<p>Targeted Toxin Gene Therapy Of Breast Cancer Stem Cells Using CXCR1 Promoter And bFGF 5′UTR</p>

PE38-based gene therapy of HER2-positive breast cancer stem cells via VHH-redirected polyamidoamine dendrimers

Scientific Reports ◽

10.1038/s41598-021-93972-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Cobra Moradian ◽

Fatemeh Rahbarizadeh

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Gene Therapy ◽

Growth Factor ◽

Cancer Stem Cells ◽

Toxin Gene ◽

Breast Cancer Stem Cells ◽

Hanging Drop ◽

Her2 Positive ◽

Mammosphere Formation

AbstractBreast cancer stem cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2+ BCSCs. Plasmids containing CXCR1 promoter, PE38 toxin, and 5′UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with anti-HER2 VHHs were used for plasmid delivery. Stem cell proportion of MDA-MB-231, MDA-MB-231/HER2+ and MCF-10A were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression, and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. MDA-MB-231/HER2+ had higher ability to form mammosphere compared to MCF-10A. Our multi-targeted nanosystem efficiently inhibited the mammosphere formation of MDA-MB-231 and MDA-MB-231/HER2+ cells, while it was unable to prevent the mammosphere formation of MCF-10A. In the hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression, and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However, the efficacy of our targeted toxin gene therapy was lower in MDA-MB-231/HER2+ spheroids compared with that in the monolayer culture. the combination of the cell surface, transcriptional, and translational targeting increased the stringency of the treatment.

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Combined transcriptional, translational and cell surface targeted gene therapy of HER2-positive breast cancer stem cells in three-dimentional cell culture

10.21203/rs.3.rs-184507/v1 ◽

2021 ◽

Author(s):

Cobra Moradian ◽

Fatemeh Rahbarizadeh

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Gene Therapy ◽

Growth Factor ◽

Cancer Stem Cells ◽

Monolayer Culture ◽

Breast Cancer Stem Cells ◽

Hanging Drop ◽

Her2 Positive ◽

Targeted Gene Therapy

Abstract Purpose Breast Cancer Stem Cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2-positive BCSCs. Methods Plasmids containing CXCR1 promoter, PE38 toxin and 5′UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with an anti-HER2 VHH were used for plasmid delivery. Stem proportion of MCF-10A and MDA-MB-231/HER2+ (a cellular model of HER2 overexpression, developed in our lab) were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. Results MDA-MB-231/HER2+ had more than 3 times higher ability to form mammosphere compared to MCF-10A, representing higher stem proportion in MDA-MB-231/HER2+. In hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However efficacy of our targeted toxin gene therapy were lower in MDA-MB-231/HER2+ spheroid than those in monolayer. Conclusions Our novel multi-targeted nanosystem resulted in synergistic cytotoxic effects in HER2-positive BCSCs. This results demonstrate the necessity to use a combinatorial tumor targeting approach, to restrict the expression of a killer gene to target cells. Lower efficacy of our targeted gene therapy in spheroid models vs. monolayer culture suggest that, anticancer therapy assessments using spheroid might be more predictive of clinical efficacy than conventional monolayer culture.

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