Isolation,Characterization,and Application Of Apoptotic Bodies

Apoptosis is widely known as programmed cell death eliciting no inflammatory responses.It has been at the center of an array of researches,accumulating a wealth of knowledge which led to not only a better understanding of the fundamental process,but also potent therapeutic measures of diseases.The classic intrinsic and extrinsic signaling pathways of apoptosis,along with a variety of regulatory factors and molecular mechanisms have been well delineated.Drugs and therapeutic measures designed based on current understanding of apoptosis have long been employed.Small-molecule apoptosis-inducers have been clinically used for eliminating morbid cells and therefore treating diseases such as cancer.Biologics with improved apoptotic efficacy and selectivity,such as recombinant proteins and antibodies,are being massively researched and some have been approved by the FDA.Apoptosis also produces membrane-bound vesicles derived from disassembly of apoptotic cells,now known as apoptotic bodies(ApoBDs).These little sealed sacs containing information and substances from dying cells were previously regarded as garbage bags until they were discovered to be capable of delivering useful materials to healthy recipient cells(e.g.autoantigens).As a highly coordinated cellular process in multicellular organisms,apoptosis confers profound advantages to organisms in development,immune responses,maintaining homeostasis.Research on apoptosis has been ongoing for decades and recent research spotlight has shifted from pathways and relevant transducers leading to apoptosis to the detailed process of apoptotic disassembly.A wealth of knowledge on apoptotic cellular disassembly has been growing and ApoBDs are central to the disassembly process.Many questions regarding ApoBDs remained unanswered and hold the key to unlocking the ultimate puzzle of apoptosis.In this study,we investigated the biological nature of ApoBDs with a focus on the RNA content.We believe non-coding RNAs,such as miRNA,lncRNA and circRNA,contained within ApoBDs derived from cancer cells are highly functional in recipient cells upon engulfment.We induced apoptosis in A549 cells using two apoptosis-inducers,Etoposide and RGD-TRAIL.We employed centrifugation to isolated ApoBDs whose identity and purity was confirmed by scanning electron microcopy and flow cytometry.We also attempted to extract RNA from ApoBDs whereas the traditional Trizol method was proven ineffective.We then used the mirVANA RNA isolation kit with which RNA of ApoBDs was successfully extracted.RNA-seq results showed that ApoBDs derived from A549 cells contain a large amount of RNAs of different varities.However,ApoBDs derived from Etoposide and RGD-TRAIL-induced apoptosis showed different RNA content in all four categories of RNAs(circRNA,lncRNA,miRNA,and mRNA).According to previously published studies,these two inducers induce two distinct types of apoptosis with drastically different biology.Etoposide induce intrinsic apoptosis while RGD-TRAIL,which is an enhanced version of TRAIL,induces extrinsic apoptosis.Our findings indicate that cells induced with different types of apoptosis-inducers package their cellular content differently into ApoBDs.Furthermore,we performed qPCR-based confirmation of RNAs obtained from sequencing results,especially highly expressed RNAs in ApoBDs.In qPCR experiment,we monitored the abundance of RNAs not only in ApoBDs,but also in healthy and apoptotic cells(apoptotic cells are not ApoBDs as they are much larger cell remnants than ApoBDs).Our results showed that A549 cells preferentially package tumor-promoting RNAs,such mir-21-5p,mir-1246,hsa_circ₀060927(circ0060927),RN7SL1,PAK1 mRNA etc.,into ApoBDs,as the abundance of these RNAs was much higher in ApoBDs than in healthy and apoptotic cells.Moreover,qPCR experiment also confirmed that some RNAs contained within ApoBDs derived from RGD-TRAIL-induced apoptosis of A549 cells were unique compared to its Etoposide-induced counterparts,and vice versa.Lastly,we investigated the possible functions of a highly expressed circRNA,circ0060927,in A549 ApoBDs derived from both Etoposide and RGD-TRAIL-induced apoptosis.We discovered that circ0060927 is a tumor-promoting RNA as stable overexpression of circ0060927 in A549 cells facilitated proliferation and migration of A549.Studies on the effect on apoptosis revealed that circ0060927 inhibits apoptosisWe also used an ApoBD-mimic,PS-containing liposome,to deliver siRNAs to the hard-to-transfect macrophages.We first characterized ApoBDs and PS-containing liposomes for their surface PS using flow cytometry and MST,respectively.We then screened for ideal PS-containing liposomal formulas for transfection and obtained a formula which we named Ca-PS lipopolyplex in this study.Ca-PS lipopoly was proven to be highly effective in delivering siRNA to multiple cell types.Moreover,cytotoxicity assay showed that Ca-PS lipopolyplex was less toxic to transfected cells than the widely used Lipofectamine2000(Lipo2000).Most importantly,Ca-PS lipopolyplex was shown to be capable of effecting highly efficient siRNA knockdown of genes in macrophages within a knockdown range of 65-72%,which was much higher than Lipo2000.Study on the uptake efficiency of transfection complexes by macrophages revealed the PS component of the Ca-PS lipopolyplex contributed to markedly improved uptake compared to Lipo2000.Lastly,we monitored the state of M1 or M2 polarization of macrophages upon transfection.Transfection by both Ca-PS lipopolyplex and Lipo2000 did NOT induce polarization of macrophages both in Ana-1 and BMDMs.Therefore,Ca-PS lipopolyplex is an effective liposomal formula for siRNA transfection in macrophagesIn the last part,we identified a cell culture contaminant as a new bacterial species.This contaminant was a tremendous obstacle for obtaining pure ApoBDs of mammalian cells as it has a size indistinguishable from the smaller ApoBDs and therefore would be collected along with ApoBDs using centrifugation.Had this contaminant been left untreated,RNA-seq of ApoBDs would NOT have been accomplished.We assayed a variety of antibiotics for elimination of this contaminant,which we name NJUB218 in our study and unraveled a potent combinatorial treatment using Ampicillin and Gentamicin both at a concentration of 100 ?g/mL.Furthermore,we identified this contaminant as a new bacterial species using 16S phylogenetic classification and de novo sequencing.NJUB218 belongs to the genus of Pusillimonas and therefore has current taxonomic name Pusillimonas sp.NJUB218In summary,we explored the RNA content of ApoBDs and compared the RNA content both among ApoBDs derived from different treatment groups and among ApoBDs,apoptotic cells and healthy cells.The most important conclusion is dying cells don't randomLy distribute their cellular RNAs into ApoBDs.Rather,cancer cells preferentially package tumorgenic RNAs into ApoBDs as a legacy and therefore promotes invasion and metastasis of the living cancer cells and tissues.Furthermore,different types of apoptosis-inducers give rise to different content contained within ApoBDs.For ApoBD-related application,PS-containing liposomes were shown to be highly efficient in effecting siRNA knockdown of genes in macrophages.