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Tumor Biology |
Departments of Biological and Technological Research [B. C., A. M., C. F., A. C.], and Histopathology [G. A.], San Raffaele H Scientific Institute, 20132 Milan, Italy
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CgA is abnormally expressed by various tumors, including pheochromocytoma, carcinoid tumors, medullary thyroid carcinoma, pancreatic islet cell tumors, small cell lung cancer, prostate cancer, and many others (14 , 20, 21, 22) . CgA antigen, released in high amounts in the blood of patients, has proven to be a sensitive and specific serum marker for diagnosis of various types of neuroendocrine tumors. Moreover, serum CgA is an independent marker of prognosis in patients with carcinoid tumors (23 , 24) .
The effect of this protein on tumor growth is unknown. In this work we have studied the effect of CgA secretion on tumor growth and morphogenesis in two experimental animal models. To this aim, we have transfected mouse RMA lymphoma and TS/A adenocarcinoma cells (CgA-negative) with the cDNA encoding CgA and studied the proliferation and tumorigenicity of several CgA-producing and nonproducing clones in vitro and in vivo. We show that CgA secretion affects the tumor growth and tissue architecture.
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MATERIALS AND METHODS |
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CgA Assays.
CgA quantification in cell supernatants, cell extracts, and animal sera was carried out by sandwich ELISA using mAb B4E11 and polyclonal rabbit anti-CgA IgGs, as described (28)
. Samples were diluted 2-fold with 0.15 M sodium chloride, 0.05 M sodium phosphate buffer (pH 7.3) containing 0.5% BSA, 2.5% normal goat serum, and 0.05% (v/v) Tween 20. Rabbit IgGs were detected using a goat antirabbit IgG-horseradish peroxidase conjugate and o-phenylenediamine, as a chromogenic substrate. A dose-response curve, covering the range between 0.15 and 10 nM human CgA, was obtained. Spiking serum samples with 5 µg/ml of CgA68–91 peptide, containing the B4E11 epitope (25)
, efficiently inhibited the ELISA signal, confirming the assay specificity for CgA. The assay does not detect chromogranin B (data not shown). Moreover, the assay, based on mAb B4E11 as a "capturing" reagent, efficiently detected human CgA but not murine CgA (Fig. 1)
. Detection of murine CgA in mouse adrenal gland HSF was carried out using a similar assay based on an antibody that cross-react with both species (mAb 5A8; Fig. 1
).
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Transfection of RMA Cells.
RMA lymphoma cells of C57BL/6 origin (obtained from Dr. Paolo Dellabona, San Raffaele H. Scientific Institute, Milan, Italy) were cultured in RPMI 1640, 5% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, 2 mM glutamine, and 50 µM 2-mercaptoethanol. The cDNA coding for the full-length human CgA, including the leader sequence, was prepared by reverse transcription-PCR on SK-N-BE cells total RNA as described (26)
. The amplified DNA (1389 bp) was subcloned into a SmaI-digested pUC19 vector and sequenced using an automatic DNA sequencer (Perkin-Elmer 373A). This plasmid was named pUC19/CgA. The CgA coding region was then cloned into the mammalian expression vector pRS1-neo using EcoRI and HindIII (pRS1Neo-CgA). pRS1Neo-CgA (15 µg) was electroporated into 107 RMA lymphoma cells using a Bio-Rad Gene Pulser apparatus (250 V; 960 µF). Transfected RMA cells, surviving selection with 1.8 mg/ml geneticin, were selected and subcloned. The supernatant of each clone was tested by CgA-ELISA. Five clones secreting CgA and five clones nonsecreting CgA were selected, amplified, and stored for subsequent studies.
The presence of human CgA cDNA in transfected cells was analyzed by PCR on genomic DNA using the following primers: 5'-TGCATGCGCTCCGCCGCTGTCCTGGC-3' (forward primer) and 5'-TCAGGATCCTCATCAGCCCCGCCGTAGTGCCTGC-3'(reverse primer). Sequences were designed to include the ATG start codon in the forward primer and the 3'-end coding sequence of CgA in the reverse primer. Extraction and quantification of genomic DNA were carried out according to standard protocols. The ratio 260:280 nm of genomic DNA isolated from each clone was 1.8-2. PCR reactions were carried out in 50 µl (final volumes) containing 100 ng of genomic DNA, 1 µM primers, 0.25 unit of Taq DNA polymerase, 0.2 mM of deoxynucleotide triphosphates, 50 mM potassium chloride, 1.5 mM magnesium chloride, 5% DMSO, and 20 mM Tris-HCl (pH 8.3). After an initial incubation at 94°C for 3 min, the following temperature cycling was performed: 94°C for 45 s, 65°C for 45 s, and 72°C for 1 min (30 cycles), followed by 72°C for 10 min. The PCR products were analyzed by 0.9% agarose gel electrophoresis using ethidium bromide staining.
Transfection of TS/A Cells.
TS/A cells from a BALB/c spontaneous mammary adenocarcinoma (29)
were cultured in RPMI 1640, 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 2 mM glutamine. pRS1Neo-CgA (4 µg) was mixed with 200 µl of 0.15 mg/ml of Lipofectin Reagent (Life Technologies, Inc.) in 0.15 M sodium chloride and incubated for 15 min at room temperature. Then, 100 µl of this mixture was added to 104 TS/A cells and plated 1 day before in 200 µl of culture medium. After 48 h of incubation, 1 mg/ml geneticin was added to the culture. One week later, cells surviving selection were subcloned. The supernatant of each clone was tested by CgA-ELISA. Four CgA-secreting clones were obtained.
TS/A cells transfected with the cDNA coding for the Thy 1.1 antigen were also prepared using the pRS1Neo-Thy 1.1 vector (30) .
In Vitro Proliferation Assay.
Various clones of transfected cells were seeded into 96-wells microtiter plates at various densities (5, 10, 20, and 40 x 103 cells/well) in 200 µl of RPMI 1640 with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 0.5 mg/ml geneticin, and left to incubate at 37°C, 5% CO2, for 0 or 3 days. Living cells were stained with a 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (Calbiochem, San Diego, CA; 10 µl/well, 2 h at 37°C). The medium was gently aspirated using a multichannel pipette and replaced with 200 µl/well of DMSO. The absorbance was read using a microplate reader at 570 nm (reference wavelength, 670 nm). In parallel, calibration curves for each clone (cell number versus absorbance) were obtained with known amounts of freshly seeded cells. The number of cells in each well was then obtained by interpolating the absorbance of each sample on the relevant calibration curve. The proliferation index of each clone was calculated by dividing the number of cells in wells cultured for 3 and 0 days.
Cell Adhesion Assays.
Adhesion of various RMA or TS/A clones to the plastic surface of microtiter plates was carried out as described (17
, 18)
. Cells were stained with crystal violet.
In Vivo Studies.
In vivo studies on animals were performed according to the prescribed guidelines of the Ethical Committee of the San Raffaele H Scientific Institute. CD-1 nu/nu BR (nude) mice (Charles River Laboratories, Calco, Italy) were challenged with 2 x 106 RMA or TS/A (CgA-negative or CgA-positive) cells, s.c. in the left flank. The tumor growth was monitored by measuring the size with calipers. The tumor volume was estimated by calculating r1 x r2 x r3 x 4/3 
, where r1 and r2 are the longitudinal and lateral radii, and r3 is the thickness of tumors protruding from the skin surface. Animals were killed at day 15. The tumor of each animal was then excised for morphological and immunohistochemical examination.
Histochemistry and Immunohistochemistry.
Each RMA tumor sample was sectioned in two parts. One part was fixed with formalin and embedded in paraffin. The other part was frozen in isopentane-liquid nitrogen and embedded in OCT (BDH Italia, Milan, Italy) for immunohistochemical analysis. Paraffin sections were cut (thickness, 4 µm) and stained with H&E or Gomori silver stain for morphological and histochemical evaluation of tumors. The presence of necrotic areas, tumor nodules, and protein casts was evaluated by microscopy analysis of H&E-stained sections and scored from 0 to 4. The amount (density-intensity) of collagen fibers was evaluated on Gomori-stained sections and graded from 0 to 3. Statistical analysis of the results was performed using the Student unpaired t test (GraphPad software).
The microvessels density of each tumor was assessed by immunohistochemical analysis of criocut sections (thickness, 6 µm) using the rat-antimouse CD31 mAb 01951 (PharMingen, San Diego, CA) as follows: acetone fixed sections were incubated with 0.3% hydrogen peroxide for 15 min to quench endogenous peroxidase and rinsed with 0.05 M Tris-HCl buffer, (pH 7.5; TBS). Each section was then incubated (15 min) with TBS containing 5% normal goat serum followed by 10 µg/ml anti-CD31 mAb in TBS containing 2% BSA (30 min at room temperature). Bound antibody was detected using a biotinylated goat-antirat immunoglobulins secondary antibody and avidin-biotin complexes (Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA) followed by a 3,3'-diamino-benzidine-tetrahydrocloride chromogenic solution (Biogenex, San Ramon, CA). The sections were slightly counterstained with Harris hematoxylin.
Microvessels density was then evaluated by counting the vessels stained by CD31 in seven different fields/sections at various magnification (x100, x200, and x400). In addition, microvessels density was evaluated in areas with a high vessel density, identified in each section at low magnification (x40), and counted at high magnification (x400).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). No evidence of CgA production by CgA-negative clones was obtained by Western blot analysis and ELISA of cell extracts (not shown).
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. These results suggest that the geneticin-resistant CgA-negative clones, despite the fact that they were efficiently transfected, do not produce human CgA, because they lost the CgA cDNA.
The in vitro proliferation and cell morphology of each clone were then studied. No significant difference in proliferation of CgA-positive and -negative clones was detected in 3-day culture assays (Fig. 2B)
. Moreover, no difference in morphology or adhesion to culture plates, depending on CgA secretion, was observed (not shown).
Because these cells were subjected to identical transfection, cloning, and culture conditions, they may represent a good model for investigating the effect of CgA secretion on tumor progression in vivo.
CgA Production by RMA Tumor Cells Correlates with Tumor Growth and Morphogenesis in Vivo.
To investigate the effect of CgA expression on the tumorigenicity of lymphoma cells, each RMA CgA-positive and -negative clone was implanted s.c. in nude mice. Immunodeficient animals were used to avoid T-cell-dependent immune responses against murine-transfected cells, because these cells express human CgA. Two separate experiments were done with RMA cells (called "Exp. 1" and "Exp. 2"), using 80 mice in total. All of the animals bearing RMA CgA-positive tumors had elevated serological levels of human CgA by ELISA (Fig. 3, A–C)
. The CgA levels were variable, ranging from 2 to 
40 nM, whereas no CgA was detected in the serum of animals bearing CgA-negative tumors.
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. Macroscopically, CgA-positive tumors presented multinodular growth patterns, whereas CgA-negative tumors appeared frequently compact and similar to WT RMA tumors (Figs. 4B
and 5, A–D
). Interestingly, we observed a significant correlation between serum CgA levels and tumor nodules, in both Exp. 1 and 2 (Table 1)
. A weak correlation was observed also between tumor volumes and CgA levels in cell supernatants (r = 0.27; P = 0.012) but not with CgA levels in animal sera. This could be explained by the fact that serum levels may directly depend on the tumor mass, which is lower for clones that secrete high amounts of CgA.
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. Tumor macro- and micronodules, completely or partially surrounded by extracellular matrix containing septa, were also visible. Tumor necrosis and nodule formation, scored by microscopy analysis, were significantly different between CgA-positive and CgA-negative tumors (Fig. 5G)
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To investigate the effect of CgA secretion on tumor stroma formation, we evaluated the amount of collagen fibers and vessel density in each section. The amount of stromal fibers in non-necrotic areas of CgA-positive and -negative tumors was similar, as judged by semiquantitative evaluation of Gomori silver-stained sections (Fig. 5G)
. Of note, thicker fibers were observed in CgA-positive tumors within or close to necrotic areas (Fig. 5H)
and, to a lower extent, also in CgA-negative tumors (Fig. 5I)
. The vessel density, assessed by counting the CD31-positive vessels in different areas of each tumor (Fig. 5, L and M)
, was not significantly different in the two groups. Protein casts were also observed in both CgA-positive and -negative tumors to a similar level (Fig. 5G)
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Transfection of TS/A Cells with CgA cDNA Inhibits Tumor Growth in Vivo.
To investigate the effect of CgA expression on the tumorigenicity of adenocarcinoma cells, TS/A cells were transfected with cDNAs coding for CgA or Thy 1.1 (as a negative control). Four CgA-secreting clones were obtained (A6A, A6B, A5B, and A5C; Fig. 6A
). Of note, clone A6B secreted >14 nM of CgA in the supernatant. In contrast, no CgA was detected in the supernatant of a clone transfected with the Thy 1.1 antigen (Thy 1.1) and of WT TS/A cells. The in vivo growth rate of CgA-positive clones was markedly lower than that of controls, particularly in the case of clone A6B (Fig. 6B)
. The tumor dimension of CgA-positive tumors at day 17 was very small, and histological examination at this stage revealed a single nodule in most cases (not shown). To assess whether the reduced growth rate was directly related to changes in cell proliferation and the adhesion, we analyzed the in vitro proliferation index and adhesion of each clone. The proliferation index of clone A6B but not of A6A, A58, and A5C was significantly lower than that of WT or Thy 1.1 controls (Fig. 6C)
. The adhesion of A6A and A5B but not of A6B and A5C to microtiter wells was stronger than that of controls (Fig. 6D)
. Thus, also in the case of TS/A cells no significant correlation was observed between tumorigenicity and in vitro proliferation, or between tumorigenicity and in vitro cell adhesion.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies on the mechanisms of action showed that CgA expression does not affect the in vitro proliferation index of RMA cells, whereas it affects the in vivo growth. This suggests that the effect is indirect and host-mediated. One possibility is that CgA affects the complex interplay between neoplastic cells and tumor stroma, which is critical for tumor growth. Components of the tumor stroma include blood vessels, inflammatory leukocytes, extracellular matrix molecules, and the cells necessary for their production. Noteworthy, we have found recently that CgA and its NH2-terminal fragments can inhibit vascular permeability.4 Thus, it is possible that CgA affects tumor growth by affecting the vascular compartment of the tumor, e.g., by decreasing the transport of macromolecules critical for tumor cell proliferation across the endothelial barrier. In recent studies, we also observed that CgA regulates the adhesion of fibroblasts and smooth muscle cells to solid phases (17 , 18) . Other studies showed that CgA at a nanomolar concentration may increase deposition of basement membrane components, such as collagen type IV, laminin, and perlecan, by mammary ductal epithelial cells and alter ductal morphogenesis in vitro (19) . Thus, it is possible that CgA could affect the tumor architecture also by modulating the physiology of stromal fibroblasts within the tumor, which in turn are important for the production of other extracellular matrix proteins. The presence of thicker stromal fibers in CgA-positive tumors within or close to necrotic areas is apparently in line with this hypothesis. However, because this could also be explained by an increased fibrotic response to damaged tissues, it is difficult to draw conclusions on this point.
The question is raised as to which is the cause of increased necrosis in RMA CgA-positive tumors. The finding that in vitro production of CgA does not affect its proliferation index, hence it is not cytotoxic, might suggests that the mechanism of necrosis is indirect. Hypoxia-related mechanisms dependent on changes in vascular density are unlikely, given the similar microvessel density in CgA-positive and -negative tumors. Possibly, necrosis could be a consequence of decreased vessel permeability and reduced supply of macromolecules critical for tumor cell survival. Alternatively, CgA could affect the production of other mediators by cells present in the tumor stroma, which, in turn, are responsible for the increased necrosis.
The results of this study could have some pathophysiological implications. CgA is present in the blood of normal subjects at 0.4–2 nM (31
, 32)
. Increased levels of CgA (
200 nM) have been detected in the blood of a variety of patients with different neuroendocrine tumors (33
, 34)
. Given that serum CgA levels in our animal models were 2–40 nM, it is possible that the amount of CgA secreted by neuroendocrine tumors in patients is sufficient to affect tumor morphogenesis as we observed in our models.
Several studies have been carried out thus far to assess the correlation among neuroendocrine differentiation, CgA expression, and prognosis in patients with neuroendocrine and nonendocrine tumors. For instance, it has been shown that the expression of CgA decreases with increasing malignancy in neuroendocrine tumors, being higher in well-differentiated carcinomas (low grade) and lower in poorly differentiated (high grade) carcinomas (31) . Interestingly, one study showed that the number of CgA-immunoreactive neuroendocrine cells remarkably decreases in invasive breast carcinomas compared with noninvasive breast carcinomas (32) . On the other hand, other studies showed that neuroendocrine differentiation in prostate tumors could be associated with poorer prognosis (33) and that large cell carcinomas of the lung with neuroendocrine features are more clinically aggressive than classic large cell carcinomas (34) . Because neuroendocrine differentiation and CgA production could be associated with secretion of many other hormonal peptides, it is difficult to speculate on the role of CgA simply on the basis of these correlations. Our results, showing slower progression of mouse mammary adenocarcinoma after transfection with the CgA cDNA, suggest that indeed CgA may contribute to regulate the growth of neuroendocrine tumors in a negative manner.
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FOOTNOTES |
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1 Supported by Associazione Italiana Ricerca sul Cancro and Ministero della Sanità of Italy (Ricerca Finalizzata RF99.54). 
2 To whom requests for reprints should be addressed, at San Raffaele H Scientific Institute, via Olettina 58, 20132 Milan, Italy. Phone: 39-02-26-43-48-02; Fax 39-02-26-43-47-86; E-mail: corti.angelo{at}hsr.it
3 The abbreviations used are: CgA, chromogranin A; mAb, monoclonal antibody; HSF, heat stable fraction; TBS, Tris-buffered saline; WT, wild-type.
4 E. Ferrero, E. Magni, C. Foglieni, F. Curnis, A. Villa, M. E. Ferrero, and A. Corti. Chromogranin A protects vessels against tumor necrosis factor-2-induced vascular leakage, submitted for publication.
Received 8/ 1/01. Accepted 11/30/01.
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REFERENCES |
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. Cancer Res., 57: 1922-1928, 1997.This article has been cited by other articles:
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  D. Belloni, S. Scabini, C. Foglieni, L. Veschini, A. Giazzon, B. Colombo, A. Fulgenzi, K. B. Helle, M. E. Ferrero, A. Corti, et al. The vasostatin-I fragment of chromogranin A inhibits VEGF-induced endothelial cell proliferation and migration FASEB J, October 1, 2007; 21(12): 3052 - 3062. [Abstract] [Full Text] [PDF]
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 R. Connolly, D. Gates, N. Loh, D. Baban, R. Thakker, B. Johnston, D. McCance, J. Ardill, D. T. O'Connor, L. Taupenot, et al. Cox-2 Promotes Chromogranin A Expression and Bioactivity: Evidence for a Prostaglandin E2-Dependent Mechanism and the Involvement of a Proximal Cyclic Adenosine 5'-Monophosphate-Responsive Element Endocrinology, September 1, 2007; 148(9): 4310 - 4317. [Abstract] [Full Text] [PDF]
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