Reovirus oncolysis of human breast cancer

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.     

Pinostrobin inhibits proliferation and induces apoptosis in cancer stem-like cells through a reactive oxygen species-dependent mechanism

Current treatments and targeted therapies for malignancies are limited due to their severe toxicity and the development of resistance against such treatments, which leads to relapse. Past evidence has indicated that a number of plant-derived dietary agents possess biological activity against highly tumorigenic and resistant cell populations associated with cancer relapse. These subpopulations, termed cancer stem-like cells (CSCs), have been targeted with plant-derived dietary flavonoids. The present study was undertaken to assess the anti-proliferative potential of pinostrobin, a dietary flavonoid, against CSCs. Sphere-forming cells were developed from HeLa cell lines using specific culture conditions. The existence of a CSC population was confirmed by the morphological examination and analysis of surface markers using confocal microscopy and flow cytometry. The effect of pinostrobin on the cell viability of the CSC population, evaluated through MTT reduction assays and the expression levels of surface markers (CD44⁺ and CD24⁺), was studied through various biological assays. HeLa-derived CSCs showed higher CD44⁺ and lower CD24⁺ expression. Pinostrobin inhibited the self-renewal capacity and sphere formation efficiency of CSCs in a dose-dependent manner. Increased ROS production, and decreased mitochondrial membrane potential and CD44⁺ expression indicated that pinostrobin promoted ROS-mediated apoptosis in CSCs. These results thus demonstrate the therapeutic potential and effectiveness of pinostrobin in the chemoprevention and relapse of cancer by targeting the CSC population. Thus, pinostrobin, in combination with currently available chemo and radiation therapies, could possibly be used as a safe strategy to alleviate adverse treatment effects, together with enhancing the efficacy.

The anti-proliferative potential of pinostrobin, a dietary flavonoid, is evaluated against cancer stem-like cells.     

Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis

Cancer stem cells (CSCs) are a small subpopulation of cancer cells that have increased resistance to conventional therapies and are capable of establishing metastasis. However, only a few biomarkers of CSCs have been identified. Here, we report that ganglioside GD2 (a glycosphingolipid) identifies a small fraction of cells in human breast cancer cell lines and patient samples that are capable of forming mammospheres and initiating tumors with as few as 10 GD2+ cells. In addition, the majority of GD2+ cells are also CD44hiCD24lo, the previously established CSC-associated cell surface phenotype. Gene expression analysis revealed that GD3 synthase (GD3S) is highly expressed in GD2+ as well as in CD44hiCD24lo cells and that interference with GD3S expression, either by shRNA or using a pharmacological inhibitor, reduced the CSC population and CSC-associated properties. GD3S knockdown completely abrogated tumor formation in vivo. Also, induction of epithelial-mesenchymal transition (EMT) in transformed human mammary epithelial cells (HMLER cells) dramatically increased GD2 as well as GD3S expression in these cells, suggesting a role of EMT in the origin of GD2+ breast CSCs. In summary, we identified GD2 as a new CSC-specific cell surface marker and GD3S as a potential therapeutic target for CSCs, with the possibility of improving survival and cure rates in patients with breast cancer.     

CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts

Recent evidence suggests that breast cancer and other solid tumors possess a rare population of cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. We report here the development of a strategy to target these breast cancer stem cells (CSCs) through blockade of the IL-8 receptor CXCR1. CXCR1 blockade using either a CXCR1-specific blocking antibody or repertaxin, a small-molecule CXCR1 inhibitor, selectively depleted the CSC population in 2 human breast cancer cell lines in vitro. Furthermore, this was followed by the induction of massive apoptosis in the bulk tumor population via FASL/FAS signaling. The effects of CXCR1 blockade on CSC viability and on FASL production were mediated by the FAK/AKT/FOXO3A pathway. In addition, repertaxin was able to specifically target the CSC population in human breast cancer xenografts, retarding tumor growth and reducing metastasis. Our data therefore suggest that CXCR1 blockade may provide a novel means of targeting and eliminating breast CSCs.     

Cytokine Conditioning Enhances Systemic Delivery and Therapy of an Oncolytic Virus

Optimum clinical protocols require systemic delivery of oncolytic viruses in the presence of an intact immune system. We show that preconditioning with immune modulators, or loading virus onto carrier cells ex vivo, enhances virus-mediated antitumor activity. Our early trials of systemic reovirus delivery showed that after infusion reovirus could be recovered from blood cells—but not from plasma—suggesting that rapid association with blood cells may protect virus from neutralizing antibody. We therefore postulated that stimulation of potential carrier cells directly in vivo before intravenous viral delivery would enhance delivery of cell-associated virus to tumor. We show that mobilization of the CD11b⁺ cell compartment by granulocyte macrophage-colony stimulating factor immediately before intravenous reovirus, eliminated detectable tumor in mice with small B16 melanomas, and achieved highly significant therapy in mice bearing well-established tumors. Unexpectedly, cytokine conditioning therapy was most effective in the presence of preexisting neutralizing antibody. Consistent with this, reovirus bound by neutralizing antibody effectively accessed monocytes/macrophages and was handed off to tumor cells. Thus, preconditioning with cytokine stimulated recipient cells in vivo for enhanced viral delivery to tumors. Moreover, preexisting neutralizing antibody to an oncolytic virus may, therefore, even be exploited for systemic delivery to tumors in the clinic.     

Attraction and Activation of Dendritic Cells at the Site of Tumor Elicits Potent Antitumor Immunity

Tumor cells harbor unique genetic mutations, which lead to the generation of immunologically foreign antigenic peptide repertoire with the potential to induce individual tumor-specific immune responses. Here, we developed an in situ tumor vaccine with the ability to elicit antitumor immunity. This vaccine comprised an E1B-deleted oncolytic adenovirus expressing β-defensin-2 (Ad-BD2-E1A) for releasing tumor antigens, recruiting and activating plasmacytoid dendritic cells (pDCs). Intratumoral injections of Ad-BD2-E1A vaccine inhibited primary breast tumor growth and blocked naturally occurring metastasis in mice. Ad-BD2-E1A vaccination induced potent tumor-specific T-cell responses. Splenic and intratumoral DCs isolated from Ad-BD2-E1A-immunized mice were able to stimulate or promote the differentiation of naive T cells into tumor-specific cytotoxic T cells. We further found that the increased numbers of mature CD45RA⁺CD8α⁺CD40⁺ pDCs infiltrated into Ad-BD2-E1A-treated tumors. The antitumor effect of Ad-BD2-E1A vaccination was abrogated in toll-like receptor 4 (TLR4) deficient mice, suggesting the critical role of TLR4 in the induction of antitumor immunity by Ad-BD2-E1A. The results of this study indicate that in situ vaccination with the oncolytic BD2-expressing adenovirus preferentially attracts pDCs and promotes their maturation, and thus elicits potent tumor-specific immunity. This vaccine represents an attractive therapeutic strategy for the induction of individualized antitumor immunity.     

Multifaceted Therapeutic Targeting of Ovarian Peritoneal Carcinomatosis Through Virus-induced Immunomodulation

Immunosuppression associated with ovarian cancer (OC) and resultant peritoneal carcinomatosis (PC) hampers the efficacy of many promising treatment options, including immunotherapies. It is hypothesized that oncolytic virus-based therapies can simultaneously kill OC and mitigate immunosuppression. Currently, reovirus-based anticancer therapy is undergoing phase I/II clinical trials for the treatment of OC. Hence, this study was focused on characterizing the effects of reovirus therapy on OC and associated immune microenvironment. Our data shows that reovirus efficiently killed OC cells and induced higher expression of the molecules involved in antigen presentation including major histocompatibility complex (MHC) class I, β2-microglobulin (β2M), TAP-1, and TAP-2. In addition, in the presence of reovirus, dendritic cells (DCs) overcame the OC-mediated phenotypic suppression and successfully stimulated tumor-specific CD8+ T cells. In animal studies, reovirus targeted local and distal OC, alleviated the severity of PC and significantly prolonged survival. These therapeutic effects were accompanied by decreased frequency of suppressive cells, e.g., Gr1.1+, CD11b+ myeloid derived suppressor cells (MDSCs), and CD4+, CD25+, FOXP3+ Tregs, tumor-infiltration of CD3+ cells and higher expression of Th1 cytokines. Finally, reovirus therapy during early stages of OC also resulted in the postponement of PC development. This report elucidates timely information on a therapeutic approach that can target OC through clinically desired multifaceted mechanisms to better the outcomes.     

CD26 a cancer stem cell marker and therapeutic target

Cancer stem cells (CSCs) comprise a tumor subpopulation responsible for tumor maintenance, resistance to chemotherapy, recurrence and metastasis. The identification of this cell group is very important, but there is still no consensus on its characterization. Several CSC markers have been described, like CD133, CD24, CD44 and ALDH1, but more research to identify new markers to facilitate the identification of CSC in a heterogeneous tumoral mass is required. Thus, this article describes the CD26 expression as a CSC marker and the role that it plays in different types of cancer. CD26 expression correlates with some characteristics of CSCs, like the formation of spheres in vitro, formation of new tumors, and resistance to chemotherapy. CD26 is therefore suggested as an auxiliary marker for CSC in different types of cancer, and as a potential therapeutic target.     

Is CD133 a marker of metastatic colon cancer stem cells?

The concept of the so-called cancer stem cell (CSC) holds that only a minority of cells within a tumor have the ability to generate a new tumor. Over the last decade, a large body of literature has implicated the protein CD133 as a marker of organ-specific adult stem cells and in some cancers as a bona fide CSC marker. In this issue of the JCI, Shmelkov et al. challenge the view that CD133 is a marker of CSCs in colon cancer (see the related article beginning on page 2111). CD133 was thought previously to have a very restricted distribution within tissues; the authors have used genetic knock-in models to demonstrate that CD133 in fact is expressed on a wide range of differentiated epithelial cells in adult mouse tissues and on spontaneous primary colon tumors in mice. In primary human colon tumors, all of the epithelial cells also expressed CD133, whereas metastatic colon cancers isolated from liver had distinct CD133+ and CD133- epithelial populations. Intriguingly, the authors demonstrate that the CD133+ and CD133- populations were equally capable of tumor initiation in xenografts. In light of these new findings, the popular notion that CD133 is a marker of colon CSCs may need to be revised.     

Suppression of Antiviral Innate Immunity by Sunitinib Enhances Oncolytic Virotherapy

The use of lytic viruses to preferentially infect and eliminate cancer cells while sparing normal cells is a promising experimental therapeutic approach for treating cancer. However, the efficacy of oncolytic virotherapy is often limited by two innate immunity pathways, the protein kinase PKR and the 2′-5′-oligoadenylate (OAS)/RNase L systems, which are widely present in many but not all tumor cell types. Previously, we reported that the anticancer drug, sunitinib, an inhibitor of VEGF-R and PDGF-R, has off-target effects against both PKR and RNase L. Here we show that combining sunitinib treatments with infection by an oncolytic virus, vesicular stomatitis virus (VSV), led to the elimination of prostate, breast, and kidney malignant tumors in mice. In contrast, either virus or sunitinib alone slowed tumor progression but did not eliminate tumors. In prostate tumors excised from treated mice, sunitinib decreased levels of the phosphorylated form of translation initiation factor, eIF2-α, a substrate of PKR, by 10-fold while increasing median viral titers by 23-fold. The sunitinib/VSV regimen caused complete and sustained tumor regression in both immunodeficient and immunocompetent animals. Results indicate that transient inhibition of innate immunity with sunitinib enhances oncolytic virotherapy allowing the recovery of tumor-bearing animals.     

Cancer Stem Cell�CDirected Therapies: Recent Data From the Laboratory and Clinic

Cancer stem cells (CSCs) are defined by their ability to (i) fully recapitulate the tumor of origin when transplanted into immunodeficient mouse hosts, and (ii) self-renew, demonstrated by their ability to be serially transplanted. These properties suggest that CSCs are required for tumor maintenance and metastasis; thus, it has been predicted that CSC elimination is required for cure. This prediction has profoundly altered paradigms for cancer research, compelling investigators to prospectively isolate CSCs to characterize the molecular pathways regulating their behavior. Many potential strategies for CSC-directed therapy have been proposed, but few studies have rigorously demonstrated their efficacy using in vivo models. Herein, we highlight recent studies that demonstrate the utility of CSC-directed therapies and discuss the implications of the CSC hypothesis to experimental design and therapeutic strategies.     

Adaptive Antiviral Immunity Is a Determinant of the Therapeutic Success of Oncolytic Virotherapy

Oncolytic virotherapy, the selective killing of tumor cells by oncolytic viruses (OVs), has emerged as a promising avenue of anticancer research. We have previously shown that KM100, a Herpes simplex virus type-1 (HSV) deficient for infected cell protein 0 (ICP0), possesses substantial oncolytic properties in vitro and has antitumor efficacy in vivo, in part by inducing antitumor immunity. Here, we illustrate through T-cell immunodepletion studies in nontolerized tumor-associated antigen models of breast cancer that KM100 treatment promotes antiviral and antitumor CD8⁺ cytotoxic T-cell responses necessary for complete tumor regression. In tolerized tumor-associated antigen models of breast cancer, antiviral CD8⁺ cytotoxic T-cell responses against infected tumor cells correlated with the induction of significant tumoristasis in the absence of tumor-associated antigen-specific CD8⁺ cytotoxic T-cells. To enhance oncolysis, we tested a more cytopathic ICP0-null HSV and a vesicular stomatitis virus M protein mutant and found that despite improved in vitro replication, oncolysis in vivo did not improve. These studies illustrate that the in vitro cytolytic properties of OVs are poor prognostic indicators of in vivo antitumor activity, and underscore the importance of adaptive antiviral CD8⁺ cytotoxic T-cells in effective cancer virotherapy.     

A recombinant protein TmSm(T34A) can inhibit proliferation and proapoptosis to breast cancer stem cells(BCSCs) by down-regulating the expression of Cyclin D1

Cancer stem cells (CSCs), a small fraction of cancer cells lines proved with stem cell characteristics, were regarded as “bad seeds” related to recurrence, metastasis and chemotherapy resistance of breast carcinoma in recent years. So inhibiting the growth or inducing the differentiation and apoptosis of CSCs were considered as one of the effective pathways to fight against breast cancer. Based on the recombinant protein TmSm(T34A) that was designed and prepared in our previous experiments for targeting survivin, an inhibitor of apoptosis protein(IAP), in this study, we explored the effects of TmSm(T34A) on BCSCs obtained by enriching in serum-free suspension, sorting and characterizing of MCF-7/ADM. The results showed that TmSm(T34A) could not only inhibit the proliferation and growth of BCSCs by decreasing CD44⁺CD24⁻ proportion and down-regulating the expression of Cyclin D1 significantly, but also induce BCSCs apoptosis evidently. Furthermore, in BCSCs xenograft nude mice administrated TmSm(T34A), the tumor growth was slower than that of the control obviously. Thus it can be seen TmSm(T34A) would be a promising potential protein for treatment of breast cancer by effecting on BCSCs.     

Cancer stem cells as therapeutic targets of pancreatic cancer

The discovery of stem cells and their potential abilities in self-renewal and differentiation has opened a new horizon in medicine. Scientists have found a small population of stem cells in some types of cancers with the same functions as normal stem cells. There are two models for tumor progression: clonal (stochastic) and cancer stem cell (CSCs) models. According to the first model, all transformed cells in the tumor have carcinogenic potential and are able to proliferate and produce the same cells. The latter model, which has received more attention recently, considers the role of CSCs in drug resistance and tumor metastasis. Following the model, researchers have found that targeting CSCs may be a promising way in cancer therapy. This review describes CSC characteristics in general, while also focusing on CSC properties in the context of pancreatic cancer.     

Active Immunotherapy Combined With Blockade of a Coinhibitory Pathway Achieves Regression of Large Tumor Masses in Cancer-prone Mice

Vaccines that aim to expand tumor-specific CD8⁺ T cells have yielded disappointing results in cancer patients although they showed efficacy in transplantable tumor mouse models. Using a system that more faithfully mimics a progressing cancer and its immunoinhibitory microenvironment, we here show that in transgenic mice, which gradually develop adenocarcinomas due to expression of HPV-16 E7 within their thyroid, a highly immunogenic vaccine expressing E7 only induces low E7-specific CD8⁺ T-cell responses, which fail to affect the size of the tumors. In contrast, the same type of vaccine expressing E7 fused to herpes simplex virus (HSV)-1 glycoprotein D (gD), an antagonist of the coinhibitory B- and T-lymphocyte attenuator (BTLA)/CD160-herpes virus entry mediator (HVEM) pathways, stimulates potent E7-specific CD8⁺ T-cell responses, which can be augmented by repeated vaccination, resulting in initial regression of even large tumor masses in all mice with sustained regression in more than half of them. These results indicate that active immunization concomitantly with blockade of the immunoinhibitory HVEM-BTLA/CD160 pathways through HSV-1 gD may result in sustained tumor regression.     

Oncolytic adenoviruses kill breast cancer initiating CD44+CD24-/low cells.

Cancer stem cells have been indicated in the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. In breast cancer, they may reside in the CD44(+)CD24(-/low) population. The use of oncolytic adenoviruses presents an attractive anti-tumor approach for eradication of these cells because their entry occurs through infection and they are, therefore, not susceptible to those mechanisms that commonly render stem cells resistant to many drugs. We isolated CD44(+)CD24(-/low) cells from patient pleural effusions and confirmed stem cell-like features including oct4 and sox2 expression and Hoechst 33342 exclusion. CD44(+)CD24(-/low) cells, including the Hoechst excluding subpopulation, could be effectively killed by oncolytic adenoviruses Ad5/3-Delta24 and Ad5.pk7-Delta24. In mice, CD44(+)CD24(-/low) cells formed orthotopic breast tumors but virus infection prevented tumor formation. Ad5/3-Delta24 and Ad5.pk7-Delta24 were effective against advanced orthotopic CD44(+)CD24(-/low)-derived tumors. In summary, Ad5/3-Delta24 and Ad5.pk7-Delta24 can kill CD44(+)CD24(-/low), and also committed breast cancer cells, making them promising agents for treatment of breast cancer.     

Noninvasive Imaging and Radiovirotherapy of Prostate Cancer Using an Oncolytic Measles Virus Expressing the Sodium Iodide Symporter

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 (¹²⁵I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of ¹²³I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 × 10⁶ TCID₅₀) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of ¹³¹I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by ¹³¹I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.     

The Clinical Potential of Targeted Nanomedicine: Delivering to Cancer Stem-like Cells

Cancer stem-like cells (CSCs) have been implicated in recurrence and treatment resistance in many human cancers. Thus, a CSC-targeted drug delivery strategy to eliminate CSCs is a desirable approach for developing a more effective anticancer therapy. We have developed a tumor-targeting nanodelivery platform (scL) for systemic administration of molecular medicines. Following treatment with the scL nanocomplex carrying various payloads, we have observed exquisite tumor-targeting specificity and significant antitumor response with long-term survival benefit in numerous animal models. We hypothesized that this observed efficacy might be attributed, at least in part, to elimination of CSCs. Here, we demonstrate the ability of scL to target both CSCs and differentiated nonstem cancer cells (non-CSCs) in various mouse models including subcutaneous and intracranial xenografts, syngeneic, and chemically induced tumors. We also show that systemic administration of scL carrying the wtp53 gene was able to induce tumor growth inhibition and the death of both CSCs and non-CSCs in subcutaneous colorectal cancer xenografts suggesting that this could be an effective method to reduce cancer recurrence and treatment resistance. This scL nanocomplex is being evaluated in a number of clinical trials where it has been shown to be well tolerated with indications of anticancer activity.     

CD13 is a therapeutic target in human liver cancer stem cells

Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. However, neither their existence nor their identity within many cancers has been well defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13⁺ cells predominated in the G₀ phase of the cell cycle and typically formed cellular clusters in cancer foci. Following treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. 5-FU inhibited CD90⁺ proliferating CSCs, some of which produce CD13⁺ semiquiescent CSCs, while CD13 inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.