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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 mice with HBCx. Upper part: STxB-Cy3 accumulation in tumor and organs after ESL or intravenous injection. A, B: Gb3 expression in fine-needle aspirates of HBCx with accumulation of STxB-Cy3 in tubular structures (A), or individualized cells (B). C: Diffuse and high expression of Gb3 in tubular structures of renal medulla, D: Diffuse and low expression in liver parenchyma in cells with endothelial morphology lining liver sinusoids, E: Gb3 expression in HBCx. Note a diffuse and high STxB-Cy3 accumulation in tumor cells with epithelial morphology compared to the absence of Gb3 expressing cells in tumor stroma (*). Lower part: FACS analysis of Gb3 expressing tumor cells in different HBCx. Note the lack of Gb3 positivity in HBCx-11 (left), the presence of two populations of Gb3 expressing cells in HBCx-52 (middle), and of one Gb3 positive population in HBCx-162 (right).
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Fig4: Gb3 expression in mice with HBCx. Upper part: STxB-Cy3 accumulation in tumor and organs after ESL or intravenous injection. A, B: Gb3 expression in fine-needle aspirates of HBCx with accumulation of STxB-Cy3 in tubular structures (A), or individualized cells (B). C: Diffuse and high expression of Gb3 in tubular structures of renal medulla, D: Diffuse and low expression in liver parenchyma in cells with endothelial morphology lining liver sinusoids, E: Gb3 expression in HBCx. Note a diffuse and high STxB-Cy3 accumulation in tumor cells with epithelial morphology compared to the absence of Gb3 expressing cells in tumor stroma (*). Lower part: FACS analysis of Gb3 expressing tumor cells in different HBCx. Note the lack of Gb3 positivity in HBCx-11 (left), the presence of two populations of Gb3 expressing cells in HBCx-52 (middle), and of one Gb3 positive population in HBCx-162 (right).

Mentions: Breast cancer xenografts are useful tools for preclinical assessment of new treatments because of their similar molecular profile to the corresponding patient tumors [26]. Recent studies with these and similar breast cancer xenografts completed in our institution show that human breast cancer xenografts maintain the overall histologic, genomic and gene expression profile of the corresponding patient tumors and remains stable throughout sequential in vivo generations [20, 27]. ESL and FACS used for the evaluation of Gb3 expression in 11 HBCx. Concordant results between ESL and FACS were found in all tested HBCx: 6 were STxB-positive, and 5 negative (Table 2). Furthermore, using flow cytometry it was found that 4 out of 6 positive HBCx had two different tumor cell populations with high or low Gb3 expression levels. As illustrated in Figure 4A, Gb3 expressing cells were mainly morphologically well differentiated (i.e. cohesive polygonal epithelial cells forming lobular structures) and showed granular, often perinuclear intracytoplasmic labeling. Furthermore, less differentiated cells (i.e. groups of cells without particular formation) less frequently accumulated STxB (Figure 4B). In addition, 8 HBCx were available for Gb3 extraction. ESL and Gb3 extraction showed concordant results in 6 from 8 tested HBCx, including 4 positive and 2 negative xenografts. 2 HBCx were positive in Gb3 extraction and negative in ESL (Table 2). ER, PR and HER2 status was studied in all xenografts and the results are summarized in Table 2. Tested panel of HBCx included 1 ER+/PR-/HER2- and 2 HER2+ xenografts as well as 8 triple negative HBCx. There were no correlation between ER, PR or HER2 status and Gb3 expression.Table 2


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 mice with HBCx. Upper part: STxB-Cy3 accumulation in tumor and organs after ESL or intravenous injection. A, B: Gb3 expression in fine-needle aspirates of HBCx with accumulation of STxB-Cy3 in tubular structures (A), or individualized cells (B). C: Diffuse and high expression of Gb3 in tubular structures of renal medulla, D: Diffuse and low expression in liver parenchyma in cells with endothelial morphology lining liver sinusoids, E: Gb3 expression in HBCx. Note a diffuse and high STxB-Cy3 accumulation in tumor cells with epithelial morphology compared to the absence of Gb3 expressing cells in tumor stroma (*). Lower part: FACS analysis of Gb3 expressing tumor cells in different HBCx. Note the lack of Gb3 positivity in HBCx-11 (left), the presence of two populations of Gb3 expressing cells in HBCx-52 (middle), and of one Gb3 positive population in HBCx-162 (right).
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Related In: Results  -  Collection

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Fig4: Gb3 expression in mice with HBCx. Upper part: STxB-Cy3 accumulation in tumor and organs after ESL or intravenous injection. A, B: Gb3 expression in fine-needle aspirates of HBCx with accumulation of STxB-Cy3 in tubular structures (A), or individualized cells (B). C: Diffuse and high expression of Gb3 in tubular structures of renal medulla, D: Diffuse and low expression in liver parenchyma in cells with endothelial morphology lining liver sinusoids, E: Gb3 expression in HBCx. Note a diffuse and high STxB-Cy3 accumulation in tumor cells with epithelial morphology compared to the absence of Gb3 expressing cells in tumor stroma (*). Lower part: FACS analysis of Gb3 expressing tumor cells in different HBCx. Note the lack of Gb3 positivity in HBCx-11 (left), the presence of two populations of Gb3 expressing cells in HBCx-52 (middle), and of one Gb3 positive population in HBCx-162 (right).
Mentions: Breast cancer xenografts are useful tools for preclinical assessment of new treatments because of their similar molecular profile to the corresponding patient tumors [26]. Recent studies with these and similar breast cancer xenografts completed in our institution show that human breast cancer xenografts maintain the overall histologic, genomic and gene expression profile of the corresponding patient tumors and remains stable throughout sequential in vivo generations [20, 27]. ESL and FACS used for the evaluation of Gb3 expression in 11 HBCx. Concordant results between ESL and FACS were found in all tested HBCx: 6 were STxB-positive, and 5 negative (Table 2). Furthermore, using flow cytometry it was found that 4 out of 6 positive HBCx had two different tumor cell populations with high or low Gb3 expression levels. As illustrated in Figure 4A, Gb3 expressing cells were mainly morphologically well differentiated (i.e. cohesive polygonal epithelial cells forming lobular structures) and showed granular, often perinuclear intracytoplasmic labeling. Furthermore, less differentiated cells (i.e. groups of cells without particular formation) less frequently accumulated STxB (Figure 4B). In addition, 8 HBCx were available for Gb3 extraction. ESL and Gb3 extraction showed concordant results in 6 from 8 tested HBCx, including 4 positive and 2 negative xenografts. 2 HBCx were positive in Gb3 extraction and negative in ESL (Table 2). ER, PR and HER2 status was studied in all xenografts and the results are summarized in Table 2. Tested panel of HBCx included 1 ER+/PR-/HER2- and 2 HER2+ xenografts as well as 8 triple negative HBCx. There were no correlation between ER, PR or HER2 status and Gb3 expression.Table 2

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