| BackgroundOsteosarcoma is a bone malignancy that occurs most frequently in children and adolescents,typically in the metaphyses of long bones,such as the distal femur and proximal tibia.Currently,adequate surgical resection plus postoperative reconstruction,combined with preoperative and postoperative chemotherapy,remains the most common treatment modality.For non-metastatic osteosarcoma,the five-year overall survival rate is around 60.17%,with the five-year survival rate for appendicular bone osteosarcoma higher at 64.43%compared to 45.97%for axial bone and 45.57%for other sites.However,the survival rate after metastasis drops to less than 25%.Despite progress in improving surgical techniques and early diagnosis sensitivity,the long-term survival rate of osteosarcoma patients has not improved significantly over the past 15 years.Thus,there is a pressing need to develop novel therapeutic strategies to enhance the long-term survival rate of patients suffering from recurrent or metastatic osteosarcoma.Since the discovery of specific tumor antigens using chemical carcinogen-induced tumor models in the 1950s,tumor immunotherapy has attracted considerable attention from scientists.Increasing numbers of scholars are now dedicating their efforts to study the tumor immune microenvironment,including its non-tumor components and their relationship with tumor cells,with the aim of discovering novel strategies to enhance therapeutic outcomes for osteosarcoma.Multiple studies have confirmed that macrophages occupy more than 50%of the tumor immune microenvironment(TME)in osteosarcoma,with M2 macrophages predominating and playing a critical role in the interaction between cancer cells and their microenvironment.This contributes to tumor growth and metastasis and is generally associated with poor prognosis in cancer patients.The development and utilization of therapeutic modalities targeting tumor-associated macrophages(TAMs),such as promoting TAM polarization from the pro-tumor and metastasis M2 subtype to the tumor progression-suppressing M1 subtype,may be an effective measure to improve the survival of patients with advanced osteosarcoma.Previous research has indicated that tumor cells in the bone microenvironment can generate a plethora of cytokines,such as transforming growth factor(TGF)-βand transforming growth factor-α(TGF-α),that stimulate osteoclast activity.This increased osteoclast activity can facilitate the production of multiple lymphokines and growth factors,establishing a self-catalyzing process that promotes tumor cell proliferation.To break this vicious cycle,osteoclast activity must be inhibited.Alendronate(ALN),a third-generation bisphosphonate drug,is widely used in clinics for treating osteoporosis,as it helps inhibit bone resorption and reduce osteoclast activity.Moreover,it serves as a significant adjunct in the treatment of bone metastasis in various malignant tumors,including breast and prostate cancers and multiple myeloma.Nevertheless,several side effects come with alendronate,such as gastroesophageal irritation,nausea,secondary hyperparathyroidism,and musculoskeletal pain,limiting its use in some patients.Although many studies use alendronate’s bone-targeting ability and affinity to osteoclasts to couple it with polymers,loaded in hydrogels or nanoparticles to precisely deliver drugs,few have explored the direct effects of alendronate and its polymers on the tumor microenvironment.ObjectiveWe prepared ALN,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)–hydroxysuccinimide ester(DSPE-PEG-NHs),using a straightforward synthetic process,termed Synthesis of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-alendronate(DSPE-PEG-ALN,DPA).The objective was to extend the blood circulation time of free ALN,investigate the polarization of ALN and its polymers to macrophages and establish a mouse orthotopic osteosarcoma model to clarify its inhibitory effect on osteosarcoma growth.Additionally,our aim was to determine whether ALN and its polymers might impact the phenotype of TAMs in TME.We hope that this study will serve as a valuable experimental basis for future studies focusing on alendronate in osteosarcoma immunotherapy.MethodsThis experiment was divided into three parts,namely,the synthesis and characterization of DPA,exploring the biocompatibility of ALN and its polymer DPA using in vitro cell experiments and the ability to polarize macrophages,observing the osteosarcoma growth and metastasis inhibitory effect of free ALN and DPA using in vivo animal experiments and the regulatory effect on TAMs in the TME.The specific study protocol is described below:(1)According to the previous synthesis steps of our group,DPA was synthesized and verified by proton nuclear magnetic resonance(~1H-NMR),Fourier transform infrared spectroscopy(FT-IR)and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).(2)Mouse derived RAW264.7 cell line and mouse k7M2 osteosarcoma cell line were selected to study the biocompatibility of different concentrations of free ALN as well as DPA by CCK8 assay;Bone marrow-derived macrophages(BMDMs)from BABL/C mice were extracted,induced to differentiate into M2 type macrophages and the polarization of M2 type macrophages by free ALN as well as DPA was verified by flow cytometry,and the contents of the relevant cytokines in the cell culture supernatants were determined using an ELISA factor assay kit;(3)Murine orthotopic osteosarcoma models were established and treated with different concentrations of free ALN and DPA,and their inhibitory effects on osteosarcoma growth,metastasis,and effects on TAMs in the TME were evaluated by H-E staining,immunofluorescence,and TUNEL staining.ResultsIn this study,we employed the aforementioned methodology to synthesize DPA,which was subsequently confirmed via nuclear magnetic resonance hydrogen spectroscopy,Fourier transform infrared spectroscopy,and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.Our CCK8 assays demonstrated the cytotoxic effects of both high concentrations of ALN and DPA on RAW264.7 and K7M2 cells,with polymer DPA exhibiting relatively stronger toxicity than free ALN at the equivalent concentrations.Flow cytometry analysis revealed that both free ALN and DPA could drive M2-like macrophage polarization,but the polarizing effect of DPA was more potent than that of free ALN under comparable ALN concentration conditions.Using animal models,we found that when compared with free ALN,high concentrations of DPA could inhibit the growth of osteosarcoma,while also reducing the nonspecific toxicity of free ALN,as evidenced by changes in mouse weight and tumor volume.Histopathological studies via H-E and TUNEL staining illustrated that high-dosage DPA effectively inhibited lung metastasis of osteosarcoma and promoted apoptosis in the tumor region,while no such outcomes were observed for free ALN.Immunofluorescence investigations further revealed that high concentrations of DPA led to M1-like macrophage polarization in TME,thus improving the immunosuppressive microenvironment of osteosarcoma.ConclusionPolymer DPA based on ALN can regulate the immune microenvironment by polarizing M2-like macrophages with tumor-promoting effects into M1-like macrophages with tumor-suppressing effects,thereby improving the immunosuppressive microenvironment of osteosarcoma,and inhibiting its progression and metastasis. |
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