The Role Of Hyaluronan And CD44in Targeted Therapy Of Breast Cancer

Objective：This study aimed to determine the crucial role of activated-CD44in selectivetargeting to breast cancer, the role of the HA-coat as a “fortress” fencing off tumor cellsfrom drugs applied in the circulation, and the use of HA oligosaccharide to breach theendogenous HA-coat for breast cancer therapy.Methods:1.Clinial patient-derived tumor specimens and cell lines were analyzedthrough immunohistochemistry analysis and flow cytometric analysis to observe theactive states of CD44between normal and cancerous cells.2. Nanoparticles wereself-assembled with the ligand-HA coated on the surface.Induction of internalization wasanalyzed by confocal imaging system, and cytotoxicity to breast cancer cell wasdetermined by MTT.3. In vivo imaging was used to prove the selective targeting ofHA-coated nanoparticles to activated-CD44but not to resting CD44in normal tissues invivo.4. We next investigated if the treatment of breast tumor with the HA-coatednanoparticles would inhibit the growth of the xenografts and sensitize them to paclitaxeltreatment.5. The expression of hyaluronan was analyzed by immunofluorescence assayand a particle-exclusion assay. MTT assay was used to examine the effects of HA-coat onchemoresistance of breast cancer cells.6. The fluorescence resonance energy transfer(FRET) technique and immunocytochemistry analysis and chemical cross-linking wasperformed to observe the receptors clustering induced by exogenous and endogenous HA.7.The immunofluorescence assay and a particle-exclusion assay were performed toexamine the effects of oHA on HA coat.3D culture system was used to mimic in vivomicroenvironment to confrm the effect of oHA on HA coat in vivo. Hoechst staining andMTT assay were performed to determine the effects of oHA on chemoresistance ofHA-coated cells.8. Nanoparticles were prepared with oHA loaded on the surface. Toconfrm the function of oHA loaded on the surface of the nanoparticles was determinedby the immunofluorescence assay and MTT assay. To evaluate the anti-tumor activity in vivo, oHA-loaded nanoparticles were administered i. v. into mice xenografted withMDA-MB-231cells and tumor growth was monitored.Results:1.For the first time, we provided solid evidence showing that CD44is in activestate on breast cancer cells whereas inactive on normal cells.2. Further studies showedthat the activation state of CD44between normal and cancer cells was different, revealingstriking similarities to observations in clinical tissues.3. Further studies showed thathyaluronan (HA), the CD44principal ligand, selectively targeted the cancerous cellsrather than normal cells both in vitro and in vivo. When HA was used as a drug-carrier,severe cytotoxicity in human breast cancer cells was induced while no toxicity effect wasfound in normal cells.4. In this study we demonstrated that an unusually abundantsecreted HA contributed to drug resistance in breast cancer.5. Further results suggest thatHA binding to CD44selectively induces CD44clustering, which could be inhibited byoHA.6. However, oligosaccharides of HA (oHA) treatment disrupted thecell-associated HA-coat and rendered breast cancer cells profoundlyvulnerable to paclitaxel.7.Next the anti-tumor activity of self-assembled oHA-loadednanoparticles was evaluated. Results showed that the nanoparticles induced an anti-tumorresponse both in vitro andin vivo. Systemic application of the nanoparticles dramaticallyincreased the activity of chemotherapies and reduced tumor growth in breasttumor-bearing mouse model, possibly as a result of reduced accumulationof HAin the extracellular matrix surrounding xenografts tumor.Conclusion: Our results suggested that the active state of CD44may serve as a selectivetarget for anti-cancer therapy by avoiding nonspecific toxicity on quiescent normal cells.Moreover, we provided direct evidence suggesting that oHA possesses the advantages ofideal drug carrier and drug targeting, and oHA-loaded nanoparticles have great potential toovercome HAassociated chemoresistance and improve cancer therapy.