CT26 Tumor Cells Research Articles

Tumor cell loaded thermosensitive hydrogel for photodynamic therapy associated tumor antigens release

Background: Immunotherapy is a powerful strategy for treating cancer and can be used to inhibit the post-surgical relapse of tumors. Methods: To achieve this, a Cell@hydrogel was developed as a template using a mixture of CT26 tumor cells and Pluronic® F-127/gelatin. Results: The proposed mixture has a solution-to-gelation functionality and vice versa. The morphology of the Cell@hydrogel was characterized by scanning electron microscopy and confocal microscopy. For photodynamic immunotherapy, the Cell@hydrogel was functionalized with Cy7 (Cy7-Cell@hydrogel) to quantify reactive oxygen species in CT26 tumor cells. Gel electrophoresis and membrane integrity tests were performed to determine the efficiency of the Cy7-Cell@hydrogel following photodynamic therapy. Conclusions: This protocol provides an alternative approach that mechanistically inhibits the post-surgical relapse of solid tumors based on immunotherapy.

NaCe0.7FX:Gd0.1,Tb0.2 nanoscintillator as a new sonosensitizer for potentiating cancer sonodynamic-immune therapy

Trends in sonodynamic therapy (SDT) have stimulated greater research efforts toward the development of novel sonosensitizers due to their crucial roles in increasing reactive oxygen species (ROS). Herein, a new scintillator, NaCe0.7FX:Gd0.1,Tb0.2, was explored as an ultrasmall inorganic sonosensitizer for glutathione (GSH) depletion-enhanced SDT of cancer. Specifically, fluorine vacancy-donated defective NaCe0.7FX:Gd0.1,Tb0.2 scintillating nanodots (ScNDs) were prepared via a high-temperature reaction method. PEGylated ScNDs, namely ScND-PEG, exhausted GSH at first, which further augmented the sonodynamic performance of ROS generation. In vitro studies demonstrated that US-irradiated ScND-PEG exerted significant cytotoxicity on CT26 tumor cells by triggering immunogenic cell death (ICD). In vivo studies revealed that ScND-PEG-mediated enhanced SDT substantially inhibited tumor development by activating cytotoxic T lymphocytes and secreting antitumor proinflammatory cytokines. With the assistance of immune checkpoint blockade (ICB), improved antitumor outcomes were further obtained with prolonged survival time. Notably, no significant acute or chronic toxicity occurred during our treatment schedule based on ScND-PEG. Overall, our findings paved the way for identifying a new kind of ScND-based inorganic sonosensitizer as a reserve, which might open up new opportunities for advancing sonodynamic-immune oncotherapy.

Catalase-positive Staphylococcus epidermidis based cryo-millineedle platform facilitates the photo-immunotherapy against colorectal cancer via hypoxia improvement

The synergistic anti-tumor impact of phototherapy and a cascading immune response are profoundly limited by hypoxia and a weakened immune response. Intravenous and intratumoral injection of therapeutic drugs also cause pain, rapid drug clearance and low utilization rates. Here, a novel cryo-millineedle platform for intratumoral delivery of a phototherapy system, S.epi@IR820, is developed in this work, combining the properties of Staphylococcus epidermidis (S. epidermidis) and IR820 for photo-immunotherapy of colorectal cancer. In this cryo-millineedle platform, S. epidermidis enhances the near-infrared absorption and light stability of IR820 and catalyzes the decomposition of H2O2 into O2 via an endogenous catalase to relieve tumor hypoxia, improve phototherapy and enhance immunogenic cell death (ICD). More interestingly, the native immunogenicity of S. epidermidis and ICD elicited by phototherapy achieved a potent anti-tumor immune response. To the best of our knowledge, this is the first study to utilize native S. epidermidis to relieve hypoxia and facilitate phototherapy. Both in vitro and in vivo experiments showed that the millineedle based phototherapy system can efficiently catalyse the decomposition of H2O2 into O2, facilitate phototherapeutic killing of CT26 tumor cells by S.epi@IR820 and enhance ICD, thus successfully activated the immune response and achieved the photo-immunotherapy against colorectal cancer. In conclusion, this study provides a novel strategy for enhanced anti-tumor efficiency of photo-immunotherapy, and develops an effective method for orthotopic administration of tumors.

Self-Delivery Nanobooster to Enhance Immunogenic Cell Death for Cancer Chemoimmunotherapy.

Inducing immunogenic cell death (ICD) is a promising strategy for cancer immunotherapy. Shikonin (SHK), a naphthoquinone compound from Lithospermum erythrorhizon, can stimulate antitumor immunity by inducing ICD. Nevertheless, the immunogenicity of tumor cells killed by SHK is weak. Endoplasmic reticulum (ER) stress is an important intracellular pathway of the ICD effect. Curcumin (CUR) can directly induce ER stress by disrupting Ca2+ homeostasis, which might enhance SHK-induced ICD effect. A self-delivery ICD effect nanobooster (CS-PEG NPs) was developed by the self-assembly of SHK (ICD inducer) and CUR (ICD enhancer) with the assistance of DSPE-PEG2K for cancer chemoimmunotherapy. CS-PEG NPs possessed effective CT26 tumor cell cellular uptake and tumor accumulation ability. Moreover, enhanced cytotoxicity against tumor cells and apoptosis promotion were achieved due to the synergistic effect of CUR and SHK. Notably, CS-PEG NPs induced obvious Ca2+ homeostasis disruption, ER stress, and ICD effect. Subsequently, the neoantigens produced by the robust ICD effect in vivo promoted dendritic cell maturation, which further recruited and activated cytotoxic T lymphocytes. Superior antitumor efficacy and systemic antitumor immunity were observed in the CT26-bearing BALB/c mouse model without side effects in major organs. This study offers a promising self-delivery nanobooster to induce strong ICD effect and antitumor immunity for cancer chemoimmunotherapy.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway.

Nanosized outer membrane vesicles (OMVs) from Gram-negative bacteria have attracted increasing interest because of their antitumor activity. However, the antitumor effects of MVs isolated from Gram-positive bacteria have rarely been investigated. MVs of Staphylococcus aureus USA300 were prepared and their antitumor efficacy was evaluated using tumor-bearing mouse models. A gene knock-in assay was performed to generate luciferase Antares2-MVs for bioluminescent detection. Cell counting kit-8 and lactic dehydrogenase release assays were used to detect the toxicity of the MVs against tumor cells in vitro. Active caspase-1 and gasdermin D (GSDMD) levels were determined using Western blot, and the tumor inhibition ability of MVs was determined in B16F10 cells treated with a caspase-1 inhibitor. The vesicular particles of S. aureus USA300 MVs were 55.23 ± 8.17 nm in diameter, and 5 μg of MVs remarkably inhibited the growth of B16F10 melanoma in C57BL/6 mice and CT26 colon adenocarcinoma in BALB/c mice. The bioluminescent signals correlated well with the concentrations of the engineered Antares2-MVs (R2 = 0.999), and the sensitivity for bioluminescence imaging was 4 × 10-3 μg. Antares2-MVs can directly target tumor tissues in vivo, and 20 μg/mL Antares2-MVs considerably reduced the growth of B16F10 and CT26 tumor cells, but not non-carcinomatous bEnd.3 cells. MV treatment substantially increased the level of active caspase-1, which processes GSDMD to trigger pyroptosis in tumor cells. Blocking caspase-1 activation with VX-765 significantly protected tumor cells from MV killing in vitro and in vivo. S. aureus MVs can kill tumor cells by activating the pyroptosis pathway, and the induction of pyroptosis in tumor cells is a promising strategy for cancer treatment.

Reactive oxygen species-sensitive chondroitin sulfate A-cholesteryl hemisuccinate micelles for targeted doxorubicin delivery in tumor therapy

The efficient drug release triggered by the tumor microenvironment is challenging in the development of stimuli-sensitive nanomedicine. In this study, we developed novel polymeric micelles sensitive to reactive oxygen species (ROS) for targeted delivery of doxorubicin (DOX) in tumor therapy. Hydrophilic chondroitin sulfate A (CSA) was conjugated with cholesteryl hemisuccinate (CHS) via a ROS-cleavable thioketal (TK) bond. The resulting CSA-TK-CHS could form self-assembled micelles, and DOX-loaded CSA-TK-CHS (CSA-TK-CHS/DOX) micelles displayed a nearly spherical shape with an average hydrodynamic diameter of 324 nm. The bioresponsive DOX release of CSA-TK-CHS/DOX micelles was evaluated in an H2O2-containing phosphate buffer solution (PBS). CSA-TK-CHS/DOX micelles showed higher cellular uptake than free DOX in 4T1 cells by confocal laser scanning microscopy (CLSM) observation. And CSA-TK-CHS/DOX improved DOX cytotoxicity against CD44-overexpressed 4T1 and CT26 tumor cells. Notably, CSA-TK-CHS/DOX micelles displayed significantly stronger potency (P < 0.01) in antitumor activity than DOX·HCl in orthotropic 4T1-bearing Balb/c mice. Overall, these findings indicate that amphiphilic CSA-TK-CHS micelles are a promising targeted and intelligent drug carrier for tumor therapy.

F1 fraction isolated from Mesobuthus eupeus scorpion venom induces macrophage polarization toward M1 phenotype and exerts anti-tumoral effects on the CT26 tumor cell line

Scorpion venoms identified as agents with anti-tumor and anti-angiogenic features. Tumor microenvironment (TME) plays a pivotal role in the process of tumorigenesis, tumor development, and polarization of M2 phenotype tumor associated macrophages (TAMs). M2 polarized cells are associated with tumor growth, invasion, and metastasis. The fractionation process was performed by gel filtration chromatography on a Sephadex G50 column. To elucidate whether scorpion venom can alter macrophage polarization, we treated interleukin (IL)-4-polarized M2 cells with isolated fractions from Mesobuthus eupeus. Next, we evaluated the cytokine production and specific markers expression for M2 and M1 phenotype using enzyme linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR), respectively. The phagocytic capacity of macrophages was also assessed. In addition, the migration assay and MTT analysis were performed to investigate the effects of reprogrammed macrophages on the CT-26 colon cancer cells. The results indicated that F1 fraction of venom significantly upregulated the levels and expression of M1-associated cytokines and markers, including tumor necrosis factor-alpha (TNF-α) (p < 0.001), IL-1 (p < 0.01), interferon regulatory factor 5 (IRF5) (p < 0.0001), induced nitric oxide synthase (iNOS) (p < 0.0001), and CD86 (p < 0.0001), and downregulated M2-related markers, including transforming growth factor-beta (TGF-β) (p < 0.05), IL-10 (p < 0.05), Fizz1 (p < 0.0001), arginase-1 (Arg-1) (p < 0.0001), and CD206 (p < 0.001). The macrophage phagocytic capacity was enhanced after treatment with F1 fraction (p < 0.01). Moreover, incubation of CT-26 cell line with conditioned media of F1-treated macrophages suppressed migration (p < 0.0001) and proliferation (p < 0.01) of tumor cells. In conclusion, our findings demonstrated the potential of Mesobuthus eupeus venom in M2-to-M1 macrophage polarization as a promising therapeutic approach against proliferation and metastasis of colon cancer cells.

Abstract 3740: Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor

Abstract Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC) targeted to carcino-embryonic antigen-related cell adhesion molecule 5 (CEACAM5) and delivering DM4, a maytansinoid cytotoxic. CEACAM5 is highly expressed in several tumor types, including non-squamous non-small cell lung cancer (NSQ NSCLC). It is currently being evaluated in patients with advanced CEACAM5-positive NSQ NSCLC, in a phase II study in combination with pembrolizumab, and a phase III trial as single agent in comparison with docetaxel. Preclinical investigations were performed to determine whether SAR408701 could be beneficially combined with immune checkpoint inhibitors. We, first, demonstrated that the tusamitamab ravtansine payload, DM4, is able to induce robust immunogenic cell death (ICD) using an in vivo vaccination assay. In this assay, immunocompetent mice were “vaccinated” by DM4-treated CT26 cells and then later rechallenged with CT26 tumor cells. Tumor rejection was observed in more than 50% of “vaccinated” mice. The ICD was characterized by the expression or release of danger-associated molecular patterns (DAMPs), and we observed DAMP modulation such as calreticulin (CRT) exposure at the cell surface of dying tumor cells, and the release of the High-Mobility Group Box 1 (HMGB1) in the extracellular space by CT26 cells in response to DM4 treatment. We also showed that DM4-treated CT26 tumor cells implanted into mice or SAR408701 treatment of MC38 tumor-bearing mice induced an elevation of plasmatic level of murine IL-6 and INFγ, revealing potential antitumor immunity modulation by the ADC and its payload. Immunohistochemistry analysis of MC38 tumors also revealed that tusamitamab ravtansine treatment induced a dose-dependent increase of CD8 T-cell infiltration in tumors correlating to dose-dependent efficacy. Finally, tusamitamab ravtansine was combined with immune checkpoint inhibitors (ICIs) in immunocompetent mice bearing MC38 tumor model. In this model, the combination of tusamitamab ravtansine with anti-PD-1 or anti-PD-L1 antibody led to complete response and, even, tumor-free survivors at 120 days post tumor implantation while single agents were inactive or transiently active. In summary, these preclinical data show that tusamitamab ravtansine could be beneficially combined with immune checkpoint and strongly support their evaluation in clinical development. Citation Format: Celine Nicolazzi, Anne-Marie Lefebvre, Nicolas Moindrot, Christelle Larois, Marion Classe, Sukhvinder Sidhu. Tusamitamab ravtansine induces immunogenic cell death and synergizes with anti-PD-1 or anti-PD-L1 antibody combination in solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3740.

Abstract 1913: KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine

Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with diverse biological functions, including apoptosis, cell survival, inflammation, and immunity. While recombinant human TNFα has demonstrated therapeutic efficacy in isolated limb perfusion for soft tissue sarcoma and melanoma, its systemic administration is limited by severe toxicities, including hypotension. Tumor-specific targeting and enhancement of TNFα's tumor cell killing activity hold promise for improving its therapeutic index. To address these challenges, we developed KD5005, a novel HER-2-targeted immunocytokine fusion protein comprising an anti-HER-2 IgG1 monoclonal antibody (trastuzumab) and human TNFα. Tissue distribution studies revealed that KD5005 specifically accumulated in HER-2-expressing tumors, demonstrating its tumor-targeting capability. In vitro, KD5005 exhibited reduced TNFα-mediated apoptosis in L929 cells compared to recombinant TNFα, indicating its lower non-specific toxicity. Notably, KD5005 induced apoptosis of HER-2-positive human gastric cancer NCI-N87 cells at significantly lower concentrations than trastuzumab, TNFα, or their combination, demonstrating its enhanced tumor cell killing activity. The potent apoptotic activity of KD5005 was HER-2-dependent, as it did not show increased apoptotic activity in WT mouse forestomach carcinoma (MFC) cells and had similar apoptotic activity compared to the combination of trastuzumab and TNFα. This HER-2 dependency further supports the tumor-specific targeting of KD5005.In vivo, KD5005 demonstrated superior anti-tumor efficacy compared to the combination of trastuzumab and TNFα in syngeneic tumor models using CT26 tumor cells transfected with the human HER-2 gene. KD5005 induced robust tumor regression and durable cure in 7 of 8 mice, while the combination of trastuzumab and TNFα elicited weaker therapeutic responses, resulting in tumor rejection in only 3 of 8 mice. Pharmacokinetic and toxicity studies of KD5005 were conducted in cynomolgus monkeys. KD5005 exhibited a significantly prolonged in vivo half-life compared to TNFα, indicating its improved pharmacokinetic profile. KD5005 was administered intravenously every two weeks to the same animal in a dose-escalation study at 0.3 mg/kg, 1.0 mg/kg, 3 mg/kg, 10 mg/kg, and 30 mg/kg. The toxicity study results showed that the maximum tolerated dose for KD5005 in cynomolgus monkeys is 10 mg/kg, indicating its favorable toxicity profile. In summary, we have developed KD5005, a HER-2-targeted immunocytokine fusion protein with enhanced tumor-killing activity, improved pharmacokinetic properties, and a favorable toxicity profile. These preclinical findings support the further clinical development of KD5005 as a promising therapeutic agent for HER-2-positive cancers. Citation Format: YI CHEN, PENG LI, GUOBO FANG, HENG WU, CHI ZHANG, ZELING CAI. KD5005: HER-2 dependent strong potent anti-tumor activity of an anti-HER-2 mAb-TNFaimmunocytokine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1913.

MHY1485 potentiates immunogenic cell death induction and anti-cancer immunity following irradiation.

Recent in vitro experiments showed that combined treatment with MHY1485, a low-molecular-weight compound, and X-ray irradiation significantly increased apoptosis and senescence in tumor cells, which was associated with oxidative stress, endoplasmic reticulum (ER) stress and p21 stabilization, compared to radiation treatment alone. However, evidence for MHY1485 treatment-mediated suppression of tumor growth in animals is still lacking. Furthermore, it has been shown that ER stress enhances immunogenic cell death (ICD) in tumor cells, as it can exert a favorable influence on the anti-cancer immune system. In the present study, we examined whether co-treatment of MHY1485 and X-ray irradiation induces ICD and in vivo tumor growth suppression using the CT26 and Lewis lung carcinoma murine tumor cell lines. We found that MHY1485 + X-ray treatment promotes ICD more effectively than X-ray treatment alone. MHY1485 suppresses tumor growth in vivo under co-treatment with X-rays and increases INF-γ, tumor necrosis factor, interleukin-2 and interleukin-12 levels in the spleen as well as the presence of CD8+ cells in the tumor. The results suggest that MHY1485 treatment leads to the conversion of irradiated tumors into effective vaccines. Thus, MHY1485 is a promising lead compound for use in combination with radiotherapy.

Novel nanotherapeutics for cancer immunotherapy by albumin nanoparticles functionalized with PD-1 and PD-L1 aptamers

BackgroundPD-1/PD-L1 blockade plays a crucial role in cancer immunotherapy. Exploration of new technologies to further enhance the efficacy of PD-1/PD-L1 blockade is therefore of potential medical importance. Nanotherapeutics can accumulate in tumor tissues due to enhanced permeability and retention (EPR) effects. In this study, a novel nanotherapeutic for cancer immunotherapy was implemented with albumin nanoparticles functionalized by both PD-1 and PD-L1 aptamers.ResultsAlbumin nanoparticles (NP) were functionalized with either PD-1 aptamers (PD1-NP), PD-L1 aptamers (PDL1-NP), or both types of aptamers (PD1-NP-PDL1). Average sizes of PD1-NP, PDL1-NP, and PD1-NP-PDL1 were 141.8 nm, 141.8 nm, and 164.2 nm, respectively. PD1-NP had good affinity for activated T cells that expresses PD-1. Similarly, PDL1-NP could bind with MDA-MB-231 or CT26 tumor cells that express PD-L1. Moreover, the bispecific PD1-NP-PDL1 could bind with both the activated T cells and the PD-L1-expressing tumor cells, and tether the two type of cells together. Functionally, aptamer-modified nanoparticles exhibited stronger immune-stimulating effects vs. free aptamers. Specifically, PD1-NP or PDL1-NP induced stronger lymphocyte-mediated cytotoxicity against PD-L1-expressing tumor cells in vitro vs. free PD-1 or PD-L1 aptamers. Animal studies also showed that PD1-NP or PDL1-NP significantly improved antitumor efficacy against CT26 colon cancer in vivo vs. free PD-1 or PD-L1 aptamers. Importantly, the bispecific PD1-NP-PDL1 further boosted the in vivo antitumor efficacy compared with PD1-NP or PDL1-NP, without raising systemic toxicity.ConclusionThe results suggest that the bispecific PD1-NP-PDL1 is a promising nanotherapeutic to improve the efficacy of PD-1/PD-L1 blockade, and may have application potential in colon cancer treatment.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

Macrophage-membrane-coated hybrid nanoparticles with self-supplied hydrogen peroxide for enhanced chemodynamic tumor therapy.

Addressing the challenges of chemodynamic therapies (CDTs) relying on Fenton reactions in malignant tumors is an active research area. Here, we report a method to develop pH-responsive hybrid nanoparticles for enhanced chemodynamic tumor treatment. Reactive CaO2 nanoparticles (core) are isolated by biocompatible ZIF-8 doped with Fe2+ (shell), and then encapsulated by macrophage membranes (symbolized as CaO2@Fe-ZIF-8@macrophage membrane or CFZM), thus endowed with high stability under normal physiological conditions. Our design features active tumor-homing by the macrophage-membrane coating, tumor microenvironment (TME)-responsive cargo release, and self-supplied hydrogen peroxide for promotion of the Fenton reaction. We demonstrate the improved delivery/tumor cell uptake of CFZM, the efficient production of toxic ˙OH with self-supplied H2O2 in CFZM, and high-efficacy tumor ablation on BALB/c mice bearing CT26 tumor cells. This offers a translational strategy to develop active tumor-targeting and TME-responsive nanotherapeutics with enhanced CDT against malignant tumors.

Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer

Background: Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer. Methods: We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells. Results: ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors. Conclusions: Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.

Enhancement of cancer immunotherapy using CRT valgus tumor cell membranes coated bacterial whole peptidoglycan combined with radiotherapy

Immunotherapy has achieved some success in preclinical and clinical studies, but the immunosuppressive tumor microenvironment (TME) leads to a low response rate of this therapy. In this paper, we describe a calreticulin (CRT) valgus CT-26 tumor cell membranes-coated bacterial whole peptidoglycan (WPG) from P. aeruginosa (CPW/SR) with a high rate of the STING agonist loading. In the construct, WPG from P. aeruginosa (P.WPG) was used as a carrier with the immunoadjuvant function while synergistically promoting the maturation of dendritic cells (DCs) through the delivery of the STING agonist SR-717. CRT valgus tumor cell membranes were identified and internalized by DCs via CRT on the surface. In addition, this construct was able to reverse the immunosuppressive TME in vivo and achieve synergies with radiotherapy by creating a personalized tumor vaccine, therefore achieving more resultful antitumor efficacy. In conclusion, CPW/SR constructed in this paper provides a new approach for achieving efficient cancer immunotherapy and combination therapy.

Tumor cell-intrinsic c-Myb upregulation stimulates antitumor immunity in a murine colorectal cancer model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. While no differences in proliferation, apoptosis, and angiogenesis of tumors were evident tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAMs). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding 2-microglobulin and IFNβ, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

Radiofrequency Ablation–Induced Tumor Growth Is Suppressed by MicroRNA-21 Inhibition in Murine Models of Intrahepatic Colorectal Carcinoma

PurposeTo investigate the role of microRNA-21 (miR21) in radiofrequency (RF) ablation–induced tumor growth and whether miR21 inhibition suppresses tumorigenesis. Material and MethodsStandardized liver RF ablation was applied to 35 C57/BL6 mice. miR21 and target proteins pSTAT3, PDCD4, and PTEN were assayed 3 hours, 24 hours, and 3 days after ablation. Next, 53 Balb/c and 44 C57BL/6 mice received Antago-miR21 or scrambled Antago-nc control, followed by intrasplenic injection of 10,000 CT26 or MC38 colorectal tumor cells, respectively. Hepatic RF ablation or sham ablation was performed 24 hours later. Metastases were quantified and tumor microvascular density (MVD) and cellular proliferation were assessed at 14 or 21 days after the procedures, respectively. ResultsRF ablation significantly increased miR21 levels in plasma and hepatic tissue at 3 and 24 hours as well as target proteins at 3 days after ablation (P < .05, all comparisons). RF ablation nearly doubled tumor growth (CT26, 2.0 SD ± 1.0 fold change [fc]; MC38, 1.9 SD ± 0.9 fc) and increased MVD (CT26, 1.9 SD ± 1.0 fc; MC38, 1.5 ± 0.5 fc) and cellular proliferation (CT26, 1.7 SD ± 0.7 fc; MC38, 1.4 SD ± 0.5 fc) compared with sham ablation (P < .05, all comparisons). RF ablation–induced tumor growth was suppressed when Antago-miR21 was administered (CT26, 1.0 SD ± 0.7 fc; MC38, 0.9 SD ± 0.4 fc) (P < .01, both comparisons). Likewise, Antago-miR21 decreased MVD (CT26, 1.0 SD ± 0.3 fc; MC38, 1.0 SD ± 0.2 fc) and cellular proliferation (CT26, 0.9 SD ± 0.3 fc; MC38, 0.8 SD ± 0.3 fc) compared with baseline (P < .05, all comparisons). ConclusionsRF ablation upregulates protumorigenic miR21, which subsequently influences downstream tumor-promoting protein pathways. This effect can potentially be suppressed by specific inhibition of miR21, rendering this microRNA a pivotal and targetable driver of tumorigenesis after hepatic thermal ablation.

Murine cytotoxic CD4+ T cells in the tumor microenvironment are at a hyper-maturation stage of Th1 CD4+ T cells sustained by IL-12.

The roles of tumor-infiltrating CD4+Foxp3- T cells are not well characterized due to their plasticity of differentiation, and varying levels of activation or exhaustion. To further clarify this issue, we used a model featuring subcutaneous murine colon cancer and analyzed the dynamic changes of phenotype and function of the tumor-associated CD4+ T-cell response. We found that, even at a late stage of tumor growth, the tumor-infiltrating CD4+Foxp3- T cells still expressed effector molecules, inflammatory cytokines and molecules that are expressed at reduced levels in exhausted cells. We used microarrays to examine the gene-expression profiles of different subsets of CD4+ T cells and revealed that the tumor-infiltrating CD4+Foxp3- T cells expressed not only type 1 helper (Th1) cytokines, but also cytolytic granules such as those encoded by Gzmb and Prf1. In contrast to CD4+ regulatory T cells, these cells exclusively co-expressed natural killer receptor markers and cytolytic molecules as shown by flow-cytometry studies. We used an ex vivo killing assay and proved that they could directly suppress CT26 tumor cells through granzyme B and perforin. Finally, we used pathway analysis and ex vivo stimulation to confirm that the CD4+Foxp3- T cells expressed higher levels of IL12rb1 genes and were activated by the IL-12/IL-27 pathway. In conclusion, this work finds that, in late-stage tumors, the tumor-infiltrating lymphocyte population of CD4+ cells harbored a sustained, hyper-maturated Th1 status with cytotoxic function supported by IL-12.