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Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds.

Stimmer L, Dehay S, Nemati F, Massonnet G, Richon S, Decaudin D, Klijanienko J, Johannes L - BMC Cancer (2014)

Bottom Line: Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04).Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates.

View Article: PubMed Central - PubMed

Affiliation: Endocytic Trafficking and Therapeutic Delivery Group, UMR3666 CNRS - U1143 INSERM, Institut Curie-Centre de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France. ludger.johannes@curie.fr.

ABSTRACT

Background: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen.

Methods: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model.

Results: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.

Conclusion: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

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Gb3 expression in fine-needle aspirates of human primary breast cancers and lymph node metastases. A, B, C, F: Fine-needle aspirates from different patients. A: Tubular structure with Gb3 (red) expression within the cytokeratin expressing epithelial cells (green); B: isolated cytokeratin expressing epithelial cells (green) with no Gb3 expression; C: low number of Gb3 (red) expressing individualized epithelial cells (green); F: Gb3 (red) expression in cells with endothelial morphology forming a capillary-like structure (center). D, E: Gb3 expression (red) in lobular structures of a primary tumor (D), and in tubular formations in lymph node metastases of the same patient (E). Note similar morphological appearance, i.e. cohesive, polygonal cells forming lobular structures in both specimens. Bars: 20 μm.
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Fig3: Gb3 expression in fine-needle aspirates of human primary breast cancers and lymph node metastases. A, B, C, F: Fine-needle aspirates from different patients. A: Tubular structure with Gb3 (red) expression within the cytokeratin expressing epithelial cells (green); B: isolated cytokeratin expressing epithelial cells (green) with no Gb3 expression; C: low number of Gb3 (red) expressing individualized epithelial cells (green); F: Gb3 (red) expression in cells with endothelial morphology forming a capillary-like structure (center). D, E: Gb3 expression (red) in lobular structures of a primary tumor (D), and in tubular formations in lymph node metastases of the same patient (E). Note similar morphological appearance, i.e. cohesive, polygonal cells forming lobular structures in both specimens. Bars: 20 μm.

Mentions: In order to assess which cells express Gb3 in FNA of primary and secondary mammary cancers we used standard cytochemical MGG labeling as well as combined immunocytochemistry and ESL techniques. Roughly 100 to 500 cells with epithelial morphology were subjected to STxB accumulation. Patients with lower cell count were excluded from the study. Cell density and morphology was judged on MGG stained slides and on ESL slides using DAPI nuclear stain. The epithelial nature of STxB-positive cells was confirmed using immunocytochemical staining for pan-cytokeratins, showing a cytoplasmic co-localization of STxB-Cy3 and AE1/AE3 signals (Figure 3A). As shown in Figure 3A, D and E, labeling by STxB was noticed mainly in epithelial cells (i.e. larger epithelial tumor cells forming lobular, tubular and/or acinic structures). Isolated malignant cells were usually less positive than clustered cells (Figure 3B and C). The STxB signal was distributed mainly in the perinuclear region, or diffusely throughout cytoplasm, showing fine granular appearance. Furthermore, STxB staining was less frequently observed in neutrophils and rare in macrophages. Here, the signal was mostly cytoplasmic and appeared coarse granular. Structures with capillary morphology, which were sometimes seen, were strongly positive for STxB labeling (Figure 3F).Figure 3


Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds.

Stimmer L, Dehay S, Nemati F, Massonnet G, Richon S, Decaudin D, Klijanienko J, Johannes L - BMC Cancer (2014)

Gb3 expression in fine-needle aspirates of human primary breast cancers and lymph node metastases. A, B, C, F: Fine-needle aspirates from different patients. A: Tubular structure with Gb3 (red) expression within the cytokeratin expressing epithelial cells (green); B: isolated cytokeratin expressing epithelial cells (green) with no Gb3 expression; C: low number of Gb3 (red) expressing individualized epithelial cells (green); F: Gb3 (red) expression in cells with endothelial morphology forming a capillary-like structure (center). D, E: Gb3 expression (red) in lobular structures of a primary tumor (D), and in tubular formations in lymph node metastases of the same patient (E). Note similar morphological appearance, i.e. cohesive, polygonal cells forming lobular structures in both specimens. Bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289340&req=5

Fig3: Gb3 expression in fine-needle aspirates of human primary breast cancers and lymph node metastases. A, B, C, F: Fine-needle aspirates from different patients. A: Tubular structure with Gb3 (red) expression within the cytokeratin expressing epithelial cells (green); B: isolated cytokeratin expressing epithelial cells (green) with no Gb3 expression; C: low number of Gb3 (red) expressing individualized epithelial cells (green); F: Gb3 (red) expression in cells with endothelial morphology forming a capillary-like structure (center). D, E: Gb3 expression (red) in lobular structures of a primary tumor (D), and in tubular formations in lymph node metastases of the same patient (E). Note similar morphological appearance, i.e. cohesive, polygonal cells forming lobular structures in both specimens. Bars: 20 μm.
Mentions: In order to assess which cells express Gb3 in FNA of primary and secondary mammary cancers we used standard cytochemical MGG labeling as well as combined immunocytochemistry and ESL techniques. Roughly 100 to 500 cells with epithelial morphology were subjected to STxB accumulation. Patients with lower cell count were excluded from the study. Cell density and morphology was judged on MGG stained slides and on ESL slides using DAPI nuclear stain. The epithelial nature of STxB-positive cells was confirmed using immunocytochemical staining for pan-cytokeratins, showing a cytoplasmic co-localization of STxB-Cy3 and AE1/AE3 signals (Figure 3A). As shown in Figure 3A, D and E, labeling by STxB was noticed mainly in epithelial cells (i.e. larger epithelial tumor cells forming lobular, tubular and/or acinic structures). Isolated malignant cells were usually less positive than clustered cells (Figure 3B and C). The STxB signal was distributed mainly in the perinuclear region, or diffusely throughout cytoplasm, showing fine granular appearance. Furthermore, STxB staining was less frequently observed in neutrophils and rare in macrophages. Here, the signal was mostly cytoplasmic and appeared coarse granular. Structures with capillary morphology, which were sometimes seen, were strongly positive for STxB labeling (Figure 3F).Figure 3

Bottom Line: Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04).Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates.

View Article: PubMed Central - PubMed

Affiliation: Endocytic Trafficking and Therapeutic Delivery Group, UMR3666 CNRS - U1143 INSERM, Institut Curie-Centre de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France. ludger.johannes@curie.fr.

ABSTRACT

Background: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen.

Methods: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model.

Results: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor.

Conclusion: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

Show MeSH
Related in: MedlinePlus