Anti-CD44 monoclonal antibody, clone IM7

Breast cancer is not only one of the most prevalent types of cancer, but also it is a prime cause of death in women aged between 20 and 59. Although chemotherapy is the most common therapy approach, multiple side effects can result from lack of specificity and the use of overdose as safe doses may not completely cure cancer. Therefore, we aimed in this study is to combine the merits of NF-kappa B inhibiting potential of celastrol (CST) with glutathione inhibitory effect of sulfasalazine (SFZ) which prevents CST inactivation and thus enhances its anti-tumor activity. Inspired by the CD44-mediated tumor targeting effect of the hydrophilic polysaccharide chondroitin sulphate (ChS), we chemically synthesized amphiphilic zein-ChS micelles. While the water insoluble SFZ was chemically coupled to zein, CST was physically entrapped within the hydrophobic zein/SFZ micellar core. Moreover, physical encapsulation of oleic acid-capped SPIONs in the hydrophobic core of micelles enabled both magnetic tumor targeting as well as MRI theranostic capacity. Combining magnetic targeting to with the active targeting effect of ChS resulted in enhanced cellular internalization of the micelles in MCF-7 cancer cells and hence higher cytotoxic effect against MCF-7 and MDA-MB-231 breast cancer cells. In the in vivo experiments, magnetically-targeted micelles (154.4 nm) succeeded in achieving the lowest percentage increase in the tumor volume in tumor bearing mice, the highest percentage of tumor necrosis associated with significant reduction in the levels of TNF-alpha, Ki-67, NF-kappa B, VEGF, COX-2 markers compared to non-magnetically targeted micelles-, free drug-treated and positive control groups. Collectively, the developed magnetically targeted micelles pave the way for design of cancer nano-theranostic drug combinations.

We previously demonstrated that both CD44 and TLR4, but predominately TLR4, mediated HA stimulation of Lgr(5+) stem cell proliferation, crypt fission, and intestinal growth in postnatal mice. Here we address the questions of which cell type expresses the relevant TLR4 in driving intestinal growth and what are the downstream events from TLR4 activation. Studies were done in 14-day-old mice: wild type (WT), mice deficient in cyclooxygenase 2 (COX2), mice deficient in myeloid cell TLR4, and mice deficient in epithelial cell epidermal growth factor receptor (EGFR). Biological end points included crypt fission and Lgr5 cell proliferation. In WT mice, treatment with NS-398 (a COX2 inhibitor), clodronate (a macrophage-depleting agent), or tyrphostin (an EGFR inhibitor) resulted in 30% reductions in crypt fission and Lgr5(+) stem cell proliferation compared with control mice. Mice deficient in COX2 or myeloid TLR4 or epithelial cell EGFR all had 30% reductions in crypt fission and Lgr5(+) stem cell proliferation compared with WT mice. Administration of dimethyl PGE2, a stable PGE2 analog, increased crypt fission and Lgr5(+) stem cell proliferation. Administration of dimethyl PGE2 reversed the effects of NS-398, clodronate, COX2 deficiency, and myeloid TLR4 deficiency but had no effect on mice treated with tyrphostin or mice deficient in epithelial cell EGFR. We conclude that, in postnatal mice, similar to 30% of intestinal growth as manifested by crypt fission and Lgr5(+) stem cell proliferation is driven by a novel pathway: Extracellular HA binds TLR4 on pericryptal macrophages, inducing the production of PGE2 through COX2. PGE2 transactivates EGFR in Lgr5(+) epithelial stem cells, resulting in Lgr5(+) stem cell proliferation and crypt fission. NEW & NOTEWORTHY This study, in newborn mice, describes a novel molecular pathway regulating Lgr5(+) epithelial stem cell proliferation and normal intestinal elongation, as assessed by crypt fission. In this pathway, endogenous extracellular hyaluronic acid binds to Toll-like receptor 4 on pericryptal macrophages releasing PGE2 which binds to epidermal growth factor receptor on Lgr5(+) stem cells resulting in proliferation. Lgr5(+) stem cell proliferation leads to crypt fission and intestinal elongation. The demonstration that normal growth requires microbial-independent Toll-like receptor activation is novel.