Immunobiological properties of cancer stem cells isolated from colorectal cancer patients

Colorectal cancer (CRC) is the third most common form of cancer and the second cause of cancer-related death in the Western world. Despite the emergence of new targeted agents and the use of various therapeutic combinations, none of the treatments options available is curative in patients with advanced cancer. In the last decade, several studies have shown that a small population of “stem cell like” cells, with the capacity for self-renewal, multipotency and high tumorigenic potential, could be isolated from tumors with different histological origin, including human CRC. These peculiar stem cells are believed to be responsible for tumor initiation, progression and resistance to therapeutic agents. Therefore, treatments that are design to eradicate tumor masses should be also targeted to CSCs. Recently in our laboratory cancer stem cells (CSCs) lines and their fetal bovine serum (FBS)-cultured non-CSC pair lines were in vitro isolated from surgical specimens of patients with primary CRC cancer. In this study we performed an immunobiological characterization of CRC CSCs and investigated whether these cells can represent suitable targets for immunotherapy protocols. The phenotype and functional characterization of these cell lines was examined by IF and cytofluorimetric analysis. Firstly the expression levels of CRC cancer and/or stem cell-associated molecules such as: Ep-CAM, CEA, HCAM, CD44, Aldefluor, CD133, SOX-2, Nanog and Oct-4 was assessed. Notably, the tumorigenic potency of CSCs was higher than that of FBS cells as shown by the serial dilutions and serial transplantations cell injection in immunodeficient mice. Moreover the xenografts generated by CRC CSC showed large areas of necrosis than tumor tissues generated by FBS cells and better matched the phenotype of the original tumor, as revealed by an IHC analysis of CRC and CSC-associated molecules. In addition, the expression of MHC-I and MHC-II molecules, NKG2D ligands, immune regulatory molecules, antigen-processing machinery (APM) and tumor associated antigens (TAA) was examined. Both CRC CSC and FBS lines were found weakly positive for MHC-I and negative for MHC-II, while NKG2D ligands were commonly detected in both cells types. The APM was mainly defective in CRC CSC versus FBS cell lines. We also investigated whether the expression of immune-regulatory molecules (CTLA-4, PD-1, B7-H3) can affect the immunogenicity of these cells. All CSCs and FBS cell lines were homogeneously positive for CTLA-4, PD-1 and, although at higher levels for PD-L1 and B7-H3. All these molecules acts as negative modulator of T-cells response because of the inhibition of the vital ‘second signal’ required for optimal T cell recognition and activation. Interestingly, IL-4 was expressed by all the cell lines both in the cytoplasm and in the membrane while IL-4R was weakly detectable on the membrane of CSC and was absent in FBS lines counterparts. These data may confirm the possibility of an autocrine production by CRC CSC and also FBS line of IL-4, that may thus protect themselves by apoptosis as recently described by Stassi’s group (Todaro M et al, 2007), this citockine may also induce an immunomodulatory role for T-cells mediated responses against CRC. Unexpectedly FBS tumor lines released in the supernatants high levels of prostaglandin-E2 (PGE2) that, on the contrary was weakly or absent in the supernatant of CSCs. In addition, T lymphocytes isolated from the peripheral blood of CRC patients were stimulated in vitro with autologous CSCs or FBS, in order to assess of anti-CRC reactivity. In patient #1076 we could isolate mostly TH1-mediated responses (detected by INF-γ release), showing weak reactivity against CSCs. On the contrary a more efficiently INF-γ release was detectable when T-lymphocytes were stimulated with FBS tumor cells. In a second patient (#1247), a MHC-independent recognition of CSCs was observed, suggesting an NK-mediated response. This result was also confirmed by the phenotype analysis of these lymphocytes that revealed the presence of 20-30% NK cells (CD3-CD16*CD56+).These findings correlate with the defective expression of molecules involved in antigen presenting and processing machinery observed in CSCs. Taken together our data we can conclude that we have isolate from CRC tissues cells with stemness properties, thus showing a lower immunogenicity compared with the FBS counterpart. T cell responses could be obtained in CRC patients directed against CSC and FBS cells, though with higher reactivity for FBS tumor cells, while in another patient NK-mediated responses could be isolated These findings may be useful for the identification of new agents that can efficiently rescue the immunogenicity of CSCs in order to be targeted by T cell-mediated immune response.