Potential Perturbations of Critical Cancer-regulatory Genes in Triple-Negative Breast Cancer Cells Within the Humanized Microenvironment of Patient-derived Xenograft Models.

PURPOSE: In this study, we aimed to establish humanized patient-derived xenograft (PDX) models for triple-negative breast cancer (TNBC) using cord blood (CB) hematopoietic stem cells (HSCs). Additionally, we attempted to characterize the immune microenvironment of the humanized PDX model to understand the potential implications of altered tumor-immune interactions in the humanized PDX model on the behavior of TNBC cells. METHODS: To establish a humanized mouse model, high-purity CD34+ HSCs from CB were transplanted into immunodeficient NOD scid γ mice. Peripheral and intratumoral immune cell compositions of humanized and non-humanized mice were compared. Additionally, RNA sequencing of the tumor tissues was performed to characterize the gene expression features associated with humanization. RESULTS: After transplanting the CD34+ HSCs, CD45+ human immune cells appeared within five weeks. A humanized mouse model showed viable human immune cells in the peripheral blood, lymphoid organs, and in the tumor microenvironment. Humanized TNBC PDX models showed varying rates of tumor growth compared to that of non-humanized mice. RNA sequencing of the tumor tissue showed significant alterations in tumor tissues from the humanized models. tumor necrosis factor receptor superfamily member 11B (TNFRSF11B) is a shared downregulated gene in tumor tissues from humanized models. Silencing of TNFRSF11B in TNBC cell lines significantly reduced cell proliferation, migration, and invasion in vitro. Additionally, TNFRSF11B silenced cells showed decreased tumorigenicity and metastatic capacity in vivo. CONCLUSION: Humanized PDX models successfully recreated tumor-immune interactions in TNBC. TNFRSF11B, a commonly downregulated gene in humanized PDX models, may play a key role in tumor growth and metastasis. Differential tumor growth rates and gene expression patterns highlighted the complexities of the immune response in the tumor microenvironment of humanized PDX models.

Triple-Negative Breast Cancer-Derived Extracellular Vesicles Promote a Hepatic Premetastatic Niche via a Cascade of Microenvironment Remodeling.

Patients with triple-negative breast cancer (TNBC) often develop metastases in visceral organs including the liver, but the detailed molecular mechanisms of TNBC liver metastasis is not clearly understood. In this study, we tried to dissect the process of premetastatic niche formation in the liver by using patient-derived xenograft (PDX) models of TNBC with different metastatic propensity. RNA sequencing of TNBC PDX models that successfully metastasized to liver showed upregulation of the Cx3cr1 gene in the liver microenvironment. In syngeneic breast cancer models, the Cx3cr1 upregulation in liver preceded the development of cancer cell metastasis and was the result of recruitment of CX3CR1-expressing macrophages. The recruitment was induced by the CX3CL1 production from the liver endothelial cells and this CX3CL1-CX3CR1 signaling in the premetastatic niche resulted in upregulation of MMP9 that promoted macrophage migration and cancer cell invasion. In addition, our data suggest that the extracellular vesicles derived from the breast cancer cells induced the TNFα expression in liver, which leads to the CX3CL1 upregulation. Lastly, the plasma CX3CL1 levels in 155 patients with breast cancer were significantly associated with development of liver metastasis. IMPLICATIONS: Our data provides previously unknown cascades regarding the molecular education of premetastatic niche in liver for TNBC.

AKR1C2 Promotes Metastasis and Regulates the Molecular Features of Luminal Androgen Receptor Subtype in Triple Negative Breast Cancer Cells.

PURPOSE: Patients with triple-negative breast cancer (TNBC) have an increased risk of distant metastasis compared to those with other subtypes. In this study, we aimed to identify the genes associated with distant metastasis in TNBC and their underlying mechanisms. METHODS: We established patient-derived xenograft (PDX) models using surgically resected breast cancer tissues from 31 patients with TNBC. Among these, 15 patients subsequently developed distant metastases. Candidate metastasis-associated genes were identified using RNA sequencing. In vitro wound healing, proliferation, migration, and invasion assays and in vivo tumor xenograft and metastasis assays were performed to determine the functional importance of aldo-keto reductase family 1 member C2 (AKR1C2). Additionally, we used the METABRIC dataset to investigate the potential role of AKR1C2 in regulating TNBC subtypes and their downstream signaling activities. RESULTS: RNA sequencing of primary and PDX tumors showed that genes involved in steroid hormone biosynthesis, including AKR1C2, were significantly upregulated in patients who subsequently developed metastasis. In vitro and in vivo assays showed that silencing of AKR1C2 resulted in reduced cell proliferation, migration, invasion, tumor growth, and incidence of lung metastasis. AKR1C2 was upregulated in the luminal androgen receptor (LAR) subtype of TNBC in the METABRIC dataset, and AKR1C2 silencing resulted in the downregulation of LAR classifier genes in TNBC cell lines. The androgen receptor (AR) gene was a downstream mediator of AKR1C2-associated phenotypes in TNBC cells. AKR1C2 expression was associated with gene expression pathways that regulate AR expression, including JAK-STAT signaling or interleukin 6 (IL-6). The levels of phospho-signal transducer and activator of transcription and IL-6, along with secreted IL-6, were significantly downregulated in AKR1C2-silenced TNBC cells. CONCLUSION: Our data indicate that AKR1C2 is an important regulator of cancer growth and metastasis in TNBC and may be a critical determinant of LAR subtype features.

An integrative approach for exploring the nature of fibroepithelial neoplasms.

BACKGROUND: Malignant phyllodes tumour (MPT) is a rare breast malignancy with epithelial and mesenchymal features. Currently, there are no appropriate research models or effective targeted therapeutic approaches for MPT. METHODS: We collected fresh frozen tissues from nine patients with MPT and performed whole-exome and RNA sequencing. Additionally, we established patient-derived xenograft (PDX) models from patients with MPT and tested the efficacy of targeting dysregulated pathways in MPT using the PDX model from one MPT. RESULTS: MPT has unique molecular characteristics when compared to breast cancers of epithelial origin and can be classified into two groups. The PDX model derived from one patient with MPT showed that the mouse epithelial component increased during tumour growth. Moreover, targeted inhibition of platelet-derived growth factor receptor (PDGFR) and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) by imatinib mesylate and PKI-587 showed in vivo tumour suppression effects. CONCLUSIONS: This study revealed the molecular profiles of MPT that can lead to molecular classification and potential targeted therapy, and suggested that the MPT PDX model can be a useful tool for studying the pathogenesis of fibroepithelial neoplasms and for preclinical drug screening to find new therapeutic strategies for MPT.

MUC1 expressing tumor growth was retarded after human mucin 1 (MUC1) plasmid DNA immunization.

INTRODUCTION: Naked DNA is one of the attractive tools for vaccination studies. We studied naked DNA vaccination against the human tumor antigen, mucin, which is encoded by the MUC1 gene. METHODS: We constructed the pcDNA3.0-MUC1 (pcDNA-MUC1) plasmid expressing an underglycosylated MUC1 protein. BALB/c mice were immunized intradermally thrice at 2-weeks intervals with pcDNA-MUC1. Two weeks after the last immunization, tumor challenge experiments were performed using either the CT26 or TA3HA tumor cell lines, both of which transduce human MUC1. RESULTS: Immune cell population monitoring from pcDNA-MUC1-immunized animals indicated that immune cell activation was induced by MUC1-specific immunization. Using intracellular fluorescence activated cell sorting and enzyme-linked immunosorbent spot assay, we reported that interferon-γ secreting CD8+ T cells were mainly involved in MUC1-specific immunization. In all mice immunized with MUC1 DNA, tumor growth inhibition was observed, whereas control mice developed tumors (p < 0.001). CONCLUSION: Our results suggest that intradermal immunization with MUC1 DNA induces MUC1-specific CD8+ T cell infiltration into tumors, elicits tumor-specific Th1-type immune response, and inhibits tumor growth.

Immune Evasion Mechanism and AXL.

Extensive interest in cancer immunotherapy is reported according to the clinical importance of CTLA-4 and (PD-1/PD-L1) [programmed death (PD) and programmed death-ligand (PD-L1)] in immune checkpoint therapies. AXL is a receptor tyrosine kinase expressed in different types of cancer and in relation to resistance against various anticancer therapeutics due to poor clinical prognosis. AXL and its ligand, i.e., growth arrest-specific 6 (GAS6) proteins, are expressed on many cancer cells, and the GAS6/AXL pathway is reported to promote cancer cell proliferation, survival, migration, invasion, angiogenesis, and immune evasion. AXL is an attractive and novel therapeutic target for impairing tumor progression from immune cell contracts in the tumor microenvironment. The GAS6/AXL pathway is also of interest immunologically because it targets fewer antitumor immune responses. In effect, several targeted therapies are selective and nonselective for AXL, which are in preclinical and clinical development in multiple cancer types. Therefore, this review focuses on the role of the GAS6/AXL signaling pathway in triggering the immunosuppressive tumor microenvironment as immune evasion. This includes regulating its composition and activating T-cell exclusion with the immune-suppressive activity of regulatory T cells, which is related to one of the hallmarks of cancer survival. Finally, this article discusses the GAS6/AXL signaling pathway in the context of several immune responses such as NK cell activation, apoptosis, and tumor-specific immunity, especially PD-1/PDL-1 signaling.

JAK2 regulates paclitaxel resistance in triple negative breast cancers.

We investigated the molecular mechanisms of paclitaxel resistance in TNBC using seven patient-derived xenograft (PDX) models and TNBC cell lines. Among the seven PDX models, four models showed resistance to paclitaxel. Dysregulation of JAK/STAT pathways and JAK2 copy number gains were observed in the four paclitaxel-resistant PDX tumors. In TNBC cell lines, silencing the JAK2 gene showed a significant but mild synergistic effect when combined with paclitaxel in vitro. However, JAK1/2 inhibitor treatment resulted in restoration of paclitaxel sensitivity in two out of four paclitaxel-resistant PDX models and JAK1/2 inhibitor alone significantly suppressed the tumor growth in one out of the two remaining PDX models. Transcriptome data derived from the murine microenvironmental cells revealed an enrichment of genes involved in the cell cycle processes among the four paclitaxel-resistant PDX tumors. Histologic examination of those PDX tumor tissues showed increased Ki67-positive fibroblasts in the tumor microenvironment. Among the four different cancer-associated fibroblast (CAF) subtypes, cycling CAF exhibiting features of active cell cycle was enriched in the paclitaxel-resistant PDX tumors. Additionally, fibroblasts treated with the conditioned media from the JAK2-silenced breast cancer cells showed downregulation of cell cycle-related genes. Our data suggest that the JAK2 gene may play a critical role in determining responses of TNBC to paclitaxel by modulating the intrinsic susceptibility of cancer cells against paclitaxel and also by eliciting functional transitions of CAF subtypes in the tumor microenvironment. KEY MESSAGES : We investigated the molecular mechanisms of paclitaxel resistance in TNBC. JAK2 signaling was associated with paclitaxel resistance in TNBC PDX models. Paclitaxel-resistant PDX tumors were enriched with microenvironment cCAF subpopulation. JAK2 regulated paclitaxel-resistant CAF phenotype transition.

S100A8/A9 mediate the reprograming of normal mammary epithelial cells induced by dynamic cell-cell interactions with adjacent breast cancer cells.

To understand the potential effects of cancer cells on surrounding normal mammary epithelial cells, we performed direct co-culture of non-tumorigenic mammary epithelial MCF10A cells and various breast cancer cells. Firstly, we observed dynamic cell-cell interactions between the MCF10A cells and breast cancer cells including lamellipodia or nanotube-like contacts and transfer of extracellular vesicles. Co-cultured MCF10A cells exhibited features of epithelial-mesenchymal transition, and showed increased capacity of cell proliferation, migration, colony formation, and 3-dimensional sphere formation. Direct co-culture showed most distinct phenotype changes in MCF10A cells followed by conditioned media treatment and indirect co-culture. Transcriptome analysis and phosphor-protein array suggested that several cancer-related pathways are significantly dysregulated in MCF10A cells after the direct co-culture with breast cancer cells. S100A8 and S100A9 showed distinct up-regulation in the co-cultured MCF10A cells and their microenvironmental upregulation was also observed in the orthotropic xenograft of syngeneic mouse mammary tumors. When S100A8/A9 overexpression was induced in MCF10A cells, the cells showed phenotypic features of directly co-cultured MCF10A cells in terms of in vitro cell behaviors and signaling activities suggesting a S100A8/A9-mediated transition program in non-tumorigenic epithelial cells. This study suggests the possibility of dynamic cell-cell interactions between non-tumorigenic mammary epithelial cells and breast cancer cells that could lead to a substantial transition in molecular and functional characteristics of mammary epithelial cells.

Assessing biological aging following systemic administration of bFGF-supplemented adipose-derived stem cells with high efficacy in an experimental rat model.

Biological aging (BA) is a tool for comprehensive assessment of individual health status. A rat model was developed for measuring BA by intravenously administering adipose-derived stem cells (ADSCs) into rats and evaluating several biochemical parameters. In addition, the effect of basic fibroblast growth factor (bFGF) on the differentiation potential of ADSCs was analyzed. A total of 12 male Sprague Dawley rats were divided into autologous ADSC administration (n=6) and saline administration (n=6) groups. The ADSC administration group was further divided into the bFGF supplemented (n=3) and bFGF non-supplemented (n=3) groups. Biochemical parameters and antioxidant potential were evaluated prior to fat harvest and ADSC administration, as well as 1, 3, and 5 weeks following ADSC administration. ADSC administration regulated inflammation, renal and hepatic functions, and levels of antioxidant enzymes. The cell doubling time of the bFGF-supplemented group was shorter (P=0.0001) than that of the bFGF non-supplemented group. Renal and hepatic functions were maintained with bFGF supplementation, which possibly enhanced the effect of ADSCs. The rat model developed in the present study may promote better understanding of BA in the context of bFGF-supplemented ADSC administration.

Adipose-derived stem cells attenuate atopic dermatitis-like skin lesions in NC/Nga mice.

There is an unmet need in novel therapeutics for atopic dermatitis (AD). We examined the effects of autologous adipose-derived stem cells (ADSCs) on AD-like skin lesions induced by the application of 2,4-dinitrochlorobenzene (DNCB) in NC/Nga mice. Autologous ADSCs and ADSC-conditioned medium (ADSC-CM) were injected intralesionally three times. Clinical severity and histopathologic findings were compared in sham naïve control, saline-treated, ADSC-treated, ADSC-CM-treated and 2.5% cortisone lotion-applied animals. The severity index, skin thickness, mast cell number, as well as expression levels of thymic stromal lymphopoietin, CD45, chemoattractant receptor-homologous molecule, chemokine ligand 9 and chemokine ligand 20 were significantly lower in mice treated with ADSC, ADSC-CM, or 2.5% cortisone lotion. Tissue levels of interferon-γ as well as serum levels of interleukin-33 and immunoglobulin E levels were also decreased in those groups. We conclude that autologous ADSCs improved DNCB-induced AD-like skin lesions in NC/Nga mice by reducing inflammation associated with Th2 immune response and interferon-γ.

Biological Aging Parameters Can Be Improved After Autologous Adipose-Derived Stem Cell Injection.

Biological aging (BA) is a comprehensive assessment tool for elderly persons. The authors aimed to develop a rat model that can be used to assess BA by evaluating various blood, biochemical, and hormonal parameters and demonstrate that the intravenous administration of autologous adipose-derived stem cells (ADSCs) improves BA. Twelve elderly (aged 20 months) male Sprague-Dawley rats were used in this study and divided into 2 groups: autologous ADSC administration (n = 6) and saline administration (n = 6). The complete blood count, biochemical and hormonal parameters, and antioxidant potential were evaluated before harvesting the rat inguinal fat tissue and intravenous ADSC administration as well as at 1, 3, and 5 weeks after ADSC administration. Adipose-derived stem cells administration regulated blood content, biochemical parameters, renal function, and antioxidant enzymes in elderly rats. Furthermore, changes in several hormonal levels were identified in the ADSC administration group compared with the saline administration group. An assessment model of BA in elderly rats was successfully developed after the intravenous administration of autologous ADSCs. The authors suggest that intravenously injected ADSC treatment may be a valuable method to improve BA.

The Role of Periostin in Capsule Formation on Silicone Implants.

Although silicone implants are widely used in breast and other reconstructive surgeries, the limited biocompatibility of these materials leads to severe complications, including capsular contracture. Here, we aimed to clarify the relationship between periostin and the process of capsule formation after in vivo implantation. Seven-week-old wild-type (WT) C57BL/6 mice and periostin-deficient mice were used. Round silicone implants were inserted into a subcutaneous pocket on the dorsum of the mice. After 8 weeks, the fibrous capsule around the implant was harvested and histologically examined to estimate capsular thickness and the number of inflammatory cells. Additionally, immunohistochemical analysis (periostin, α-SMA, and collagen type I) and western blotting (CTGF, TGF-ß, VEGF, and MPO) were performed for a more detailed analysis of capsule formation. The capsules in periostin-knockout mice (PN-KO) were significantly thinner than those in WT mice. PN-KO mice showed significantly lower numbers of inflammatory cells than WT mice. Fibrous tissue formation markers (α-SMA, periostin, collagen type I, and CTGF) were significantly reduced in PN-KO mice. We also confirmed that inflammatory reaction and angiogenesis indicators (TGF-ß, MPO, and VEGF) had lower expression in PN-KO mice. Inhibition of periostin could be important for suppressing capsule formation on silicone implants after in vivo implantation.

Protective efficacy of a plasmid DNA vaccine against transgene-specific tumors by Th1 cellular immune responses after intradermal injection.

With DNA vaccines, it is important to monitor the movement of transfectants and to overcome immune deviations. We used a pCMV-LacZ plasmid (expressing ß-galactosidase) and a pcDNA-hNIS plasmid (expressing the human sodium/iodide symporter [hNIS] gene) as non-secreted visual-imaging markers. Transfectants carrying the hNIS or LacZ gene migrated to peripheral lymphoid tissues. hNIS-expressing cells were observed specifically in the LNs and spleen. Anti-ß-galactosidase was detected in LacZ DNA immunized mice after boosting twice, suggestive of Th2 humoral immune responses. Antibody isotyping defined the humoral immune response. A dominant IgG2a type occurred in hNIS-immunized mice in ELISAs. IgG2a/IgG1 ratios increased after hNIS DNA vaccination. High levels of INF-γ-secreting cells were identified in ELISpot and increased IFN-γ levels were found in cytokine ELISAs. Tumor growth decreased in hNIS DNA-immunized mice. In conclusion, humoral immune responses switched to the Th1 cellular immune response, even though we administered plasmid DNA by intra dermal injection.

Mannosylated T/Tn with Freund's adjuvant induces cellular immunity.

Inducing cancer-specific cellular immune responses has become an attractive strategy in cancer treatment. In this study, we investigated the role of several adjuvants in eliciting T/Tn-specific cellular immunity and protection against T/Tn expressing tumor challenge. T/Tn (9:1) antigen was purified from blood type "O" erythrocytes donated from healthy Korean volunteers. Immunization was performed using: T/Tn only, T/Tn mixed with Freund's adjuvant (T/Tn + FA), keyhole limpet hemocyanin (KLH)-conjugated T/Tn mixed with FA (KLH-T/Tn + FA), and oxidized mannan-conjugated T/Tn mixed with FA (ox-M-T/Tn + FA). Mice immunized with ox-M-T/Tn + FA generated T/Tn-specific CD3, helper T (Th) cells, major histocompatibility complex (MHC) II, and MHC I; T/Tn presentation was significantly high and tolerogenic CD11b+ was the lowest among the tumor models. To verify Th type, we stained intracellular cytokines (interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and IL-10) using CD3 co-staining. Th1 (IFN-γ and GM-CSF) cytokines were highly expressed and showed high FasL/Fas ratios, cytotoxic T lymphocyte (CTL) activity, and cytotoxic T lymphocyte precursor (CTLp) activity in mice immunized with ox-M-T/Tn + FA. Lymphocyte infiltration was highest in mice immunized with ox-M-T/Tn + FA. Additionally, we monitored FasL, MHC I, CD301, and T/Tn expression levels using immunohistochemistry (IHC) on macrophage and tumor sites. The expression of all markers was highest in the ox-M-T/Tn + FA group. Furthermore, tumor retardation and survival rate were highest in the ox-M-T/Tn + FA group. These results demonstrate that a vaccine formulation of T/Tn conjugated with ox-M and mixed with FA-induced cellular immunity and sustained a humoral immune response without over-activating the immune system, thus effectively inhibiting tumor growth.

T/Tn immunotherapy avoiding immune deviation.

Tumor immunotherapy, capable of inducing both cellular and humoral immune responses, is an attractive treatment strategy for cancer. It has been reported that the inactivation of cell-mediated immunity by hyper-activation of humoral immunity-referred to as immune deviation-does not inhibit tumor growth. We investigated the ability of several adjuvants to elicit Thomsen-Friedenreich (T/Tn)-specific humoral immunity while avoiding immune deviation and conferring protection against tumorigenesis. T/Tn (9:1) antigen was purified from blood type O erythrocytes donated by healthy Korean volunteers. Immunization was performed using T/Tn only, T/Tn mixed with Freund's adjuvant (T/Tn+FA), keyhole limpet hemocyanin (KLH)-conjugated T/Tn mixed with FA (KLH-T/Tn+FA), or oxidized mannan-conjugated T/Tn mixed with FA (ox-M-T/Tn+FA). Anti-T/Tn antibodies were generated in the T/Tn+FA, KLH-T/Tn+FA, and ox-M-T/Tn+FA groups. The antibody level was highest in the KLH-T/Tn+FA group. Mice immunized with ox-M-T/Tn+FA showed specific complement-dependent cytotoxicity, and were protected against T/Tn-positive mammary adenocarcinoma cell challenge, although anti-T/Tn antibody levels were the highest in the KLH-T/Tn+FA group. These results demonstrate that an ox-M-conjugated T/Tn vaccine mixed with FA can promote cellular immunity while moderating the humoral immune response, thereby effectively inhibiting tumor growth.

In vivo monitoring of transfected DNA, gene expression kinetics, and cellular immune responses in mice immunized with a human NIS gene-expressing plasmid.

In assessing the effectiveness of DNA vaccines, it is important to monitor: (1) the kinetics of target gene expression in vivo; and (2) the movement of cells that become transfected with the plasmid DNA used in the immunization of a subject. In this study, we used, as a visual imaging marker, expression of the transfected human sodium/iodide symporter (hNIS) gene, which enhances intracellular radio-pertechnetate (TcO4-) accumulation. After intradermal (i.d.) and systemic injection of mice with pcDNA-hNIS and radioactive Technetium-99m (Tc-99m), respectively, whole-body images were obtained by nuclear scintigraphy. The migration of mice cells transfected with the hNIS gene was monitored over a 2-week period by gamma-radioactivity counting of isolated cell populations and was demonstrated in peripheral lymphoid tissues, especially in the draining lymph nodes (dLNs). Beginning at 24 h after DNA inoculation and continuing for the 2-week monitoring period, hNIS-expressing cells were observed specifically in the T-cell-rich zones of the paracortical area of the dLNs. Over the same time period, high levels of INF-γ-secreting CD8 T-cells were found in the dLNs of the pcDNA-hNIS immunized mice. Tumor growth was also significantly retarded in the mice that received hNIS DNA immunization followed by inoculation with CT26 colorectal adenocarcinoma cells that had been transfected with the rat NIS gene (rNIS), which is 93% homologous to the hNIS gene. In conclusion, mouse cells transfected with hNIS DNA after i.d. immunization were found to traffic to the dLNs, and hNIS gene expression in these cells continued for at least 2 weeks post immunization. Furthermore, sequential presentation of NIS DNA to T-cells by migratory antigen presenting cells could induce NIS DNA-specific Th1 immune responses and thus retard the growth of NIS-expressing tumors.

Oxidized and reduced mannan mediated MUC1 DNA immunization induce effective anti-tumor responses.

DNA immunization is an attractive form of vaccination, which has shown promising results only in small animal models. There is a need to develop efficient gene delivery systems. We previously demonstrated that oxidized (OM) and reduced mannan (RM) complexed to ovalbumin DNA via poly-l-lysine (PLL), were able to generate potent immune responses in mice. Herein, we further investigated the suitability of OMPLL and RMPLL as carriers for mucin 1 (MUC1) DNA vaccination for cancer immunotherapy. Studies presented here showed that immune responses in C57BL/6 mice induced by OMPLL-MUC1 DNA and RMPLL-MUC1 DNA immunization were more immunogenic compared to MUC1 DNA alone. Moreover, tumor protection was evident at a dose as low as 0.5 microg. In addition, strong T cell responses were induced in HLA-A2 transgenic and human MUC1 transgenic mice. These results demonstrate the potential of OM and RM as efficient non-viral gene delivery carriers for DNA vaccines for use in cancer immunotherapy.

Exosomes: a new delivery system for tumor antigens in cancer immunotherapy.

Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC type-distinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other well-described exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1-expressing tumor growth in a MUC1-specific and dose-related manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free tumor vaccine.