Intratumoral Dendritic Cells Research Articles

Abstract 3624: Activation of tumor specific antibody response following systemic delivery of receptor targeted nanoparticles into Balb/c mice bearing mouse mammary tumors

Abstract Targeted nanoparticles (NP) offer a great opportunity for developing new cancer imaging agents and targeted drug delivery carriers. Although increasing evidence supports the potential of the targeted NP for early cancer detection and drug delivery, the impact of systemic delivery of targeted NP on the immune response in the tumor has yet to be addressed. The possible immune enhancement effect of NP has attracted great attention to apply various NP for vaccine development since NP can be nonspecifically taken up by macrophages. In this study, we developed epidermal growth factor receptor (EGFR) receptor-targeted dual imaging modality magnetic iron oxide NP (IONP) using single chain antibody to EGFR (ScFvEGFR) without or with chemotherapy drug doxorubicin (Dox) for targeted tumor imaging and therapy of triple negative breast cancer. The NP were injected into normal Balb/c mice or mice bearing 4T1 mammary tumors once per week for three weeks, then serum and tumors were collected. Systemic delivery of the receptor-targeted NP led to accumulation of NP in the tumors and inhibited tumor growth. Using ELISA on tumor cell lysates and intact tumor cells, we found that mouse serum from targeted IONP had the highest level of mammary tumor specific antibody titers compared to mice that received Dox only, non-targeted NP and no treatment control. However, the antibody titer was decreased in the mice that received ScFvEGFR-IONP-Dox. To understand the antibody activation mechanism by targeted delivery of ScFvEGFR-IONP but not ScFvEGFR-IONP-Dox, we examined changes in the amount, phenotype, and localization of antigen presenting macrophages using a pan-macrophage (CD68) and M2 macrophage (CD163) and also dendritic cells (CD83) markers in frozen tumor tissue sections. Results of double labeling immunofluorescence against CD68 and CD163 showed the majority macrophage subtype at the tumor edge was M2 for the tumors treated with ScFvEGFR-IONP with and without Dox. However, in the central tumor area M1 macrophages was the main phenotype in ScFvEGFR -IONP-Dox treated tumors while ScFvEGFR -IONP still expressed majority M2. Also, a higher level of CD83+ cells infiltrated into the central tumor area of ScFvEGFR-IONP compared to ScFvEGFR-IONP-Dox. Results of our study suggest that targeted delivery of ScFvEGFR-IONP into tumor is able to activate intratumoral macrophages and dendritic cells to antigen presentation and stimulate tumor specific antibody responses while ScFvEGFR-IONP-Dox had decreased amounts of CD163 and CD83. Therefore, targeted delivery of NP into tumors and infiltration of antigen-presenting macrophages into the center of tumors are important for the activation of tumor specific antibody responses. This study could have clinical implications in development of NP imaging and therapy agents for cancer detection and treatment. Citation Format: Christina Ward, Weiping Qian, Emmy Yang, Erica Bozeman, Y. Andrew Wang, Lily Yang. Activation of tumor specific antibody response following systemic delivery of receptor targeted nanoparticles into Balb/c mice bearing mouse mammary tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3624. doi:10.1158/1538-7445.AM2014-3624

Modulated electro-hyperthermia enhances dendritic cell therapy through an abscopal effect in mice.

The aim of this study was to assess whether modulated electro-hyperthermia (mEHT) can induce an abscopal effect and thereby enhance the antitumor effects of immunotherapy. We used an intratumoral dendritic cell (DC) injection and mEHT to treat C3H/He mice inoculated with squamous cell carcinoma SCCVII cells in the left leg, and we assessed the whole body antitumor effects. Tumors were examined every two or three days in order to assess growth inhibition. The tumor-draining lymph nodes were removed to enable flow cytometric analysis of CD3+ and CD8+ cells, whereas immunohistochemistry was used to assess CD8, S100 and Foxp3 expression in the tumors. Additionally, GP96 expression in the tumors from the different treatment groups was measured. In the control group, the mean tumor volume was larger than that in other groups. These results indicated that the combination therapy of an intratumoral DC injection and mEHT evoked systemic antitumor activity. A larger number of CD3+ and CD8+ cells were detected by flow cytometric analysis in the DC plus mEHT treatment group. Tumor tissue immunostaining showed that CD8 and S100 were more strongly expressed in the DC plus mEHT treatment group, although Foxp3 expression was much higher in the control group. The GP96 gene expression level in the mEHT group was significantly different from the expression level in the control group. An abscopal effect may be induced by mEHT, and the effect of immunotherapy with DCs was strongly enhanced by the overexpression of GP96. GP96 is thought to be one of the molecules explaining the abscopal effect. Direct intratumoral administration of DCs and mEHT may be a feasible future treatment strategy.

Cancer chemotherapy agents target intratumoral dendritic cells to potentiate antitumor immunity

Cytotoxic drugs capable of killing cancer cells in conjunction with targeted conversion of tumor resident, tolerogenic dendritic cells (DCs) into efficient antigen presenting cells (APCs) are highly complementary therapeutic routes to boost antitumor immunity. Our data suggest that the microtubule-depolymerizing compounds Dolastatin 10 and Ansamitocin P3 may serve as prototypes for a class of agents that display this binary mode of action.

Clinical relevance of immune parameters in the tumor microenvironment of head and neck cancers.

The tumour microenvironment is a highly complex region where multiple interactions occur between host and cancer cells. The host response is defined by the presence and function of different tumour infiltrating lymphocytes and cytokines. Head and neck squamous cell carcinomas comprise a subgroup of human cancers with significant morbidity and mortality. A literature review was performed to identify studies, which have investigated the impact of immune factors within the tumour microenvironment of head and neck squamous cell carcinomas on clinical outcomes. Higher counts of intratumoral dendritic cells and CD8+ T-lymphocytes are associated with a favorable prognosis in head and neck cancers. However, contrasting results have been found with T-regulatory cells whereby higher intratumoral counts appear favorable particularly in tumours of oropharyngeal origin. A number of studies have found a link between various biomarkers, e.g. IL-10 in oral cancers, and clinical outcome. We have identified immune factors within the tumour microenvironment, which are critically related to prognosis and also those, which require further work to elucidate their full relevance. Furthermore it is clear that if immunotherapy is to be introduced as an adjunct in the treatment of head and neck cancers, different immunological parameters will need to be selected for each distinct subsite.

Neoadjuvant administration of radiation therapy and intratumoral autologous dendritic cells in patients with localized high-risk soft tissue sarcomas.

10519 Background: Patients with high grade, large (<5 cm) soft tissue sarcomas (STS) can develop metastases after surgical therapy with no proven benefit to adjuvant chemotherapy. Our in vivo murin...

Local and systemic antitumor effects of activated autologous dendritic cells for intratumoral injection: A phase I/II trial.

TPS3133 Background: Dendritic cells (DC) are antigen presenting cells proficient in inducing de novo immune responses, and the presence of intratumoral DCs confers a survival advantage to patients,...

Dendritic cell-based cancer immunotherapy: the stagnant approach and a theoretical solution

Anticancer dendritic cells (DC) therapy currently uses in vitro propagation of the patient's DC and pulsing with tumor antigens. However, clinical achievements are far from desirable. Here, I suggest that the lack of anticipated responses could be because cancer cells continuously mutate, whereas the population of tumor antigens from the excised tumor is genetically static, and because there is an absence of biologic mechanisms to facilitate intratumoral DC retention, which is needed for DC pulsing. I hypothesize that stable tumor transfection with fetal liver tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) DNAs will induce homing, propagation and maturation of intratumoral DC. This must be followed by drug-induced apoptosis of tumor cells, to ensure the release of tumor antigens for DC pulsing. Then, regardless of any mutation of tumor cells, they would always incite DC propagation and maturation, pulsing and antitumor immunity.

Relationship of dendritic cell density, HMGB1 expression, and tumor-infiltrating lymphocytes in non-small cell lung carcinomas.

Lung cancer is the leading cause of cancer death worldwide and non-small cell lung carcinoma (NSCLC) is the most common type of lung carcinomas. In adenocarcinomas, the most frequent histologic type of NSCLC, dendritic cells (DCs) are localized in close contact with tumor cells, and tumor-infiltrating lymphocytes (TILs) are observed in the peritumoral zones. In NSCLC, no studies investigating the density of intratumoral DCs and their impact on the density of TILs have been performed. In addition, the role of the alarmin high-mobility group box1 (HMGB1) in intratumoral DCs recruitment has not been analyzed. In the present study, a total of 82 cases of advanced stages of NSCLC were included. Tissue samples were obtained from biopsies and autopsies. DCs in biopsies or combinations of DCs and NK cells, CD3 T lymphocytes, or CD8 T lymphocytes from autopsy specimens were quantified in high power fields. Also, distribution of HMGB1 in tumor cells was detected. In lung adenocarcinomas, irrespective of subclassification, high densities of infiltrating DCs directly associated to high densities of peritumoral TILs. A 2.5-fold increase in TILs was found in specimens with high densities of infiltrating DCs compared with TILs from adenocarcinomas with low densities of infiltrating DCs. High densities of infiltrating DCs were associated with lung adenocarcinomas expressing cytoplasmic or nuclear-cytoplasmic HMGB1. Our results suggest that in adenocarcinoma patients, HMGB1 produced by tumor cells recruits DCs, which associate to an increase of TILs. Encouraging tumor-DCs-T lymphocytes interactions should improve the quality of life and survival of NSCLC patients.

Targeting the Tumor Microenvironment with Interferon-β Bridges Innate and Adaptive Immune Responses

Antibodies (Abs) that preferentially target oncogenic receptors have been increasingly used for cancer therapy, but tumors often acquire intrinsic Ab resistance after prolonged and costly treatment. Herein we armed the Ab with IFNβ and observed that it is more potent than the first generation of Ab for controlling Ab-resistant tumors. This strategy controls Ab resistance by rebridging suppressed innate and adaptive immunity in the tumor microenvironment. Mechanistically, Ab-IFNβ therapy primarily and directly targets intratumoral dendritic cells, which reactivate CTL by increasing antigen cross-presentation within the tumor microenvironment. Additionally, blocking PD-L1, which is induced by Ab-IFNβ treatment, overcomes treatment-acquired resistance and completely eradicates established tumors. This study establishes a next-generation Ab-based immunotherapy that targets and eradicates established Ab-resistant tumors.

Interferon response factor 3 is crucial to poly-I:C induced NK cell activity and control of B16 melanoma growth

Interferon Response Factor 3 (IRF3) induces several NK-cell activating factors, is activated by poly-I:C, an experimental cancer therapeutic, but is suppressed during many viral infections. IRF3 Knockout (KO) mice exhibited enhanced B16 melanoma growth, impaired intratumoral NK cell infiltration, but not an impaired poly-I:C therapeutic effect due to direct suppression of B16 growth. IRF3 was responsible for poly-I:C decrease in TIM-3 expression by intratumoral dendritic cells, induction of NK-cell Granzyme B and IFN-γ, and induction of macrophage IL-12, IL-15, IL-6, and IRF3-dependent NK-activating molecule (INAM). Thus, IRF3 is a key factor controlling melanoma growth through NK-cell activities, especially during poly-I:C therapy.

Enhanced therapeutic effect of B cell-depleting anti-CD20 antibodies upon combination within-situdendritic cell vaccination in advanced lymphoma

The present standard of care for B cell non-Hodgkin's lymphoma includes the anti-CD20 monoclonal antibody rituximab. Although combination treatments with chemotherapy and rituximab improved the duration of remissions and overall survival in indolent B cell lymphoma, the disease is essentially incurable. Thus, new therapeutic approaches are needed. One such approach is active immunization. Given that rituximab depletes both malignant and normal B cells, it is expected to impair humoral immune responses in vaccinated patients. Hence, optimal vaccination strategies for rituximab-treated patients require induction of effector T cells, which can be achieved by dendritic cell (DC) vaccines. We have demonstrated in a mouse model that chemotherapy combined with DC vaccines was therapeutically effective. However, efficacy was related to tumour size at the onset of treatment, decreasing in correlation with increasing tumour burdens. We therefore examined whether, in spite of its low efficacy in advanced disease, DC vaccination may synergize with anti-CD20 antibodies to enhance therapy. Lymphoma-bearing mice were treated with cyclophosphamide, anti-CD20 antibodies and an intratumoral DC vaccine. Results clearly demonstrated the enhanced therapeutic effect of this combination treatment. Thus, under conditions of disseminated disease, when either anti-CD20 antibody treatment or vaccination showed insufficient efficacy, their combination resulted in synergism that mediated long-term survival. We demonstrated further that the combination of antibody and vaccine induced T cell-mediated anti-tumour immune responses with long-term memory. Combination treatments including tumour cell-loaded DC vaccines may therefore provide a strategy for enhancing therapy in rituximab-treated patients.

Clinical Trial of the Intratumoral Administration of Labeled DC Combined With Systemic Chemotherapy for Esophageal Cancer

Esophageal cancer is a highly aggressive disease, and improved modalities for its treatment are needed. We performed chemoimmunotherapy involving the intratumoral administration of 111In-labeled dendritic cells (DC) in combination with preoperative chemotherapy in 5 esophageal cancer patients. Mature DC were generated and traced by scintigraphy after their administration. No adverse events that were directly related to the intratumoral DC administration were observed. Delayed-type hypersensitivity skin tests against keyhole limpet hemocyanin, which was added to the culture medium, detected a positive response in 3 patients, and keyhole limpet hemocyanin antibody production was observed in 4 patients, suggesting that intratumorally administered DC migrate to the lymph nodes, where they function as antigen-presenting cells. However, scintigraphic images obtained after the DC administration demonstrated that the DC remained at the esophageal tumor injection sites in all cases, and no DC accumulation was observed elsewhere. The accumulation of CD83+ cells in the primary tumor was also observed in 2 out of 4 patients in an immunohistochemical analysis using surgically resected specimens. Although the induction of tumor-specific immune responses during chemoimmunotherapy was also analyzed in enzyme-linked immunosorbent assay against 28 tumor antigens, none of the antibodies against the antigens displayed enhanced titers. No changes of NY-ESO-1-specific cellular immune response was observed in a patient who displayed NY-ESO-1 antibody production before the DC administration. These results suggest that the intratumoral administration of 111In-labeled mature DC during chemotherapy does not lead to detectable DC migration from the primary tumor to the draining lymph nodes, and therefore, might not achieve an optimal clinical response.

Serial Assessment of Lymphocytes and Apoptosis in the Prostate During Coordinated Intraprostatic Dendritic Cell Injection and Radiotherapy

Local radiotherapy plus intratumoral syngeneic dendritic cell injection can mediate apoptosis/cell death and immunological tumor eradication in murine models. A novel method of coordinated intraprostatic, autologous dendritic cell injection together with radiation therapy was prospectively evaluated in five HLA-A2(+) subjects with high-risk, localized prostate cancer, using androgen suppression, 45 Gy external beam radiation therapy in 25 fractions over 5 weeks, dendritic cell injections after fractions 5, 15 and 25 and then interstitial radioactive seed placement. Serial prostate biopsies before and during treatment showed increased apoptotic cells and parenchymal distribution of CD8(+) cells. CD8(+) T-cell responses to test peptides were assessed using an enzyme-linked immunosorbent spot IFN-γ production assay, demonstrating some prostate cancer-specific protein-derived peptides associated with increased titer. In conclusion, the technique was feasible and well-tolerated and specific immune responses were observable. Future trials could further test the utility of this approach and improve on temporal coordination of intratumoral dendritic cell introduction with particular timelines of therapy-induced apoptosis.

Clinical opportunities in combining immunotherapy with radiation therapy.

Preclinical work in murine models suggests that local radiotherapy plus intratumoral syngeneic dendritic cells (DC) injection can mediate immunologic tumor eradication. Radiotherapy affects the immune response to cancer, besides the direct impact on the tumor cells, and other ways to coordinate immune modulation with radiotherapy have been explored. We review here the potential for immune-mediated anticancer activity of radiation on tumors. This can be mediated by differential antigen acquisition and presentation by DC, through changes of lymphocytes’ activation, and changes of tumor susceptibility to immune clearance. Recent work has implemented the combination of external beam radiation therapy (EBRT) with intratumoral injection of DC. This included a pilot study of coordinated intraprostatic, autologous DC injection together with radiation therapy with five HLA-A2(+) subjects with high-risk, localized prostate cancer; the protocol used androgen suppression, EBRT (25 fractions, 45 Gy), DC injections after fractions 5, 15, and 25, and then interstitial radioactive implant. Another was a phase II trial using neo-adjuvant apoptosis-inducing EBRT plus intra-tumoral DC in soft tissue sarcoma, to test if this would increase immune activity toward soft tissue sarcoma associated antigens. In the future, radiation therapy approaches designed to optimize immune stimulation at the level of DC, lymphocytes, tumor and stroma effects could be evaluated specifically in clinical trials.

Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy

Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C′-PSA) and α-fetoprotein (rHSP70C′-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor immune response were measured on CT26/PSA and CT26/AFP mice model. Intratumoral co-injection of rHsp70 and DC into the irradiated tumor site induced a more potent anti-tumor immune response than injection of DC alone. rHsp70 was as effective as rHsp70C′-PSA or rHsp70C′-AFP in inducing a tumor-specific cytotoxic T lymphocyte response or tumor growth delay. These results demonstrate that co-administration with rHsp70 and RT could be a simple and effective source of tumor antigens to achieve RT-DC immunotherapy protocol and easy to apply in clinical use.

Intratumoral plasmacytoid dendritic cells associate with increased survival in patients with follicular lymphoma

Gene array studies on follicular lymphoma (FL) have associated non-malignant tumor-infiltrating immune cells with patient survival. We examined the role of such cells detectable by immunohistochemistry in a tissue microarray, focusing on plasmacytoid dendritic cells (pDCs). These cells physiologically produce interferon-α, which has been used in the therapy of FL. High numbers of pDCs are associated with increased survival, and so are high numbers of CD3+ T cells. The regular distribution of pDCs within T cell areas is reflected by a weak but significant correlation between pDCs and T cells. However, in multivariate Cox models, CD123 proved to be an independent prognostic factor. These findings support the hypothesis of an association of pDCs with better prognosis by producing interferon. Furthermore, due to a linear relationship between pDCs and survival shown by means of Kaplan–Meier plots, immunohistochemical staining of CD123 could serve as a prognostic tool for patients with FL.

Optimization of hyperthermia and dendritic cell immunotherapy for squamous cell carcinoma

The aims of this study were to explore the feasibility of a novel combination therapy comprising hyperthermia (HT) and dendritic cell (DC) application for squamous cell carcinoma (SCC), and to optimize the conditions of this therapy. In vitro, the correlation between maturation of DCs by co-culture with SCCVII cells and HT was investigated. DCs did not mature in simple HT (43 °C for 30 min) with SCCVII cells. On the other hand, DC maturation occurred in additional mild HT (mHT: 41 °C for 30 min) with simple HT. To assess whether additional mHT was effective, in vivo combined treatment was performed using tumor-bearing C3H/HeJ mice. A more suppressive effect of tumor growth was observed, and cytotoxic T cell infiltration was significantly increased by adding mHT compared to conventional only simple HT with DCs. These phenomena also occurred in non-treated contralateral tumors as well as treated ones. Our data suggest that the combination of 43 °C preheated simple HT SCCVII tumors and additional 41 °C heat mHT promotes DC maturation, resulting in suppression of tumor growth systemically and lifetime prolongation in mice. A three-step process of additional mHT after local HT and intratumoral immature DC (iDC) injection could be a more effective and novel method for the treatment of SCC.

Combination of External Beam Radiotherapy (EBRT) With Intratumoral Injection of Dendritic Cells as Neo-Adjuvant Treatment of High-Risk Soft Tissue Sarcoma Patients

The goal of this study was to determine the effect of combination of intratumoral administration of dendritic cells (DC) and fractionated external beam radiation (EBRT) on tumor-specific immune responses in patients with soft-tissue sarcoma (STS). Seventeen patients with large (>5 cm) high-grade STS were enrolled in the study. They were treated in the neoadjuvant setting with 5,040 cGy of EBRT, split into 28 fractions and delivered 5 days per week, combined with intratumoral injection of 10(7) DCs followed by complete resection. DCs were injected on the second, third, and fourth Friday of the treatment cycle. Clinical evaluation and immunological assessments were performed. The treatment was well tolerated. No patient had tumor-specific immune responses before combined EBRT/DC therapy; 9 patients (52.9%) developed tumor-specific immune responses, which lasted from 11 to 42 weeks. Twelve of 17 patients (70.6%) were progression free after 1 year. Treatment caused a dramatic accumulation of T cells in the tumor. The presence of CD4(+) T cells in the tumor positively correlated with tumor-specific immune responses that developed following combined therapy. Accumulation of myeloid-derived suppressor cells but not regulatory T cells negatively correlated with the development of tumor-specific immune responses. Experiments with (111)In labeled DCs demonstrated that these antigen presenting cells need at least 48 h to start migrating from tumor site. Combination of intratumoral DC administration with EBRT was safe and resulted in induction of antitumor immune responses. This suggests that this therapy is promising and needs further testing in clinical trials design to assess clinical efficacy.

Know when to say when: Serial assessment of apoptosis and lymphocyte infiltrates with combined intraprostatic autologous dendritic cell immunotherapy/radiation therapy in high-risk prostate cancer.

149 Background: Previous work suggested combining local radiotherapy (XRT) with intratumoral syngeneic dendritic cell (DC) injection could result in apoptosis/cell death mediated induction of cytotoxic T lymphocytes (CTL) based anti-tumor immunity. However, the presence, timing, and effectiveness of effector CTL prior, during, and following combined XRT/intratumoral autologous DC injection in humans is unknown. Methods: Herein, we report on timing concerns of intraprostatic DC injection in vivo, in human; five HLA-A2+, high risk localized prostate cancer patients were treated on protocol using androgen suppression therapy (AS), external beam radiation therapy (EBRT, 45 Gy) followed by brachytherapy permanent interstitial implant with addition of autologous intraprostatic DC injections during EBRT, after fractions 5, 15, and 25 of 25. Multiple serial prostate biopsies were collected before initiation of treatment, during EBRT and at 3, 12, 24 and 36 months after completion of treatment. Biopsies were stained for CD3, CD4, CD8, and cleaved caspase 3, and evaluated in a blinded manner. Specific anti-tumor immunity was assessed via ELISpot IFN-gamma production by CTL stimulated by HLA-A2 peptides derived from sequences of proteins associated with prostate cancer. Results: Apheresis, DC injections, and biopsies were well tolerated. The pattern of distribution of CD8+ cells was consistent with prostate cancer antigen targeting, rather than non-specific organ infiltration. There was not immediate obvious intraprostratic infiltrate by CTL after DC injection in vivo, in humans. Measurable, induced increases in ELISpot titers in peripheral blood CTL were observed for some subjects and for some antigens, but non-specific immunity fluctuated. Conclusions: This initial translational experience demonstrates safety and timing of DC injection coordinated with combined AS and XRT. Design of future trials employing combination XRT and DC will consider timing concerns to match therapy-induced apoptosis/cell death. No significant financial relationships to disclose.

Myeloid-derived Suppressor Cell and Regulatory T Cell Immune Monitoring of Soft Tissue Sarcoma Patients Undergoing Combination External Beam Radiation and Intratumoral Dendritic Cell Vaccination

Myeloid-derived Suppressor Cell and Regulatory T Cell Immune Monitoring of Soft Tissue Sarcoma Patients Undergoing Combination External Beam Radiation and Intratumoral Dendritic Cell Vaccination