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Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only 10–30% of patients with various cancers. Literature has demonstrated that a “hot tumor” which contains high T lymphocytes in the tumor microenvironment exhibits a better response to immunotherapies than a “cold tumor.” This study aimed to investigate whether tumor-intrinsic IFNα and CXCL10 determine the recruitment and activation of CD8+ T cells to become “hot tumor.” In this study, we found that CXCL10 overexpressed in a variety of tumors including lung, colon, and liver tumors with a correlation with PD-L1. High PD-L1 and CXCL10 are associated with better survival rates in tumor patients receiving immunotherapies. IFNs-downstream transcriptional factor IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression. We demonstrated that IRF-1 and STAT1 were both bound with the promoters of PD-L1 and CXCL10, sharing the same signaling pathway and determining IFNs-mediated PD-L1 and CXCL10 expression. In addition, IFNα significantly increased activation marker IFNγ in PBMCs, promoting M1 type monocyte differentiation, CD4+ T, and CD8+ T cell activation. Particularly, we found that CD8+ T lymphocytes abundantly expressed CXCR3, a receptor of CXCL10, by flow cytometry, indicating that tumor-intrinsic CXCL10 potentially recruited CD8+ T in tumor microenvironment. To demonstrate the hypothesis, immunotherapy-sensitive CT26 and immunotherapy-resistant LL/2 were used and we found that CT26 cells exhibited higher IFNα, IFNγ, CXCL10, and PD-L1 levels compared to LL/2, leading to higher IFNγ expression in mouse splenocytes. Moreover, we found that CD8+ T cells were recruited by CXCL10 in vitro, whereas SCH546738, an inhibitor of CXCR3, inhibited T cell migration and splenocytes-mediated anti-tumor effect. We then confirmed that CT26-derived tumor was sensitive to αPD-L1 immunotherapy and LL/2-tumor was resistant, whereas αPD-L1 significantly increased T lymphocyte activation marker CD107a in CT26-derived BALB/c mice. In conclusion, this study revealed that CXCL10 expression is correlated with PD-L1 in tumors, sharing the same signaling pathway and associating with better immunotherapeutic efficacy. Further evidence in the syngeneic tumor models demonstrated that immunotherapy-sensitive CT26 intrinsically exhibited higher IFNα and CXCL10 compared to immunotherapy-resistant LL/2 to recruit and activate CD8+ T cells in the tumor microenvironment, exhibiting “hot tumor” characteristic of sensitizing αPD-L1 immunotherapies.

BackgroundImmunotherapy using checkpoint inhibitors, especially PD-1/PD-L1 inhibitors, has now evolved into the most promising therapy for cancer patients. However, most of these inhibitors are monoclonal antibodies, and their large size may limit their tumor penetration, leading to suboptimal efficacy. As a result, there has been a growing interest in developing low-molecular-weight checkpoint inhibitors.MethodsWe developed a novel biopanning strategy to discover small peptide-based anti-PD-L1 inhibitors. The affinity and specificity of the peptides to PD-L1 were examined using various assays. Three-dimensional (3D) spheroid penetration study was performed to determine the tumor penetration capability of the peptides. Anti-tumor activity of the peptides was evaluated in mice bearing CT26 tumor cells.ResultsWe discover several anti-PD-L1 peptide inhibitors to block PD-1/PD-L1 interaction. The peptides exhibit high affinity and specificity to human PD-L1 protein as well as PD-L1-overexpressing human cancer cells MDA-MB-231 and DU-145. Molecular docking studies indicate that the peptide CLP002 specifically binds to PD-L1 at the residues where PD-L1 interacts with PD-1. The peptide also blocks the CD80/PD-L1 interaction, which may further enhance the immune response of tumor-infiltrating T cells. Compared to antibody, the peptide CLP002 exhibits better tumor penetration in a 3D tumor spheroid model. The peptide CLP002 restores proliferation and prevents apoptosis of T cells that are co-cultured with cancer cells. The peptide CLP002 also inhibits tumor growth and increases survival of CT26 tumor-bearing mice.ConclusionsThis study demonstrated the feasibility of using phage display to discover small peptide-based checkpoint inhibitors. Our results also suggested that the anti-PD-L1 peptide represents a promising low-molecular-weight checkpoint inhibitor for cancer immunotherapy.

The present study reflected on high-priority biological activities of novel silver nanoparticles (AgNPs) synthesized via two cold-tolerant strains; namely, Streptomyces sp.OSIP1 and Streptomyces sp.OSNP14. These AgNPs were synthesized through a green method using culture supernatant of bacteria at 20 °C and characterized by several instrumental techniques. The TEM results revealed that the NPs obtained from OSIP1 were smaller (8 nm, average) than those taken from OSIP14 (15 nm, average). Both AgNPs-OSP1 and AgNPs-OSNP14 also posed the strongest growth inhibitory effect against several pathogenic bacteria alone and especially in combination with antibiotics. Smaller NPs especially at 3.9–31.25 µg/ml concentrations were assumed more effective biofilm inhibitors of Pseudomonas aeruginosa. Cytotoxic activity of both AgNPs (at 25 and 50 µg/mL concentrations) on mouse colorectal carcinoma cells (CT26) were then studied using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The findings demonstrated that smaller AgNPs at a 50 µg/mL concentration had 7% more cytotoxicity effects. In conclusion; although AgNPs produced by diverse strains of cold-adapted Streptomyces had close characteristics and biological activities, they showed some multifarious properties.

The numerous side effects of platinum based chemotherapy has led to the design of new therapeutics with platinum replaced by another transition metal. Here, we investigated the interactions of previously reported copper(II) complexes containing S-isoalkyl derivatives, the salicylic acid with guanosine-5′-monophosphate and calf thymus DNA (CT-DNA) and their antitumor effects, in a colon carcinoma model. All three copper(II) complexes exhibited an affinity for binding to CT-DNA, but there was no indication of intercalation or the displacement of ethidium bromide. Molecular docking studies revealed a significant affinity of the complexes for binding to the minor groove of B-form DNA, which coincided with DNA elongation, and a higher affinity for binding to Z-form DNA, supporting the hypothesis that the complex binding to CT-DNA induces a local transition from B-form to Z-form DNA. These complexes show a moderate, but selective cytotoxic effect toward colon cancer cells in vitro. Binuclear complex of copper(II) with S-isoamyl derivative of thiosalicylic acid showed the highest cytotoxic effect, arrested tumor cells in the G2/M phase of the cell cycle, and significantly reduced the expression of inflammatory molecules pro-IL-1β, TNF-α, ICAM-1, and VCAM-1 in the tissue of primary heterotopic murine colon cancer, which was accompanied by a significantly reduced tumor growth and metastases in the lung and liver.

IntroductionImmuno-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.MethodsUsing 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.ResultsWe 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.DiscussionThese differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.

CCR8 chemokine receptor is a selective marker of tumor-infiltrating regulatory T cells (ti-Tregs) which interfere with the efficacy of checkpoint inhibitor immunotherapy (ICI) in many types of cancer. Eliminating CCR8+ ti-Tregs dramatically improves the results of subsequent ICI. We have recently reported using 225Actinium-labeled anti-CCR8 IgG for killing CCR8+ ti-Tregs in murine colorectal tumors which synergized with subsequent anti-CTLA4 ICI. Here, we aimed to compare the in vivo behavior of anti-CCR8 full-sized IgG and its Fab fragments to select the best antibody format for the pre-clinical development of this combination modality. Anti-CCR8 Fab fragments were generated by papain digest of the whole IgG. The whole IgG and Fab were conjugated to bifunctional chelating agent DOTA and labeled with 111Indium (111In). MC8 and CT6 murine colorectal tumor-bearing C57Bl6 and Balb/c mice, respectively, were administered 111In-DOTA-IgG or 111In-DOTA-Fab and imaged with microSPECT/CT at 2–72 h post-injection. A biodistribution was performed after the last imaging time point. Both 111In-DOTA-IgG and 111In-DOTA-Fab demonstrated high tumor uptake in both MC38 and CT26 tumors, with 111In-DOTA-IgG uptake being significantly higher from the 24 h time point and onwards. 111In-DOTA-Fab also exhibited pronounced kidney uptake which persisted even at 72 h. The kidney clearance and retention of 111In-DOTA-Fab might represent a problem during therapy employing 225Actimium or other long-lived therapeutic radionuclides by potentially causing a dose-limiting kidney toxicity. This imaging/biodistribution evaluation not only determined that full-size anti-CCR8 IgG is the optimal antibody format for pre-clinical development but also informed on the timing of immunotherapy administration in future radioimmunotherapy and immunotherapy combination studies.

A series of heterocyclic compounds containing spirofused barbiturate and 3-azabicyclo[3.1.0]hexane frameworks have been studied as potential antitumor agents. Antiproliferative activity of products was screened in human erythroleukemia (K562), T lymphocyte (Jurkat), and cervical carcinoma (HeLa) as well as mouse colon carcinoma (CT26) and African green monkey kidney epithelial (Vero) cell lines. The most effective among the screened compounds show IC50 in the range from 4.2 to 24.1 μM for all tested cell lines. The screened compounds have demonstrated a significant effect of the distribution of HeLa and CT26 cells across the cell cycle stage, with accumulation of cells in SubG1 phase and induced apoptosis. It was found, using a confocal microscopy, that actin filaments disappeared and granular actin was distributed diffusely in the cytoplasm of up to 90% of HeLa cells and up to 64% of CT26 cells after treatment with tested 3-azaspiro[bicyclo [3.1.0]hexane-2,5′-pyrimidines]. We discovered that the number of HeLa cells with filopodium-like membrane protrusions was reduced significantly (from 91% in control cells to 35%) after treatment with the most active compounds. A decrease in cell motility was also noticed. Preliminary in vivo experiments on the impact of the studied compounds on the dynamics of CT26 tumor growth in Balb/C mice were also performed.

Ecklonia cava (E. cava) is well known as one of edible alga that contains various unique polyphenols. The anti-tumor activity of an aqueous extract of E. cava (AEC) against colon carcinoma was evaluated by analyzing the alterations in tumor growth, histopathological structure and molecular mechanisms in CT26 tumor-bearing BALB/cKorl syngeneic mice after administrating AEC for five weeks. AEC contained high total phenolic contents and demonstrated significant scavenging activity against 2,2-diphenyl-1-picrylhydrazyl radicals. Marked anti-tumor effects were demonstrated in the AEC-treated CT26 cells. In the in vivo syngeneic model, the AEC treatment decreased the volume and weight of CT26 tumors, and expanded the necrotic region in the hematoxylin and eosin stained tumor sections. The inhibitory effects of AEC on tumor growth were reflected by the increased level of apoptotic proteins, inhibition of cell proliferation, suppression of metastasis ability and increase in tumor-suppressing activity in CT26 tumor-bearing BALB/cKorl syngeneic mice. The potential function of phlorotannin (PT), one of the primary active compounds in AEC, was demonstrated by the increased cytotoxicity, apoptosis and suppression of cell proliferation in PT-treated CT26 cells. Overall, the results of the present study provide novel scientific evidence that AEC can suppress the growth of CT26 colon cancer by activating apoptosis, suppressing cell proliferation, inhibiting cell migration and enhancing the tumor-suppressing activity.

Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin 12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory.

Simultaneous targeting of several mutations can be useful in colorectal cancer (CRC) due to its heterogeneity and presence of somatic mutations. As CT26 mutations and expression profiles resemble those of human CRC, we focused on designing a polyepitope vaccine based on CT26 neoepitopes. Due to its low immunogenicity, outer membrane vesicles (rOMV) as an antigen delivery system and adjuvant was applied. Herein, based on previous experimental and our in silico studies four CT26 neoepitopes with the ability to bind MHC-I and MHC-II, TCR, and induce IFN-α production were selected. To increase their immunogenicity, the gp70 and PADRE epitopes were added. The order of the neoepitopes was determined through 3D structure analysis using ProSA, Verify 3D, ERRAT, and Ramachandran servers. The stable peptide-protein docking between the selected epitopes and MHC alleles strengthen our prediction. The CT26 polytope vaccine sequence was fused to the C-terminal of cytolysin A (ClyA) anchor protein and rOMVs were isolated from endotoxin-free ClearColi™ strain. The results of the C-ImmSim server showed that the ClyA-CT26 polytope vaccine could induce T and B cells immunity.The ClyA-CT26 polytope was characterized as a soluble, stable, immunogen, and non-allergen vaccine and optimized for expression in ClearColi™ 24 h after induction with 1 mM IPTG at 25 °C. Western blot analysis confirmed the expression of ClyA-CT26 polytope by ClearColi™ and also on ClearColi™-derived rOMVs. In conclusion, we found that ClearColi™-derived rOMVs with CT26 polytope can deliver CRC neoantigens and induce antitumor immunity, but in vivo immunological studies are needed to confirm vaccine efficacy.