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[Cancer Research 62, 941-946, February 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Chromogranin A Expression in Neoplastic Cells Affects Tumor Growth and Morphogenesis in Mouse Models1

Barbara Colombo, Flavio Curnis, Chiara Foglieni, Antonella Monno, Gianluigi Arrigoni and Angelo Corti2

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


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chromogranin A (CgA), a secretory protein expressed by many neuroendocrine cells, has been recognized as a useful tissue and serum marker of neuroendocrine tumors. To investigate the effect of CgA secretion on neoplastic morphogenesis and progression, we have transfected mouse RMA lymphoma and TS/A adenocarcinoma cells with the cDNA encoding human CgA and selected several CgA-positive (secreting) and CgA-negative (nonsecreting) clones. In both models, the growth rate of CgA-positive clones implanted s.c. in nude mice was slower than that of CgA-negative clones. Histological analysis of each RMA tumor showed that CgA-expression was associated with multinodular growth patterns, whereas CgA-negative tumors appeared more compact and similar to wild-type RMA tumors. Moreover, CgA production was associated with increased tumor necrosis. The number of nodules in each RMA tumor correlated with the serum levels of CgA (n = 40, r = 0.537, P = 0.0004). The reduced growth rate of CgA-positive RMA and TS/A tumors was not related to reduced in vitro proliferation or to changes in cell adhesion and shape, suggesting that the mechanism is indirect and host-mediated. These results suggest that abnormal secretion of CgA by neuroendocrine neoplastic cells could affect neoplastic growth and morphogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CgA3 is a glycoprotein expressed by many neuroendocrine cells and neurons (1, 2, 3) . Under physiological conditions CgA is concentrated and stored within secretory granules, and is released in the extracellular environment together with coresident hormones. After secretion, CgA can reach the blood stream via the capillaries or the lymphatic vessels (2) . Biochemical studies have shown that CgA is a polypeptide of 439 amino acids (4, 5, 6, 7) , characterized by several post-translational modifications including glycosylation, sulfation, and phosphorylation (2 , 3) . Although the extracellular function of CgA is not yet clearly understood, it is believed that this protein is a multivalent precursor of several polypeptides that may exert autocrine, paracrine, and endocrine effects (8) . For instance, NH2-terminal fragments released from the adrenal medulla and from sympathetic nerve terminals (9 , 10) can suppress vasoconstriction in isolated blood vessels (8 , 11 , 12) . Other fragments inhibit the secretion of hormones, such as insulin, parathormone, and catecholamines, from neuroendocrine cells (13, 14, 15, 16) . Recent works have also shown that CgA and its NH2-terminal fragments can regulate the adhesion of fibroblasts and smooth muscle cells in vitro (17 , 18) and may increase deposition of basement membrane components by mammary ductal epithelial cells in vitro (19) .

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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Natural CgA and Anti-CgA Antibodies.
Mouse antihuman CgA mAbs (mAb 5A8, B4E11, and A11) and rabbit anti-CgA polyclonal antibodies were described previously (25, 26, 27) . HSFs of human pheochromocytoma tissues and mouse adrenal glands were prepared as described previously (27) . Human CgA was purified from pheochromocytoma tissues by immunoaffinity chromatography on the mAb A11-agarose column as described (28) .

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)Citation . 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. 1Citation ).



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  		Fig. 1. Cross-reactivity of mAb B4E11 and 5A8 with human and murine CgA. The assay was carried out by ELISA using mAb B4E11 or 5A8 in the capturing step. Samples include various amounts of protein extracts (HSF) of mouse adrenal glands (A) or human pheochromocytoma (B). mAb B4E11 does not cross-react with murine CgA.

 
Western blot analysis was carried out using mAb B4E11 and the ECL Western Blotting kit (Amersham), as described (28) .

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 {pi}, 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).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transfection of RMA Cells with CgA cDNA Does Not Affect Their in Vitro Proliferation.
To investigate the effect of CgA secretion on tumor growth and morphogenesis, we have transfected mouse RMA lymphoma cells with the cDNA coding for residues 1–439 of human CgA. Among the various clones that acquired geneticin-resistance, we selected five clones (1E1, 1D3, 4C1, 2G10, and 3A6) that secrete CgA in the culture supernatants (termed RMA "CgA-positive" clones) and five clones (4H4, 3C3, 1F10, 4D10, and 1D10) that do not secrete CgA (termed RMA "CgA-negative" clones; ELISA detection limit, 0.3 nM; Fig. 2ACitation ). 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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  		Fig. 2. In vitro production of CgA (A) and proliferation (B) of RMA lymphoma clones transfected with human CgA cDNA. A, CgA production was assessed by measuring the CgA levels in the supernatant of each clone by ELISA after 3 days of culture (20 x 103 cells seeded in 200 µl of culture medium). B, in vitro proliferation index of clones 1E1, 1D3, 4C1, 2G10, and 3A6 (CgA+), and clones 4H4, 3C3, 1F10, 4D10, and 1D105 (CgA-; n = 5); bars, ±SE. , ELISA detection limit (0.3 nM). N.S., not significant by unpaired Student t test. C, PCR analysis of WT RMA cells, CgA-negative clones, and clone 3A6. PCR was carried out using primers specific for the human CgA cDNA (see "Materials and Methods"). The plasmid pRS1neo-CgA was included as positive control; molecular markers (M).

 
To assess whether the CgA cDNA was present in the CgA-negative clones we analyzed each clone by PCR using CgA-specific primers. A DNA fragment corresponding to CgA cDNA (1389 bp) was amplified in clone 3A6 (CgA-positive) but not in the CgA-negative clones (Fig. 2C)Citation . 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)Citation . 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)Citation . 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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  		Fig. 3. Circulating levels of CgA in animals bearing RMA CgA-positive and -negative clones. A, CgA serum levels of mice bearing various tumors (Exp. 1). B, cumulative data of Experiment 1 and Experiment 2; bars, ±SE.

 
The growth and morphology of each RMA tumor was then evaluated. The volume of CgA-positive tumors was smaller than that of CgA-negative tumors (Fig. 4A)Citation . Macroscopically, CgA-positive tumors presented multinodular growth patterns, whereas CgA-negative tumors appeared frequently compact and similar to WT RMA tumors (Figs. 4BCitation and 5, A–DCitation ). Interestingly, we observed a significant correlation between serum CgA levels and tumor nodules, in both Exp. 1 and 2 (Table 1)Citation . 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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  		Fig. 4. Tumorigenicity of RMA CgA-positive and -negative clones. Tumor volumes (A) and number of macroscopically visible tumor nodules (B) of animals bearing CgA-negative or -positive tumors (cumulative results of Experiment 1 and 2; bars, ±SE). CgA- versus CgA+, P = 0.006 (*), by unpaired t test.

 


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  		Fig. 5. Morphology of RMA CgA-negative and -positive tumors after H&E staining (A–F) or Gomori-silver staining (H and I). WT RMA (A), RMA CgA-negative (B), and RMA CgA-positive tumors (C and D) at low magnification (x100); microphotographs of RMA CgA-positive tumors at higher magnification (x400, E, F, H, and I). Note the presence of nodules (nod) in C and D separated from the rest of the tumor mass by septa (s). F, intranodular necrotic areas (n). G, results of semiquantitative evaluation of various parameters (Experiment 1, bars, ±SE). H and I, Gomori-silver stained CgA-positive and CgA-negative sections, respectively (x200) showing collagen fibers (arrows). L and M, representative microphotographs of CgA-positive (L) and CgA-negative (M) tissues immunostained with anti-CD31 mAb 01951 showing tumor vessels (arrowheads). P = 0.0003 (**); P = 0.0011 (*).

 

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  		Table 1 Correlation between serum CgA, nodule formation, and tumor volume of animals bearing various clones of RMA cells transfected with the cDNA coding for CgA

 
Histological examination of tissue sections showed the presence of necrotic areas and stroma collagen fibers in all of the samples, though to a different extent. In CgA-positive samples, the necrotic areas resulted more extended and frequently presented a big-branched core accompanied by other smaller areas of necrosis (Fig. 5, C–F)Citation . 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)Citation .

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)Citation . Of note, thicker fibers were observed in CgA-positive tumors within or close to necrotic areas (Fig. 5H)Citation and, to a lower extent, also in CgA-negative tumors (Fig. 5I)Citation . The vessel density, assessed by counting the CD31-positive vessels in different areas of each tumor (Fig. 5, L and M)Citation , 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)Citation .

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. 6ACitation ). 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)Citation . 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)Citation . The adhesion of A6A and A5B but not of A6B and A5C to microtiter wells was stronger than that of controls (Fig. 6D)Citation . 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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  		Fig. 6. CgA secretion (A), tumorigenicity (B), in vitro proliferation (C), and adhesion (D) of TS/A adenocarcinoma clones transfected with human CgA cDNA or Thy 1.1 antigen. A, CgA secretion was assessed by measuring the CgA levels in the supernatant of each clone by ELISA as described for RMA cells in the legend of Fig. 1Citation . B, tumor growth of TS/A CgA-positive clones (A6A, A6B, A5A, and A5B), WT TS/A cells, and TS/A cells transfected with the Thy 1.1 cDNA. C, in vitro proliferation index of each clone. D, adhesion of TS/A clones to the plastic surface of microtiter plates after 3 h of incubation in the presence of 0.2% FCS.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study suggest that CgA expression by tumor cells can affect tumor development and architecture. The study has been carried out using genetically engineered mouse RMA lymphoma and TS/A adenocarcinoma cells expressing variable levels of human CgA implanted in nude mice. The most striking observation is that CgA expression was associated with a decreased tumorigenicity in mice. Moreover, CgA production was associated with increased tumor necrosis and multinodular growth pattern in RMA tumors but not in TS/A tumors. Interestingly, the number of tumor nodules significantly correlated with the serum levels of CgA. Because the CgA ELISA used in this study does not detect murine CgA, it is very likely that the circulating CgA antigen reflects the secretory activity of tumor cells and not the production of CgA by the neuroendocrine system of the animals. Moreover, given that the half life of CgA is 18.4 min (20) , the presence of CgA in animal sera 15 days after tumor implantation indicates that this protein was secreted by tumor cells throughout the duration of the experiments. These notions, together with the observed correlation between tumor morphology and circulating CgA, suggest that the different growth patterns of CgA-positive and -negative tumors are related to locally produced CgA.

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.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Associazione Italiana Ricerca sul Cancro and Ministero della Sanità of Italy (Ricerca Finalizzata RF99.54). Back

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 Back

3 The abbreviations used are: CgA, chromogranin A; mAb, monoclonal antibody; HSF, heat stable fraction; TBS, Tris-buffered saline; WT, wild-type. Back

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. Back

Received 8/ 1/01. Accepted 11/30/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rosa P., Gerdes H. H. The granin protein family: markers for neuroendocrine cells and tools for the diagnosis of neuroendocrine tumors. J. Endocrinol. Invest., 17: 207-225, 1994.[Medline]
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