Effect of Cellular and Microenvironmental Multidrug Resistance on Tumor-Targeted Drug Delivery in Triple-Negative Breast cancer.

Multidrug resistance (MDR) reduces the efficacy of chemotherapy. Besides inducing the expression of drug efflux pumps, chemotherapy treatment alters the composition of the tumor microenvironment (TME), thereby potentially limiting tumor-directed drug delivery. To study the impact of MDR signaling in cancer cells on TME remodeling and nanomedicine delivery, we generated multidrug-resistant 4T1 triple-negative breast cancer (TNBC) cells by exposing sensitive 4T1 cells to gradually increasing doxorubicin concentrations. In 2D and 3D cell cultures, resistant 4T1 cells are presented with a more mesenchymal phenotype and produced increased amounts of collagen. While sensitive and resistant 4T1 cells showed similar tumor growth kinetics in vivo, the TME of resistant tumors was enriched in collagen and fibronectin. Vascular perfusion was also significantly increased. Fluorophore-labeled polymeric (∼10 nm) and liposomal (∼100 nm) drug carriers were administered to mice with resistant and sensitive tumors. Their tumor accumulation and penetration were studied using multimodal and multiscale optical imaging. At the whole tumor level, polymers accumulate more efficiently in resistant than in sensitive tumors. For liposomes, the trend was similar, but the differences in tumor accumulation were insignificant. At the individual blood vessel level, both polymers and liposomes were less able to extravasate out of the vasculature and penetrate the interstitium in resistant tumors. In a final in vivo efficacy study, we observed a stronger inhibitory effect of cellular and microenvironmental MDR on liposomal doxorubicin performance than free doxorubicin. These results exemplify that besides classical cellular MDR, microenvironmental drug resistance features should be considered when aiming to target and treat multidrug-resistant tumors more efficiently.

Pancreatic tumor microenvironmental acidosis and hypoxia transform gold nanorods into cell-penetrant particles for potent radiosensitization.

Coating nanoparticles with stealth epilayers increases circulation time by evading opsonization, macrophage phagocytosis, and reticuloendothelial sequestration. However, this also reduces internalization by cancer cells upon reaching the tumor. We designed gold nanorods (GNRs) with an epilayer that retains stealth properties in circulation but transforms spontaneously in the acidotic tumor microenvironment to a cell-penetrating particle. We used a customized stoichiometric ratio of l-glutamic acid and l-lysine within an amphiphilic polymer of poly(l-glutamic acid-co-l-lysine), or P(Glu-co-Lys), to effect this transformation in acidotic environments. P(Glu-co-Lys)-GNRs were internalized by cancer cells to facilitate potent in vitro radiosensitization. When administered intravenously in mice, they accumulate in the periphery and core of tumors without any signs of serum biochemical or hematological alterations, normal organ histopathological abnormalities, or overt deterioration in animal health. Furthermore, P(Glu-co-Lys)-GNRs penetrated the tumor microenvironment to accumulate in the hypoxic cores of tumors to potently radiosensitize heterotopic and orthotopic pancreatic cancers in vivo.

Differential regulation of Lipocalin 2 (LCN2) in doxorubicin-resistant 4T1 triple negative breast cancer cells.

Chemoresistance is a multifactorial and complex phenomenon, leading to re-adjustment of several intracellular signaling pathways and expression patterns which compromises the efficacy of cancer drug chemo-therapy. Via comparative analysis of sensitive and doxorubicin-resistant 4T1 cells, here we show that Lipocalin 2 (LCN2) is downregulated at the mRNA and protein level in resistant cells. The pro-inflammatory cytokine, IL-1ß was found to be a potent inducer of LCN2 expression most likely involving STAT3 activation. Upregulation in both sensitive and resistant 4T1 cells argues against complete silencing of the LCN2 gene. Coinciding with LCN2 downregulation, we observed an increased activation of bone morphogenetic protein (BMP)-signaling in resistant cells, as evidenced by higher Smad1/5/9 phosphorylation and Id1 target gene expression. Blockade of the BMP-pathway by Dorsomorphin increased the expression of LCN2. Conversely, BMP2, which is known to be a pro-tumorigenic ligand in breast cancer, potently inhibited LCN2 expression at both the mRNA and protein level in resistant cells. These findings indicate that in doxorubicin-resistant 4T1 cells, LCN2 expression is negatively regulated by BMP signaling.

Vimentin silencing effect on invasive and migration characteristics of doxorubicin resistant MCF-7 cells.

Chemotherapy is one of the well-known treatments in cancer therapy. The effectiveness of chemotherapy is limited by several factors one of which is the emergence of multidrug resistance (MDR). One of the major mechanisms of MDR is the activity of several ATP binding cassette (ABC) transporters that pump drugs out of the cells. Doxorubicin intercalates and inhibits DNA replication; it is a powerful chemotherapeutic agent. However, it causes development of MDR in tumor cells. Vimentin is a type III intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and also poor prognosis of cancer. There are several studies that have shown the connection between expression level of vimentin and invasiveness of tumor cells. In this study, MCF-7 cell line which is a model for human mammary carcinoma, and a doxorubicin resistant subline (MCF-7/Dox) were used. The resistant subline was previously obtained by stepwise selection in our laboratory. In the resistant cells, high levels of vimentin expression were observed. The main purpose of this study was to investigate changes in invasive and migration characteristics of MCF-7/Dox cell line, after transient silencing of vimentin gene by specific siRNA.