A new aggressive xenograft model of human colon cancer using cancer-associated fibroblasts.

BACKGROUND: Colorectal cancer is the second leading cause of cancer death. Almost half of the patients present recurrence within 5 years after the treatment of the primary tumor, the majority, with metastasis. On the other hand, in the search for new animal models that simulate metastatic cancer, it has been suggested that fibroblasts immersed in the peritumoral stroma (cancer-associated fibroblasts (CAFs)), play a relevant role in the development of cancer. The objective of this study was to identify an adequate animal model to study metastatic colon cancer and the application of new treatments. METHODS: Human CAFs and normal fibroblasts (NF) for transplant and culture were obtained from surgical fresh samples of patients with adenocarcinoma of sigmoid colon. Stromal cell purity was evaluated by morphology and immunostaining with vimentin (VIM) as a fibroblast marker and anti-proColXIα1 as a specific human CAF marker. Phenotypic characterization of cultured stromal cells was performed by co-staining with mesenchymal and epithelial cell markers. For identification in mice, human CAFs were labeled with the PKH26 red fluorescence dye. Cell line HT-29 was used as tumor cells. Transplant in the head of the pancreas of 34 SCID mice was performed in four different groups, as follows: I. 150,000 CAFS (n = 12), IIa. 1.5 million HT29 cells (n = 7), IIb. 150,000 NF+1.5 million HT29 cells (n = 5), III. 150,000 CAFS+1.5 million HT29 cells (n = 10). After euthanasia performed one month later, histological analysis was made using hematoxylin-eosin and anti-proColXIα1. A histopathological score system based on three features (tumor volume, desmoplasia and number of metastasized organs) was established to compare the tumor severity. RESULTS: The CAFs and NF cultured were proColXIα1+/VIM+, proColXIα1/alphaSMA+ and proColXIα1+/CK19+ in different proportions without differences among them, but the CAFs growth curve was significantly larger than that of the NF (p < 0.05). No tumor developed in those animals that only received CAFs. When comparing group II (a + b) vs. group III, both groups showed 100% hepatic metastases. Median hepatic nodules, tumor burden, lung metastases and severity score were bigger in group III vs group II (a + b), although without being significant, except in the case of the median tumor volume, that was significantly higher in group III (154.8 (76.9-563.2) mm3) vs group II (46.7 (3.7-239.6) mm3), p = 0.04. A correlation was observed between the size of the tumor developed in the pancreas and the metastatic tumor burden in the liver and with the severity score. CONCLUSION: Our experiments demonstrate that cultured CAFs have a higher growth than NF and that when human CAFs are associated to human tumor cells, larger tumors with liver and lung metastases are generated than if only colon cancer cells with/without NF are transplanted. This emphasizes the importance of the tumor stroma, and especially the CAFs, in the development of cancer.

Response to chemoradiotherapy and lymph node involvement in locally advanced rectal cancer.

AIM: To establish the association between lymph node involvement and the response to neoadjuvant therapy in locally advanced rectal cancer. METHODS: Data of 130 patients with mid and low locally advanced rectal adenocarcinoma treated with neoadjuvant chemoradiation followed by radical surgery over a 5-year period were reviewed. Tumor staging was done by endorectal ultrasound and/or magnetic resonance imaging. Tumor response to neoadjuvant therapy was determined by T-downstaging and tumor regression grading (TRG). Pathologic complete response (pCR) is defined as the absence of tumor cells in the surgical specimen (ypT0N0). The varying degrees TRG were classified according to Mandard's scoring system. The evaluation of the response is based on the comparison between previous clinico-radiological staging and the results of pathological evaluation. χ (2) and Spearman's correlation tests were used for the comparison of variables. RESULTS: Pathologic complete response (pCR, ypT0N0, TRG1) was observed in 19 cases (14.6%), and other 18 (13.8%) had only very few residual malignant cells in the rectal wall (TRG2). T-downstaging was found in 63 (48.5%). Mean lymph node retrieval was 9.4 (range 0-38). In 37 cases (28.5%) more than 12 nodes were identified in the surgical specimen. Preoperative lymph node involvement was seen in 77 patients (59.2%), 71 N1 and 6 N2. Postoperative lymph node involvement was observed in 41 patients (31.5%), 29 N1 and 12 N2, while the remaining 89 were N0 (68.5%). In relation to ypT stage, we found nodal involvement of 9.4% in ypT0-1, 22.2% in ypT2 and 43.7% in ypT3-4. Of the 37 patients considered "responders" to neoadjuvant therapy (TRG1 and 2), there were only 4 N+ (10.8%) and the remainder N0 (89.2%). In the "non responders" group (TRG 3, 4 and 5), 37 cases were N+ (39.8%) and 56 (60.2%) were N0 (P < 0.001). CONCLUSION: Response to neoadjuvant chemoradiation in rectal cancer is associated with lymph node involvement.

COL11A1/(pro)collagen 11A1 expression is a remarkable biomarker of human invasive carcinoma-associated stromal cells and carcinoma progression.

The COL11A1 human gene codes for the α1 chain of procollagen 11A1 and mature collagen 11A1, an extracellular minor fibrillar collagen. Under regular conditions, this gene and its derived products are mainly expressed by chondrocytes and mesenchymal stem cells as well as osteoblasts. Normal epithelial cells and quiescent fibroblasts from diverse locations do not express them. Mesenchyme-derived tumors and related conditions, such as scleroderma and keloids, are positive for COL11A1/(pro)collagen 11A1 expression, as well as high-grade human gliomas/glioblastomas. This expression is almost absent in benign pathological processes such as breast hyperplasia, sclerosing adenosis, idiopathic pulmonary fibrosis, cirrhosis, pancreatitis, diverticulitis, and inflammatory bowel disease. By contrast, COL11A1/(pro)collagen 11A1 is highly expressed by activated stromal cells of the desmoplastic reaction of different human invasive carcinomas, and this expression is correlated with carcinoma aggressiveness and progression, and lymph node metastasis. COL11A1 upregulation has been shown to be associated to TGF-ß1, Wnt, and Hh signaling pathways, which are especially active in cancer-associated stromal cells. At the front of invasive carcinomas, neoplastic epithelial cells, putatively undergoing epithelial-to-mesenchymal transition, and carcinoma-derived cells with highly metastatic capabilities, can express COL11A1. Thus, in established metastases, the expression of COL11A1/(pro)collagen 11A1 could rely on both the metastatic epithelial cells and/or the accompanying activated stromal cells. COL11A1/(pro)collagen 11A1 expression is a remarkable biomarker of human carcinoma-associated stromal cells and carcinoma progression.

Predictive markers of response to neoadjuvant therapy in rectal cancer.

BACKGROUND: Neoadjuvant therapy followed by radical surgery is the standard treatment in locally advanced rectal cancer. It is important to predict the response because the treatment has side effects and is costly. The aim of this study was to establish the relationship among clinical, pathologic, and molecular biomarkers and the response to neoadjuvant therapy. METHOD: A total of 130 patients with locally advanced mid and low rectal cancer who underwent long-course radiotherapy with 5-FU based chemotherapy followed by radical surgical resection were included in the study. Clinical and pathologic data were collected. Paraffin-embedded sections obtained in diagnostic biopsies were assessed by immunohistochemical staining for molecular markers and classified using a semiquantitative method. Results were related with T-downstaging and tumor regression grade using Mandard scoring system on surgical specimens. RESULTS: Pathologic complete response was found in 19 patients (14.6%), while in another 18 (13.8%) only minor residual disease was seen in the rectal wall. T-downstaging was observed in 63 (48.5%). The average of lymph node retrieval in the surgical specimens was 9.4. Regarding predictive markers of response, there was significant correlation between the expression of B-cell lymphoma 2 (P = 0.005), ß-catenin (P = 0.03), vascular endothelial growth factor (P = 0.048) and apoptotic protease activating factor 1 (P = 0.03), tumor differentiation grade (P < 0.001), and response in the univariate analysis. T-downstaging was associated with vascular endothelial growth factor expression (P = 0.03) and tumor differentiation grade (P < 0.001). Significant parameters found in the multivariate analysis were tumor differentiation grade and Bcl-2 expression. CONCLUSIONS: Pathologic and molecular biomarkers in the diagnostic biopsies may help us predict tumor response to chemoradiation in rectal cancer patients.

Validation of COL11A1/procollagen 11A1 expression in TGF-ß1-activated immortalised human mesenchymal cells and in stromal cells of human colon adenocarcinoma.

BACKGROUND: The human COL11A1 gene has been shown to be up-regulated in stromal cells of colorectal tumours, but, so far, the immunodetection of procollagen 11A1, the primary protein product of COL11A1, has not been studied in detail in human colon adenocarcinomas. Some cancer-associated stromal cells seem to be derived from bone marrow mesenchymal cells; the expression of the COL11A1 gene and the parallel immunodetection of procollagen 11A1 have not been evaluated in these latter cells, either. METHODS: We used quantitative RT-PCR and/or immunocytochemistry to study the expression of DES/desmin, VIM/vimentin, ACTA2/αSMA (alpha smooth muscle actin) and COL11A1/procollagen 11A1 in HCT 116 human colorectal adenocarcinoma cells, in immortalised human bone marrow mesenchymal cells and in human colon adenocarcinoma-derived cultured stromal cells. The immunodetection of procollagen 11A1 was performed with the new recently described DMTX1/1E8.33 mouse monoclonal antibody. Human colon adenocarcinomas and non-malignant colon tissues were evaluated by immunohistochemistry as well. Statistical associations were sought between anti-procollagen 11A1 immunoscoring and patient clinicopathological features. RESULTS: Procollagen 11A1 was immunodetected in human bone marrow mesenchymal cells and in human colon adenocarcinoma-associated spindle-shaped stromal cells but not in colon epithelial or stromal cells of the normal colon. This immunodetection paralleled, in both kinds of cells, that of the other mesenchymal-related biomarkers studied: vimentin and alpha smooth muscle actin, but not desmin. Thus, procollagen 11A1(+) adenocarcinoma-associated stromal cells are similar to "activated myofibroblasts". In the series of human colon adenocarcinomas here studied, a high procollagen 11A1 expression was associated with nodal involvement (p = 0.05), the development of distant metastases (p = 0.017), and advanced Dukes stages (p = 0.047). CONCLUSION: The immunodetection of procollagen 11A1 in cancer-associated stromal cells could be a useful biomarker for human colon adenocarcinoma characterisation.

Characterization of a novel mouse monoclonal antibody, clone 1E8.33, highly specific for human procollagen 11A1, a tumor-associated stromal component.

A novel IgG1, κ mouse monoclonal antibody (clone 1E8.33) to human procollagen 11A1 has been generated. This antibody is poorly mutated, essentially in germ line configuration; its complementarity determining regions (CDRs) are especially rich in tyrosine and serine residues. The epitope recognized is encompassed in the YNYGTMESYQTEAPR amino acid stretch within the variable region of human procollagen 11A1. Human procollagens 5A1 and 11A1 are very similar. However, this antibody does not cross-react with human procollagen 5A1. In human breast tumors, only the activated peritumoral myofibroblasts show a strong intracytoplasmic staining with this antibody. As procollagen 11A1 is overexpressed in the stroma of human tumors with desmoplastic reaction, this antibody represents a valuable tool for diagnostic purposes.

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