Development of Allogeneic Stem Cell-Based Platform for Delivery and Potentiation of Oncolytic Virotherapy.

We describe the repurposing and optimization of the TK-positive (thymidine kinase) vaccinia virus strain ACAM1000/ACAM2000™ as an oncolytic virus. This virus strain has been widely used as a smallpox vaccine and was also used safely in our recent clinical trial in patients with advanced solid tumors and Acute Myeloid Leukemia (AML). The vaccinia virus was amplified in CV1 cells and named CAL1. CAL1 induced remarkable oncolysis in various human and mouse cancer cells and preferentially amplified in cancer cells, supporting the use of this strain as an oncolytic virus. However, the therapeutic potential of CAL1, as demonstrated with other oncolytic viruses, is severely restricted by the patients' immune system. Thus, to develop a clinically relevant oncolytic virotherapy agent, we generated a new off-the-shelf therapeutic called Supernova1 (SNV1) by loading CAL1 virus into allogeneic adipose-derived mesenchymal stem cells (AD-MSC). Culturing the CAL1-infected stem cells allows the expression of virally encoded proteins and viral amplification prior to cryopreservation. We found that the CAL1 virus loaded into AD-MSC was resistant to humoral inactivation. Importantly, the virus-loaded stem cells (SNV1) released larger number of infectious viral particles and virally encoded proteins, leading to augmented therapeutic efficacy in vitro and in animal tumor models.

Novel Survivin Peptides Screened With Computer Algorithm Induce Cytotoxic T Lymphocytes With Higher Cytotoxic Efficiency to Cancer Cells.

The identification of novel biomarkers and therapeutic targets in advanced cancer is critical for improving cancer diagnosis and therapeutics. Survivin (SV) is highly expressed predominantly in most cancer cells and tissues but is absent or undetectable in terminally differentiated normal adult tissues. Therefore, it functions as an almost universal tumor antigen. Peptides are short chains of amino acids linked by peptide bonds. To obtain novel SV decamers that are able to induce SV-specific cytotoxic T lymphocytes (CTLs) with a higher cytotoxic efficiency against cancer cells, major histocompatibility complex (MHC) peptide binding algorithms were conducted to predict nine modified SV95 decamers (from SV95-2 to SV95-10) based on the natural SV95-104 peptide sequence of ELTLGEFLKL (here defined as SV95-1). The fluorescent density of each SV95 peptide was determined by a MHC stability assay, followed by the generation of SV95-specific CTLs with each SV95 peptide (from SV95-1 to SV95-10) and human dendritic cells (DCs) loaded with Poly(lactic-co-glycolic) acid (PLGA) nanoparticles encapsulated with SV95 peptide. Finally, IFN-γ ELISpot and CytoTox 96® Non-Radioactive Cytotoxicity Assays were employed to verify their cytotoxic efficiency of the SV95-specific CTLs generated with the corresponding artificial antigen presenting cells (aAPCs) containing SV95 (SV95-1 to SV95-10) peptide. Furthermore, the cytotoxicity of the SV95 specific CTLs generated with nine mutated SV95 peptides was compared to the one generated with natural SV95-1 peptide and TIL2080 cells. The results indicated that the HLA-A2-restricted mutated SV95 epitope decamers (SV95-6 and SV95-7) showed significant higher binding ability compared to natural peptide SV95-1 in MHC stability assay. More importantly, SV95-specific CTLs with higher cytotoxicity were successfully induced with both SV95-6 and SV95-7 peptides, which significantly eliminated target cells (not only SV95-1 peptide pulsed T2 cells, but also both HLA-A2 and SV positive cancer cells) when compared to those generated with natural SV95-1 peptide and TIL2080 cells. These findings suggest that the SV95-6 and SV95-7 peptides are two novel HLA-A2-restricted CTL epitopes and may be useful for the immunotherapy for patients with survivin expressing cancer.

Rationale for the clinical use of adipose-derived mesenchymal stem cells for COVID-19 patients.

In late 2019, a novel coronavirus (SARS-CoV-2) emerged in Wuhan, capital city of Hubei province in China. Cases of SARS-CoV-2 infection quickly grew by several thousand per day. Less than 100 days later, the World Health Organization declared that the rapidly spreading viral outbreak had become a global pandemic. Coronavirus disease 2019 (COVID-19) is typically associated with fever and respiratory symptoms. It often progresses to severe respiratory distress and multi-organ failure which carry a high mortality rate. Older patients or those with medical comorbidities are at greater risk for severe disease. Inflammation, pulmonary edema and an over-reactive immune response can lead to hypoxia, respiratory distress and lung damage. Mesenchymal stromal/stem cells (MSCs) possess potent and broad-ranging immunomodulatory activities. Multiple in vivo studies in animal models and ex vivo human lung models have demonstrated the MSC's impressive capacity to inhibit lung damage, reduce inflammation, dampen immune responses and aid with alveolar fluid clearance. Additionally, MSCs produce molecules that are antimicrobial and reduce pain. Upon administration by the intravenous route, the cells travel directly to the lungs where the majority are sequestered, a great benefit for the treatment of pulmonary disease. The in vivo safety of local and intravenous administration of MSCs has been demonstrated in multiple human clinical trials, including studies of acute respiratory distress syndrome (ARDS). Recently, the application of MSCs in the context of ongoing COVID-19 disease and other viral respiratory illnesses has demonstrated reduced patient mortality and, in some cases, improved long-term pulmonary function. Adipose-derived stem cells (ASC), an abundant type of MSC, are proposed as a therapeutic option for the treatment of COVID-19 in order to reduce morbidity and mortality. Additionally, when proven to be safe and effective, ASC treatments may reduce the demand on critical hospital resources. The ongoing COVID-19 outbreak has resulted in significant healthcare and socioeconomic burdens across the globe. There is a desperate need for safe and effective treatments. Cellular based therapies hold great promise for the treatment of COVID-19. This literature summary reviews the scientific rationale and need for clinical studies of adipose-derived stem cells and other types of mesenchymal stem cells in the treatment of patients who suffer with COVID-19.

First-in-human study of TK-positive oncolytic vaccinia virus delivered by adipose stromal vascular fraction cells.

BACKGROUND: ACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML). METHODS: Twenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment. RESULTS: No serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days-an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients' blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition. CONCLUSIONS: Treatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility. Trial registration Retrospectively registered (ISRCTN#10201650) on October 22, 2018.

Tumor growth inhibition by mSTEAP peptide nanovaccine inducing augmented CD8+ T cell immune responses.

Poly(lactic-co-glycolic) acid (PLGA) has been successfully used in drug delivery and biomaterial applications, but very little attention has been directed towards the potential in vivo effects of peptide-loaded PLGA nanoparticles (NPs), specifically the potency of intravenous (IV) STEAP peptide-loaded PLGA-NP (nanovaccine) dosing and whether STEAP-specific CD8+ T cells directly play a key role in tumor inhibition. To address these concerns, syngeneic prostate cancer mouse models were established and treated with either mSTEAP peptide emulsified in incomplete Freund's adjuvant (IFA) via subcutaneous (SC) injection or mSTEAP peptide nanovaccine containing the same amount of peptide via IV or SC injection. Meanwhile, mice were treated with either CD8b mAb followed by nanovaccine treatment, free mSTEAP peptide, or empty PLGA-NPs. Immune responses in these mice were examined using cytotoxicity assays at 14 days after treatment. Tumor size and survival in various treatment groups were measured and monitored. The results demonstrated that mSTEAP peptide nanovaccine resulted in tumor inhibition by eliciting a significantly stronger CD8+ T cell immune response when compared with the controls. Moreover, the survival periods of mice treated with mSTEAP nanovaccine were significantly longer than those of mice treated with mSTEAP peptide emulsified in IFA or the treatment controls. Additionally, it was observed that the peptide nanovaccine was mainly distributed in the mouse liver and lungs after IV injection. These findings suggest that the peptide nanovaccine is a promising immunotherapeutic approach and offers a new opportunity for prostate cancer therapies.

Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers.

BACKGROUND: Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties. METHODS: To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations. RESULTS: Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor. CONCLUSIONS: Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.

The ratio of ADSCs to HSC-progenitors in adipose tissue derived SVF may provide the key to predict the outcome of stem-cell therapy.

BACKGROUND: Stromal vascular fraction (SVF) represents an attractive source of adult stem cells and progenitors, holding great promise for numerous cell therapy approaches. In 2017, it was reported that 1524 patients received autologous SVF following the enzymatic digestion of liposuction fat. The treatment was safe and effective and patients showed significant clinical improvement. In a collaborative study, we analyzed SVF obtained from 58 patients having degenerative, inflammatory, autoimmune diseases, and advanced stage cancer. RESULTS: Flow analysis showed that freshly isolated SVF was very heterogeneous and harbored four major subsets specific to adipose tissue; CD34high CD45- CD31- CD146- adipose-derived stromal/stem cells (ADSCs), CD34low CD45+ CD206+CD31- CD146- hematopoietic stem cell-progenitors (HSC-progenitors), CD34high CD45- CD31+CD146+ adipose tissue-endothelial cells and CD45-CD34-CD31-CD146+ pericytes. Culturing and expanding of SVF revealed a homogenous population lacking hematopoietic lineage markers CD45 and CD34, but were positive for CD90, CD73, CD105, and CD44. Flow cytometry sorting of viable individual subpopulations revealed that ADSCs had the capacity to grow in adherent culture. The identity of the expanded cells as mesenchymal stem cells (MSCs) was further confirmed based on their differentiation into adipogenic and osteogenic lineages. To identify the potential factors, which may determine the beneficial outcome of treatment, we followed 44 patients post-SVF treatment. The gender, age, clinical condition, certain SVF-dose and route of injection, did not play a role on the clinical outcome. Interestingly, SVF yield seemed to be affected by patient's characteristic to various extents. Furthermore, the therapy with adipose-derived and expanded-mesenchymal stem cells (ADE-MSCs) on a limited number of patients, did not suggest increased efficacies compared to SVF treatment. Therefore, we tested the hypothesis that a certain combination, rather than individual subset of cells may play a role in determining the treatment efficacy and found that the combination of ADSCs to HSC-progenitor cells can be correlated with overall treatment efficacy. CONCLUSIONS: We found that a 2:1 ratio of ADSCs to HSC-progenitors seems to be the key for a successful cell therapy. These findings open the way to future rational design of new treatment regimens for individuals by adjusting the cell ratio before the treatment.

Artificial human antigen-presenting cells are superior to dendritic cells at inducing cytotoxic T-cell responses.

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T-cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I-peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T-cell response, we generated artificial antigen-presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen-specific CTLs were then induced using either peptide-loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen-specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA-A2-positive tumour antigen-bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA-NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.

Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis.

Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb's in vitro binding ability and functionality in cell culture, beside the significant increase of CD56bright NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded ß-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our "patient-like" humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system.

IFNγ enhances cytotoxic efficiency of the cytotoxic T lymphocytes against human glioma cells.

Cytotoxic T lymphocytes (CTLs) are a key player in cancer immunotherapies, and MHC class I molecules on the cell surface are crucial for cellular recognition. However, the aberrant expression of MHC class I molecules is frequently found in various malignancies. IFNγ has dual functions in cancer progression, and its effect on tumor immunity is controversial. To investigate whether IFNγ can enhance cytotoxic efficiency of the tumor antigen-specific CTLs, we generated the CTLs using modified human dendritic cells as antigen presenting cells, then studied the activities of CTLs on human leukocyte antigen (HLA)-A2 positive glioma cells treated with, or without IFNγ. The results from both ELISpot and cytotoxicity assays demonstrated that the CTLs recognized and eliminated the HLA-A2 positive glioma cells treated with IFNγ more effectively when compared to the glioma cells deprived of IFNγ treatment. In addition, in vitro experiments showed that the levels of MHC class I molecules were upregulated in all of the HLA-A2 positive glioma cells. Using the publicly accessed TCGA data of low-grade glioma, we found significantly positive associations between IFNγ and both MHC class I molecules and CD8+ T cell activation score (p<0.0001). Furthermore, we found a significantly reduced risk of death in the glioma patients with high T cell activation score in comparison to those with low score (p=0.022). These findings suggest that a clinical application of IFNγ treatment may have potential benefits.

Colonization of xenograft tumors by oncolytic vaccinia virus (VACV) results in enhanced tumor killing due to the involvement of myeloid cells.

BACKGROUND: The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment. METHODS: Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS+ myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student's t test was used for comparison between groups in all of the experiments. RESULTS: Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b+ ly6G+ myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS+ MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7-10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments. CONCLUSIONS: We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.

Overcoming tumor immune evasion with an unique arbovirus.

Combining dendritic cell vaccination with the adjuvant effect of a strain of dengue virus may be a way to overcome known tumor immune evasion mechanisms. Dengue is unique among viruses as primary infections carry lower mortality than the common cold, but secondary infections carry significant risk of hypovolemic shock. While current immuno-therapies rely on a single axis of attack, this approach combines physiological (hyperthermic reduction of tumor perfusion), immunological (activation of effector cells of the adaptive and innate immune system), and apoptosis-inducing pathways (sTRAIL) to destroy tumor cells. The premise of using multiple mechanisms of action in synergy with a decline in the ability of the tumor cells to employ resistance methods suggests the potential of this combination approach in cancer immunotherapy.

Expression of anti-VEGF antibody together with anti-EGFR or anti-FAP enhances tumor regression as a result of vaccinia virotherapy.

The tumor microenvironment plays an important role in tumor growth and progression. Here we demonstrate that vaccinia virus-mediated, constitutively expressed intratumoral antibodies against vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), and fibroblast activation protein (FAP) significantly improved tumor regression and oncolytic virotherapy through suppression of angiogenesis, cell proliferation, and stromagenesis in virus-colonized tumors. In contrast to the tumor growth inhibition by the three tumor growth-inhibiting antibodies individually, when two of the three antibodies were expressed simultaneously by single vaccinia virus strains tumor regression was further enhanced. These findings strongly indicate that interference with the two tumor growth-stimulating mechanisms did in fact result in enhanced therapeutic efficacy in tumor xenograft models and may lead to an effective therapy in patients with cancer.

TLR4-dependent activation of dendritic cells by an HMGB1-derived peptide adjuvant.

High mobility group box protein 1 (HMGB1) acts as an endogenous danger molecule that is released from necrotic cells and activated macrophages. We have previously shown that peptide Hp91, whose sequence corresponds to an area within the B-Box domain of HMGB1, activates dendritic cells (DCs) and acts as an adjuvant in vivo. Here we investigated the underlying mechanisms of Hp91-mediated DC activation. Hp91-induced secretion of IL-6 was dependent on clathrin- and dynamin-driven endocytosis of Hp91 and mediated through a MyD88- and TLR4-dependent pathway involving p38 MAPK and NFκB. Endosomal TLR4 has been shown to activate the MyD88-independent interferon pathway. Hp91-induced activation of pIRF3 and IL-6 secretion was reduced in IFNαßR knockout DCs, suggesting an amplification loop via the IFNαßR. These findings elucidate the mechanisms by which Hp91 acts as immunostimulatory peptide and may serve as a guide for the future development of synthetic Th1-type peptide adjuvants for vaccines.

Intravenous ascorbic acid as an adjuvant to interleukin-2 immunotherapy.

Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA (JTM 9:1-7, 2011). In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy.

Optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains.

Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.

Biotherapeutic approaches to target cancer stem cells.

With the cementing of the cancer stem cell (CSC) concept, cancer biology and cancer drug discovery have attained a new avenue to target cancer. Studying the hierarchy of tumor tissue organization and how to inhibit the cell that resides at the very top of this hierarchy has opened up a new branch of tumor biology and given the opportunity to develop novel cancer-targeting strategies. With the discovery of CSCs in majority of cancer indications there seems to be a universal applicability of the concept. However, the CSC field is still at an early fledgling state and a lot more needs to be done in terms of understanding their emergence, maintenance, role in metastasis and their function in shaping the tumor architecture. CSCs are considered to be responsible for tumor initiation, metastasis and resistance to conventional radio and chemotherapy. Therefore, different approaches to targeting these tumorigenic and rare cells are urgently needed in order to improve the efficacy of anti-cancer therapy. We outline here the cancer stem cell concept and its relevance as well as biotherapeutic approaches to CSC targeting, including oncolytic viruses, monoclonal antibodies, cytokines and cytotoxic T lymphocytes.

Functional role of solid tumor stem cells in disease etiology and susceptibility to therapeutic interventions.

Cancer stem cells (CSCs) are defined by their innate stem cell like properties and can be identified by specific markers that include antigens, molecules and signaling pathways. Like stem cells, CSC divide indefinitely giving rise to both more CSCs and differentiated cell progeny. CSCs can give rise to tumors that phenotypically resemble their origin, either morphologically or by expression of tissue specific genes. Tumors arise from a single cell, the CSC, but the cells that constitute the tumor are not identical to each other. Evidence of heterogeneous populations within a tumor has led to an investigation of the cellular hierarchy of cancers. This review gives an overview of cancer stem cells, from breast, cervical, lung, prostate, head and neck, glioblastoma, pancreatic and colorectal cancers and mechanisms implicated in tumor development and therapeutic interventions.

Characterization of iNOS(+) Neutrophil-like ring cell in tumor-bearing mice.

BACKGROUND: Myeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset. METHODS: Single-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS(+) ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS(+) ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro. RESULTS: The results showed that intracellular iNOS staining distinguished a granular iNOS(+) SSC(hi) CD11b(+) Gr-1(dim) F4/80(+) subset with ring-shaped nuclei (ring cells) among the CD11b(+) Gr-1(+) cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8(+) T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells. CONCLUSIONS: In this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.