Dendritic Cell-based Immunotherapy Research Articles

Preparation and Formulation of a New Therapeutic Supplement by the Combination of Dendritic Cells and IPI-549 (Eganelisib) for the Treatment of Breast Cancer in BALB/c Mice

: Dendritic cell-based cancer immunotherapy is considered an innovative and promising approach aimed at enhancing the host's immune response to combat tumors. Additionally, IPI-549 has been identified as a first-line therapeutic option for breast cancer treatment. The objective of this research was to develop and formulate a novel therapeutic supplement by combining dendritic cells with IPI-549 (Eganelisib) for breast cancer treatment. The concurrent administration of dendritic cells and IPI-549 (DC-IPI) was utilized to treat mice and elicit immunological responses triggered by vaccination. Tumor regression and overall survival rates were evaluated across five distinct experimental groups. The administration of tumor cell lysate alongside DC-IPI resulted in a significant reduction in tumor growth and a two- to three-fold increase in the survival duration of treated mice. DC-IPI, whether decorated with mannan or not, elicited stronger responses in terms of delayed-type hypersensitivity, lymphocyte proliferation, and CD107a expression. Moreover, our findings demonstrated a reduction in IL-4 production in the supernatants of splenocyte cultures. A significant reduction in BRCA1 mRNA levels was also observed following treatment with DC-IPI. In conclusion, our results suggest that the DC-IPI antigen delivery system exhibited substantial anti-tumor efficacy in a breast cancer mouse model, representing a potential advancement in immunotherapy for human breast cancer.

Nitroxide radical conjugated ovalbumin theranostic nanosystem for enhanced dendritic cell-based immunotherapy and T1 magnetic resonance imaging

Melanoma, known for its aggressive metastatic nature, presents a formidable challenge in cancer treatment, where conventional therapies often fall short. This study introduces a pioneering approach utilizing metal-free nanosystem as tumor vaccines, spotlighting their potential in revolutionizing melanoma treatment. This work employed organic nitroxides, specifically 4-carboxy-TEMPO, in combination with chitosan (CS), to create a novel nanocomposite material - the CS-TEMPO-OVA nanovaccines. This composition not only improves biocompatibility and extends blood circulation time of TEMPO but also marks a significant departure from traditional gadolinium-based contrast agents in MRI technology, addressing safety concerns. CS-TEMPO-OVA nanovaccines demonstrate excellent biocompatibility at both the cellular and organoid level. They effectively stimulate bone marrow-derived dendritic cells (BMDCs), which in turn promote the maturation and activation of T cells. This ultimately leads to a strong production of essential cytokines. These nanovaccines serve a dual purpose as both therapeutic and preventive. By inducing an immune response, activating cytotoxic T cells, and promoting macrophage M1 polarization, they effectively inhibit melanoma growth and enhance survival in mouse models. When combined with αPD-1, the CS-TEMPO-OVA nanovaccines significantly bolster the infiltration of cytotoxic T lymphocytes (CTLs) within tumors, sparking a powerful systemic antitumor response that effectively curbs tumor metastasis. The ability of these nanovaccines to control both primary (subcutaneous) and metastatic B16-OVA tumors highlights their remarkable efficacy. Furthermore, the CS-TEMPO-OVA nanovaccine can be administered in vivo via both intravenous and intramuscular routes, both of which effectively enhance the T1 contrast of magnetic resonance imaging in tumor tissue. This study offers invaluable insights into the integrated application of these nanovaccines in both clinical diagnostics and treatment, marking a significant stride in cancer research and patient care.

Dendritic Cell-Based Immunotherapy in Patients With Resected Pancreatic Cancer.

Immunotherapies have shown limited responses in patients with advanced pancreatic cancer. Recently, we reported that dendritic cell (DC)-based immunotherapy induced T-cell responses against pancreatic cancer antigens. The primary objective of this study was to determine the efficacy of DC-based immunotherapy to prevent recurrence of disease. This was a single-center, open-label, single-arm, combined phase I/II trial. The primary end point was the 2-year recurrence-free survival (RFS) rate. A 2-year RFS rate of ≥60% was defined as a clinically meaningful improvement. We included patients with pancreatic cancer after resection and completion of standard-of-care (SOC) treatment without recurrent disease on cross-sectional imaging. Patients were treated with autologous DCs pulsed with an allogeneic mesothelioma tumor cell lysate, comprising antigens also expressed in pancreatic ductal adenocarcinoma. Thirty-eight patients were included in the analysis of the primary end point (47% male, 53% female). The median age was 62 years (IQR, 55-68). Twenty-eight patients (74%) received five DC vaccinations and completed the study protocol. Three patients (8%) received four vaccinations, and seven patients (16%) received three vaccinations. After a median follow-up of 25.5 months, 26 patients (68%) had not developed recurrence of disease. The estimated 2-year RFS was 64%. Vaccination led to the enrichment of circulating activated CD4+ T cells and the detection of treatment-induced immune responses in vitro. T-cell receptor-sequencing analyses of a resected solitary lung metastasis showed influx of vaccine-specific T cells. This study reached its primary end point of a 2-year RFS rate of ≥60% following pancreatectomy after SOC treatment and adjuvant DC-based immunotherapy in patients with pancreatic cancer. These results warrant a future randomized trial.

Activation of dendritic cells by a synthesized small molecule for breast cancer vaccination.

e13149 Background: Dendritic cell-based vaccination is a promising immunotherapy for treating various types of cancer. However, tumor-loaded dendritic cells alone have limited treatment efficacy. We have designed a small molecule mimicking the action of damage-associated molecular pattern molecules as an adjuvant for activating dendritic cells in vitro, which demonstrated augmented treatment efficacy. Methods: We aimed at exploring the roles of the synthesized small molecule in augmenting the efficacy of dendritic cell-based cancer vaccination to target triple negative breast cancer. Breast cancer mouse model is constructed using a 4T1 tumor cell line. Data were analyzed using a two-tailed unpaired t-test. Results: Mouse dendritic cells could be activated in vitro by the small molecule as an adjuvant evidenced by increased expression of MHC-I, MHC-II and CD40. Augmented motility of dendritic cells was also observed after treatment with the adjuvant. The signaling of NFkB is remarkably activated in dendritic cells by the adjuvant, which facilitates the maturation of dendritic cells. Engraftment of adjuvant-primed dendritic cells loaded with 4T1 tumor antigen into 4T1 tumor-bearing mice efficiently reduced the tumor size and improved survival, compared with the group treated with dendritic cells only. After injection of adjuvant-activated dendritic cells, both CD4 T cell and CD8 T cell populations were expanded in the spleen and lymph nodes draining the tumor sites. Furthermore, the adjuvant could promote the production of IFN-γ and IL-2, cytokines required for T cell activation and expansion. Next, splenocytes were obtained from individual mice after dendritic cell-based treatment. Upon re-encounter with the tumor antigen in vitro, remarkable expansion of T cell populations with a memory cell feature was confirmed in splenocytes derived from mice that received adjuvant-treated dendritic cells. Consistently, production of TNF-α, IFN-γ and granzyme B, was profoundly increased in these T cells associated with the adjuvant treatment. Conclusions: Our work suggests a novel dendritic cell-based cancer immunotherapy by using a small molecule mimicking the action of damage-associated molecular pattern molecules, which have documented immune potentiation activities.

The Improved Antigen Uptake and Presentation of Dendritic Cells Using Cell-Penetrating D-octaarginine-Linked PNVA-co-AA as a Novel Dendritic Cell-Based Vaccine.

Cancer immunotherapy using antigen-pulsed dendritic cells can induce strong cellular immune responses by priming cytotoxic T lymphocytes. In this study, we pulsed tumor cell lysates with VP-R8, a cell-penetrating D-octaarginine-linked co-polymer of N-vinylacetamide and acrylic acid (PNVA-co-AA), into the DC2.4 murine dendritic cell line to improve antigen uptake and then determined the anti-tumor effect in tumor-bearing mice. DC2.4 cells were pulsed with the cell lysate of EL4, a murine lymphoma cell line, and VP-R8 to generate the DC2.4 vaccine. For the in vivo study, DC2.4 cells pulsed with EL4 lysate and VP-R8 were subcutaneously injected into the inguinal lymph node to investigate the anti-tumor effect against EL4 and EL4-specific T cell immune responses. VP-R8 significantly improved antigen uptake into DC2.4 compared to conventional keyhole limpet hemocyanin (p < 0.05). The expression of MHC class I, MHC class II, and CD86 in DC2.4 cells significantly increased after pulsing tumor lysates with VP-R8 compared to other treatments (p < 0.05). The intra-lymph node injection of DC2.4 pulsed with both VP-R8 and EL4 lysate significantly decreased tumor growth compared to DC2.4 pulsed with KLH and lysates (p < 0.05) and induced tumor-infiltrating CD8T cells. The DC2.4 vaccine also remarkably increased the population of IFN-gamma-producing T cells and CTL activity against EL4 cells. In conclusion, we demonstrated that VP-R8 markedly enhances the efficiency of dendritic cell-based vaccines in priming robust anti-tumor immunity, suggesting its potential as a beneficial additive for dendritic cell-based immunotherapy.

Ex Vivo-Generated Tolerogenic Dendritic Cells: Hope for a Definitive Therapy of Autoimmune Diseases.

Current therapies for autoimmune diseases are immunosuppressant agents, which have many debilitating side effects. However, dendritic cells (DCs) can induce antigen-specific tolerance. Tolerance restoration mediated by ex vivo-generated DCs can be a therapeutic approach. Therefore, in this review, we summarize the conceptual framework for developing ex vivo-generated DC strategies for autoimmune diseases. First, we will discuss the role of DCs in developing immune tolerance as a foundation for developing dendritic cell-based immunotherapy for autoimmune diseases. Then, we also discuss relevant findings from pre-clinical and clinical studies of ex vivo-generated DCs for therapy of autoimmune diseases. Finally, we discuss problems and challenges in dendritic cell therapy in autoimmune diseases. Throughout the article, we discuss autoimmune diseases, emphasizing SLE.

Combinational Pulsing of TAAs Enforces Dendritic Cell-Based Immunotherapy through T-Cell Proliferation and Interferon-γ Secretion in LLC1 Mouse Model.

NSCLC, the most common type of lung cancer, is often diagnosed late due to minimal early symptoms. Its high risk of recurrence or metastasis post-chemotherapy makes DC-based immunotherapy a promising strategy, offering targeted cancer destruction, low side effects, memory formation, and overcoming the immune evasive ability of cancers. However, the limited response to DCs pulsed with single antigens remains a significant challenge. To overcome this, we enhanced DC antigen presentation by pulsing with TAAs. Our study focused on enhancing DC-mediated immune response specificity and intensity by combinatorial pulsing of TAAs, selected for their prevalence in NSCLC. We selected four types of TAAs expressed in NSCLC and pulsed DCs with the optimal combination. Next, we administered TAAs-pulsed DCs into the LLC1 mouse model to evaluate their anti-tumor efficacy. Our results showed that TAAs-pulsed DCs significantly reduced tumor size and promoted apoptosis in tumor tissue. Moreover, TAAs-pulsed DCs significantly increased total T cells in the spleen compared to the unpulsed DCs. Additionally, in vitro stimulation of splenocytes from the TAAs-pulsed DCs showed notable T-cell proliferation and increased IFN-γ secretion. Our findings demonstrate the potential of multiple TAA pulsing to enhance the antigen-presenting capacity of DCs, thereby strengthening the immune response against tumors.

Dendritic cell-based immunotherapy in non-small cell lung cancer: a comprehensive critical review.

Despite treatment advances through immunotherapies, including anti-PD-1/PD-L1 therapies, the overall prognosis of non-small cell lung cancer (NSCLC) patients remains poor, underscoring the need for novel approaches that offer long-term clinical benefit. This review examined the literature on the subject over the past 20 years to provide an update on the evolving landscape of dendritic cell-based immunotherapy to treat NSCLC, highlighting the crucial role of dendritic cells (DCs) in immune response initiation and regulation. These cells encompass heterogeneous subsets like cDC1s, cDC2s, and pDCs, capable of shaping antigen presentation and influencing T cell activation through the balance between the Th1, Th2, and Th17 profiles and the activation of regulatory T lymphocytes (Treg). The intricate interaction between DC subsets and the high density of intratumoral mature DCs shapes tumor-specific immune responses and impacts therapeutic outcomes. DC-based immunotherapy shows promise in overcoming immune resistance in NSCLC treatment. This article review provides an update on key clinical trial results, forming the basis for future studies to characterize the role of different types of DCs in situ and in combination with different therapies, including DC vaccines.

Dendritic cell-based immunotherapy in pancreatic cancer

Dendritic cell-based immunotherapy in pancreatic cancer

NKT-Licensed In Vivo Dendritic Cell-Based Immunotherapy as Cellular Immunodrugs for Cancer Treatment.

With the advent of new therapies, immunotherapy has gained attention as a critical modality. After the discovery of the natural killer T (NKT) cells ligand, ex vivo cultured dendritic cells (DCs) loaded with NKT ligand (especially α-galactosylceramide (α-GalCer) (DC/Gal) or ex vivo expanded NKT transfer studies were clinically examined in several institutes. To prevent tumoral immune escape, the link between innate and adaptive immunity, in situ selective targeting of DCs has been attempted; however, protocol optimization was required. As a type of DC targeting therapy that combines the benefits of invariant natural killer T (iNKT) cells, we established an all-in-one, off-the-shelf drug, named the artificial adjuvant vector cell (aAVC), which consists of the tumor antigen and the CD1d-iNKT ligand complex. Here, to our knowledge, we first demonstrate the DC/GalCer therapy and NKT transfer therapy. Next, we introduce and discuss the use of aAVC therapy not only for efficient innate and adaptive immunity induction using fully matured DC in situ but also the characterization necessary for locally reprogramming the tumor microenvironment and systemically inducing long-term memory in T cells. We also discuss how the immune network mechanism is controlled by DCs. Next, we performed the first human clinical trial using WT1 antigen-expressing aAVC against relapse and refractory acute myelogenous leukemia. Thus, we highlight the challenges of using aAVCs as prodrugs for actively energizing DCs in vivo, underpinning immunological networks, and developing strategies for providing maximal benefits for patients.

Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing

Chronic wounds impose a significant healthcare burden to a broad patient population. Cell-based therapies, while having shown benefits for the treatment of chronic wounds, have not yet achieved widespread adoption into clinical practice. We developed a CRISPR/Cas9 approach to precisely edit murine dendritic cells to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing of tolerogenic dendritic cells, we identified N-myc downregulated gene 2 (Ndrg2), which marks a specific population of dendritic cell progenitors, as a promising target for CRISPR knockout. Ndrg2-knockout alters the transcriptomic profile of dendritic cells and preserves an immature cell state with a strong pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a therapeutic hydrogel for in vivo cell delivery and developed an effective translational approach for dendritic cell-based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in dendritic cells for treating various types of chronic wounds.

Adjuvant dendritic cell-based immunotherapy after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in patients with malignant peritoneal mesothelioma: a phase II clinical trial

BackgroundMalignant peritoneal mesothelioma (MPM) is an aggressive malignancy with a poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) improves survival outcomes, but recurrence rates remain high. Dendritic cell-based...

Oncodrivers for Immunotherapies in Triple-Negative Breast Cancer

Breast Cancer: Breast CancerMany advances have been made in therapies for patients having breast cancer driven by the estrogen receptor, the progesterone receptor, or the human epidermal growth factor 2 receptor (HER2). However, for patients with breast cancer not driven by any of these mechanisms, the disease, termed triple-negative, is characterized by an aggressive clinical course with poor patient outcomes. In a recent poster presented at the AACR Annual Meeting 2023, Saurabh K. Garg, PhD, a research scientist in the Czerniecki Lab at Moffitt Cancer Center, presented data from a retrospective study of triple-negative breast cancer patients who had undergone surgery at the Moffitt Cancer Center between January 2010 and February 2019 (Abstract 962). “Our team at the Czerniecki Lab and Moffitt Breast Clinic utilizes the power of dendritic cells, the primary antigen-presenting cells of the body, to present major histocompatibility complex Class II antigens from tumor-associated oncodrivers to prime the immune response against breast cancer,” Garg noted. “In our clinical trials, we are seeing a robust targeted response with dendritic cell-based therapies in HER2-positive breast cancer. This prompted us to identify immunogenic oncodrivers to develop targeted immunotherapies for triple-negative breast cancer, which accounts for 15-20 percent of breast cancer cases and presents as a more aggressive disease, with poor outcomes from current standard-of-care treatment options.” Regarding their study, Garg noted, “This is an institutional review board-approved, single institution retrospective study of 90 patients with triple-negative breast cancer who have undergone standard of care at Moffitt Cancer Center between January 2010 and February 2019. “Surgical biopsy tissue blocks of various stages, along with patient outcome data, were available to perform this downstream analysis,” he stated. “We utilized multiplex tissue microarray analyses, where multiple tissue core biopsies are arranged on a single slide for this immunofluorescence assay,” Garg explained. The tissue microarray analyses were divided into two panels: one for the oncodrivers and the other for tumor-infiltrating lymphocytes. In the first panel, “the expression of three oncodrivers: MET (MET proto-oncogene, receptor tyrosine kinase), EGFR (epidermal growth factor receptor), and HER3 (erb-b2 receptor tyrosine kinase 3) on surgical tissue blocks was scored by tumor microarray analysis,” Garg explained. “We also performed a second tissue microarray analysis to examine tumor-infiltrating lymphocytes expressing CD4, CD8, CD20, CD45, and CD56,” Garg noted about the second panel. “Subsequently, we correlated the expression of oncodrivers with patient outcome data and lymphocyte infiltration to catalog the immunogenicity of MET, EGFR, and HER-3 expression in triple-negative breast cancer settings.” Research Results When queried as to their findings, Dr. Garg answered, “The main finding of this study is that high expression of MET, EGFR, and HER3 correlated with higher occurrence of CD4, CD8, and CD45, respectively. This provided us invaluable information, indicating that these tumor-driving proteins have immunogenic potential to recruit lymphocytes to the tumor site, which could be harnessed to design targeted immunotherapies.” In addition, the elevated expression of HER3 was observed in the samples obtained from chemotherapy-treated patients, possibly implying a role in disease resistance. Interestingly, there was no correlation of prognoses with increased expression levels of the oncodrivers explored in this study. Another relevant observation was that better survival was observed in patients who had intermediate expression levels for EGFR with higher numbers of activated tumor-infiltrating lymphocytes. Future Directions “The present study provided a perfect segue for future targeted therapies that will be further developed and utilized in the clinic for triple-negative breast cancer patients,” Garg noted. “The identification of these immunogenic oncodrivers for triple-negative breast cancer will assist researchers at Moffitt and all over the world to target them with innovative therapies. “The next step in this study will be to identify the immunogenic regions of MET and EGFR proteins using our in-house peptide screening pipeline,” he stated. “This pipeline utilizes pre-clinical investigative tools to screen immunogenic major histocompatibility complex class II peptides to design dendritic cell-based cancer immunotherapies. Once identified and produced, we will test the efficacy of these treatments utilizing murine models of triple-negative breast cancer. “Furthermore, we have already performed peptide screening for the HER3 protein and have validated it in murine models of triple-negative breast cancer,” Garg said. “This HER3-based dendritic cell immunotherapy has been translated to a clinical trial, which is now underway at Moffitt for triple-negative breast cancer patients. Overall, successful completion of the pre-clinical translational research on all three triple-negative breast cancer oncodrivers will open the door for them to be incorporated in future dendritic cell immunotherapy clinical trials at the Moffitt Cancer Center with the potential of making significant improvements to the current triple-negative breast cancer standard of care and patient outcomes.” Richard Simoneaux is a contributing writer.

Dendritic cell-based immunotherapy: a potential player in oral cancer therapeutics.

Background & aim: Dendritic cell (DC)-based immunotherapy modulates a patient's immune system for recognition and subsequent removal of tumor cells. DC-mediated anticancer therapy has been considered in several studies/ongoing trials for multiple types of cancer. Our aim is to describe the potential and current status of DC-based immunotherapy in oral cancer therapeutics. Materials & methods: An internet-based literature search with relevant search terms from 2012 to 2022 post-screening resulted in 58articles that were considered for systematic review. Results & conclusion: Evaluation of DC based immunotherapyexploiting the critical immune cellsin well-equipped laboratories with adequately trained and skilled experts alongwitha synergistic approach that is affordable and approachable to the patients can prove asan efficientanticancer therapy.

LW-1 MY LIFE WORK : MY RESEARCH WORK FOCUSING ON TUMOR-ASSOCIATED ENDOTHELIAL CELLS ALONG WITH VEGF FOR 30 YEARS

Abstract When I was a medical student, I happened to see the typical histopathology of glioblatoma (GBM) containing expanding necrosis surrounded by enormous glomerulous microvasculature with endothelial proliferation resembling glomeruli in the kidney. It looks like tumor cells starving out due to lack of blood supply with accelerating growth. Since then, I have been fascinated by tumor angiogenesis that might be an attractive therapeutic target.In 1989, vascular endothelial growth factor (VEGF) and vascular permeability factor (VPF) turned out to be identical. In 1992, VEGF expression was distributed surrounding necrosis area in GBM. In 1993, anti-tumor effect of monoclonal antibody for VEGF via inhibition of angiogenesis on various tumor models including glioma was verified in vivo. In 1996, VEGF was found to inhibit the functional maturation of dendritic cells, suggesting that therapeutic blockade of VEGF may improve prospect for immunotherapy as well as inhibition of angiogenesis. Those findings made me excited with visionary idea that combination therapy of anti-VEGF therapy and dendritic cell-based immunotherapy might be a potential therapeutic approach for GBM. Currently, I am dedicated in clinical study of dendritic cell-based immunotherapy and bevacizumab for GBM.Since graduating from medical school and discovery of VEGF in the same era, my clinic and research have been supported by serendipity of a vast of experiences and many people. I would give take-home message to next generation that originality and creativity were cultivated in real-world, and that serendipity and integration were precious for development of basic and clinical research.

MRI Response Assessment in Patients with Glioblastoma Treated with Dendritic Cell-Based Immunotherapy

MRI Response Assessment in Patients with Glioblastoma Treated with Dendritic Cell-Based Immunotherapy

Dendritic Cell-Based Immunotherapies and their Potential use in Colorectal Cancer Immunotherapy.

Dendritic cells (DCs) are professional antigen-presenting cells, which are resident or proliferating in organs. Major histocompatibility complex (MHC) Class I and II on DCs in normal steady conditions process and present antigens including cancer antigens. Many approaches are used to enhance antigen presentation process of DCs and capture cancer cells. DCs are harvested from cancer patients and manipulated ex vivo in DC-based cancer immunotherapy. In addition, DCs' vaccines and other anticancer therapy combinations were discussed to optimize DCs' efficiency for cancer immunotherapy. This review addressed the use of the human conventional type-1 DCs, OX40+ plasmacytoid DCs, and DCs-derived exosomes. In addition, different combinations with DCs therapy such as combination with the monoclonal antibody, cytokine-induced killer cells, adjuvants, chemotherapy (DCs-based chemoimmunotherapy), and nanoparticles were listed and explored for their effectiveness against cancer, and mainly against colorectal cancer.

Dendritic Cell-Based Immunotherapy in Hot and Cold Tumors.

Dendritic cells mediate innate and adaptive immune responses and are directly involved in the activation of cytotoxic T lymphocytes that kill tumor cells. Dendritic cell-based cancer immunotherapy has clinical benefits. Dendritic cell subsets are diverse, and tumors can be hot or cold, depending on their immunogenicity; this heterogeneity affects the success of dendritic cell-based immunotherapy. Here, we review the ontogeny of dendritic cells and dendritic cell subsets. We also review the characteristics of hot and cold tumors and briefly introduce therapeutic trials related to hot and cold tumors. Lastly, we discuss dendritic cell-based cancer immunotherapy in hot and cold tumors.

The grace of aging with ART: Ex vivo dendritic cell-based immunotherapy restores HIV-specific functional CD8+ T-cells.

The Nobel Prize in Physiology or Medicine 1953 was shared between Sir Hans Adolf Krebs, a German-British biochemist, and Fritz Albert Lipmann, a German-American biochemist for their pioneer work on metabolism.1 The former earned it for his discovery of tricarboxylic (TCA) or Krebs cycle, and the later earned it for his discovery of coenzyme A that completed the gap in the initial Krebs cycle. TCA cycle being a major source of energy for cells plays an important role in aerobic respiration in the mitochondria.

Rapid Isolation of Gastric Adenocarcinoma Cancer Stem Cells as a Target for Autologous Dendritic Cell-Based Immunotherapy.

Gastric cancer (GC) is a malignancy cause associated with a high death rate in the world. Cancer stem cells (CSCs) are a rare immortal subpopulation of cells within tumors with characteristics of the ability to self-renew, initiate tumor, and differentiate into defined progenies as well as and high resistance to conventional therapies. Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Therefore, rapid isolation of CSCs in order to therapeutic targets, especially immunotherapy is very important. Cancerous cell suspension isolated from patients with GC was cultured in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions to generate spheres. Expression of mRNA level stemness transcription factors (OCT4, SOX2, SALL4, and Cripto-1), CD44 variable isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) of spheroid-forming single cells compared with gastric normal tissue cells using real time PCR and molecules of CD44, CD54, and EpCAM as gastric CSC markers, and stemness factor Oct4 using flow cytometry, as well as tumorgenicity using subcutaneous injection of sphere-forming cells to nude mice were investigated. Few cancerous cells isolated from patients with GC were able to generate three-dimensional spheroid colonies in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions, and form xenograft tumors in immunodeficient nude mice after subcutaneous injection. Spheroid-forming single cells upregulated stemness transcription factors OCT4, SOX2, SALL4, and Cripto-1 that are associated with pluripotency and self-renewal and CD44 isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) compared with gastric normal tissue cells. Finally, molecules of CD44, CD54, and EpCAM as gastric CSC markers and stemness factor Oct4 were expressed in sphere-forming cells. We suggested that the sphere formation and tumorigenicity assays are two procedures, leading to the rapid isolation of cancer cells with certain stem-like properties in order to target CSCs using autologous dendritic cell therapy, especially in patients with advanced disease.