Novel AC133 Isoforms Distinguish Circulating from Incorporated AC133 Cells during Tumor Growth.

AC133 is a surface glycoprotein first described on stem/ progenitor cells. Although AC133⁺ cells have been identified in cancer patients, their role during tumorigenesis is not well understood. In the present study, we analyzed the presence of bone-marrow (BM), circulating (blood) and tumor-incorporated (tumor biopsies) AC133⁺ cells in patients with different neoplasias: Non-Hodgkin Lymphomas (NHL), Acute Myeloid Leukemia (AML), Acute Lymphoid Leukemia (ALL), Breast cancer (BC), Prostate cancer (PC), Thyroid cancer (TC), and Colorectal carcinoma. AC133 expression was analyzed, by RT-PCR, with primers specific for AC133 mRNA coding the N-terminal tail (putative signaling domain) of the protein (exons 32–37). The number of AC133⁺ samples varied among the tested tumors, ranging from 37.5% in PC samples to 100% in TC biopsies. Three AC133-3′ mRNA isoforms, confirmed by DNA sequencing, were differentially expressed in the tested samples (blood vs. biopsies). BM and blood-derived AC133⁺ cells, of distinct origins (NHL, ALL, AML, BC), exclusively expressed AC133-3C isoform (depleted of exon 35: 262 bp). In contrast, AC133-3B, depleted of exons 34 and 35 (238 bp), was only detected in some tumor biopsies (NHL, BC and PC). AC133-3′ complete isoform (with exons 32 to 37), AC133-3A, was expressed in all positive samples. Taken together, these results imply that, regardless of the tumor type, different AC133-3′ isoforms may be used to distinguish circulating (BM-derived) AC133⁺ cells from those present within tumor tissues.

To characterize phenotipically the AC133⁺ cells in tumors, we performed flow cytometry analysis for lineage markers. Circulating AC133⁺ cells (from NHL and BC patients) coexpressed CD34 and CD45 antigens, and in BC patients, AC133⁺ KDR⁺ cells were also detected. On the other hand, tumor-incorporated AC133⁺ cells, from PC and NHL biopsies, expressed epithelial (cytokeratin antigens CAM 5.2 and MNF 116) and hematopoietic/ lymphoid (CD45/ CD19) markers, respectively. These data suggest that AC133-3′ isoforms may be used to discriminate circulating hematopoietic or vascular stem/ precursor cells (BM-derived) from tissue stem/ cancer stem cells.

To study the molecular characteristics of circulating and tumor-incorporated AC133⁺ cells we are in the process of determining their gene expression profiles by Affymetrix arrays. Preliminary results show that, when compared to cord blood derived AC133⁺ cells, NHL circulating AC133⁺ cells differ in the expression of genes involved in signaling pathways, apoptosis, cell cycle and biosynthetic pathways. Interestingly, the majority of genes differing between these 2 types of AC133⁺ cells map to chromosome region 3q, a chromosomal region usually altered in different tumor types.

This study reports for the first time that, in tumors, AC133-3′ mRNA isoforms identify lineage specific AC133⁺ cells, and so their identification may have clinical relevance. While in some tumors the presence of engrafted cancer stem cells may be important for tumor progression or response to therapy, in other cases, BM-derived AC133⁺ cells may represent stem/ progenitor cells with hematopoietic or vascular differentiation potential.