Gemei, Marica (2012) Isolation and characterization of cancer stem cell subsets by identification of new molecular determinants in colon cancer. [Tesi di dottorato] (Inedito)

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Abstract

The initial aim of our study was to dissect the mosaic of cell populations within the CD133+ putative stem cell compartment in human colon cancer. While addressing this question we built up a panel of 20 monoclonal antibodies chosen on the basis of information provided by selecting in a literature search molecules somewhat involved in the overall malignant behavior of tumor cells. We used an innovative seven-color flow cytometric approach to characterize cells obtained from the mechanic and enzymatic digestion of fresh normal colon and colon cancer tissues. We subdivided our panel into different tubes analyzing two selected markers for each tube and using other fluorescence channels available to keep fixed two putative stem cell markers (CD133 and CD44), a hematopoietic specificity (CD45), an epithelial specific marker (CD326) and a vital dye (Sytox blue). Tumoral population to be analyzed was selected as CD45-/CD326+/Sytox Blue-. This gating was fundamental for the subsequent evaluation of the other markers because of the amount of contaminants composing the cell suspension derived from the digestion of a fresh tissue. These contaminants could easily confuse results leading to ambiguous, misleading and incorrect data. A first analysis of our data revealed a distribution of analyzed markers into three main categories: 1) highly expressed antigens, 2) bimodally expressed antigens and 3) low expressed antigens. A first interesting finding was the observation of a small subset of double positive CD133+/CD44+ cells. The existence of this subset could explain similar results obtained by the three groups which first isolated colorectal cancer stem cells using apparently different approaches (CD133+ or CD44+/CD326+). At the same time we observed this population rapidly appeared in literature studies enforcing the idea that the CD133+/CD44+ population could represent a more purified stem cell subset in human colon cancer. We analyzed the presence of CD133+, CD44+ and CD133+/CD44+ cells in 36 patients correlating these markers to tumor progression and disease free survival. From our analysis CD133 alone seems to be correlated to tumor progression while only the CD133+/CD44+ subset’s presence affected disease free survival in the analyzed patients, suggesting that this cell population was not the main responsible for tumor growth but it was involved in cancer recurrence. From a first analysis of the expression of the analyzed markers another molecule got our attention: CD66c. CD66c is a GPI-linked molecule belonging to the family of CEA antigen known to be involved in carcinogenesis and in colon cancer pathogenesis, however no complete and exhaustive informations are available. From our data, CD66c showed an extremely reproducible behavior and interesting correlations with other cancer related molecules. Its expression correlated with that of all proposed colon cancer stem cell markers (CD133, CD44, CD166, CD24). A correlation was also observed with CD151 (PETA3, involved in the extravasion of cancerous cells), CD227 (MUC1, known as prognostic factor in colon cancer), CD182 (CXCR2, involved in cell migration), CD221 (insulin-like growth factor 1, linked to cell proliferation) and CD55 (Decay accelerating factor, a complement inhibiting molecule). We carefully analyzed the expression of CD66c in normal colon and in colon cancer tissues finding this antigen to be a cancer associate molecule. In fact it was significantly expressed at higher levels in cancer than in normal tissue. We also found a gradient of expression from normal tissues to adenomas and to carcinomas with an increase related to the severity of the lesion. As expected a significantly higher expression was also observed in advanced stages (T3-4 and M1) of colon cancer and in in vitro sphere cultures, which promote the proliferation of stem-like cells. We further analyzed CD66c expression in CD133+ putative stem cell compartment and found a surprisingly sharp distinction between normal and cancer tissues. In normal tissues CD133+ cells are completely CD66c negative while a correlation between the two antigens was observed in cancerous samples. When CD66c was silenced in Caco2 cells, which express high levels of this molecule, their proliferation resulted slowed down while their apoptosis and necrosis appeared increased. Moreover, the in vitro clonogenic and tumorigenic potential of Caco2 cells resulted impaired. Initial hints about the possible use of CD66c as a therapeutic target are suggested by the cCD66c mRNA silencing experiments in Caco2 cells, further studies will be needed to unveil this possibility.

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