Inducing immunogenic cell death in immuno-oncological therapies

Immunotherapy has revolutionized cancer treatment and substantially improved patient outcomes with respect to multiple types of tumors.However,most patients cannot benefit from such therapies,mainly due to the intrinsic low immunogenicity of cancer cells(CCs)that allows them to escape recognition by immune cells of the body.Immunogenic cell death(ICD),which is a form of regulated cell death,engages in a complex dialogue between dying CCs and immune cells in the tumor microenvironment(TME),ultimately evoking the damage-associated molecular pattern(DAMP)signals to activate tumor-specific immunity.The ICD inducers mediate the death of CCs and improve both antigenicity and adjuvanticity.At the same time,they reprogram TME with a“cold-warmhot”immune status,ultimately amplifying and sustaining dendritic cell-and T cell-dependent innate sensing as well as the antitumor immune responses.In this review,we discuss how to stimulate ICD based upon the biological properties of CCs that have evolved under diverse stress conditions.Additionally,we highlight how this dynamic interaction contributes to priming tumor immunogenicity,thereby boosting anticancer immune responses.We believe that a deep understanding of these ICD processes will provide a framework for evaluating its vital role in cancer immunotherapy.

Immune response induced by hematoporphyrin derivatives mediated photodynamic therapy:Immunogenic cell death and elevated costimulatory molecules

Photodynamic therapy(PDT)not only destroys tumor cells directly but also induced anti-tumor immune response through damage-associated molecular patterns(DAMPs).It is reported that anti-tumor response was associated with light dose and photosensitizer used in PDT.In this study,4T1 tumor cells were implanted on both the right and left flanks of mice.Only the right tumor was treated by HpD-PDT,while the left tumor was not irradiated.The anti-tumor immune response induced by HpD-PDT was investigated.The expression of DAMPs and costimulatory molecules induced by HpD-PDT were tested by immuno fluorescence and flow cytometry in vivo.Different light doses of PDT were designed to treat 4T1 cells.The killing effect was assessed by CCK-8 kit and apoptosis kit.The expression of DAMPs on 4T1 cells after HpDPDT were evaluated by flow cytometry,western blot and ATP kit.This study showed that CD4^(+)T,CD8^(+)T and the production of IFN-γwere increased significantly on day 10 in righttumor after PDT treatment compared with control group.HpD-PDT enhanced the expression of calreticulin(CRT)on tumor tissue.Importantly,co-stimulatory molecular OX-40 and 4-1BB were elevated on CD8^(+)T cells.In vitro,immunogenic death of 4T1 cells was induced after PDT.Besides,the expression of DAMPs increased with the increasing of energy density.This study indicates that anti-tumor immune effect was induced by HpD-PDT.The knowledge of the involvement of CRT,ATP and co-stimulatory molecules uncovers important mechanistic insight into the anti-tumor immunogenicity.It was the first time that co-stimulatory molecules were investigated and found to elevate after PDT.

Marine natural product lepadin A as a novel inducer of immunogenic cell death via CD91-dependent pathway

Immunogenic Cell Death(ICD)represents a mechanism of enhancing T cell-driven response against tumor cells.The process is enabled by release of damage-associated molecular patterns(DAMPs)and cytokines by dying cells.Based on molecular studies and clinical marker assessment,ICD can be a new target for cancer chemotherapy hitherto restricted to a few conventional anticancer drugs.In view of the development of small molecules in targeted cancer therapy,we reported the preliminary evidence on the role of the natural product lepadin A(1)as a novel ICD inducer.Here we describe the ICD mechanism of lepadin A(1)by proving the translocation of the protein calreticulin(CRT)to the plasma membrane of human A2058 melanoma cells.CRT exposure is an ICD marker in clinical studies and was associated with the activation of the intrinsic apoptotic pathway in A2058 cells with lepadin A(1).After the treatment,the tumour cells acquired the ability to activate dendritic cells(DCs)with cytokine release and costimulatory molecule expression that is consistent with a phenotypic profile committed to priming T lymphocytes via a CD91-dependent mechanism.The effect of lepadin A(1)was dose-dependent and comparable to the response of the chemotherapy drug doxorubicin(2),a well-established ICD inducer.

Immunogenic cell death-related long noncoding RNA influences immunotherapy against lung adenocarcinoma

Lung adenocarcinoma(LUAD)is the leading cause of cancer-related deaths,accounting for over a million deaths worldwide annually.Immunogenic cell death(ICD)elicits an adaptive immune response.However,the role of ICD-related long noncoding RNAs(lncRNAs)in LUAD is unknown.In this study,we investigated the characteristics of the tumor microenvironment in LUAD,the prognostic significance of ICD-related lncRNAs,and the half-maximal inhibitory concentration(IC50)of possible chemotherapeutic drugs.We sorted prognostic lncRNAs using univariate Cox regression and constructed a risk signature based on them.We then confirmed the model’s accuracy and generated a nomogram.Additionally,we performed immune microenvironment analysis,somatic mutation calculation,Tumor Immune Dysfunction and Exclusion(TIDE)analysis,and anticancer pharmaceutical IC50 prediction.Least absolute shrinkage and selection operator Cox regression identified 27 prognostic lncRNAs related to ICD,and a unique risk signature using 10 ICD-related lncRNAs was constructed.The risk score was confirmed to be a reliable predictor of survival,with the highest c-index score.The signature had a remarkable predictive performance with clinical applicability and could accurately predict the overall survival in LUAD.Furthermore,the lncRNA signature was closely associated with immunocyte invasion.We also analyzed the correlation between the risk score,tumor-infiltrating immune cells,and prognosis and identified high immune and ESTIMATE scores in low-risk patients.Moreover,we observed elevated checkpoint gene expression and low TIDE scores in high-risk patients,indicating a good immunotherapy response.Finally,high-risk patients were shown to be susceptible to anticancer medications.Therefore,our unique risk signature comprising 10 ICD-related lncRNAs was demonstrated to indicate the characteristics of the tumor-immune microenvironment in LUAD,predict patients’overall survival,and guide individualized treatment.

An Immunogenic Cell Death-Related Classification Predicts Prognosis and Response to Immunotherapy in Glioblastoma

To investigate the immunogenic Cell Death gene’s potential mechanism and prognostic value in glioblastoma. Information on GBM samples from The Cancer Genome Atlas database was downloaded, ICD genes were obtained, genotyping, integrated bioinformatics to verify the prognostic value of genotyping, and finally, prognostic model construction. Two subtypes associated with the ICD gene were obtained by consensus clustering, and the high ICD subtype (risk) group was associated with poor prognosis, high mutations in the PTEN gene, high stromal score, and high immune score. We also constructed a new classification system for GBM based on ICD characteristics. This study is the first to use immunogenic cell death genes for genotyping and successfully build a prognostic model.

Endoplasmic reticulum-targeted delivery of celastrol and PD-L1 siRNA for reinforcing immunogenic cell death and potentiating cancer immunotherapy

The prospect of employing chemoimmunotherapy targeted towards the endoplasmic reticulum(ER)presents an opportunity to amplify the synergistic effects of chemotherapy and immunotherapy.In this study,we initially validated celastrol(CEL)as an inducer of immunogenic cell death(ICD)by promoting ER stress and autophagy in colorectal cancer(CRC)cells.Subsequently,an ER-targeted strategy was posited,involving the codelivery of CEL with PD-L1 small interfering RNAs(siRNA)using KDEL peptide-modified exosomes derived from milk(KME),to enhance chemoimmunotherapy outcomes.Our findings demonstrate the efficient transportation of KME to the ER via the Golgi-to-ER pathway.Compared to their non-targeting counterparts,KME exhibited a significant augmentation of the CEL-induced ICD effect.Additionally,it facilitated the release of danger signaling molecules(DAMPs),thereby stimulating the antigen-presenting function of dendritic cells and promoting the infiltration of T cells into the tumor.Concurrently,the ER-targeted delivery of PD-L1 siRNA resulted in the downregulation of both intracellular and membrane PD-L1 protein expression,consequently fostering the proliferation and activity of CD8^(+)T cells.Ultimately,the ER-targeted formulation exhibited enhanced anti-tumor efficacy and provoked anti-tumor immune responses against orthotopic colorectal tumors in vivo.Collectively,a robust ER-targeted delivery strategy provides an encouraging approach for achieving potent cancer chemoimmunotherapy.

Mitochondria-targeted nano-AIEgens as a powerful inducer for evoking immunogenic cell death

AIEgens can serve as an effective platform for the construction of photosensitizer-based immunogenic cell death(ICD)inducers.To date,several mitochondria or endoplasmic reticulum(ER)-targeted aggregationinduced emission(AIE)molecules have been developed and have evoked massive ICD in cells.However,due to the complex physicochemical environment in cells,these small AIE molecules cannot maintain a stable aggregate state,which not only affects the fluorescence intensity of the photosensitizer but also decreases the generation of reactive oxygen species(ROS),and thus reducing the effect of the photosensitizer to elicit ICD.AIEgen-based nanomicelles,which maintain a stable micellar structure,can prevent defects of AIE molecules in photodynamic therapy(PDT)applications.Therefore,in this study,a mitochondria-targeted AIE nanophotosensitizer was synthesized and used as a highly potent ICD inducer for vaccine preparation and tumor prevention.

Nanomedicine-mediated immunogenic cell death and its combination with immune checkpoint blockade therapy

Cancer immunotherapies, which train the natural immune system to specifically kill tumor cells while sparing the healthy cells,have helped revolutionize cancer treatments and demonstrated promising clinical therapeutic benefits for decades. However, the therapeutic outcome of immunotherapies, even for the most successful immune checkpoint blockade(ICB) therapy, remains unsatisfactory in the clinical practice, mainly due to the low immunogenicity of solid tumors and its immunosuppressive tumor microenvironment(TME). Notably, several cancer treatment modalities, including chemotherapy, radiotherapy, and phototherapy, have been revealed to evoke tumor immunogenicity and reverse immunosuppressive TME via inducing immunogenic cell death(ICD) of tumor cells, which synergistically sensitized tumors to ICB therapy. Nanomedicines have been extensively applied to augment ICD-inducing treatment modalities and potentiate ICB therapeutic efficacy therapy due to the opportune convergence of immunotherapy and nanotechnology. Here, we discuss the recent advances in nanomedicine-mediated ICD and its combination with ICB therapy.

MMAE-loaded PLGA nanomedicine with improved biosafety to achieve efficient antitumor treatment

Monomethyl auristatin E(MMAE)is a derivative of the marine peptide Dolastatin 10,which has therapeutic effects against various cancers according to its antimitotic activity in multiple clinical trials.The antibody drug conjugate(ADC)of MMAE is currently used in clinical practice.However,the safety issues of MMAE-based ADC,such as high drug toxicity and poor bioavailability,still exist when using it for anticancer therapy.A sustained release of drug delivery approach should be used to reduce toxicity and achieve sufficient anticancer effects.Herein,PLGA-b-PEG 2000 with excellent biocompatibility and slow degradation ability was adopted to construct MMAE-loaded nanoparticles for safe and effective chemotherapy.The sustained release effect and the immunogenic cell death(ICD)effect of PLGA-MMAE nanoparticles were assessed by in vitro experiments.The PLGA-MMAE nanoparticles effectively accumulated in the tumor through the enhanced permeability and retention(EPR)effect,inducing cell apoptosis and causing a certain degree of immune response.The sustained drug release of PLGA-MMAE improved the bioavailability and effectively reduced the toxicity and development of the tumor compared to the effect of free MMAE or ADC.Overall,this study provides a safe and effective chemotherapeutic approach,as well as a simple and effective synthetic process for MMAE-based nanoparticles,improving their therapeutic efficacy and safety.

NIR-Ⅱ responsive Janus nanoparticles amplify immunogenic cell death for enhanced cancer immunotherapy

Immunotherapy brings new hope for tumor treatment by inducing immunogenic cell death(ICD)of tumor cells.However,insufficient immunogenicity and low immune response rate greatly limit antitumor immunity.Herein,by optimizing the composition and morphology,the rational design of Janus nanoparticles composed of Fe_(3)O_(4) nanospheres and SiO_(2)nanorods was realized for enhanced cancer immunotherapy through amplified ICD.After glucose oxidase(GOx)was loaded by the Janus nanoparticles,the resultant M-FS-GOx consumes glucose at tumor sites to generate gluconic acid and hydrogen peroxide(H_(2)O_(2))for starvation therapy while the H_(2)O_(2)supply promotes the production of highly toxic·OH to achieve effective chemodynamic therapy(CDT).Under a 1064 nm light irradiation,the photothermal effect of M-FS-GOx enhances the enzyme activity of GOx for improved starvation therapy.Furthermore,both tumor-associated antigens released during the process of ICD and the intrinsic immunoadjuvant property of M-FS-GOx stimulate dendritic cell maturation to activate antitumor immune responses.This work provides a promising strategy for the construction of Janus nanoparticles to achieve enhanced cancer immunotherapy through combination therapy-amplified ICD.

Investigating the Immunogenic Cell Death‑Dependent Subtypes and Prognostic Signature of Triple‑Negative Breast Cancer

Recently,immunotherapy has emerged as a promising and efective method for treating triple-negative breast cancer(TNBC).However,challenges still persist.Immunogenic cell death(ICD)is considered a prospective treatment and potential combinational treatment strategy as it induces an anti-tumor immune response by presenting the antigenic epitopes of dead cells.Nevertheless,the ICD process in TNBC and its impact on disease progression and the response to immunotherapy are not well understood.In this study,we observed dysregulation of the ICD process and verifed the altered expression of prognostic ICD genes in TNBC through quantitative real-time polymerase chain reaction(qRT-PCR)analysis.To investigate the potential role of the ICD process in TNBC progression,we determined the ICD-dependent subtypes,and two were identifed.Analysis of their distinct tumor immune microenvironment(TIME)and cancer hallmark features revealed that Cluster 1 and 2 corresponded to the immune“cold”and“hot”phenotypes,respectively.In addition,we constructed the prognostic signature ICD score of TNBC patients and demonstrated its clinical independence and generalizability.The ICD score could also serve as a potential biomarker for immune checkpoint blockade and may aid in the identifcation of targeted efective agents for individualized clinical strategies.

Pulsed Modulation in Electro-Hyperthermia

Modulated electro-hyperthermia (mEHT) is one of the novel oncological treatments with many preclinical and clinical results showing its advantages. The basis of the method is the synergy of thermal and nonthermal effects, similar to the thermal action of conventional hyperthermia combined with ionizing radiation (radiotherapy). The electric field and the radiofrequency current produced both the thermal and nonthermal processes. The thermal effects produce the elevated temperature as a thermal background to optimize the nonthermal impacts. The low frequency amplitude modulation ensures accurate targeting and promotes immunogenic cell death to develop the tumor specific memory T cells disrupting the malignant cells by immune surveillance. This process (abscopal effect) works like a vaccination. The low frequency amplitude modulation is combined in the new method with the high power pulses for short time, increasing the tumor distortion ability of the electric field. The new modulation combination has much deeper penetration triplicating the active thickness of the effective treatment. The short pulse absorption increases the safety and decreases the thermal toxicity of the treatment, making the treatment safer. The increased power allows for reduced treatment time with the prescribed dose.

Gastro-intestinal toxicity of chemotherapeutics in colorectal cancer:The role of inflammation

Chemotherapy-induced diarrhea(CID)is a common and often severe side effect experienced by colorectal cancer(CRC)patients during their treatment.As chemotherapy regimens evolve to include more efficacious agents,CID is increasingly becoming a major cause of dose limiting toxicity and merits further investigation.Inflammation is a key factor behind gastrointestinal(GI)toxicity of chemotherapy.Different chemotherapeutic agents activate a diverse range of pro-inflammatory pathways culminating in distinct histopathological changes in the small intestine and colonic mucosa.Here we review the current understanding of the mechanisms behind GI toxicity and the mucositis associated with systemic treatment of CRC.Insights into the inflammatory response activated during this process gained from various models of GI toxicity are discussed.The inflammatory processes contributing to the GI toxicity of chemotherapeutic agents are increasingly being recognised as having an important role in the development of anti-tumor immunity,thus conferring added benefit against tumor recurrence and improving patient survival.We review the basic mechanisms involved in the promotion of immunogenic cell death and its relevance in the treatment of colorectal cancer.Finally,the impact of CID on patient outcomes and therapeutic strategies to prevent or minimise the effect of GI toxicity and mucositis are discussed.

Immunogenic effects of chemotherapyinduced tumor cell death

Emerging evidence suggests that the clinical success of conventional chemotherapy is not solely attributed to tumor cell toxicity,but also results from the restoration of immunosurveillance,which has been largely neglected in the past preclinical and clinical research.Antitumor immune response can be primed by immunogenic cell death(ICD),a type of cell death characterized by cell-surface translocation of calreticulin(CRT),extracellular release of ATP and high mobility group box 1(HMGB1),and stimulation of type I interferon(IFN)responses.Here we summarize recent studies showing conventional chemotherapeutics as ICD inducers,which are capable of modulating tumor infiltrating lymphocytes(TILs)and reactivating antitumor immunity within an immuno-suppressive microenvironment.Such immunological effects of conventional chemotherapy are likely critical for better prognosis of cancer patients.Furthermore,combination of ICD-inducing chemotherapeutics with immunotherapy is a promising approach for improving the clinical outcomes of cancer patients.

Endoplasmic reticulum targeted AIE bioprobe as a highly efficient inducer of immunogenic cell death

Focused oxidative stress of the specific organelles(e.g.,endoplasmic reticulum(ER)and mitochondrion)of cancer cells can boost the immunogenic cell death(ICD)effect for cancer immunotherapy.Herein,an ER-targeted bioprobe with aggregationinduced emission(AIE)characteristics(TPE-PR-FFKDEL)was rationally designed and synthesized by integrating a new AIE photosensitizer with ER targeting peptide,which has been demonstrated to be able to efficiently induce ER oxidative stress to evoke ICD.Compared with the photosensitizer hypericin that is well-known as an ER-targeted ICD inducer,TPE-PR-FFKDEL can lead to more robust emission of immunostimulatory damage-associated molecular patterns such as surface-exposed calreticulin,ATP secretion,and high-mobility group protein B1(HMGB1)and heat shock protein 70(HSP 70)expression.Furthermore,a range of immune responses are activated to protect mice from the attack of cancer cells in vivo.

Combining immune checkpoint blockade with ATP-based immunogenic cell death amplifier for cancer chemo-immunotherapy

Amplifying“eat me signal”during tumor immunogenic cell death(ICD)cascade is crucial for tumor immunotherapy.Inspired by the indispensable role of adenosine triphosphate(ATP,a necessary“eat me signal”for ICD),a versatile ICD amplifier was developed for chemotherapy-sensitized immunotherapy.Doxorubicin(DOX),ATP and ferrous ions(Fe^(2+))were co-assembled into nanosized amplifier(ADO-Fe)throughπ‒πstacking and coordination effect.Meanwhile,phenylboric acid-polyethylene glycol-phenylboric acid(PBA-PEG-PBA)was modified on the surface of ADO-Fe(denoted as PADO-Fe)by the virtue of d-ribose unit of ATP.PADO-Fe could display active targetability against tumor cells via sialic acid/PBA interaction.In acidic microenvironment,PBA-PEG-PBA would dissociate from amplifier.Moreover,high H_(2)O_(2)concentration would induce hydroxyl radical(·OH)and oxygen(O_(2))generation through Fenton reaction by Fe^(2+).DOX and ATP would be released from the amplifier,which could induce ICD effect and“ICD adjuvant”to amplify this process.Together with programmed death ligands 1(PD-L1)checkpoint blockade immunotherapy,PADO-Fe could not only activate immune response against primary tumor,but also strong abscopal effect against distant tumor.Our simple and multifunctional ICD amplifier opens a new window for enhancing ICD effect and immune checkpoint blockade therapy.

Polypeptide nanoformulation-induced immunogenic cell death and remission of immunosuppression for enhanced chemoimmunotherapy

Many conventional chemotherapeutics play an immune-modulating effect by inducing immunogenic cell death(ICD)in tumor cells.However,they hardly arouse strong antitumor immune response because the immunosuppressive lymphocytes are present in the tumor microenvironment.These immunosuppressive lymphocytes include regulatory T cells(Tregs)and myeloid-derived suppressor cells(MDSCs).We used a low dose of doxorubicin(DOX)to induce ICD in combination with immune regulator 1-methylDL-tryptophan(1 MT)to suppress indoleamine 2,3-dioxygenase and overcome Treg-and MDSCassociated immune suppression.By co-encapsulation of DOX and 1 MT into a reduction-responsive polypeptide nanogel,the drugs were simultaneously released in the tumor cells and synergistically performed antitumor efficacy.After treatment,recruitment of Tregs and MDSCs was inhibited,and the frequency of tumor-infiltrating CD8+T cells was remarkably enhanced.These results demonstrated that the chemoimmunotherapy strategy effectively suppressed tumor growth without causing evident adverse effects,indicating its great potential in clinical cancer therapy.

Applying nanotechnology to boost cancer immunotherapy by promoting immunogenic cell death

Tumor immunotherapy,especially immune checkpoint blockade(ICB),has revolutionized the cancer field.However,the limited response of tumors to immunotherapy is a major obstacle.Tumor immunogenic cell death(ICD)is a death mode of tumor cells that can promote tumor immunity.ICD can induce strong an-titumor immune responses through the ectopic exposure of calreticulin on the plasma membrane surface and the release of the non-histone nuclear protein high-mobility group box 1(HMGB1),ATP,and in-terferon(IFN),thus activating an adaptive immune response against dead cell-associated antigens and enhancing the therapeutic effect of tumor immunotherapy.Chemotherapy,radiotherapy,photothermal therapy,magneto-thermodynamics therapy,nanopulse stimulation,and oncolytic virus therapy can all induce a strong antitumor immune response by ICD.In addition,the application of nanotechnology can precisely target drug delivery and improve the efficacy of immunotherapy.Here we introduce the basic concepts and molecular mechanisms underlying the induction of ICD.Then,we summarize and discuss the progress in the application of nanotechnology in immunotherapy to promote ICD.Finally,we attempt to define the challenges and future directions in this area to extend the benefits of ICD to a broader patient population.

Activation of pyroptosis by specific organelle-targeting photodynamic therapy to amplify immunogenic cell death for anti-tumor immunotherapy

Pyroptosis,a unique lytic programmed cell death,inspired tempting implications as potent anti-tumor strategy in pertinent to its potentials in stimulating anti-tumor immunity for eradication of primary tumors and metastasis.Nonetheless,rare therapeutics have been reported to successfully stimulate pyroptosis.In view of the intimate participation of reactive oxygen species(ROS)in stimulating pyroptosis,we attempted to devise a spectrum of well-defined subcellular organelle(including mitochondria,lysosomes and endoplasmic reticulum)-targeting photosensitizers with the aim of precisely localizing ROS(produced from photosensitizers)at the subcellular compartments and explore their potentials in urging pyroptosis and immunogenic cell death(ICD).The subsequent investigations revealed varied degrees of pyroptosis upon photodynamic therapy(PDT)towards cancerous cells,as supported by not only observation of the distinctive morphological and mechanistic characteristics of pyroptosis,but for the first-time explicit validation from comprehensive RNA-Seq analysis.Furthermore,in vivo anti-tumor PDT could exert eradication of the primary tumors,more importantly suppressed the distant tumor and metastatic tumor growth through an abscopal effect,approving the acquirement of specific anti-tumor immunity as a consequence of pyroptosis.Hence,pyroptosis was concluded unprecedently by our proposed organelles-targeting PDT strategy and explicitly delineated with molecular insights into its occurrence and the consequent ICD.

Stimuli-responsive nano vehicle enhances cancer immunotherapy by coordinating mitochondria-targeted immunogenic cell death and PD-L1 blockade

Induction of immunogenic cell death promotes antitumor immunity against cancer. However, majority of clinically-approved drugs are unable to elicit sufficient ICD. Here, our study revealed that mitochondria-targeted delivery of doxorubicin(DOX) massively amplified ICD via substantial generation of reactive oxygen species(ROS) after mitochondrial damage. The underlying mechanism behind increased ICD was further demonstrated to be ascribed to two pathways:(1) ROS elevated endoplasmic reticulum(ER) stress, leading to surface exposure of calreticulin;(2) ROS promoted release of various mitochondriaassociated damage molecules including mitochondrial transcription factor A. Nevertheless, adaptive upregulation of PD-L1 was found after such ICD-inducing treatment. To overcome such immunosuppressive feedback,we developed a tumor stimuli-responsive nano vehicle to simultaneously exert mitochondrial targeted ICD induction and PD-L1 blockade. The nano vehicle was self-assembled from ICD-inducing copolymer and PD-L1 blocking copolymer, and possessed long-circulating property which contributed to better tumor accumulation and mitochondrial targeting. As a result, the nano vehicle remarkably activated antitumor immune responses and exhibited robust antitumor efficacy in both immunogenic and non-immunogenic tumor mouse models.