Syngeneic Tumor Models Research Articles

E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

BackgroundAlterations in several tripartite motif-containing (TRIM) family proteins have been implicated in the pathogenesis of lung cancer. TRIM28, a member of the TRIM E3 ligase family, has been associated with tumorigenesis, cell proliferation, and inflammation. However, little is known about TRIM28 expression and its role in the immune microenvironment of non-small cell lung cancer (NSCLC).MethodsWe assessed the clinical significance of TRIM28 in tissue microarrays and TCGA cohorts. We investigated the function of TRIM28 in syngeneic mouse tumor models, the KrasLSL−G12D/+; Tp53fl/fl (KP) mouse model, and humanized mice. Immune cell composition was analyzed using flow cytometry and immunohistochemistry.ResultsOur findings revealed a positive correlation between TRIM28 expression and the infiltration of suppressive myeloid-derived suppressor cells (MDSCs) in NSCLC. Moreover, silencing TRIM28 enhanced the efficacy of anti-PD-1 immunotherapy by reshaping the inflamed tumor microenvironment. Mechanistically, we demonstrated that TRIM28 could physically interact with receptor-interacting protein kinase 1 (RIPK1) and promote K63-linked ubiquitination of RIPK1, which is crucial for sustaining activation of the NF-κB pathway. Mutagenesis of the E3 ligase domain corroborated the essential role of E3 ligase activity in TRIM28-mediated NF-κB activation. Further experiments revealed that TRIM28 could upregulate the expression of CXCL1 by activating NF-κB signaling. CXCL1 could bind to CXCR2 on MDSCs and promote their migration to the tumor microenvironment. TRIM28 knockdown increased responsiveness to anti-PD-1 therapy in immunocompetent mice, characterized by increased CD8+T tumor-infiltrating lymphocytes and decreased MDSCs.ConclusionThe present study identified TRIM28 as a promoter of chemokine-driven recruitment of MDSCs through RIPK1-mediated NF-κB activation, leading to the suppression of infiltrating activated CD8+T cells and the development of anti-PD-1 resistance. Understanding the regulation of MDSC recruitment and function by TRIM28 provides crucial insights into the association between TRIM28 signaling and the development of an immunosuppressive tumor microenvironment. These insights may inform the development of combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in NSCLC.

XFab-α4-1BB/CD40L fusion protein activates dendritic cells, improves expansion of antigen-specific T cells, and exhibits antitumour efficacy in multiple solid tumour models.

Additional immunotherapies are still warranted for non-responders to checkpoint inhibitors with refractory or relapsing cancers, especially for patients with "cold" tumours lacking significant immune infiltration at treatment onset. We developed XFab-α4-1BB/CD40L, a bispecific antibody targeting 4-1BB and CD40 for dendritic cell activation and priming of tumour-reactive T cells to inhibit tumours. XFab-α4-1BB/CD40L was developed by engineering an anti-4-1BB Fab arm into a CD40L trimer based on XFab® platform. Characterisation of the bispecific antibody was performed by cell-based reporter assays, maturation of dendritic cell assays, and mixed lymphocyte reactions. The abilities of antigen-specific T-cell expansion and antitumour efficacy were assessed in syngeneic mouse tumour models. Toxicological and pharmacodynamic profiles were investigated in non-human primates. XFab-α4-1BB/CD40L demonstrated independent CD40 agonistic activity and conditional 4-1BB activity mediated by CD40 crosslinking, leading to dendritic cell maturation and T-cell proliferation in vitro. We confirmed the expansion of antigen-specific T cells in the vaccination model and potent tumour regression induced by the bispecific antibody alone or in combination with gemcitabine in vivo, concomitant with improved tumour-reactive T-cell infiltration. XFab-α4-1BB/CD40L showed no signs of liver toxicity at doses up to 51mg/kg in a repeated-dose regimen in non-human primates. XFab-α4-1BB/CD40L is capable of enhancing antitumour immunity by modulating dendritic cell and T-cell functions via targeting 4-1BB agonism to areas of CD40 expression. The focused, potent, and safe immune response induced by the bispecific antibody supports further clinical investigations for the treatment of solid tumours.

Inter-crosslinking peptide augments 4-1BB receptor clustering for cancer immunotherapy

4–1BB as a co-stimulatory receptor represents a next-generation therapeutic target in immune-oncology, which has been targeted by agonist antibodies or embedded in CAR-T cell construction. Notably, 4–1BB activation requires cluster formation due to natural ligand association. However, precise and robust regulation of receptor clustering still encounters great challenges. Herein, we report a rationally designed self-assembled peptide ligand, composed of targeting and assembly modules, for in situ triggering ligand-receptor complex aggregation and augmenting clustering on T cells. The unique peptide ligand structure imparts ligand inter-crosslinking (LIC) effect that i) augments assembly rate by 3-fold upon binding to specific receptor of interest, ii) stabilizes ligand-receptor complex compared to both monomeric ligand and ligand aggregates, iii) triggers enhancement of pro-inflammatory cytokine release, cell viability and phosphorylation of transcription factor compared to binding-only molecule. Finally, the ligand also supplements anti-PD-1 therapy alone in CT26 syngeneic murine tumor model with controllable toxicity risk.

Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and impact response to immuno-oncology treatments.

Immuno-oncology (IO) research relies heavily on murine syngeneic tumor models. However, whilst the average age for a cancer diagnosis is 60 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response. Using aged (60-72 weeks old) mice bearing CT26 tumors, we investigated the impact of ageing on tumor growth as well as the immune composition of the tumor and peripheral lymphoid organs. We found many differences in the immune cell composition of both the tumor and tumor-draining lymph node between aged and young mice, such as a reduction in the naïve T cell population and a decreased intratumoral CD8/Treg ratio in aged animals. We hypothesized that these differences may contribute to impaired anti-cancer immune responses in aged mice and therefore assessed the anti-tumor efficacy of different IO therapies in aged mice, including both co-stimulation (using an anti-OX40 antibody) and immune checkpoint blockade (using anti-PD-L1 and anti-CTLA-4 antibodies). Whilst aged mice retained the capacity to generate anti-tumor immune responses, these were significantly attenuated when compared to the responses observed in young mice. These differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.

Interleukin-17 signaling influences CD8+ T cell immunity and tumor progression according to the IL-17 receptor subunit expression pattern in cancer cells

ABSTRACT IL-17 immune responses in cancer are controversial, with both tumor-promoting and tumor-repressing effects observed. To clarify the role of IL-17 signaling in cancer progression, we used syngeneic tumor models from different tissue origins. We found that deficiencies in host IL-17RA or IL-17A/F expression had varying effects on the in vivo growth of different solid tumors including melanoma, sarcoma, lymphoma, and leukemia. In each tumor type, the absence of IL-17 led to changes in the expression of mediators associated with inflammation and metastasis in the tumor microenvironment. Furthermore, IL-17 signaling deficiencies in the hosts resulted in decreased anti-tumor CD8+ T cell immunity and caused tumor-specific changes in several lymphoid cell populations. Our findings were associated with distinct patterns of IL-17A/F cytokine and receptor subunit expression in the injected tumor cell lines. These patterns affected tumor cell responsiveness to IL-17 and downstream intracellular signaling, leading to divergent effects on cancer progression. Additionally, we identified IL-17RC as a critical determinant of the IL-17-mediated response in tumor cells and a potential biomarker for IL-17 signaling effects in tumor progression. Our study offers insight into the molecular mechanisms underlying IL-17 activities in cancer and lays the groundwork for developing personalized immunotherapies.

THU475 Ovarian Tumor-cell Glucocorticoid Receptor Activity Modulates Cytokine Secretion Promoting Recruitment And Activation of Immunosuppressive Cells Into The Tumor Microenvironment

Abstract Disclosure: M. Taya: None. E. LeBlanc: None. L. Bennett: None. S.D. Conzen: None. Elevated levels of immunosuppressive myeloid-derived suppressor cells (MDSCs) circulate in the peripheral blood or infiltrate both tumors and ascites in ovarian cancer patients, however the cause for this observed phenotype is unknown. MDSCs contribute to tumor immune tolerance primarily via inhibition of cytotoxic T-cells or by activation of immunosuppressive T-regulatory cells. We previously showed that high tumor cell glucocorticoid receptor (GR) expression is associated with a relatively poor prognosis in ovarian cancer patients. Additionally, using primary tumor data retrieved from the ovarian cancer TCGA dataset, we show high tumor GR expression is associated with significantly higher expression of Foxp3, a marker of immunosuppressive T-regulatory cells, as well as CD33, a marker of immunosuppressive myeloid cells, further suggesting an immunosuppressive phenotype in ovarian cancer patients. We thus hypothesize that tumor-intrinsic GR activation in ovarian cancer is associated with increased MDSC infiltration into the tumor microenvironment partly due to the modulation of tumor cell cytokine secretion. To determine whether tumor-cell GR activation modulates the cytokine secretome, we previously showed that GR activation in ovarian cancer cells upregulates secretion of immunomodulatory factors including G-CSF, TGFb and CXCL5. Moreover, co-treatment of cells with a selective GR modulator (SGRM) reverses this effect. Preliminary gene expression data indicate GR mediated cytokine secretion and signaling may involve post-transcriptional or post-translational mechanisms. Considering that elevated levels of these cytokines are necessary for the production and recruitment of MDSCs, these data highlight the relevance of this evident glucocorticoid receptor-cytokine circuit in ovarian cancer cells. Furthermore, we show that this tumor-cell cytokine secretome has the capacity to promote differentiation of MDSCs from precursor myeloid cells in healthy PBMCs. Importantly, measuring expression of activation markers such as Arginase-1 and NOS2 illustrated that these MDSCs are in their functional state. To our knowledge, this is the first evidence that a nuclear receptor in ovarian cancer tumor cells is actively driving MDSC differentiation. Finally, employing ovarian cancer xenograft models as well as a syngeneic ID8 tumor model, treatment with SGRMs demonstrated reduced levels of circulating MDSCs in the peripheral blood as well as tumor infiltrated MDSCs. Using neutralizing antibodies for cytokines, we will now identify key mechanisms, specifically, which targetable immunomodulatory factors are contributing to immune suppression. Based on the insight provided by these data, we propose to determine whether targeting ovarian tumor cell-intrinsic GR activation and resulting MDSC infiltration contributes to improved anti-tumor immune response. Presentation: Thursday, June 15, 2023

Multi-Radionuclide in Vivo Imaging of Solid Tumors in Small Laboratory Animals for Experimental Oncology

Purpose: To show the feasibility of the simultaneous trimodal PET/SPECT/CT with 18F-FDG and 177Lu-DOTATATE for characterization of syngeneic subcutaneous mouse tumor model.
 Material and methods: Female C57Bl/6 mice with transplanted subcutaneous melanoma B16F10 were
 used for the study. Preclinical trimodal PET/SPECT/CT imaging system MiLabs VECTor 6 (Netherlands) was used. Mice from the first control group were intravenously injected with 177Lu-DOTATATE
 and underwent SPECT/CT. Mice from the second control group were intravenously injected with 18F-FDG and underwent PET/CT. Mice from the experimental group were intravenously injected with 18F-FDG and 177Lu-DOTATATE in one syringe and underwent simultaneous PET/SPECT/CT imaging.
 Results: Distribution pattern of each radiopharmaceutical 18F-FDG and 177Lu-DOTATATE in the tumor
 of mice from experimental group was similar to the pattern, observed in control groups. 177Lu-DOTATATE was detected in the tumor periphery, and its concentration decreased with time. 18F-FDG
 was detected in the tumor as a large homogeneous focus of hyperfixation and its concentration increased with time. Biodistribution of both tracers in the mouse organism, according to PET/SPECT/CT
 after single injection, was similar to the distribution of the tracers individually in PET and SPECT.
 Conclusion: The obtained results of the study confirm that neither physical or physiological interference nor chemical reaction between two tracers occurred. The tracers had no pharmacological influence on each other. The decrease of 177Lu-DOTATATE in experimental tumor indicated low level of SSTR
 expression, the increase of 18F-FDG revealed high level of metabolic activity in the tumor.

Oncolytic virus M1 functions as a bifunctional checkpoint inhibitor to enhance the antitumor activity of DC vaccine

Although promising, dendritic cell (DC) vaccines still provide limited clinical benefits, mainly due to the immunosuppressive tumor microenvironment (TME) and the lack of tumor-associated antigens (TAAs). Oncolytic virus therapy is an ideal strategy to overcome immunosuppression and expose TAAs; therefore, they may work synergistically with DC vaccines. In this study, we demonstrate that oncolytic virus M1 (OVM) can enhance the antitumor effects of DC vaccines across diverse syngeneic mouse tumor models by increasing the infiltration of CD8+ effector Tcells in the TME. Mechanically, we show that tumor cells counteract DC vaccines through the SIRPα-CD47 immune checkpoint, while OVM can downregulate SIRPα in DCs and CD47 in tumor cells. Since OVM upregulates PD-L1 in DCs, combining PD-L1 blockade with DC vaccines and OVM further enhances antitumor activity. Overall, OVM strengthens the antitumor efficacy of DC vaccines by targeting the SIRPα-CD47 axis, which exerts dominant immunosuppressive effects on DC vaccines.

Longitudinal evaluation of the biodistribution and cellular internalization of the bispecific CD3xTRP1 antibody in syngeneic mouse tumor models

BackgroundCD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the tumor uptake and the extracellular...

Fungal Depletion Bolsters Anti-Tumor Immune Response Elicited by Anti-PD1 Alone and in Combination with Radiation Therapy

Pembrolizumab in combination with chemotherapy has become the standard of care treatment for both metastatic and early-stage triple negative breast cancer (TNBC). Clinical trials are currently underway investigating the use of pembrolizumab with radiation in the neoadjuvant setting in early TNBC. Several groups have described a link between the microbiome and the efficacy of chemotherapy and anti-PD1 immune checkpoint inhibitors (ICIs) in preclinical models. Recent work from our lab has shown that targeting commensal fungi in the microbiome enhances the radiation induced antitumor immune response. Therefore, we hypothesized that fungal depletion might positively impact anti-PD1 therapy and combination treatment with anti-PD1 and radiation therapy (RT). This study utilized an orthotopic syngeneic breast tumor model in which the syngeneic cell line E0771 was injected into the mammary fat pad of female C57BL/6 mice. Tumor-bearing mice were then treated with and without the antifungal fluconazole, anti-PD1, and radiation (16 Gy single fraction) using the X-RAD SmART platform with CT guidance. Tumor volumes were compared using 2-way ANOVA and survival curves analyzed using log rank. In a separate set of experiments, tumor-infiltrating immune cells were isolated and analyzed by high-dimensional multiplex flow cytometry. We found that fungal depletion with fluconazole prior to treatment with anti-PD1 reduced the tumor volume and significantly improved survival in comparison to those treated with anti-PD1 alone (P = 0.0016). To identify what changes in the tumor immune microenvironment is driving this increased anti-tumor response, we performed flow cytometry on immune cells isolated from the tumors. We found that the use of fluconazole prior to anti-PD1 treatment reduced the proportion of CD11b+F4/80+ tumor-associated macrophages (TAMs) (P = 0.01) and increased tumor infiltrating cytotoxic T cell population (P = 0.04) when compared with the use of anti-PD1 alone. We also evaluated the effect of fungal depletion on combination therapy with RT and anti-PD1. Strikingly, we found that mice depleted of fungi with fluconazole prior to radiation and anti-PD1 therapy, have decreased tumor burden and significantly increased survival when compared to their fungally-intact counterparts (P = 0.043). Our data indicates that the depletion of the gut fungal populations induces an increased antitumor response following anti-PD1 alone and in combination with radiation. This increased antitumor immune response is associated with an increase in the cytotoxic CD8+ T cell compartment with concomitant decrease in immunosuppressive tumor associated macrophages.

Radiotherapy Shows Synergistic Anti-Tumor Efficacy with Treg Depletion by Reprogramming the Immune-Excluded Tumor Microenvironment

Tumor-infiltrating Treg cells (TITR) negatively regulates anti-tumor immune response and promotes tumor progression. Increased levels of TITR cells have been associated with poor prognosis in multiple cancer indications which led to the development of immunotherapies that target tumor Tregs. CCR8 antibody is one of the best-in-class therapies in clinical development for depleting TITR cells. It has demonstrated potent efficacy in tumors that are responsive to checkpoint inhibitors but failed in cold tumors that lack immune infiltration and activation. Radiotherapy (RT) has been shown to induce pro-inflammatory responses in tumors, but still failed to show consistent synergy with checkpoint inhibitors. Here we test the hypothesis that RT synergizes with Treg depletion to improve anti-tumor efficacy by reprogramming the immune-excluded tumor microenvironment. The CCR8 + RT combination therapy was tested in syngeneic mouse tumor models: 4T1 breast, LL2 lung, and B16F10 melanoma, all of which present a cold tumor phenotype. Tumor-bearing mice (n = 10 per group) received a single dose of focal irradiation by SARRP, 4 doses of anti-CCR8 depleting antibody or a combination of the two. Tumor size was measured 3 times per week, survival was monitored until day 42, and tumor metastasis was evaluated at endpoint by necropsy. Mouse tumors, spleens, draining lymph nodes, and blood were collected 1 day after the last dose of CCR8 therapy and analyzed by multi-color flow cytometry and RNA-sequencing for pharmacodynamics and mechanism studies. In the syngeneic mouse models, CCR8+RT combination therapy displayed significantly improved tumor growth inhibition, metastasis control, and prolonged survival compared with either treatment alone, without increase in toxicity. Flow cytometry showed reduced TITR population and increased infiltration and activation of CD8+ cytotoxic T cells in the tumors of CCR8 + RT treated mice. Increased activation of CD8+ T cells was also observed in the blood and draining lymph nodes in mice treated with the combination therapy. Moreover, transcriptomic analysis identified up-regulation of pathways associated with TNF-α and IFN-γ response in the combination-treated tumors, suggesting the creation of a pro-inflammatory tumor microenvironment. Interestingly, these changes were only induced by the combination therapy, but not either treatment alone. Immunotherapies targeting Treg/TITR cells have so far failed to show promise in clinical trials. Our data suggest that TITR depletion is not sufficient to treat tumors with minimal immune infiltration. Our animal data demonstrate that combining RT with CCR8 therapy significantly inhibited tumor growth and prevented metastasis by enriching the tumor microenvironment with pro-inflammatory cytokines and activated cytotoxic T cells. These findings have clinical relevance to apply TITR depletion therapy in combination with RT.

The Effect of Dose Rate on Tumor Microenvironment in External Beam Radiotherapy

The fact that treatment can be given at a narrow dose rate range in external radiotherapy (ERT) applications with crude delivery techniques has caused the dose rate to be neglected for a long time. Recently dose delivery technology has rapidly evolved and with the re-introduction of FLASH-RT to the radiation oncology stage, dose rate studies escalated. It is also known that interactions within the tumor microenvironment (TME) play a significant role in tumor prognosis. Herein we aimed to investigate the effect of three different dose rates on TME. To generate the tumor model, 1 million B16-F10 malignant melanoma cells were subcutaneously injected into the right flank of C57BL/6 mice. RT time was estimated to be 0.5 cm in diameter. Forty-two mice were randomly assigned to the control group, 2 different doses (2 Gy and 10 Gy), and 3 different dose rates (1 Gy/min, 6 Gy/min, and 14 Gy/min). Vernier scale measurements were taken every day to monitor the mice's tumor size. Euthanasia is induced on the seventh day after ERT. Tumor and metastatic organs were harvested, and evaluated histomorphological, and immunohistochemically. Intracardiac blood was collected for the comet assay. After radiation, the 10 Gy arm's mean tumor size was lower than that of the 2 Gy and control arms (p = 0.01 and p = 0.007). Tumor size did not differ between the 2 Gy and control arms (p = 0.53). Additionally, there was no difference in tumor diameters between low and high doses at different dose rates (p = 0.12, p = 0.21). Blood mononuclear cells in the control arm had less DNA damage than those in the 10 Gy arm (p = 0.04). The 2 Gy and 10 Gy arms did not differ in terms of DNA damage (p = 0.65). Each dose arm was evaluated in terms of dose rates and no significant difference was found (p = 0.79, p = 0.24). The number of mitoses per 10 HPF was assessed during the histological analysis. The number of mitoses in the 10 Gy arm was less than the 2 Gy arm (p = 0.001). There was no difference at different dose rates in the 2 Gy and 10 Gy arms (p = 0.28, p = 0.97). Pleomorphism score was evaluated as mild-moderate and severe (score 1-2-3). In comparison to the 2 Gy and control arms, the 10 Gy arm had a higher pleomorphism score (p = 0.01). The 2 Gy arm and the control arm did not vary from one another (p = 0.04). Pleomorphism scores were comparable across low and high doses at different dose rates (p = 0.35, p = 0.58). Different dose rates resulted in the same staining pattern with aSMA and SOX-10. In our study involving the B16-F10 syngeneic tumor model, we could not find a significant effect of three different dose rates on TME. With constantly improving treatment techniques, larger dose rates are going to be achieved. Owing to this, protocols should also list dose rate as a treatment variable. This project was supported by the TÜBİTAK, Short Term R&D Funding Program.

High-Dose Acetaminophen with N-acetylcysteine Rescue Inhibits M2 Polarization of Tumor-Associated Macrophages.

High-dose acetaminophen (AAP) with N-acetylcysteine (NAC) rescue is among the few treatments that has shown activity in phase I trials without achieving dose-limiting toxicity that has not progressed to evaluation in later line studies. While the anti-tumor effects of AAP/NAC appear not to be mediated by glutathione depletion and free radical injury, the mechanism of anti-tumor effects of AAP/NAC has not been definitively characterized. In vitro, the effects of AAP/NAC were evaluated on bone marrow derived macrophages. Effects of AAP on IL-4/STAT6 (M2) or IFN/LPS/STAT1 (M1) signaling and downstream gene and protein expression were studied. NAC reversed the AAP toxicity in the normal liver but did not reverse AAP cytotoxicity against tumor cells in vitro. AAP/NAC selectively inhibited IL-4-induced STAT6 phosphorylation but not IFN/LPS-induced STAT1 phosphorylation. Downstream, AAP/NAC inhibited IL-4 induction of M2-associated genes and proteins but did not inhibit the IFN/LPS induction of M1-associated genes and proteins. In vivo, AAP/NAC inhibited tumor growth in EF43.fgf4 and 4T1 triple-negative breast tumors. Flow cytometry of tumor-associated macrophages revealed that AAP/NAC selectively inhibited M2 polarization. The anti-tumor activity of high-dose AAP/NAC is lost in macrophage-depleted mouse syngeneic tumor models, suggesting a macrophage-dependent mechanism of action. In conclusion, our study is the first to show that high-dose AAP/NAC has profound effects on the tumor immune microenvironment that facilitates immune-mediated inhibition of tumor growth.

PARP14 inhibition restores PD-1 immune checkpoint inhibitor response following IFNγ-driven acquired resistance in preclinical cancer models

Resistance mechanisms to immune checkpoint blockade therapy (ICBT) limit its response duration and magnitude. Paradoxically, Interferon γ (IFNγ), a key cytokine for cellular immunity, can promote ICBT resistance. Using syngeneic mouse tumour models, we confirm that chronic IFNγ exposure confers resistance to immunotherapy targeting PD-1 (α-PD-1) in immunocompetent female mice. We observe upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in chronic IFNγ-treated cancer cell models, in patient melanoma with elevated IFNG expression, and in melanoma cell cultures from ICBT-progressing lesions characterised by elevated IFNγ signalling. Effector T cell infiltration is enhanced in tumours derived from cells pre-treated with IFNγ in immunocompetent female mice when PARP14 is pharmacologically inhibited or knocked down, while the presence of regulatory T cells is decreased, leading to restoration of α-PD-1 sensitivity. Finally, we determine that tumours which spontaneously relapse in immunocompetent female mice following α-PD-1 therapy upregulate IFNγ signalling and can also be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.

Combination of irinotecan silicasome nanoparticles with radiation therapy sensitizes immunotherapy by modulating the activation of the cGAS/STING pathway for colorectal cancer

Our previous clinical trial (Identifier: NCT02605265) revealed that addition of irinotecan (IRIN) to neoadjuvant chemoradiotherapy for rectal cancer could improve the curative effect. However, the adverse effects caused by IRIN limited the wide application of IRIN chemoradiotherapy. This study aimed to explore the mechanism under the synergistic effects of IRIN plus radiation therapy in colorectal cancer (CRC) cells and optimization of IRIN delivery via a silicasome nanocarrier in vivo. Our results revealed that compared with single IRIN or radiation treatment, IRIN combined with radiation therapy remarkably activated the intracellular cGAS/STING pathway, and promoted the expression levels of major histocompatibility complex class I (MHC-I) and programmed death ligand 1 (PD-L1). Further, a silicasome (mesoporous silica nanoparticle coated with lipid bilayer) nanocarrier was utilized to improve the delivery of IRIN with enhanced efficacy and reduced side effects. In the MC38 CRC syngeneic tumor model, IRIN silicasome combined with radiation therapy demonstrated a greater antitumor efficacy than free IRIN plus radiation therapy. Flow cytometry showed the increased number of CD4+ T cells, CD8+ T cells, and dendritic cells (DCs) in tumor in the IRIN silicasome plus radiation group. The immunofluorescence staining further confirmed the activated immune microenvironment with the elevated interferon-γ (IFN-γ) deposition. Besides, the antitumor effect of IRIN silicasome plus radiation therapy was synergistically enhanced by anti-PD-1 immunotherapy. These findings indicated that the combination of IRIN silicasome with radiation therapy could sensitize immunotherapy by manipulating the cGAS/STING pathway serving as a new strategy for CRC treatment.

The application of HER2 and CD47 CAR-macrophage in ovarian cancer

BackgroundThe chimeric antigen receptor (CAR)-T therapy has a limited therapeutic effect on solid tumors owing to the limited CAR-T cell infiltration into solid tumors and the inactivation of CAR-T cells by the immunosuppressive tumor microenvironment. Macrophage is an important component of the innate and adaptive immunity, and its unique phagocytic function has been explored to construct CAR macrophages (CAR-Ms) against solid tumors. This study aimed to investigate the therapeutic application of CAR-Ms in ovarian cancer.MethodsIn this study, we constructed novel CAR structures, which consisted of humanized anti-HER2 or CD47 scFv, CD8 hinge region and transmembrane domains, as well as the 4-1BB and CD3ζ intracellular domains. We examined the phagocytosis of HER2 CAR-M and CD47 CAR-M on ovarian cancer cells and the promotion of adaptive immunity. Two syngeneic tumor models were used to estimate the in vivo antitumor activity of HER2 CAR-M and CD47 CAR-M.ResultsWe constructed CAR-Ms targeting HER2 and CD47 and verified their phagocytic ability to ovarian cancer cells in vivo and in vitro. The constructed CAR-Ms showed antigen-specific phagocytosis of ovarian cancer cells in vitro and could activate CD8+ cytotoxic T lymphocyte (CTL) to secrete various anti-tumor factors. For the in vivo model, mice with human-like immune systems were used. We found that CAR-Ms enhanced CD8+ T cell activation, affected tumor-associated macrophage (TAM) phenotype, and led to tumor regression.ConclusionsWe demonstrated the inhibition effect of our constructed novel HER2 CAR-M and CD47 CAR-M on target antigen-positive ovarian cancer in vitro and in vivo, and preliminarily verified that this inhibitory effect is due to phagocytosis, promotion of adaptive immunity and effect on tumor microenvironment.

Discovery of selective and potent USP22 inhibitors via structure-based virtual screening and bioassays exerting anti-tumor activity

Ubiquitin-specific protease 22 (USP22) plays a prominent role in tumor development, invasion, metastasis and immune reprogramming, which has been proposed as a potential therapeutic target for cancer. Herein, we employed a structure-based discovery and biological evaluation and discovered that Rottlerin (IC50 = 2.53 μM) and Morusin (IC50 = 8.29 μM) and as selective and potent USP22 inhibitors. Treatment of HCT116 cells and A375 cells with each of the two compounds resulted in increased monoubiquitination of histones H2A and H2B, as well as reduced protein expression levels of Sirt1 and PD-L1, all of which are known as USP22 substrates. Additionally, our study demonstrated that the administration of Rottlerin or Morusin resulted in an increase H2Bub levels, while simultaneously reducing the expression of Sirt1 and PD-L1 in a manner dependent on USP22. Furthermore, Rottlerin and Morusin were found to enhance the degradation of PD-L1 and Sirt1, as well as increase the polyubiquitination of endogenous PD-L1 and Sirt1 in HCT116 cells. Moreover, in an in vivo syngeneic tumor model, Rottlerin and Morusin exhibited potent antitumor activity, which was accompanied by an enhanced infiltration of T cells into the tumor tissues. Using in-depth molecular dynamics (MD) and binding free energy calculation, conserved residue Leu475 and non-conserved residue Arg419 were proven to be crucial for the binding affinity and inhibitory function of USP22 inhibitors. In summary, our study established a highly efficient approach for USP22-specific inhibitor discovery, which lead to identification of two selective and potent USP22 inhibitors as potential drugs in anticancer therapy.

Tumor cell-expressed lipolysis-stimulated lipoprotein receptor negatively regulates T-cell function.

Lipolysis-stimulated lipoprotein receptor (LSR) is known as a lipoprotein receptor. LSR is expressed in various solid tumors, including epithelial ovarian, gastric, and colon cancers. High LSR expression is significantly associated with poor prognosis, but its role in cancer has not been fully elucidated. LSR belongs to the Ig protein superfamily, which is conserved in B7 family. Here, we assessed LSR as a novel immune checkpoint molecule. We developed a novel anti-LSR antibody (#27-6 mF-18) that defects antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. The #27-6 mF-18 cross-reacts with both human and mouse LSR. We found that LSR was expressed on 4T1 murine breast cancer cell line. The #27-6 mF-18 exhibited antitumor effects against the 4T1 syngeneic tumor model, a poor immunogenic model refractory to treatment with anti-PD-1 or anti-CTLA-4 antibodies. Compared with control antibody-treated mice, mice treated with #27-6 mF-18 showed significantly increased numbers of CD8+ T cells and a ratio of activated CD8+ T cells infiltrated in the tumor tissue. This antitumor effect was abrogated by CD8+ T-cell depletion through anti-CD8 antibody treatment, indicating that LSR negatively regulates tumor immunity by repressing CD8+ T cells. These findings show that LSR negatively regulates T-cell immune activity. LSR targeting could provide immune checkpoint inhibitors for cancer immunotherapy.

AXL receptor tyrosine kinase inhibition improves the anti-tumor effects of CD8+ T cells by inducing CD103+ dendritic cell-mediated T cell priming

AXL is a member of TAM receptor family and has been highlighted as a potential target for cancer treatment. Accumulating evidence has uncovered the critical role of the AXL signaling pathway in tumor growth, metastasis, and resistance against anti-cancer drugs, as well as its association with cancer immune escape. However, the function of AXL as a manipulator of the immune system in the tumor microenvironment (TME) remains unclear. Therefore, in this study, we investigated the impact of AXL on immune cells in the TME of a syngeneic tumor model using AXL knockout (AXL−/−) mice. Compared to AXL wild-type (AXL+/+) mice, tumor growth was significantly suppressed in AXL−/− mice, and an induced population of tumor-infiltrated CD8+ T cells and CD103+ dendritic cells (DCs) was observed. The change of CD8+ T cells and CD103+ DCs was also confirmed in tumor-draining lymph nodes (TdLN). In addition, the clonal expansion of OVA-specific CD8+ T cells was dominant in AXL−/− mice. Finally, anti-PD-1 treatment evidenced synergistic anti-cancer effects in AXL−/− mice. Overall, our data indicate that AXL signaling may inhibit the clonal expansion of tumor-specific CD8+ T cells through the regulation of the migration of CD8+ T cells and DCs in TME. Thus, AXL may be a powerful molecular target to improve anti-cancer effects through single or combined therapy with immune checkpoint inhibitors (ICI).

Enhancement of Radiation Therapy through Blockade of the Immune Checkpoint, V-domain Ig Suppressor of T Cell Activation (VISTA), in Melanoma and Adenocarcinoma Murine Models.

Radiation therapy (RT) has recently demonstrated promise at stimulating an enhanced immune response. The recent success of immunotherapies, such as checkpoint inhibitors, CART cells, and other immune modulators, affords new opportunities for combination with radiation. The aim of this study is to evaluate whether and to what extent blockade of VISTA, an immune checkpoint, can potentiate the tumor control ability of radiation therapy. Our study is novel in that it is the first comparison of two VISTA-blocking methods (antibody inhibition and genetic knockout) in combination with RT. VISTA was blocked either through genetic knockout (KO) or an inhibitory antibody and combined with RT in two syngeneic murine flank tumor models (B16 and MC38). Selected mRNA, immune cell infiltration, and tumor growth delay were used to assess the biological effects. When combined with a single 15Gy radiation dose, VISTA blockade via genetic knockout in the B16 model and via anti-VISTA antibodies in the MC38 model significantly improved survival compared to RT alone by an average of 5.5 days and 6.3 days, respectively (p < 0.05). The gene expression data suggest that the mechanism behind the enhanced tumor control is primarily a result of increased apoptosis and immune-mediated cytotoxicity. VISTA blockade significantly enhances the anti-tumor effect of a single dose of 15Gy radiation through increased expression and stimulation of cell-mediated apoptosis pathways. These results suggest that VISTA is a biologically relevant immune promoter that has the potential to enhance the efficacy of a large single radiation dose in a synergic manner.