Human colorectal tumors and metastases express Gb3 and can be targeted by an intestinal pathogen-based delivery tool.

The targeting of solid tumors requires delivery tools that resist intracellular and extracellular inactivation, and that are taken up specifically by tumor cells. We have shown previously that the recombinant nontoxic B-subunit of Shiga toxin (STxB) can serve as a delivery tool to target digestive tumors in animal models. The aim of this study was to expand these experiments to human colorectal cancer. Tissue samples of normal colon, benign adenomas, colorectal carcinomas, and liver metastases from 111 patients were obtained for the quantification of the expression of the cellular STxB receptor, the glycosphingolipid globotriaosyl ceramide (Gb(3) or CD77). We found that compared with normal tissue, the expression of Gb(3) was strongly increased in colorectal adenocarcinomas and their metastases, but not in benign adenomas. Short-term primary cultures were prepared from samples of 43 patients, and STxB uptake was studied by immunofluorescence microscopy. Of a given tumor sample, on average, 80% of the cells could visibly bind STxB, and upon incubation at 37 degrees C, STxB was transported to the Golgi apparatus, following the retrograde route. This STxB-specific intracellular targeting allows the molecule to avoid recycling and degradation, and STxB could consequently be detected on tumor cells even 5 days after initial uptake. In conclusion, the targeting properties of STxB could be diverted for the delivery of contrast agents to human colorectal tumors and their metastases, whose early detection and specific targeting remains one of the principal challenges in oncology.

Tumor-specific targeting of pancreatic cancer with Shiga toxin B-subunit.

Pancreatic carcinoma is one of the most aggressive tumor entities, and standard chemotherapy provides only modest benefit. Therefore, specific targeting of pancreatic cancer for early diagnosis and therapeutic intervention is of great interest. We have previously shown that the cellular receptor for Shiga toxin B (STxB), the glycosphingolipid globotriaosylceramide (Gb(3) or CD77) is strongly increased in colorectal adenocarcinoma and their metastases. Here, we report an upregulation of Gb(3) in pancreatic adenocarcinoma (21 of 27 cases) as compared with matched normal tissue (n = 27). The mean expression was highly significantly increased from 30 ± 16 ng Gb(3)/mg tissue in normal pancreas to 61 ± 41 ng Gb(3)/mg tissue (mean ± SD, P = 0.0006), as evidenced by thin layer chromatography. Upregulation of Gb(3) levels did not depend on tumor stage or grading and showed no correlation with clinical outcome. Tumor cells and endothelial cells were identified as the source of increased Gb(3) expression by immunocytochemistry. Pancreatic cancer cell lines showed rapid intracellular uptake of STxB to the Golgi apparatus, following the retrograde pathway. The therapeutic application of STxB was tested by specific delivery of covalently coupled SN38, an active metabolite of the topoisomerase I inhibitor irinotecan. The cytotoxic effect of the STxB-SN38 compound in pancreatic cancer cell lines was increased more than 100-fold compared with irinotecan. Moreover, this effect was effectively blocked by competing incubation with nonlabeled STxB, showing the specificity of the targeting. Thus, STxB constitutes a promising new tool for specific targeting of pancreatic cancer.

Synthesis and properties of a mitochondrial peripheral benzodiazepine receptor conjugate.

Peripheral benzodiazepine receptors are potential targets for cancer therapeutics through the use of specific ligands such as the pro-apoptotic benzodiazepine RO5-4864. However, the poor water solubility of this compound has been a limitation to its application in vivo. Herein we describe an efficient synthesis for the conjugation, via a cleavable linker arm, of RO5-4864 to a novel tumour-delivery tool, the B-subunit of Shiga toxin (STxB). The conjugate is water soluble and specifically targets cancer cells that overexpress the glycolipid Gb3, the cellular Shiga toxin receptor that is found on several human tumours. After internalisation via retrograde transport, the prodrug is cleaved inside cells to release the active principle. Delivery by STxB therefore increases the cytotoxic activity of RO5-4864 and its tumour specificity.

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)3 selectively potentiates its photodynamic activity.

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)3 into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)3 and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.