Mechanisms Of Tumor Regenerative Cells Escaping CTL Killing

Objective:Despite the unprecedented success of PD-1 blockade antibodies and CAR-T therapy in the clinic,relapse is still common in cancer patients that initially responded with extraordinary treatment outcomes.This indicates that a subpopulation of tumor cells may escape from,or be intrinsically resistant to,T cell killing.However,to date,the underlying mechanism remains a mystery.Following recognition,T cells release perforin which drills holes in the target cell membrane allowing granzymes to enter the cell,thus initiating apoptosis.However,to date,whether tumor cells can evade T cell killing at this stage has not been well documented,and the structure and morphology of the pores on these cell membranes have never really been observed.We have developed a mechanics-based culture system using 3D soft fibrin gels that can selectively amplify TRCs from primary tumor tissues and cancer cell lines.These 3D gel-amplified TRCs are much softer than their differentiated counterparts.Based on the above findings,we hypothesize that a certain level of cellular stiffness is required to support pore formation by the perforin molecules and that TRCs may use their mechanical softness to impede perforin drilling a pore,thus escaping T cell-mediated death.The aim of this project is to determine the mechanical mechanisms by TRCs escape immune killing by CTLs.This project will help answer how tumors evade the immune system,provide new insights for tumor immune evasion theory and inspire new strategies for tumor immunotherapy.Methods:Tumor cells were co-incubated with CTLs and cell apoptosis was determined by flow cytometry.Bulk cells or TRCs were treated with recombinant perforin,and then cells were analyzed by flow cytometry,confocal.Visualization of perforin/SLO-induced pore formation using AFM.Cellular stiffness were measured by AFM.Using chemical drugs or gene editing technology to knock out or overexpress Cdc42 protein,affect cytoskeleton proteins and thereby change cell stiffness,and then explore the effect on perforin pore formation.Using immunoprecipitation,western blot and mass spectrometry to identified hat certain membrane molecule(s)was able to interact with perforin,leading to the "drill" force generation.To investigate how the mechanical force was involved in the process of perforin drilling a hole,we use BLI assay.We inoculated a small number of OVA-B16 cells into C57BL/6 mice,followed by OT-1 T cell adoptive transfer,to validate highly tumorigenic cells mobilized their intrinsic softness to hinder perforin pore formation in order to evade CTL killing.Jas treatment also enhanced antitumor T cell immunity,as evidenced by a remarkable inhibition of tumor growth.Finally,we isolated tumor cells from patients and analyzed of the presence of soft tumor cells in tumor tissue that cannot be killed by T cells.Results:(1)Soft TRCs are resistant to cytolytic T cells.OVA-B16 and activated OT-1 CTLs were co-cultured for the cytotoxicity assay.Despite the killing efficiency,some tumor cells evaded death,even in the presence of exceedingly high numbers of CTLs with sufficient killing time.We found that the surviving tumor cells are TRCs and are much softer than bulk cells which can be killed by CTL,suggesting that cellular softness is a common feature for TRCs.(2)Perforin pore formation is impaired in soft TRC.To test whether TRCs evade CTL killing at the killing stage,we used isolated perforin to treat TRCs and control tumor cells.We found perforin does not effectively puncture pores in the membrane of TRCs.These data suggest that soft TRCs use certain mechanism to prevent perforin from drilling a pore,thus evading T cell killing.(3)Cellular softness interferes with perforin pore formation in TRCs.F-actin is an essential element that contributes to cellular stiffness.When we used Cytochalasin D to treat the stiff bulk tumor cells,the cells became soft,accompanied with simultaneous resistance to perforin pore formation.On the other hand,when we used Jas to increase TRC stiffness,we observed increased perforin pore formation.Forced overexpression of Cdc42 resulted in increased stiffness of TRCs and perforin pore formation.Cdc42 knockdown resulted in the decreased stiffness of bulk tumor cells and resistance to perforin pore formation.(4)MYH9 is identified as a molecule required for perforin pore formation.To investigate how the mechanical force was involved in the process of perforin drilling a hole,we used immunoprecipitation,western blot and mass spectrometry to identified that MYH9 was able to interact with perforin,leading to the "drill" force generation.Moreover,we knocked out MYH9 in tumor cells,which showed the results of impaired pore formation induced by perforin and decreased stiffness of bulk tumor cells.(5)Perforin drilling force is generated via interaction with MYH9.Next,we wondered how MYH9 mediated a low perforin-generated force in TRCs but a high force in bulk tumor cells.We found that the transmission of the perforin-generated force via MYH9 to fewer F-actins(i.e.,greater softness)led to a lower force;but transmission of the force to more F-actins(thus higher cell stiffness)caused a stronger force.Indeed,fewer F-actins were observed in TRCs,compared to their bulk counterparts.Together,these results suggest that TRCs use their softness to reduce the MYH9-mediated force generation linked to perforin and required for pore formation.(6)Cell softness hinders CTL-mediated perforin pore formation in vivo.We adoptively transferred CD8+T cells to melanoma-bearing mice.We found adoptive transfer of CD8+T cells resulted in poor entry of GrB into CD133+tumor cells and reduced pore formation.Then,we treated the mice with Jas to increase the cellular stiffness.We found that the transferred T cells following Jas treatment were now capable of killing CD133+tumorigenic cells,and the triple treatment(T cell/anti-PD-1/Jas)resulted in the best treatment outcome.(7)Softness attenuates perforin pore formation in cancer patients.Finally,we sought to validate our findings with clinical patient samples.We further confirmed cellular softness also prevents perforin from drilling holes in human tumor cells,thus mediating their immune evasion.Conclusion:TRC softness prevents membrane pore formation caused by CTL-released perforin.Perforin interacting with nonmuscle myosin heavy chain 9 transmits forces to less F-actins in soft TRCs,thus generating an inadequate contractile force for perforin pore formation.Stiffening TRCs allows perforin the ability to drill through the membrane,leading to CTLs killing TRCs.Importantly,overcoming the mechanical softness in human TRCs also enhances TRC cell death caused by human CTLs,potentiating a mechanics-based immunotherapeutics.