TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study

Background Histological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM) on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta) signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited. Methods Tissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4) in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2) vs. high-grade (i.e. grade 3 and 4)), lymph node metastasis, distant metastasis, 5 year cancer related survival) using Chi-square or Fisher's exact test, when appropriate, to compare frequencies, Kaplan-Meier method to calculate 5-year rates of distant metastases and cancer-related survival and log rank test to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed. Results High-grade tumours and those with lymph node metastases showed higher rates of TAMs and lower expression of TGF-beta1. Loss of nuclear Smad4 expression in tumor was associated with presence of lymph node metastasis, but no influence on prognosis could be demonstrated. Decrease of both TGF-beta receptors in tumour-associated stroma was associated with increased lymph node metastasis and shorter survival. Stromal TGF-beta receptor 2 expression was an independent prognostic factor for cancer related survival. Conclusion Histological phenotype and clinical behaviour of colon cancer is not only influenced by mutational incidents in tumour cells but also affected by interaction of tumour tissue with inflammatory cells like macrophages and associated stroma and TGF-beta signalling is one important part of this crosstalk. Further studies are needed to elucidate the exact mechanisms.


Background
Tumours do not exclusively consist of neoplastic epithelial cells, but are also accompanied by a stromal compartment composed of a variety of non-malignant cells, such as fibroblasts, inflammatory cells, and endothelial cells, as well as extracellular elements [1,2] Nonetheless in the past cancer research has been focused primarily on oncogenic events in tumour cells. It has, however, become increasingly clear that the tumour environment plays an important role in malignant disease, and a correlation between (chronic) inflammation and human predisposition to carcinogenesis has been demonstrated in several malignancies [3][4][5].
The majority of leukocytes that infiltrate the neoplastic stroma consist of macrophages, which are referred to as tumour-associated macrophages (TAMs) [1,4,6]. Clinical observations have shown that the presence of abundant TAMs can be associated with malignant behaviour in breast, prostatic, ovarian, and cervical carcinomas [4]. For other types of cancer, such as gastric, lung, and colorectal carcinomas, opposing data have been reported [4,[7][8][9]. Thus, the biological significance and possible clinical implications of TAMs' presence are not yet fully understood.
Maintenance of epithelial tissues needs the stroma. When the epithelium changes, the stroma inevitably follows. Crosstalk between tumour and stromal compartment is based on several signalling pathways. One important cytokine in this context is transforming growth factor beta (TGF-β). The TGF-β superfamily of secreted polypeptides consists of three 25 kDa-proteins (TGF-β1, 2 and 3) and regulates cell proliferation, differentiation, motility, apoptosis and extracellular matrix formation in a variety of different cell types [10][11][12]. TGF-β serves as a tumour suppressor pathway in the normal colon by inhibiting cell proliferation and inducing apoptosis [13][14][15]. During late stages of colorectal carcinogenesis, TGF-β serves as a tumour promoter [16,17] and is often over expressed. A high expression level of TGF-β in the primary tumour is associated with advanced stages [18], tumour recurrence [19], and decreased survival [18].
The TGF-β signal is transduced by a pair of transmembrane serine-threonine kinase receptors [11]. TGF-β binds primarily to TGF-β-R2 receptor homodimers, which then form heterotetrameric complexes with two TGF-β-R1 molecules. As a consequence, the TGF-β-R2 kinase phosphorylates TGF-β-R1, thereby activating its serine-threonine kinase. In response to receptor activation, two cytosolic proteins, Smad2 and Smad3, become transiently associated with and phosphorylated by the TGF-β-R1 kinase. After their activation, Smad2 and Smad3 form heteromeric complexes with a third homologue, Smad4. These complexes are translocated to the nucleus, bind to DNA in a sequence-specific manner, and regulate gene transcription [11]. The resulting repression of c-myc and induction of cyclin-dependent kinase inhibitors as well as cdc25A phosphatase lead to G 1 phase cell cycle arrest.
Most colorectal cancers escape the tumour suppressor effects of TGF-β as demonstrated by their resistance to the antiproliferative and apoptotic effects of TGF-β [16,17]. The molecular mechanisms by which colorectal cancers escape the tumour suppressor effects of TGF-β are an area of active investigation. A subset of colorectal cancers has been shown to have mutations or down-regulation of the type 1 receptor [20], type 2 receptor [21], Smad2 [22,23] and Smad4 [24][25][26].
Most studies so far focussed on alterations of the TGF-β pathway in the tumour cells. In the present study we investigated in addition to tumour-associated macrophages alterations of this signalling in tumour-associated desmoplastic stroma and their relation to histological tumour grade, regional and distant metastasis rates and survival in a group of colon carcinoma patients that underwent colon resection.

Tumour samples
Cases of colon adenocarcinomas were retrieved retrospectively from the files of the Department of Pathology at the University of Erlangen-Nuremberg, Germany. The specimen had been formalin-fixed, paraffin-embedded and tumour diagnosis made on haematoxylin and eosin (HE) sections. In the present study 310 tumour samples of patients were included, which underwent radical resection with formal regional lymph node dissection between 1991 and 2001 and received complete surgical resection of their colon carcinoma on clinical and pathohistological examination. Only solitary colon carcinomas except appendix tumours were included. Further exclusion criteria were: anamnestic or synchronous other malignant tumours, known familial adenomatous polyposis, colitis ulcerosa or Crohn's disease, neoadjuvant therapy, synchronous distant metastases, emergency operation, perioperative death and unknown tumour stage at the end of follow up. Clinical data is summarized in table 1. The study was carried out in compliance with the Helsinki Declaration. Ethics Committee, Faculty of Medicine, University of Erlangen-Nuremberg approved research on anonymized archived tumor material of patients in a general statement from January 24 th , 2005.

Tissue micro array technique
A map of the receiver blocks was prepared with coordinates for each sample to correctly identify the tumour samples. Under a microscope nonnecrotic carcinoma areas and surrounding areas of immediately adjacent desmoplastic stroma were marked with an indelible pen on the HE whole section of each donor block.
The tissue micro arrayer (Beecher Instruments, Woodland, USA) was used as follows: cores of 0.6 mm diameter were punched from the donor blocks and positioned in a recipient paraffin array block in smaller holes of 0.4 mm for best adhesion of the samples to the array block. Three cores of tonsil tissue were positioned on the upper left corner of each recipient block for correct orientation on the array of the recipient block. The array blocks were then incubated for 30 min at 37°C to improve adhesion between cores and paraffin of the recipient block. Afterwards the blocks were cut with a standard microtome (Microm, Heidelberg, Germany).

Immunohistochemistry
Sections of 5 μm from the fourteen tissue arrays were cut onto silane-treated Super Frost slides (CML, Nemours, France) and left to dry overnight. The slides were deparaffinized in xylene and rehydrated in pure ethanol. Endogenous peroxidase was blocked using 3 % hydrogen peroxide in methanol for 30 min. The slides were then placed in a microwave oven in citrate buffer (TGF-β1, TGF-β-R1, TGF-β-R2, Smad3, CD68) for 45 min at 120 to 85°C for antigen retrieval with a subsequent biotinstreptavidin-peroxidase detection technique or in TRS6 (DAKO, Hamburg, Germany) buffer (Smad4) at the same processing conditions with subsequent tyramide signal amplification coupled alkaline phosphatase (DAKO, Hamburg, Germany) -Fast Red (Sigma-Aldrich, Munich, Germany) detection technique. All slides were then proc-

Statistical analysis
The Kaplan-Meier method was used to calculate 5-year rates of distant metastases and cancer-related survival. The log rank test was used to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed. To compare frequencies Chi-square test, or Fisher's exact test when appropriate, was used. A p-value of less than 0.05 was considered to be significant. All analyses were performed using the statistical software SPSS for Windows Version 13 (SPSS Inc., Chicago, USA).

Results
The results are summarized in table 2.

TAMs
Macrophages in tumour-associated stroma were more abundant than those infiltrating tumour tissue (mean score 1.8 vs. 0.5). We found significantly higher levels of tumour-associated macrophages in high-grade tumours (p < 0.001) and tumours with lymph node metastases (p = 0.019

Components of TGF-β pathway in tumour-associated stroma
Expression of TGF-β1 in tumour-associated stroma could not be demonstrated. Expression of TGF-β receptors and Smads in stroma was restricted to spindled and sometimes stellar stromal cells apparently representing fibroblasts and myofibroblasts. In most cases TGF-β receptors were expressed in the tumour-associated stroma (60% or 188 of 310 for type 1; 60% or 186 of 309 for type 2). For both receptors no correlation to histological grade was found. Higher TGF-β receptor expression in stroma was associated with significant decrease of nodal positive tumours ( Immunohistochemical analysis of tumor and associated stroma Figure 1 Immunohistochemical analysis of tumor and associated stroma. Examples for degree of TAM infiltration in CD68 staining and levels of expression of TGF-β1 and TGF-β-R2 in tumour and associated stroma. Note change of differentiation grade with increase in TAMs. = 0.007, table 3) was an independent prognostic factor for cancer-related survival as reflected in Kaplan-Meier survival curve (see Fig. 2).

Discussion
Macrophage functions are profoundly affected by microenvironmental signals and can range from powerful induction of inflammatory responses to immunosuppression [27]. Although it has been demonstrated previously that macrophages are capable of killing tumour cells in vitro and in vivo, evidence is now emerging in support of a tumour-promoting role for macrophages, and it has recently been hypothesized that macrophages, residing within neoplastic tissues, are educated by tumour cells to perform auxiliary oncogenic functions such as matrix break-down and induction of angiogenesis [27][28][29][30]. In the present study we found higher levels of TAMs in highgrade tumours and tumours with lymph node metastasis.
Although there was a trend towards lower rates of distant metastasis and better survival rates in TAM rich tumours, this difference was not statistically significant. As this group accounts for less than 7% of tumours in our study this result might not be reproducible in larger series. Nonetheless several observations could explain these apparently contradictory effects of macrophages in disease outcome. In a recent experimental study the level of macrophage accumulation was found to correlate with macrophage functionality: high-level secretion of monocyte chemoattractant protein-1 (MCP-1) resulted in massive infiltration of macrophages with subsequent tumour regression. By contrast, low-level secretion of MCP-1 led to moderate infiltration of macrophages and melanoma progression [31]. Second, macrophage activity may alter over time as a consequence of tumour-induced immune dysfunction, since nitric oxide production, which is a major effector molecule for tumour cell killing, was suppressed in macrophages of tumour-bearing mice [32]. Third, a conceptual framework referred to as the so called macrophage balance hypothesis has been proposed, defining two different macrophage populations ranging from polarized potent killer/effector M1 cells to alternatively activated M2 macrophages with tumour-promoting characteristics. In a recently published study, the presence Kaplan-Meier survival curve for TGF-β-R2 expression in tumor-associated stroma Figure 2 Kaplan-Meier survival curve for TGF-β-R2 expression in tumor-associated stroma. Cancer-related survival of colon carcinoma patients according to TGF-β-R2 expression in tumour-associated stroma. of macrophages directed tumours towards a histologically more malignant phenotype characterized by extensive stromal reaction, disorganized matrix deposition, and neovascularization [33]. Nonetheless, depletion of macrophages deprived tumour inhibitory functions as well, resulting in enhanced growth and decreased survival, emphasizing the complexity of the proposed macrophage balance. We think balance between the following actions of tumour-associated macrophages could account for some observations in the present study: At the primary tumour site macrophage dependent and cytokine mediated promoting activity might increase local infiltrative growth, enhancement of lymphangiogenesis and lymphatic vessel infiltration. On the other hand tumouricidal activity of macrophages might be restricted to single and isolated tumour cells on invasion front and founder cells of early hematogenic metastases. Reducing the number of macrophages per se, which has been proposed as a therapeutic strategy, may therefore not constitute the most optimal approach. However, specifically counteracting tumour-promoting characteristics and/or enhancing macrophage tumouricidal activity might be promising for future therapies. The second question we addressed in this study was whether alterations in TGF-β signalling are related to histological tumour grade and clinical behaviour of colon cancers. First we analyzed important factors of TGF-β signalling pathway in the epithelial component of the tumours. Immunostainable TGF-β1 has been detected in the malignant epithelial cells in the majority of our investigated cases of colon carcinomas and expression was shown to be higher in low-grade tumours, as shown recently [34], whereas we found no correlation to lymph node metastases, tumour stage or survival, although there was a non-significant trend towards lower TGF-β1 expression in tumours with regional lymph node metastases. This is in contrast to studies, where TGF-β1 overexpression in advanced stages of colorectal cancers has been reported and the intensity of the staining seems to correlate with advancing stages of tumour progression [35,36]. However, interpretation of these studies is complicated by difficulties associated with distinguishing the biologically inactive, latent form of TGF-β1 from its activated form.
Most tumours in the present study showed no detectable TGF-β receptor expression. The loss of TGF-β receptor expression in colon cancer either through mutation or downregulation has been reported [20,21], but like in our study no correlation to histological grade or clinical outcome was found [37]. In contrast to loss of TGF-β receptors in tumour tissue we found frequent activation of Smad signalling indicated by nuclear expression of Smad3 and Smad4 in tumour and surrounding stroma, but only nuclear expression of Smad4 in the malignant epithelial component was correlated to presence of lymph node metastasis. Although experimental data suggest tumour suppressive effects of functionally active Smad4 [38,39] and loss of Smad4 in colorectal cancer is associated with advanced stage disease, presence of lymph node metastasis and poor prognosis [40][41][42][43], others reported retained Smad4 expression in high-grade colorectal carcinoma and suggested loss of Smad4 is a late event in colorectal carcinogenesis [44]. In our study no influence of reduced Smad4 expression on prognosis was found. A possible explanation could be that in our study nodal positive cases (i.e. stage III patients) had an excellent 5 year survival of almost 80% indicating good local tumor control. The reduction of local recurrences possibly through extended lymphadenectomy could eliminate the prognostic influence of reduced Smad4. Activation of Smad signalling in our cases as indicated by frequent nuclear expression in the absence of TGF-β-R expression might occur primarily through TGF-β independent mechanisms, e.g. activin signalling [45]. Most authors so far focused primarily on the malignant epithelial component in tumours, so expression data of components of TGF-β pathway in tumour-associated stroma is rare. Unlike others [21] we found no expression of TGF-β1 in stroma. In our study frequent nuclear expression of Smads showed no correlation to tumour grade or clinical outcome. The most important finding in our study was an association of expression of both TGF-β receptors to presence of regional metastases. In patients with regional metastasized tumours decreased receptor expression in tumour-associated stroma was associated with shorter survival, whereas incidence of distant metastases showed only minor nonsignificant increase. Most importantly besides the wellestablished influence of lymph node metastasis and vessel infiltration on cancer-related survival in our study we were able to show that TGF-β-R2 expression in the stroma is an independent prognostic factor. Main effector cells for TGF-β1 in tumour-associated stroma are fibroblasts and myofibroblasts and decrease of TGF-β receptor expression in stroma as in epithelial tumour tissue might occur via mutation or downregulation [20,21] and reflects functional alteration of TGF-β signalling. Paracrine effects of TGF-β in the stroma can be summarized as locally tumour promoting and implicate stimulation of angiogenesis, escape from immunosurveillance and recruitment of myofibroblasts (as reviewed in [46]). As in models of tumour escape from chemotherapy [47], disruption of TGF-β signalling in the stroma via decreased receptor expression might drive tumour evolution towards a prometastatic phenotype. This might account for decreased survival, although other mechanisms, e.g. reduction of indirect antiproliferative TGF-β feedback effects from stroma to tumour, are conceivable.

Conclusion
In summary not only mutational incidents in tumour cells but also interaction of tumour tissue with inflammatory cells like macrophages and associated stroma through TGF-β signalling modulates histological phenotype and clinical progression in colorectal cancer. Further studies are needed to clarify the underlying mechanisms.
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