The Mechanism Of Transmissible ER Stress Between Macrophages And Tumor Cells In The Tumor Microenvironment

Numberous intrinsic and extrinsic factors,such as hypoxia,viral infection,nutrient deficiency,low p H and proto-oncogene activation,lead to aberrant endoplasmic reticulum(ER)stress activation in the tumor microenvironment(TME).To restore ER homeostasis,the unfolded protein response(UPR)increases ER chaperone content to elevate protein folding rate.Tumor cells persistently undergo ER stress and could transmit it to the surroundings,such as neighboring macrophages.Transmissible ER stress has been shown to play a critical role in intercellular communication in the pathogenesis of various diseases,especially in malignant cancers.And it plays an important role in tumor cell growth and the tumor microenvironment maintenance.However,whether ER stress could be transferred from non-tumor cells,like macrophages,to tumor cells has not yet been studied.Therefore,the transmissible ER stress as an intracellular communication in TME was worth investigating.Tumorigenesis is unceasingly occurred along with correlative chronic inflammatory responses,and the abundant tumor-associated inflammatory immune cells infiltration was observed in clinical tumor biopsies,such as macrophages.Moreover,the phenotypes of macrophages are suggested to be responsible for promoting activities of tumors,including tumor initiation,progression,metastasis and killing of tumors.Tumor infiltrating macrophages can also adapt to the microenvironment variations to fulfill their highly energy-demanding and biological functions via ER stress.Macrophages have continuous functional activity states with two extremely distinct polarized conditions,mostly named proinflammatory M1-or anti-inflammatory M2-phenotypes.Therefore,the regulation of different polarization states of macrophages in TME can largely inhibit the survival,proliferation and migration of tumor cells,and contribute to anti-cancer immunotherapy,indicating that macrophage polarization status could be an indispensable factor when studying intratumoral cell communications.However,whether the different macrophage populations differentially sense ER stress and transmit ER stress to surrounding tumor cells has not yet been elucidated.This project aims to study the role of transmissible ER stress,a novel regulator of intercellular communication in the TME,and to investigate whether the interaction between tumor cells and macrophages in the TME can be regulated by transmissible ER stress to inhibit tumor growth and metastasis,which will provide support for clinical immunotherapy of cancer.In this study,the differentiation of mouse Bone marrow-derived macrophage(BMDM)model based on different inducers was used to characterize the two extreme states of macrophages.The two different polarized macrophage populations were stimulated with different inducers to trigger ER stress and construct the ER stress transmission system.The UPR signaling activity was verified by multiple molecular biological techniques.2D and 3D cell systems were also performed to verify the exactitude and stability of the transfer system.The subsequent studies showed that tumor cells could receive the transmissible ER stress from two differentially polarized macrophage populations with different extents of ER stress activation.The proinflammatory M1-like macrophages respond to ER stress with less extent,however they could transmit more ER stress to tumor cells.While,M2-like macrophages with anti-inflammatory properties are more sensitive to ER stress but transmit less ER stress to tumor cells.Transcriptome sequencing analysis also showed that after receiving ER stress transmitted by M1-like macrophages,tumor cells highly activated signaling pathways of promoting inflammatory response and cell death,while after receiving ER stress delivered by M2-like macrophages,tumor cells significantly upregulated functions of repair and angiogenesis which contribute to tumor growth.Moreover,further exploration of the mechanism involved in the process of ER stress transmission,we then analyzed related proteins secreted by two macrophage populations under ER stress.By analyzing the protein mass spectrometry,we identified certain damage-associated molecular patterns(DAMPs),including S100A8 and S100A9,which are dominantly stored and secreted by M1-like macrophages could lead to significant recipient tumor cells death in synergy with transferred ER stress.This study systemically investigated ER stress transmission,a novel intercellular communication,was bidirectional between macrophages and tumor cells.ER stress transmitted by different polarized macrophages had different effects on recipient tumor cells,and the synergistic enhancement of DAMPs and ER stress was a potential mechanism promoting the induction of tumor cell death under ER stress transmission.The study illustrated that M1-like macrophages not only secreted substantial proinflammatory factors that made more inflammatory responses involved in the tumor microenvironment.The main effect was tumor cell death.Meanwhile,particularly under ER stress M1-like macrophages released abundant DAMPs acted synergistically with transmissible ER stress transfer caused massive tumor cell death.In contrast,M2-like macrophages delivered anti-inflammatory factors and had more extent ER stress sensitivity but transfered less ER stress.With less DAMP store and secretion,M2-like macrophages could help tumor survival and metastasis.This study enriches our knowledge and understanding of intercellular communication in the TME and provids a solid theoretical basis for the subsequent development of ER stress transmission to help clinical immunotherapy of cancer patients.