The B subunit of bacterial
Shiga toxin (STxB) is nontoxic and has low immunogenicity. Its receptor, the
glycosphingolipid Gb3/CD77, is overexpressed on the cell surface of human
colorectal cancer. We tested whether genetic porcine models, closely resembling human anatomy and pathophysiology, can be used to exploit the
tumor-targeting potential of STxB. In accordance with findings on human
colorectal cancer, the pig model APC1311 bound STxB in
colorectal tumors, but not in normal colon or jejunum, except for putative enteroendocrine cells. In primary
tumor cells from endoscopic biopsies, STxB was rapidly taken up along the retrograde intracellular route to the Golgi, whereas normal colon organoids did not bind or internalize STxB. Next, we tested a porcine model (TP53LSL-R167H) for
osteosarcoma, a
tumor entity with a dismal prognosis and insufficient treatment options, hitherto not known to express Gb3. Pig
osteosarcoma strongly bound StxB and expressed the
Gb3 synthase 1,4-galactosyltransferase (A4GALT). Primary
osteosarcoma cells, but not normal osteoblasts, rapidly internalized fluorescently labeled STxB along the retrograde route to the Golgi. Importantly, six of eight human
osteosarcoma cell lines expressed A4GALT
mRNA and showed prominent intracellular uptake of STxB. The physiologic role of A4GALT was tested by CRISPR/Cas9 mutagenesis in porcine LLC-PK1 kidney epithelial cells and RNAi in MG-63 human
osteosarcoma cells. A4GALT deficiency or knockdown abolished STxB uptake and led to significantly reduced cell migration and proliferation, hinting toward a putative
tumor-promoting role of Gb3. Thus, pig models are suitable tools for STxB-based
tumor targeting and may allow "reverse-translational" predictions on human
tumor biology.