Inhibiting Autophagy By Brucine Enhances Immunogenic Cell Death And Activates Adaptive Immune System For Tumor Cell Vaccine

Cancer remains one of the leading causes of death in the world,and cancer treatment strategies are still hampered by the complexity of cancer cells metabolic pathways and the patients' variability in their immune system.Whether the targeted therapy or personalized cancer therapeutics need to be put into place,they will also still be hampered by tumor microenvironment(TME)and metabolic process of cancer cells itself.Thus,Immune score of the TME modulation will require the assistance of intracellular metabolic pathways modulator for efficiently improve cancer treatment methods.Some of the methods that are being investigated are modulating immune system by chemotherapeutic agents in order to improve adaptive immune response to cancer development.Adaptive immune system activation induced by chemotherapeutic agents is partly related to their ability to induce immunogenic cell death(ICD).Anticancer drugs that induce ICD are reported to induce cell death,and the dying cells expose and/or release some of their intracellular contents;which are then exposed and/or released outside the cell and induce immune cell stimulation and activation.The released substances are known as damage associated molecular patterns(DAMPs)which are sensed by special cells called antigen presenting cells(APC),where the major APC are dendritic cells(DC),macrophages,and B cells.However,among these APC,DC are more specialized to present antigen to different immune cells like CD4 and CD8 T cells.DC process the antigen through different pathways:Vacuolar pathway and cytosolic pathway.All antigens are internalized through phagocytosis by DC and directed to different pathways.But the conditions that determine the fate of antigens to either pathway are yet to be determined.For cytosolic pathway,the phagocytosed antigens are directed by phagosome and delivered to proteasome for degradation into small peptide or polypeptides that are either transported to the endoplasmic reticulum(ER)through a transporter associated with antigen processing(TAP)or back to the phagosome in order to be loaded to MHC molecules and presented to DC membrane.To the contrary,for vacuolar pathway,antigens are conducted by phagosomes and degraded through other cellular degradation pathways such as autophagic degradation pathway.There are still many points for debate on this antigen processing and presentation pathways.The vacuolar pathway,is not yet clearly understood as it was believed to be proteasome and TAP independent,but later discovered that some proteasome independent may also be TAP independent.DC present antigens to T cells through their major histocompatibility complex(MHC)type ? and ?.Although autophagic molecular pathway is common in all mammalian cells,the mechanism and the fate of the cells undergoing autophagy are widely different and therefore,become more complex to clear elucidate canonical autophagy regulation and fate in all mammalian cells.Autophagy was discovered as early as 1950s,and later coined the term autophagy in 1963 by Christian de Duve;the development and discoveries of more fascinating applications of autophagy process and its involvement in may cellular metabolism,autophagy discovery was awarded a noble prize in 2016.Thus,now autophagy is known as a critical metabolic process that control cellular homeostasis by turning over many intracellular contents such damaged organelles and misfolded proteins among others.Nowadays,autophagy has become a tool for anticancer drugs discovery,as it was shown to be used by many cancer cells to satisfy their nutrients needs.Therefore,dysregulated autophagy in cancer cells would likely lead to dysfunctional metabolic pathways for cancer cells and sensitized them to other anticancer drugs such as drugs with target directly or indirectly linked to autophagy signaling pathways.By having this idea in mind,we thought to exploit the phenomenon to sensitize cancer cells to ICD by using a small molecule known as Brucine also known as 2,3dimethoxystrychnidin-10-one.Brucine is an alkaloid very much similar in chemical structure to that of a very poisonous alkaloid called strychnine,both of them present in the tree called Strychnos nux-vomica L.(Loganiaceae)and most of the time are isolated together which make people confuse brucine toxicity to that of strychnine.In this study,we have shown brucine as a potent autophagy modulator,functioning by blocking autolysosome cargo degradation in cancer cells.Autophagy impairment by brucine,caused dephosphorylation of ERK1/2,mTOR and p70S6K,which indicated that brucine disrupted ERK-mTOR-p70S6K pathway,and increased the accumulation of autophagy marker LC3 and autophagy substrate p62.The impaired autophagy has increased intracellular stress and induced damage associated molecular patterns(DAMPs)release which are known to stimulate immune system.The DAMPs released by brucine-treated cancer cells,are HMGB1,Ecto-CRT exposure and TNFa as shown by immunoblotting and immunofluorescence and flow cytometry analysis.The dying CT26 cells treated with Brucine induced ICD in mice and were able to vaccinate mice with CT26 live tumor cells rechallenge.The release of these DAMPs,were completely abolished when autophagy related gene(ATG5)was knocked down by short hairpin RNA(shRNA),which confirmed that these DAMPS were released due to autophagy impairment by Brucine.To confirm if autophagy inhibition was essential for dying cell to initiate immune cell stimulation and activation,we treated ATG5 knocked down cells with brucine and injected these dying cells into the mice and the showed a decrease of CD4 and CD8 T cells for ATG5 knocked down treated mice compared to wild type treated mice.We further investigated the immune cells involved in immunizing mice against tumor rechallenge,we realized that CD4 T cells were necessary for cytotoxicity to tumor cells rechallenge and CD8 T cells were necessary for memory formation.Our results showed that autophagy inhibition can also be a promising alternative to chloroquine for future development of autophagy inhibitors related anti-cancer drugs,and autophagy inhibition with brucine can induce ICD in mice model by improving their CD4 cytotoxicity and CD8 and memory T cells formation and development.As the link between autophagy and ICD is still less investigated,our methodology also can serve as tool to elucidate a yet to be clearly understood ICD mechanism.