with luxol fast blue prior to light microscopy examination. Computer‑assisted morphometry Morphological examination of immunostained tissue sections was carried out on a Zeiss Axioplan microscope equipped with a color CCD camera (ProgRes C10plus, Jenoptik, Jena, Germany). Morphometric analysis was achieved using KS 400 imaging software (Carl Zeiss Vision, Hallbergmoos, Germany), as described previously (Kindt et al. 2012). Data from the quantitative analysis of immunostaining were expressed as the labeling index (LI), corresponding to the percentage of positive cells. Cell line and culture The squamous cell carcinoma cell line SCCVII was a kind gift from Dr. Shulin Li (Louisiana State University) (Khurana et al. 2001). Routine cell propagation and experimental studies were carried out at 37 °C in a cell incubator with humid atmosphere at 5 % CO2. Unless specified otherwise, cells were cultured in T-flasks containing Dulbecco’s modified essential medium (DMEM) supplemented with phenol red, 10 % FBS, 25 mM N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. Knockdown of CD74 expression (CD74KD) was achieved using anti-CD74 shRNA (mouse) lentiviral particles from Santa Cruz (Santa Cruz, CA), according to manufacturer’s instructions. Controls (CD74sc) were obtained by transduction with shRNA lentiviral particles encoding a scrambled (sc) shRNA sequence. Transduced cells were selected by addition of 4 μg/ml puromycin to the culture medium. Preliminary experiments showed this puromycin concentration to be toxic toward non-transduced SCCVII cells. As shown previously, CD74 knockdown resulted in a decrease of approx. 50 % in the expression of the protein (Kindt et al. 2013b). Measurement of cell culture growth by cell counting and by crystal violet staining assay CD74KD and CD74sc cells were plated at a density of 104 cells/cm2 in 12-well dishes (n = 6). Measurement of
939
cell culture density was performed daily, beginning 24 h after seeding. Cells were dislodged from the vessel bottom by treatment with trypsin–EDTA solution. After vigorous pipetting, concentrations of cells in suspension were determined in an electronic cell counter (model Z1 Coulter counter, Beckman Coulter, Fullerton CA). Alternatively, cell culture density was assessed by colorimetry after crystal violet staining, as described previously (Journe et al. 2004). Cells were seeded in 96-well plates (500– 4000 cells/well, n = 6). After 4 days of exposure, the culture medium was discarded; cultures were rinsed with DPBS, and cells were fixed with 1 % glutaraldehyde. Following fixation, cells were stained with 1 % crystal violet. Destaining was achieved under gently running tap water, and cell monolayers were lysed in 0.2 % Triton X-100. The absorbance of cell lysates was measured at 570 nm using a Labsystems Multiskan MS microplate reader (Labsystems, Helsinki, Finland). Colony formation assay Cells were plated at a density of 20 cells/cm2 in 50-mmdiameter Petri dishes (n = 4). Eight days after seeding, cell cultures were rinsed with DPBS, and cells were fixed with 1 % glutaraldehyde in DPBS. After fixation, cell colonies were stained with 0.1 % crystal violet. Colonies on Petri dishes were digitized using a HP Scanjet 3800 (HewlettPackard, Palo Alto, CA). Numbers of colonies in digital images of dishes were determined using the public domain ImageJ software (Wayne Rasband, NIH). Cell cycle analysis Cells were plated at a density of 104 cells/cm2 in T75 flasks. Cell cultures were processed for cell cycle analysis 1, 2 and 3 days after seeding. After trypsinization, cells were transferred into centrifuge tubes and sedimented by centrifugation (300×g, 5 min). Cells were rinsed once with DPBS (same centrifugation conditions), and cell pellets were suspended and fixed in ice-cold 70 % ethanol. Before flow cytometry analysis, ethanol-fixed cells were centrifuged, rinsed in DPBS and resuspended in a mixture of propidium iodide and RNAse A (Muse™ DNA staining reagent). Cell cycle analysis by flow cytometry was carried out in a Muse® Cell Analyzer (Millipore, Billerica, MA). Cell migration assay Cell invasiveness was assessed by a Boyden chamber assay consisting a 24-well plates (lower chambers) with cell culture inserts (upper chambers), both chambers being separated by a polycarbonate membrane (8 μm size) coated with an artificial extracellular matrix (Chemicon Cell Invasion Assay kit, Millipore, Billerica, MA). The kit was used
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940
according to manufacturer’s instructions. CD74 KD and control SCCVII cells were seeded in cell culture inserts (1.5 × 105 cells/insert) in serum-free medium (DMEM, 0.1 % BSA). The lower chambers were filled with complete medium (DMEM, 10 % FBS). After 96 h, cells were wiped off the upper surface of cell culture inserts with a cotton-tipped swab, and the migrating cells were stained with crystal violet and counted in a total of 71 microscopic fields (10× magnification) using a Zeiss Axio scope A1 microscope (Carl Zeiss, Germany). Determination of matrix metallopeptidase 9 by enzyme‑linked immunosorbent assay The level of matrix metallopeptidase 9 (MMP-9) was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA) using commercial kit (Mouse MMP-9 ELISA KIT, Boster Biological Technology Co., Ltd). Plates were pre-coated with an antibody raised against MMP-9. Known amounts of MMP-9 protein (reference curve) and 50-fold diluted samples were added to the plate. After an incubation of 90 min at 37 °C, 100 μl of detection antibody was added to each well. A 1-h exposure to the detection antibody at 37 °C was followed by the addition of 100 μl of horseradish peroxidase-conjugated avidin. Immunocomplexes were revealed by the addition of 100 μl substrate solution (3,3′,5,5′-tetramethylbenzidine). After stopping the reaction with sulfuric acid solution, the absorbance was measured at 450 nm (reference wavelength: 570 nm). VEGF determination by enzyme‑linked immunosorbent assay The level of VEGF protein was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA) using commercial kit (Mouse Vascular Endothelial cell Growth Factor (VEGF) ELISA KIT, Cusabio Biotech Co., LTD). Plates were pre-coated with an antibody specific to VEGF. Known amounts of VEGF protein (reference curve) and 20-fold diluted samples were added to the plate. After an incubation of 2 h at 37 °C, 100 μl of detection antibody was added to each well. A 1-h exposure to the detection antibody at 37 °C was followed by the addition of 100 μl of horseradish peroxidase-conjugated avidin. Immunocomplexes were revealed as described above. Animal studies Animal studies in vivo were conducted on female C3H/ HeN mice (Charles River Laboratory, L’Arbresle, France). The animals were maintained and handled in compliance with the guidelines issued by the Belgian Ministry of Trade and Agriculture (Agreement No. LA1500549).
13
J Cancer Res Clin Oncol (2014) 140:937–947
For the inoculation of tumor SCC cells, CD74KD and CD74sc cells were grown to confluency and detached by trypsinization. Cells were centrifuged at 200×g for 10 min and suspended in Hank’s balanced salt solution at a concentration of 5 × 106 cells/ml. Prior to tumor cell inoculation, mice were anesthetized by i.p. injection of a ketamine (100 mg/kg)/xylazine (10 mg/kg) mixture (n = 39). Each animal received an injection of 100 μl cell suspension (i.e., 105 cells/mouse) in the mylohyoid muscle. Animals were monitored for tumor onset, and tumors were measured with a digital caliper every 2 days. They were terminated when they exhibited a tumor more than 15 mm diameter or a weight loss of more than 20 %. Statistical analyses SigmaPlot® 11 software was used for statistical analyses. Parametric analyses were achieved by the Student’s t test. Nonparametric analyses were performed by the Mann– Whitney U test (2 groups) or Kruskall–Wallis test (more than two groups). When the latter test was significant, the Dunn’s procedure (post hoc test) was used to compare each pair of groups (to avoid multiple comparison effects).
Results Clinical observations Tissue CD74 expression increases with tumor progression in oral cavity carcinomas The intensity of CD74 immunostaining was evaluated in normal oral epithelium of patients who had undergone tonsillectomy (normal epithelium in the margin of tonsils, N_E, 20 cases, Fig. 1a), in specimens exhibiting preneoplastic lesions (low-grade dysplasia (LGD), 7 cases, Fig. 1b; high-grade dysplasia (HGD), 26 cases, Fig. 1c) and in specimens of oral cavity carcinoma (CA, 46 cases, Fig. 1d). Computer-aided morphometric analysis of immunohistochemical staining in histological sections revealed a significant increase in CD74 expression during tumoral progression, as depicted by LI (Fig. 1e, Kruskal–Wallis test, p
Original Article – Cancer Research
Involvement of CD74 in head and neck squamous cell carcinomas Nadège Kindt · Jérôme R. Lechien · Denis Nonclercq · Guy Laurent · Sven Saussez
Received: 9 December 2013 / Accepted: 11 March 2014 / Published online: 25 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose While macrophage migration inhibitory factor (MIF) has been extensively studied in the context of inflammation and inflammatory disorders, less work has been devoted to its involvement in cancer, notably in neoplastic progression. In a previous study, we have found evidence that MIF plays a role in head and neck squamous cell carcinomas (HNSCC). The current investigations were undertaken in order to estimate the importance of the MIF receptor, CD74 in the progression of HNSCC. Methods and results In a cohort of 46 cases of oral cavity carcinomas, immunohistochemical staining revealed an increase in CD74 expression during progression from benign lesions to carcinoma. As shown by cell culture experiments using squamous carcinoma cell line (SCCVII) transduced with anti-CD74 shRNA, the amount of cellproduced VEGF was lower in SCCVII CD74KD cell line Guy Laurent and Sven Saussez have contributed equally to this work and should be regarded as joint last authors. N. Kindt · J. R. Lechien · S. Saussez (*) Laboratory of Anatomy and Cellular Biology, Faculty of Medicine and Pharmacy, Research Institute for Health Sciences and Technology, University of Mons (UMons), Pentagone 2A, 6 Ave du Champ de Mars, 7000 Mons, Belgium e-mail: [email protected] D. Nonclercq · G. Laurent Laboratory of Histology, Faculty of Medicine and Pharmacy, Research Institute for Health Sciences and Technology, University of Mons, 6 Ave du Champ de Mars, 7000 Mons, Belgium S. Saussez Department of Oto‑Rhino‑Laryngology, Faculty of Medicine, Free University of Brussels (ULB), CHU St‑Pierre, Rue Haute 322, 1000 Brussels, Belgium
compared with control SCCVII CD74sc cell line, suggesting that CD74 could be implicated in angiogenesis in vivo. Furthermore, knockdown of CD74 decreased proliferation of SCCVII cells in vitro. The migration of SCCVII cells, as well as the cell secretion of matrix metallopeptidase 9, was also negatively affected by CD74 knockdown. These observations in vitro were confirmed in an orthotopic mouse model of SCC where tumors produced by SCCVII CD74KD cell inoculation were found to grow more slowly than tumors generated by SCCVII CD74sc cells. Conclusion The clinical observations and experimental data reported here suggest that CD74, as well as MIF, plays a pivotal role in HNSCC progression. Keywords CD74 · MIF · Oral carcinoma · HNSCC · SCCVII
Introduction Head and neck squamous cell carcinomas (HNSCC), which account for 90 % of head and neck cancers, have been reported as the sixth leading cause of cancer-related deaths worldwide. Primary risk factors associated with HNSCC include tobacco and alcohol consumption in the western world, as well as betel quid chewing in Southeast Asia and the Indian subcontinent. Beside, one has identified populations of HNSCC patients without these typical risk factors. In these cases, the major etiological factor seems to be human papilloma virus infection (in particular HPV-16 and 18). Of note, HPV infection might be responsible for the increase in the global incidence of HNSCC observed during the last decade. Despite the fact than HNSCCs are particularly aggressive, they are often diagnosed at advanced stages,
13
938
accounting for high mortality rates in patients. Even in cases of successful curative treatments, patients generally suffer from severe and debilitating adverse effects resulting from surgery and/or chemoradiotherapy. Thus, there is an obvious need for a better knowledge of the disease, as well as for new biomarkers and alternative targeted therapeutic strategies. A number of growth factors and cytokines have been shown to contribute to the pathogenesis of HNSCC (Pries and Wollenberg 2006; Molinolo et al. 2009; Wang et al. 2009). In particular, recent work of our group has enlightened the importance of macrophage migration inhibitory factor (MIF) in the progression of this disease. Thus, MIF expression in tumor tissue, as assessed by immunohistochemistry, increases during neoplastic progression in hypopharyngeal, laryngeal and oral cavity carcinomas (Cludts et al. 2010; Kindt et al. 2012, 2013a). Moreover, an increase in MIF serum levels was also noted in patients with HNSCC as compared to non-cancer individuals (Kindt et al. 2012). Finally, we found that increased MIF expression in laryngeal carcinoma was associated with a worse prognosis in terms of local recurrence and cancer metastasis (Kindt et al. 2012). These clinical observations were complemented by experimental studies showing that MIF is somehow involved in cell proliferation and invasiveness in a squamous carcinoma cell line (SCCVII) (Kindt et al. 2012). MIF is thought to modulate target cell activity by interacting with the cell surface receptor CD74, triggering thereby intracellular cascades such as ERK 1/2 MAPK and PI3K/Akt cascades (Leng et al. 2003; Lue et al. 2006, 2007). It is noteworthy that both MIF and CD74 can be expressed on the same cells, providing the opportunity for autocrine stimulation (Kindt et al. 2013b). On the other hand, some effects of MIF might result from intracrine activity. While the importance of MIF in HNSCC pathogenesis seems well established, there is a lack of information concerning the expression of CD74 in these neoplasms. The current study was undertaken to examine clinical tumor specimens for CD74 expression and disclose possible relationship with prognosis. Furthermore, a SCCVII was used to evaluate the contribution of CD74 to cell proliferation and invasiveness, and tumor cell-mediated angiogenesis.
J Cancer Res Clin Oncol (2014) 140:937–947 Table 1 Patient population characteristics Mean age: 58 years (range between 36 and 81 years) Number of cases Gender Male Female
37 9
Under localization Tongue Mouth floor Others
5 17 24
Histological grade Well differentiated Moderately differentiated Poorly differentiated Unknown
14 23 6 3
TNM stage T1–T2 T3–T4 N0 N+ M0
27 19 25 21 46
Recurrence Yes No
21 25
Clinical follow-up (month) Range
1–120
Average
23
p value
N.S.
N.S. N.S.
N.S.
(December 1996 to February 2008) from the records of CHU Erasme (Brussels, Belgium), RHMS Baudour (Baudour, Belgium) and CHU Saint-Pierre (Brussels, Belgium). The description of the tumor status was based on the histopathological grade of tumor differentiation and the TNM staging classification (Hyams et al. 1988; Wittekind et al. 2004). Detailed information on the patients’ age, gender, tumor histopathology and follow-up data up to the last contact with the patient together with the status of the disease at the last examination was available for all patients (see Table 1). Twenty cases of tonsillectomy were used for the normal epithelium group. This study was approved by the local Institutional Review Board. Immunohistochemistry
Materials and methods Patients’ characteristics A total of 46 patients with oral cavity carcinoma who underwent surgery aimed at curative tumor resection were studied. The patient files were compiled retrospectively
13
CD74 immunohistochemistry was performed using a procedure similar to that described previously for MIF (Kindt et al. 2012). After antigen retrieval by microwave treatment, sections were pretreated with hydrogen peroxide to block endogenous peroxidase activity. Thereafter, they were successively exposed to avidin and biotin in order to avoid
J Cancer Res Clin Oncol (2014) 140:937–947
false-positive staining due to the presence of endogenous biotin. These steps were followed by sequential incubations with (1) primary antibody (rabbit polyclonal anti-human CD74 FL-296 from Santa Cruz Biotechnology, Santa Cruz, CA), (2) biotinylated secondary antibody (polyclonal goat anti-rabbit IgG), (3) avidin–biotin–peroxidase (ABC) complex. Immunocomplexes were finally visualized by exposure to the chromogen diaminobenzidine in the presence of H2O2. Sections were counterstained with luxol fast blue prior to light microscopy examination. Computer‑assisted morphometry Morphological examination of immunostained tissue sections was carried out on a Zeiss Axioplan microscope equipped with a color CCD camera (ProgRes C10plus, Jenoptik, Jena, Germany). Morphometric analysis was achieved using KS 400 imaging software (Carl Zeiss Vision, Hallbergmoos, Germany), as described previously (Kindt et al. 2012). Data from the quantitative analysis of immunostaining were expressed as the labeling index (LI), corresponding to the percentage of positive cells. Cell line and culture The squamous cell carcinoma cell line SCCVII was a kind gift from Dr. Shulin Li (Louisiana State University) (Khurana et al. 2001). Routine cell propagation and experimental studies were carried out at 37 °C in a cell incubator with humid atmosphere at 5 % CO2. Unless specified otherwise, cells were cultured in T-flasks containing Dulbecco’s modified essential medium (DMEM) supplemented with phenol red, 10 % FBS, 25 mM N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. Knockdown of CD74 expression (CD74KD) was achieved using anti-CD74 shRNA (mouse) lentiviral particles from Santa Cruz (Santa Cruz, CA), according to manufacturer’s instructions. Controls (CD74sc) were obtained by transduction with shRNA lentiviral particles encoding a scrambled (sc) shRNA sequence. Transduced cells were selected by addition of 4 μg/ml puromycin to the culture medium. Preliminary experiments showed this puromycin concentration to be toxic toward non-transduced SCCVII cells. As shown previously, CD74 knockdown resulted in a decrease of approx. 50 % in the expression of the protein (Kindt et al. 2013b). Measurement of cell culture growth by cell counting and by crystal violet staining assay CD74KD and CD74sc cells were plated at a density of 104 cells/cm2 in 12-well dishes (n = 6). Measurement of
939
cell culture density was performed daily, beginning 24 h after seeding. Cells were dislodged from the vessel bottom by treatment with trypsin–EDTA solution. After vigorous pipetting, concentrations of cells in suspension were determined in an electronic cell counter (model Z1 Coulter counter, Beckman Coulter, Fullerton CA). Alternatively, cell culture density was assessed by colorimetry after crystal violet staining, as described previously (Journe et al. 2004). Cells were seeded in 96-well plates (500– 4000 cells/well, n = 6). After 4 days of exposure, the culture medium was discarded; cultures were rinsed with DPBS, and cells were fixed with 1 % glutaraldehyde. Following fixation, cells were stained with 1 % crystal violet. Destaining was achieved under gently running tap water, and cell monolayers were lysed in 0.2 % Triton X-100. The absorbance of cell lysates was measured at 570 nm using a Labsystems Multiskan MS microplate reader (Labsystems, Helsinki, Finland). Colony formation assay Cells were plated at a density of 20 cells/cm2 in 50-mmdiameter Petri dishes (n = 4). Eight days after seeding, cell cultures were rinsed with DPBS, and cells were fixed with 1 % glutaraldehyde in DPBS. After fixation, cell colonies were stained with 0.1 % crystal violet. Colonies on Petri dishes were digitized using a HP Scanjet 3800 (HewlettPackard, Palo Alto, CA). Numbers of colonies in digital images of dishes were determined using the public domain ImageJ software (Wayne Rasband, NIH). Cell cycle analysis Cells were plated at a density of 104 cells/cm2 in T75 flasks. Cell cultures were processed for cell cycle analysis 1, 2 and 3 days after seeding. After trypsinization, cells were transferred into centrifuge tubes and sedimented by centrifugation (300×g, 5 min). Cells were rinsed once with DPBS (same centrifugation conditions), and cell pellets were suspended and fixed in ice-cold 70 % ethanol. Before flow cytometry analysis, ethanol-fixed cells were centrifuged, rinsed in DPBS and resuspended in a mixture of propidium iodide and RNAse A (Muse™ DNA staining reagent). Cell cycle analysis by flow cytometry was carried out in a Muse® Cell Analyzer (Millipore, Billerica, MA). Cell migration assay Cell invasiveness was assessed by a Boyden chamber assay consisting a 24-well plates (lower chambers) with cell culture inserts (upper chambers), both chambers being separated by a polycarbonate membrane (8 μm size) coated with an artificial extracellular matrix (Chemicon Cell Invasion Assay kit, Millipore, Billerica, MA). The kit was used
13
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according to manufacturer’s instructions. CD74 KD and control SCCVII cells were seeded in cell culture inserts (1.5 × 105 cells/insert) in serum-free medium (DMEM, 0.1 % BSA). The lower chambers were filled with complete medium (DMEM, 10 % FBS). After 96 h, cells were wiped off the upper surface of cell culture inserts with a cotton-tipped swab, and the migrating cells were stained with crystal violet and counted in a total of 71 microscopic fields (10× magnification) using a Zeiss Axio scope A1 microscope (Carl Zeiss, Germany). Determination of matrix metallopeptidase 9 by enzyme‑linked immunosorbent assay The level of matrix metallopeptidase 9 (MMP-9) was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA) using commercial kit (Mouse MMP-9 ELISA KIT, Boster Biological Technology Co., Ltd). Plates were pre-coated with an antibody raised against MMP-9. Known amounts of MMP-9 protein (reference curve) and 50-fold diluted samples were added to the plate. After an incubation of 90 min at 37 °C, 100 μl of detection antibody was added to each well. A 1-h exposure to the detection antibody at 37 °C was followed by the addition of 100 μl of horseradish peroxidase-conjugated avidin. Immunocomplexes were revealed by the addition of 100 μl substrate solution (3,3′,5,5′-tetramethylbenzidine). After stopping the reaction with sulfuric acid solution, the absorbance was measured at 450 nm (reference wavelength: 570 nm). VEGF determination by enzyme‑linked immunosorbent assay The level of VEGF protein was assessed by a sandwich enzyme-linked immunosorbent assay (ELISA) using commercial kit (Mouse Vascular Endothelial cell Growth Factor (VEGF) ELISA KIT, Cusabio Biotech Co., LTD). Plates were pre-coated with an antibody specific to VEGF. Known amounts of VEGF protein (reference curve) and 20-fold diluted samples were added to the plate. After an incubation of 2 h at 37 °C, 100 μl of detection antibody was added to each well. A 1-h exposure to the detection antibody at 37 °C was followed by the addition of 100 μl of horseradish peroxidase-conjugated avidin. Immunocomplexes were revealed as described above. Animal studies Animal studies in vivo were conducted on female C3H/ HeN mice (Charles River Laboratory, L’Arbresle, France). The animals were maintained and handled in compliance with the guidelines issued by the Belgian Ministry of Trade and Agriculture (Agreement No. LA1500549).
13
J Cancer Res Clin Oncol (2014) 140:937–947
For the inoculation of tumor SCC cells, CD74KD and CD74sc cells were grown to confluency and detached by trypsinization. Cells were centrifuged at 200×g for 10 min and suspended in Hank’s balanced salt solution at a concentration of 5 × 106 cells/ml. Prior to tumor cell inoculation, mice were anesthetized by i.p. injection of a ketamine (100 mg/kg)/xylazine (10 mg/kg) mixture (n = 39). Each animal received an injection of 100 μl cell suspension (i.e., 105 cells/mouse) in the mylohyoid muscle. Animals were monitored for tumor onset, and tumors were measured with a digital caliper every 2 days. They were terminated when they exhibited a tumor more than 15 mm diameter or a weight loss of more than 20 %. Statistical analyses SigmaPlot® 11 software was used for statistical analyses. Parametric analyses were achieved by the Student’s t test. Nonparametric analyses were performed by the Mann– Whitney U test (2 groups) or Kruskall–Wallis test (more than two groups). When the latter test was significant, the Dunn’s procedure (post hoc test) was used to compare each pair of groups (to avoid multiple comparison effects).
Results Clinical observations Tissue CD74 expression increases with tumor progression in oral cavity carcinomas The intensity of CD74 immunostaining was evaluated in normal oral epithelium of patients who had undergone tonsillectomy (normal epithelium in the margin of tonsils, N_E, 20 cases, Fig. 1a), in specimens exhibiting preneoplastic lesions (low-grade dysplasia (LGD), 7 cases, Fig. 1b; high-grade dysplasia (HGD), 26 cases, Fig. 1c) and in specimens of oral cavity carcinoma (CA, 46 cases, Fig. 1d). Computer-aided morphometric analysis of immunohistochemical staining in histological sections revealed a significant increase in CD74 expression during tumoral progression, as depicted by LI (Fig. 1e, Kruskal–Wallis test, p
