Monoclonal Antibodies Against Lgr5 Identify Human Colorectal Cancer Stem Cells

Abstract

In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target Lgr5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence-activated cell sorting-based detection of Lgr5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam 1 Lgr5 1 cells. Similarly, primary CRC-derived spheroid cultures, known to be enriched for CSCs, contain high levels of Lgr5 1 cells, which decrease upon in vitro differentiation of these CSCs. Selection of the Lgr5 high CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that Lgr5 expression is dependent on the Wnt pathway and show that Lgr5 overexpression induces clonogenic growth. We thus provide evidence that Lgr5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs.

Cancer-Initiating Cells from Colorectal Cancer Patients Escape from T Cell–Mediated Immunosurveillance In Vitro through Membrane-Bound IL-4

“…Surrogate markers associated with stem cell properties, such as CD133, CD24, CD44, and ALDH-1, have been commonly used for CRC CIC detection, although controversial results with the stem cell properties of these cells have been reported (1, 2, 21-23, 29). More recently for the first time a potential stem cell-specific marker, Lgr5, has been identified for both normal and tumor colorectal tissues (24). Of interest, our sphere-forming cells were homogeneously positive for the expression of this molecule, thus indicating that indeed these cells displayed CIC properties.…”

“…The existence within human CRCs of a subpopulation of cancer cells with tumor intiating/stem-like properties has been widely documented (1)(2)(3)(20)(21)(22)(23)(24). However, the detailed characterization and identification of the CIC antigenic/immunogenic profile have not yet been accomplished.…”

“…Moreover, the sphereforming cells homogeneously expressed (85-98% of positive cells; MRFI: 5-9; representative results from #1076 and 1247 are shown in Supplemental Fig. 1D, 1E) the colon stem cell-specific marker Lgr5 (24), whereas FBS tumor cells showed weak or negative expression, with 11-13% of positive cells that did not show statistically significant differences in the mean fluorescence intensity (MRFI = 1.4-1.6), compared with the negative control (Supplemental Fig. 1F, 1G).…”

“…The IF and cytofluorimetric or confocal microscopy analyses of a panel of TAAs previously reported as expressed by CRC (e.g., MAGE, NY-ESO-1, Gp100, SVV-1, COA-1, and IL-13Ra2) (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) revealed that all CICs and FBS tumor cells from patients 1076, 1247, 111011 and 14583 were negative for MAGE and Gp100 and expressed low levels (MRFI = 2-4) of NY-ESO-1 and IL-13Ra2 (data not shown). Sox2, Ep-CAM, and CEA, which in addition to their role as markers associated with CICs or CRC-derived CICs (Supplemental Fig.…”

Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-β

“…Importantly, this population dramatically expands after Apc deletion and produces differentiating progenies (13). Furthermore, recent results suggest that Lgr5 also marks a stem cell population with clonogenic potential in human CRC (42,43). Although additional stem cell populations marked with lumen-forming ability exist in human CRC (44,45), the Lgr5 + cells critically contribute to the initiation and growth of colorectal adenomas.…”