[Expression of endosialin in human hypertrophic scars and its regulation on fibroblast phenotype].

Objective: To explore the expression of endosialin, i.e., CD248 in human hypertrophic scars (HSs) and its regulatory effect on the phenotype of hypertrophic scar fibroblasts (HSFs). Methods: The method of experimental research was used. From March to May, 2023, 3 pediatric patients with HS were admitted to the Department of Burns and Cutaneous Surgery of the First Affiliated Hospital of Air Force Medical University, including 2 females and 1 male, aged one year ten months to two years. The HS tissue resected during the surgery and the remaining full-thickness skin graft, i.e., normal skin tissue after full-thickness skin grafting were collected from the aforementioned pediatric patients for subsequent experiments. Using the aforementioned two types of tissue, the histological structures were observed by hematoxylin-eosin staining, collagen distribution was observed by Masson staining, and the expression of CD248 was observed and measured by immunohistochemical staining. The primary HSFs were isolated from HS tissue using explant culture technique, and the 3rd to 5th passages of HSFs were used in subsequent experiments. According to the random number table, HSFs were divided into immunoglobulin G78 (IgG78)-treated group and IgG control group, which were treated with 200 nmol/L human CD248 monoclonal antibody IgG78 and human IgG control antibody for 24 h, respectively. The mRNA expressions of collagen type Ⅰ (Col Ⅰ) and α-smooth muscle actin (α-SMA) in HSFs were measured by real-time fluorescence quantitative reverse transcription polymerase chain reaction, the protein expressions of Col Ⅰ and α-SMA in HSFs were detected by Western blotting, and the intracellular location and protein expressions of Col Ⅰ and α-SMA were detected by immunofluorescence method. The number of samples in each experiment was 3. Data were statistically analyzed with paired sample t test and independent sample t test. Results: Compared with those in normal skin tissue, the epidermis and dermis in HS tissue were significantly thicker, with massive accumulation and disordered arrangement of collagen in the dermis. The expression of CD248 in HS tissue was significantly upregulated compared with that in normal skin tissue (t=5.29, P<0.05). At post treatment hour 24, the mRNA expressions of Col Ⅰ and α-SMA of HSFs in IgG78-treated group were 0.39±0.05 and 0.56±0.09, respectively, which were significantly lower than 1.00±0.07 and 1.00±0.08 in IgG control group, respectively (with t values of 11.87 and 6.49, respectively, P values all <0.05). The protein expressions of Col Ⅰ and α-SMA of HSFs in IgG78-treated group were 0.617±0.011 and 0.67±0.14, respectively, which were significantly lower than 1.259±0.052 and 1.23±0.16 in IgG control group, respectively (with t values of 20.92 and 4.52, respectively, P values all <0.05). At post treatment hour 24, immunofluorescence staining showed that Col Ⅰ and α-SMA mainly located in the cytoplasm of HSFs in the two groups, and the protein expressions of Col Ⅰ and α-SMA of HSFs in IgG78-treated group were obviously downregulated compared with those in IgG control group. Conclusions: The expression of CD248 is significantly upregulated in human HS. Targeted blockade of CD248 can significantly inhibit the collagen synthesis by HSFs and the transdifferentiation of HSFs into myofibroblasts.

Jacalin-Curcumin Complex Sensitizes the Breast Cancer MDA-MB-231 Cell Line.

Protein-drug interactions are crucial for understanding drug delivery and cell functions. Jacalin is a suitable molecule for such targeting, as it specifically recognizes the tumor-associated Thomsen-Friedenreich (TF) antigen that is expressed on the glycosylated proteins in cancer cells. The present paper describes the interaction of curcumin and jacalin, a possible carrier molecule for the delivery of antitumor drugs due to its ability to recognize tumor cells. Our results have shown that both steady-state fluorescence and fluorescent labelling of jacalin are two reliable methods to determine jacalin-curcumin interactions. The affinity of jacalin for curcumin is consistently within the micromolar range (using fluorescence and microscale thermophoresis) showing high-affinity binding of the complex. In vitro experiments on triple-negative breast cancer MDA-MB-231 cells indicated inhibition of cell growth after treating with the jacalin-curcumin complex for 48 h. The cell survival fraction was significantly reduced to 50% after combined treatment. In this paper, we report for the first time about the jacalin-curcumin interaction. We quantified this unique biomolecular interaction and gathered additional information on the binding event. We observed that the jacalin-curcumin complex inhibits the proliferation of the triple-negative breast cancer MDA-MB-231 cells.

A promising self-nanoemulsifying adjuvant with plant-derived saponin D boosts immune response and exerts an anti-tumor effect.

Objectives: The low immunogenicity of tumor antigens and unacceptable toxicity of adjuvants has hindered the application and development of tumor vaccines. Hence, we designed a novel anti-tumor vaccine composed of a plant-derived immunostimulant molecular nanoadjuvant (a self-nanoemulsifying system, SND) and the antigen OVA, to reinvigorate the immune response and inhibit tumor progression. Methods: In this study, this novel nanoadjuvant with Saponin D (SND) was designed and prepared by low-energy emulsification methods. Several important characteristics of the SND, including morphology, size, polymer dispersity index (PDI), zeta potential, and stability, were estimated, and the cytotoxicity of the SND was evaluated by MTT assay. Additionally, the immune response in terms of antibody titer levels and cellular immunity were evaluated in vivo after immunization with the vaccine, and the preventative and therapeutic effects of this novel vaccine against tumors were estimated. Finally, the antigen release profile was determined by IVIS imaging and by in vivo assay. Results: This SND nanoadjuvant had good characteristics including the average particle size of 26.35 ± 0.225 nm, narrow distribution of 0.221 ± 1.76, and stability zeta potential of -12.9 ± 0.83 mV. And also, it had good stability (size, PDI, zeta potential, antigen stability) and low toxicity in vitro and in vivo, and delayed antigen release in vivo. The humoral immune response (IgG, IgG1, IgG2a, and IgG2b) and cellular immune level (cytokines of splenocytes including IFN-γ, IL-4, IL-1ß andIL-17A) were both improved greatly after injected immunization at 0, 14, 28 days with the novel nanoadjuvant and antigen OVA. Importantly, this novel nanoadjuvant combined with OVA might lead to the induction of the prevent and treatment efficacy in the E.G7-OVA tumor-bearing mice. Conclusions: These results suggested that this novel nanoadjuvant encapsulated natural plant immunostimulant molecular OPD could be a good candidate of tumor vaccine adjuvant for reinvigorating the immune response and powerfully inhibiting tumor growth effect.

The Notch signaling pathway: a potential target for cancer immunotherapy.

Dysregulation of the Notch signaling pathway, which is highly conserved across species, can drive aberrant epigenetic modification, transcription, and translation. Defective gene regulation caused by dysregulated Notch signaling often affects networks controlling oncogenesis and tumor progression. Meanwhile, Notch signaling can modulate immune cells involved in anti- or pro-tumor responses and tumor immunogenicity. A comprehensive understanding of these processes can help with designing new drugs that target Notch signaling, thereby enhancing the effects of cancer immunotherapy. Here, we provide an up-to-date and comprehensive overview of how Notch signaling intrinsically regulates immune cells and how alterations in Notch signaling in tumor cells or stromal cells extrinsically regulate immune responses in the tumor microenvironment (TME). We also discuss the potential role of Notch signaling in tumor immunity mediated by gut microbiota. Finally, we propose strategies for targeting Notch signaling in cancer immunotherapy. These include oncolytic virotherapy combined with inhibition of Notch signaling, nanoparticles (NPs) loaded with Notch signaling regulators to specifically target tumor-associated macrophages (TAMs) to repolarize their functions and remodel the TME, combining specific and efficient inhibitors or activators of Notch signaling with immune checkpoint blockers (ICBs) for synergistic anti-tumor therapy, and implementing a customized and effective synNotch circuit system to enhance safety of chimeric antigen receptor (CAR) immune cells. Collectively, this review aims to summarize how Notch signaling intrinsically and extrinsically shapes immune responses to improve immunotherapy.

Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death.

Dental plaque bacteria produce high concentrations of short-chain fatty acids (SCFAs), as bacterial metabolites. SCFA-treated gingival epithelial cells undergo cell death. Our previous reports demonstrated that butyrate-induced cell death depends on autophagy and reactive oxygen species (ROS). However, the precise mechanisms underlying SCFA-induced gingival epithelial cell death is poorly understood. Butyrate is a strong histone deacetylase (HDAC) inhibitor. Therefore, we determined the involvement of HDAC inhibitory activity in SCFA-induced gingival epithelial cells. Ca9-22 cells were used as an in vitro counterpart of gingival epithelial cells. Ca9-22 cells were treated with HDAC inhibitors in the presence or absence of C646, a P300 histone acetyltransferase (HAT) inhibitor, and compared the number of dead cells, which are measured using SYTOX Green dye. Acetylation levels of histone H3 were examined using western blotting. Changes in transcriptomes during the butyrate and C646 treatment were examined using RNA sequencing analysis. The butyrate or propionate-treatment of Ca9-22 cells induced acetylation of histone H3, while the C646 treatment strongly reduced the elevated acetylation levels. Accordingly, butyrate or propionate-induced cell death was inhibited by the C646 treatment. Similar results were obtained when other HDAC inhibitors were used. Whole transcriptome analysis revealed that the expression of numerous genes was altered by butyrate-induced histone acetylation. Moreover, some autophagy and ROS-related genes found in the altered genes might induce cell death. This study suggests the need for HDAC-inhibitory activity of bacterial metabolites to induce cell death, and the effects might enhance autophagy and ROS production.

Enhancement of antitumor immune response by radiation therapy combined with dual immune checkpoint inhibitor in a metastatic model of HER2-positive murine tumor.

PURPOSE: Treatments for metastatic human epidermal growth factor receptor 2 (HER2)-positive tumors are improving but remain inadequate. We investigated activating antitumor immune response by combining radiation therapy with immune checkpoint inhibitors using mouse tumors overexpressing HER2, a pivotal driver oncogenic antigen, to develop new immunotherapies for metastatic HER2-positive tumors. MATERIALS AND METHODS: NT2.5 cells were inoculated into the two mammary fat pads of FVB/N mice, which were divided into four groups: no treatment (Non), anti-PD-1 and anti-CTLA4 antibodies (P1C4), irradiation of the large tumor (Rad), and combination (R + P1C4) groups. Tumor growth, immunostaining of tumor-infiltrating lymphocytes, and the proportion of HER2-tumor antigen-specific CD8-positive T cells in the spleen and tumor-infiltrating lymphocytes were analyzed. RESULTS: In the Rad group, unirradiated and irradiated tumors shrank after treatment. Besides the directly irradiated tumors, the unirradiated tumors in the R + P1C4 group shrank the most. In the unirradiated tumors, CD8-positive T cells and FOXP3-positive T cells accumulated significantly more in the R + P1C4 group than in the P1C4 and the Rad groups (all p < 0.001). CD4-positive helper T cells accumulated significantly more in the R + P1C4 group than in the Rad group (p < 0.05), but this was not significantly different from the P1C4 group. HER2-specific CD8-positive T cells in the spleen and tumor-infiltrating lymphocytes were significantly increased in the R + P1C4 group compared to the P1C4 and Rad groups (all p < 0.0001). CONCLUSION: Irradiation of HER2-positive tumors induced an antitumor immune effect against the unirradiated tumor, which was enhanced by the combined use of immune checkpoint inhibitors and was mediated by enhanced recruitment of HER2-tumor antigen-specific cytotoxic T lymphocytes at the tumor site in an HER2-positive mouse tumor model. Harnessing the distant antitumor immune response induced by the combination of radiation therapy and immune checkpoint inhibitors could be a promising treatment strategy for metastatic HER2-positive tumors.

Neoantigens as potential vaccines in hepatocellular carcinoma.

BACKGROUND: Neoantigens, new immunogenic sequences arising from tumor mutations, have been associated with response to immunotherapy and are considered potential targets for vaccination. Hepatocellular carcinoma (HCC) is a moderately mutated tumor, where the neoantigen repertoire has not been investigated. Our aim was to analyze whether tumors in HCC patients contain immunogenic neoantigens suitable for future use in therapeutic vaccination. METHODS: Whole-exome sequencing and RNAseq were performed in a cohort of fourteen HCC patients submitted to surgery or liver transplant. To identify mutations, single-nucleotide variants (SNV) originating non-synonymous changes that were confirmed at the RNA level were analyzed. Immunogenicity of putative neoAgs predicted by HLA binding algorithms was confirmed by using in vitro HLA binding assays and T-cell stimulation experiments, the latter in vivo, by immunizing HLA-A*02.01/HLA-DRB1*01 (HHD-DR1) transgenic mice, and in in vitro, using human lymphocytes. RESULTS: Sequencing led to the identification of a median of 1217 missense somatic SNV per patient, narrowed to 30 when filtering by using RNAseq data. A median of 13 and 5 peptides per patient were predicted as potential binders to HLA class I and class II molecules, respectively. Considering only HLA-A*02.01- and HLA-DRB1*01-predicted binders, 70% demonstrated HLA-binding capacity and about 50% were immunogenic when tested in HHD-DR1 mice. These peptides induced polyfunctional T cells that specifically recognized the mutated but not the wild-type sequence as well as neoantigen-expressing cells. Moreover, coimmunization experiments combining CD8 and CD4 neoantigen epitopes resulted in stronger CD8 T cell responses. Finally, responses against neoantigens were also induced in vitro using human cells. CONCLUSION: These results show that mutations in HCC tumors may generate immunogenic neoantigens with potential applicability for future combinatorial therapeutic strategies.

Neoantigen vaccine-induced CD4 T cells confer protective immunity in a mouse model of multiple myeloma through activation of CD8 T cells against non-vaccine, tumor-associated antigens.

BACKGROUND: Cancer-associated neoantigens (neoAg) derived from tumor genomic sequencing and predictive algorithms for mutated peptides are a promising basis for therapeutic vaccines under investigation. Although these are generally designed to bind major histocompatibility complex class I and induce CD8 cytolytic T lymphocyte (CTL) activity, results from preclinical and clinical studies demonstrate that the majority of neoAg vaccines efficiently induce CD4 T helper (Th) responses but not CTL. Despite this, these vaccines have demonstrated clinical efficacy. Therefore, understanding the mechanisms of CD4 + T cell-mediated tumor protection is critical to optimizing this immunotherapeutic strategy. METHODS: We investigated this phenomenon in the mineral oil-induced plasmacytoma (MOPC).315.BM (MOPC315) mouse model of multiple myeloma, a malignancy of plasma cells. MOPC315 cells express in their lambda chain a unique tumor-specific neoAg, an idiotypic (Id) peptide. We generated a vaccine formulated with this Id peptide fused to a heat shock protein HSC70 binding (HSB) motif co-delivered with poly (I:C). The immunogenicity of the Id-vaccine was measured in splenocytes by ELISpot. Mice were challenged with MOPC315 cells and antitumor immunity was assessed by co-incubating splenocytes and bone marrow mononuclear cells derived from vaccinated mice and controls, with the Id antigen and irradiated MOPC315 cells. The frequency of activated CD4 and CD8 T cells and their phenotype were characterized by flow cytometry. RESULTS: Id-vaccine efficiently induced antigen-specific CD4 Th activity and antitumor immunity, protecting mice from MOPC315 tumor growth. CD4 cytolytic activity was not detected under these conditions. Polyfunctional CD8 T cells homed to the bone marrow microenvironment of protected mice and preferentially expanded only when restimulated ex vivo with both Id peptide and MOPC315 cells. Protective activity was abrogated by depletion of either CD4 or CD8 lymphocytes. CONCLUSION: These results demonstrate that Id-HSB +poly (I:C) vaccine protects against MOPC315 growth by priming Id-specific CD4 Th cells that confer protection against tumor but are not directly cytotoxic. These data indicate that activation of CD8 CTL against MOPC315-associated antigens not present in the vaccine is one of the major mechanisms of tumor immunity.

AGEs/RAGE Promote Osteogenic Differentiation in Rat Bone Marrow-Derived Endothelial Progenitor Cells via MAPK Signaling.

Systemic vascular impairment is the most common complication of diabetes. Advanced glycation end products (AGEs) can exacerbate diabetes-related vascular damage by affecting the intima and media through a variety of mechanisms. In the study, we demonstrated that AGEs and their membrane receptor RAGE could induce the differentiation of EPCs into osteoblasts under certain circumstances, thereby promoting accelerated atherosclerosis. Differentiation into osteoblasts was confirmed by positive staining for DiI-acetylated fluorescently labeled low-density lipoprotein and FITC-conjugated Ulex europaeus agglutinin. During differentiation, expression of receptor for AGE (RAGE) was significantly upregulated. This upregulation was attenuated by transfection with RAGE-targeting small interfering (si)RNA. siRNA-mediated knockdown of RAGE expression significantly inhibited the upregulation of AGE-induced calcification-related proteins, such as runt-related transcription factor 2 (RUNX2) and osteoprotegerin (OPG). Additional experiments showed that AGE induction of EPCs significantly induced ERK, p38MAPK, and JNK activation. The AGE-induced upregulation of osteoblast proteins (RUNX2 and OPG) was suppressed by treatment with a p38MAPK inhibitor (SB203580) or JNK inhibitor (SP600125), but not by treatment with an ERK inhibitor (PD98059), which indicated that AGE-induced osteoblast differentiation from EPCs may be mediated by p38MAPK and JNK signaling, but not by ERK signaling. These data suggested that AGEs may bind to RAGE on the EPC membrane to trigger differentiation into osteoblasts. The underlying mechanism appears to involve the p38MAPK and JNK1/2 pathways, but not the ERK1/2 pathway.

Inhibition of integrin αvß6 sparks T-cell antitumor response and enhances immune checkpoint blockade therapy in colorectal cancer.

BACKGROUND: Integrin αvß6 is a heterodimeric cell surface protein whose cellular expression is determined by the availability of the integrin ß6 subunit (ITGB6). It is expressed at very low levels in most organs during tissue homeostasis but shows highly upregulated expression during the process of tumorigenesis in many cancers of epithelial origin. Notably, enhanced expression of integrin αvß6 is associated with aggressive disease and poor prognosis in numerous carcinoma entities. Integrin αvß6 is one of the major physiological activators of transforming growth factor-ß (TGF-ß), which has been shown to inhibit the antitumor T-cell response and cause resistance to immunotherapy in mouse models of colorectal and mammary cancer. In this study, we investigated the effect of ITGB6 expression and antibody-mediated integrin αvß6 inhibition on the tumor immune response in colorectal cancer. METHODS: Using orthotopic and heterotopic tumor cell injection, we assessed the effect of ITGB6 on tumor growth and tumor immune response in wild type mice, mice with defective TGF-ß signaling, and mice treated with anti-integrin αvß6 antibodies. To examine the effect of ITGB6 in human colorectal cancer, we analyzed RNAseq data from the colon adenocarcinoma dataset of The Cancer Genome Atlas (TCGA-COAD). RESULTS: We demonstrate that expression of ITGB6 is an immune evasion strategy in colorectal cancer, causing inhibition of the antitumor immune response and resistance to immune checkpoint blockade therapy by activating latent TGF-ß. Antibody-mediated inhibition of integrin αvß6 sparked a potent cytotoxic T-cell response and overcame resistance to programmed cell death protein 1 (PD-1) blockade therapy in ITGB6 expressing tumors, provoking a drastic increase in anti-PD-1 treatment efficacy. Further, we show that the majority of tumors in patients with colorectal cancer express sufficient ITGB6 to provoke inhibition of the cytotoxic T-cell response, indicating that most patients could benefit from integrin αvß6 blockade therapy. CONCLUSIONS: These findings propose inhibition of integrin αvß6 as a promising new therapy for colorectal cancer, which blocks tumor-promoting TGF-ß activation, prevents tumor exclusion of cytotoxic T-cells and enhances the efficacy of immune checkpoint blockade therapy.

Remodeling of the tumor microenvironment through PAK4 inhibition sensitizes tumors to immune checkpoint blockade.

PAK4 inhibition can sensitize tumors to immune checkpoint blockade (ICB) therapy, however, the underlying mechanisms remain unclear. We report that PAK4 inhibition reverses immune cell exclusion by increasing the infiltration of CD8 T cells and CD103+ dendritic cells (DCs), a specific type of DCs that excel at cross-presenting tumor antigens and constitute a source of CXCL10. Interestingly, in melanoma clinical datasets, PAK4 expression levels negatively correlate with the presence of CCL21, the ligand for CCR7 expressed in CD103+ DCs. Furthermore, we extensively characterized the transcriptome of PAK4 knock out (KO) tumors, in vitro and in vivo, and established the importance of PAK4 expression in the regulation of the extracellular matrix, which can facilitate immune cell infiltration. Comparison between PAK4 wild type (WT) and KO anti-PD-1 treated tumors revealed how PAK4 deletion sensitizes tumors to ICB from a transcriptomic perspective. In addition, we validated genetically and pharmacologically that inhibition of PAK4 kinase activity is sufficient to improve anti-tumor efficacy of anti-PD-1 blockade in multiple melanoma mouse models. Therefore, this study provides novel insights into the mechanism of action of PAK4 inhibition and provides the foundation for a new treatment strategy that aims to overcome resistance to PD-1 blockade by combining anti-PD-1 with a small molecule PAK4 kinase inhibitor.

Progesterone Induced Blocking Factor Reduces Hypertension and Placental Mitochondrial Dysfunction in Response to sFlt-1 during Pregnancy.

Preeclampsia (PE) is characterized by new onset hypertension in association with placental ischemia, reduced fetal weight, elevated soluble fms-like tyrosine kinase-1 (sFlt-1), and placental mitochondrial (mt) dysfunction and oxidative stress (ROS). Progesterone induced blocking factor (PIBF) is a product of progesterone signaling that blocks inflammatory processes and we have previously shown PIBF to lower mean arterial blood pressure (MAP) and sFlt-1 in a rat model of PE. Infusion of sFlt-1 causes hypertension and many characteristics of PE in pregnant rodents, however, its role in causing mt dysfunction is unknown. Therefore, we hypothesize that PIBF will improve mt function and MAP in response to elevated sFlt-1 during pregnancy. We tested our hypothesis by infusing sFlt-1 via miniosmotic pumps in normal pregnant (NP) Sprague-Dawley rats (3.7 µg·kg-1·day-1) on gestation days (GD) 13-19 in the presence or absence of PIBF (2.0 µg/mL) injected intraperitoneally on GD 15 and examined mean arterial blood pressure (MAP) and placental mt ROS on GD 19. sFlt-1 increased MAP to 112 + 2 (n = 11) compared to NP rats (98 + 2 mmHg, n = 15, p < 0.05), which was lowered in the presence of sFlt-1 (100 + 1 mmHg, n = 5, p < 0.05). Placental mtATP was reduced in sFlt-1 infused rats versus NP controls, but was improved with PIBF. Placental mtROS was elevated with sFlt-1 compared to NP controls, but was reduced with PIBF. Sera from NP + sFlt-1 increased endothelial cell mtROS, which was attenuated with PIBF. These data demonstrate sFlt-1 induced HTN during pregnancy reduces placental mt function. Importantly, PIBF improved placental mt function and HTN, indicating the efficacy of improved progesterone signaling as potential therapeutics for PE.

Engineered Nanovaccine Targeting Clec9a+ Dendritic Cells Remarkably Enhances the Cancer Immunotherapy Effects of STING Agonist.

Agonists of the stimulator of interferon gene (STING) are considered as promising therapeutics for cancer immunotherapy. However, drug-delivery barriers and adverse effects limit the clinical application of STING agonists. Therefore, it is an urgent need to develop an ideal delivery system to deliver STING agonists and avoid side effects. Here, we discovered that STING agonists significantly stimulated type I interferon (IFN) secretion in Clec9a+ dendritic cells (DCs). Then, we designed an engineered peptide-expressed biomimetic cancer cell membrane (EPBM)-coated nanovaccine drug-delivery system (PLGA/STING@EPBM) to deliver STING agonists and tumor antigens to Clec9a+ DCs. The PLGA/STING@EPBM nanovaccine significantly enhanced IFN-stimulated expression of genes and antigen cross-presentation of Clec9a+ DCs, thus eliciting strong antitumor effects in both anti-PD-1-responsive and -resistant tumor models without obvious cytotoxicity. Moreover, the PLGA/STING@EPBM nanovaccine combined with radiotherapy exhibited remarkable synergistic antitumor effects. Our work highlights the great potential of a EPBM-coated nanovaccine for systemic STING agonist delivery as an attractive tool for cancer immunotherapy.

Multi-Epitope-Based Vaccines for Colon Cancer Treatment and Prevention.

Background: Overexpression of nonmutated proteins involved in oncogenesis is a mechanism by which such proteins become immunogenic. We questioned whether overexpressed colorectal cancer associated proteins found at higher incidence and associated with poor prognosis could be effective vaccine antigens. We explored whether vaccines targeting these proteins could inhibit the development of intestinal tumors in the azoxymethane (AOM)-induced colon model and APC Min mice. Methods: Humoral immunity was evaluated by ELISA. Web-based algorithms identified putative Class II binding epitopes of the antigens. Peptide and protein specific T-cells were identified from human peripheral blood mononuclear cells using IFN-gamma ELISPOT. Peptides highly homologous between mouse and man were formulated into vaccines and tested for immunogenicity in mice and in vivo tumor challenge. Mice treated with AOM and APC Min transgenic mice were vaccinated and monitored for tumors. Results: Serum IgG for CDC25B, COX2, RCAS1, and FASCIN1 was significantly elevated in colorectal cancer patient sera compared to volunteers (CDC25B p=0.002, COX-2 p=0.001, FASCIN1 and RCAS1 p<0.0001). Epitopes predicted to bind to human class II MHC were identified for each protein and T-cells specific for both the peptides and corresponding recombinant protein were generated from human lymphocytes validating these proteins as human antigens. Some peptides were highly homologous between mouse and humans and after immunization, mice developed both peptide and protein specific IFN-γ-secreting cell responses to CDC25B, COX2 and RCAS1, but not FASCIN1. FVB/nJ mice immunized with CDC25B or COX2 peptides showed significant inhibition of growth of the syngeneic MC38 tumor compared to control (p<0.0001). RCAS1 peptide vaccination showed no anti-tumor effect. In the prophylactic setting, after immunization with CDC25B or COX2 peptides mice treated with AOM developed significantly fewer tumors as compared to controls (p<0.0002) with 50% of mice remaining tumor free in each antigen group. APC Min mice immunized with CDC25B or COX2 peptides developed fewer small bowel tumors as compared to controls (p=0.01 and p=0.02 respectively). Conclusions: Immunization with CDC25B and COX2 epitopes consistently suppressed tumor development in each model evaluated. These data lay the foundation for the development of multi-antigen vaccines for the treatment and prevention of colorectal cancer.

Effects of Combinatorial Ubiquitinated Protein-Based Nanovaccine and STING Agonist in Mice With Drug-Resistant and Metastatic Breast Cancer.

We previously reported that enriched ubiquitinated proteins (UPs) from tumor cells have the potential to be used as immunotherapy vaccine against cancer. Here we enriched UPs from epirubicin (EPB)-induced multi-drug-resistant cancer stem-like breast cancer cell line (4T1/EPB) and tested the efficacy of α-Al2O3-UPs-4T1/EPB (short for UPs-4T1/EPB) as therapeutic vaccine alone and in combination with the stimulator of interferon genes (STING) agonist in mice with drug-resistant and metastatic breast cancer. Vaccination with UPs-4T1/EPB exerted profound anti-tumor effects through augmented specific CD8+ T cell responses and amplified T cell receptor diversity of tumor-infiltrating lymphocytes (TILs). Importantly, the combination with STING agonist further facilitated the migration of mature CD8α+ dendritic cells to the lymph nodes and the infiltration of TILs within tumors, resulting in primary tumor regression and pulmonary metastasis eradication in mice. Moreover, the cured mice were completely resistant against a subsequent rechallenge with the same tumor. Our study indicates that this novel combinatorial immunotherapy with UPs-4T1/EPB vaccine and STING agonist is effective in mice with drug-resistant and metastatic breast cancer.

Recurrent Frameshift Neoantigen Vaccine Elicits Protective Immunity With Reduced Tumor Burden and Improved Overall Survival in a Lynch Syndrome Mouse Model.

BACKGROUND & AIMS: DNA mismatch repair deficiency drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. Individuals with LS are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a phase I/IIa clinical trial in patients with a history of MSI colorectal cancer. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not yet been demonstrated. METHODS: A genome-wide database of 488,235 mouse coding mononucleotide repeats was established, from which a set of candidates was selected based on repeat length, gene expression, and mutation frequency. In silico prediction, in vivo immunogenicity testing, and epitope mapping was used to identify candidates for FSP vaccination. RESULTS: We identified 4 shared FSP neoantigens (Nacad [FSP-1], Maz [FSP-1], Senp6 [FSP-1], Xirp1 [FSP-1]) that induced CD4/CD8 T cell responses in naïve C57BL/6 mice. Using VCMsh2 mice, which have a conditional knockout of Msh2 in the intestinal tract and develop intestinal cancer, we showed vaccination with a combination of only 4 FSPs significantly increased FSP-specific adaptive immunity, reduced intestinal tumor burden, and prolonged overall survival. Combination of FSP vaccination with daily naproxen treatment potentiated immune response, delayed tumor growth, and prolonged survival even more effectively than FSP vaccination alone. CONCLUSIONS: Our preclinical findings support a clinical strategy of recurrent FSP neoantigen vaccination for LS cancer immunoprevention.

Dispersive effects and focused biodistribution of recombinant human hyaluronidase PH20: A locally acting and transiently active permeation enhancer.

BACKGROUND: Recombinant human hyaluronidase PH20 (rHuPH20) facilitates the dispersion and absorption of subcutaneously administered therapeutic agents. This study aimed to characterize the transient, local action of rHuPH20 in the subcutaneous (SC) space using focused biodistribution and dye dispersion studies conducted in mice. MATERIALS AND METHODS: To evaluate the biodistribution of rHuPH20, mice were intradermally administered rHuPH20 (80 U). The enzymatic activity of rHuPH20 was analyzed in the skin, lymph nodes, and plasma. Animal model sensitivity was determined by intravenous administration of rHuPH20 (80 U) to the tail vein. To evaluate local dispersion, mice received an intradermal injection of rHuPH20 followed by an intradermal injection of Trypan Blue dye at a contralateral site 45 minutes later. Dye dispersion was measured using a digital caliper. RESULTS: After intradermal rHuPH20 injection, enzymatic activity was detected within the skin near the injection site with levels decreasing rapidly after 15 minutes. There was no clear evidence of systemic exposure after administration of rHuPH20, and no discernible rHuPH20 activity was observed in lymph or plasma as a function of time after dosing. In the dye dispersion study, delivery of rHuPH20 at one site did not impact dye dispersion at a distal skin site. CONCLUSION: These observations support the classification of rHuPH20 as a transiently active and locally acting permeation enhancer.

Generation of triacyl lipopeptide-modified glycoproteins by metabolic glycoengineering as the neoantigen to boost anti-tumor immune response.

The lack of tumor specific antigens (TSA) and the immune tolerance are two major obstacles for the immunotherapy of cancer. Current immune checkpoint inhibitors (ICIs) show clinical responses in only limited subsets of cancer patients, which, to some extent, depends on the mutation load of tumor cells that may generate neoantigens. Here, we aimed to generate a neoantigen MDP to exhibit stronger anti-tumor efficacy. Methods: In this study, we utilized chemically modified sialic acid precursor tetra acetyl-N-azidoacetyl-mannosamine (AC4ManNAZ) to engineer the glycoproteins on the membranes of tumor cells for the covalent ligation of hapten adjuvant Pam3CSK4 in vivo, which eventually generated a neoantigen, i.e., ManNAZ-DBCO-Pam3CSK4 (MDP), on tumor cells. The high labeling efficiency, relatively specific biodistribution in tumor tissues and the anti-tumor efficacy were confirmed in the syngeneic murine models of the breast cancer and the lung cancer. Results: The generation of MDP neoantigen in tumor-bearing mice significantly evoked both the humoral and the T-cell-dependent antitumor immune responses, resulting in a strong inhibition on the growth of the breast cancer and the lung cancer allografts and significantly prolonged survival of tumor-bearing mice. Interestingly, MDP neoantigen was able to dramatically increase the sensitivity of cancer cells to ICIs and greatly enhance the anti-tumor efficacy in the murine models of both breast cancer and the lung cancer, which showed no or low responses to the immunotherapy with anti-PD1 antibody alone. Conclusions: We developed a simple metabolic glycoengineering method to artificially generate neoantigens on tumor cells to enhance tumor cell immunogenicity, which is able to significantly improve the response and the clinical outcome of ICIs.

Neoantigen: A New Breakthrough in Tumor Immunotherapy.

Cancer immunotherapy works by stimulating and strengthening the body's anti-tumor immune response to eliminate cancer cells. Over the past few decades, immunotherapy has shown remarkable efficacy in the treatment of cancer, particularly the success of immune checkpoint blockade targeting CTLA-4, PD-1 and PDL1, which has led to a breakthrough in tumor immunotherapy. Tumor neoantigens, a new approach to tumor immunotherapy, include antigens produced by tumor viruses integrated into the genome and antigens produced by mutant proteins, which are abundantly expressed only in tumor cells and have strong immunogenicity and tumor heterogeneity. A growing number of studies have highlighted the relationship between neoantigens and T cells' recognition of cancer cells. Vaccines developed against neoantigens are now being used in clinical trials in various solid tumors. In this review, we summarized the latest advances in the classification of immunotherapy and the process of classification, identification and synthesis of tumor-specific neoantigens, as well as their role in current cancer immunotherapy. Finally, the application prospects and existing problems of neoantigens were discussed.

Progesterone-induced blocking factor improves blood pressure, inflammation, and pup weight in response to reduced uterine perfusion pressure (RUPP).

Preeclampsia (PE) is characterized by new-onset hypertension in association with elevated natural killer (NK) cells and inflammatory cytokines, which are likely culprits for decreased fetal weight during PE pregnancies. As progesterone increases during normal pregnancy, it stimulates progesterone-induced blocking factor (PIBF). PIBF has been shown to decrease inflammation and cytolytic NK cells, both of which are increased during PE. We hypothesized that PIBF reduces inflammation as a mechanism to improve hypertension in the preclinical reduced uterine perfusion pressure (RUPP) rat model of PE. PIBF (2.0 µg/mL) was administered intraperitoneally on gestational day 15 to either RUPP or normal pregnant (NP) rats. On day 18, carotid catheters were inserted. Mean arterial blood pressure (MAP) and samples were collected on day 19. MAP in NP rats (n = 11) was 100 ± 2 mmHg and 105 ± 3 mmHg in NP + PIBF rats (n = 8) and 122 ± 1 mmHg in RUPP rats (n = 10), which improved to 110 ± 2 mmHg in RUPP + PIBF rats (n = 11), P < 0.05. Pup weight was 2.4 ± 0.1 g in NP, 2.5 ± 0.1 g in NP + PIBF, 1.9 ± 0.1 g in RUPP, and improved to 2.1 ± 0.1 g in RUPP + PIBF rats. Circulating and placental cytolytic NK cells, IL-17, and IL-6 were significantly reduced while IL-4 and T helper (TH) 2 cells were significantly increased in RUPP rats after PIBF administration. Importantly, vasoactive pathways preproendothelin-1, nitric oxide, and soluble fms-Like tyrosine Kinase-1 (sFlt-1) were normalized in RUPP + PIBF rats compared with RUPP rats, P < 0.05. Our findings suggest that PIBF normalized IL-4/TH2 cells, which was associated with improved inflammation, fetal growth restriction, and blood pressure in the RUPP rat model of PE.