Mechanisms Involved in the Promoting Activity of Fibroblasts in HTLV-1-Mediated Lymphomagenesis: Insights into the Plasticity of Lymphomatous Cells.

Among the mechanisms leading to progression to Adult T-cell Leukaemia/Lymphoma in Human T-cell Leukaemia Virus type 1 (HTLV-1)-infected subjects, the contribution of stromal components remains poorly understood. To dissect the role of fibroblasts in HTLV-1-mediated lymphomagenesis, transcriptome studies, cytofluorimetric and qRT-PCR analyses of surface and intracellular markers linked to plasticity and stemness in coculture, and in vivo experiments were performed. A transcriptomic comparison between a more lymphomagenic (C91/III) and the parental (C91/PL) cell line evidenced hyperactivation of the PI3K/Akt pathway, confirmed by phospho-ELISA and 2-DE and WB analyses. C91/III cells also showed higher expression of mesenchymal and stemness genes. Short-term coculture with human foreskin fibroblasts (HFF) induced these features in C91/PL cells, and significantly increased not only the cancer stem cells (CSCs)-supporting CD10+GPR77+ HFF subpopulation, but also the percentage of ALDH1bright C91/PL cells. A non-cytotoxic acetylsalicylic acid treatment decreased HFF-induced ALDH1bright C91/PL cells, downregulated mesenchymal and stemness genes in cocultured cells, and delayed lymphoma growth in immunosuppressed mice, thus hindering the supportive activity of HFF on CSCs. These data suggest that crosstalk with HFF significantly intensifies the aggressiveness and plasticity of C91/PL cells, leading to the enrichment in lymphoma-initiating cells. Additional research is needed to better characterize these preliminary findings.

Zika Virus: A New Therapeutic Candidate for Glioblastoma Treatment.

Glioblastoma (GBM) is the most aggressive among the neurological tumors. At present, no chemotherapy or radiotherapy regimen is associated with a positive long-term outcome. In the majority of cases, the tumor recurs within 32-36 weeks of initial treatment. The recent discovery that Zika virus (ZIKV) has an oncolytic action against GBM has brought hope for the development of new therapeutic approaches. ZIKV is an arbovirus of the Flaviviridae family, and its infection during development has been associated with central nervous system (CNS) malformations, including microcephaly, through the targeting of neural stem/progenitor cells (NSCs/NPCs). This finding has led various groups to evaluate ZIKV's effects against glioblastoma stem cells (GSCs), supposedly responsible for GBM onset, progression, and therapy resistance. While preliminary data support ZIKV tropism toward GSCs, a more accurate study of ZIKV mechanisms of action is fundamental in order to launch ZIKV-based clinical trials for GBM patients.

Integrin alpha 6 as a stemness driver is a novel promising target for HPV (+) head and neck squamous cell carcinoma.

Although the incidence rates of head and neck squamous cell carcinoma (HNSCC) associated with human papilloma virus (HPV) infection have recently been on the rise, the underlying mechanism of its tumorigenesis remains largely unknown. Here, we investigated whether HNSCC cells with high expression of integrin alpha 6 (ITGα6), one of the HPV receptors, have a preference during HPV infection. In addition, we examined the gain or loss of function of the ITGα6 gene in HPV + ve HNSCC cells, as well as its prognostic value in patients with HNSCC. HPV pseudovirus was found to be more infective, with HNSCC cells featuring an overexpressed ITGα6 gene compared to the control cells. Overexpression and suppression of ITGα6 respectively increases and decreases stemness phenotypes of HPV + ve HNSCC cells. Furthermore, ITGα6 can regulate stemness by partially mediating AKT pathway in HPV + ve HNSCC cells. Finally, patients with HPV + ve HNSCC had a poor prognosis in cases of elevated ITGα6 expression; however, the expression levels of ITGα6 did not influence the survival rates of HPV-negative HNSCC patients. In conclusion, ITGα6 can serve as a potential therapeutic target for HPV + ve HNSCC cancer-like stem cells (CSCs).

Harnessing the Natural Biology of Adeno-Associated Virus to Enhance the Efficacy of Cancer Gene Therapy.

Adeno-associated virus (AAV) was first characterized as small "defective" contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer.

Multidirectional Strategies for Targeted Delivery of Oncolytic Viruses by Tumor Infiltrating Immune Cells.

Oncolytic virus (OV) immunotherapy has demonstrated to be a promising approach in cancer treatment due to tumor-specific oncolysis. However, their clinical use so far has been largely limited due to the lack of suitable delivery strategies with high efficacy. Direct 'intratumoral' injection is the way to cross the hurdles of systemic toxicity, while providing local effects. Progress in this field has enabled the development of alternative way using 'systemic' oncolytic virotherapy for producing better results. One major potential roadblock to systemic OV delivery is the low virus persistence in the face of hostile immune system. The delivery challenge is even greater when attempting to target the oncolytic viruses into the entire tumor mass, where not all tumor cells are equally exposed to exactly the same microenvironment. The microenvironment of many tumors is known to be massively infiltrated with various types of leucocytes in both primary and metastatic sites. Interestingly, this intratumoral immune cell heterogeneity exhibits a degree of organized distribution inside the tumor bed as evidenced, for example, by the hypoxic tumor microenviroment where predominantly recruits tumor-associated macrophages. Although in vivo OV delivery seems complicated and challenging, recent results are encouraging for decreasing the limitations of systemically administered oncolytic viruses and an improved efficiency of oncolytic viral therapy in targeting cancerous tissues in vitro. Here, we review the latest developments of carrier cell-based oncolytic virus delivery using tumor-infiltrating immune cells with a focus on the main features of each cellular vehicle.

Flow Cytometry Characterization of Pluripotent Transmembrane Glycoproteins on Resident Cervix Uteri Cells in Patients Screened for Cervical Cancer.

The aim of this study was to characterize both by flow cytometry analysis and immunohistochemistry cervix uteri cells of nulliparous women screened for cervical intraepithelial neoplasia (CIN) in comparison to a group without CIN by using mesenchymal stem cell-like and hematopoietic lineage markers. A significant expression for CD29, CD38, HLA-I, and HLA-II was correlated positively to the CIN degree and it was more relevant in patients positive for human papilloma virus (HPV). Thus, identification and detailed characterization of pluripotent resident in uteri cells could be a promising therapeutic target.

Hepatitis B virus X protein enhances the development of liver fibrosis and the expression of genes associated with epithelial-mesenchymal transitions and tumor progenitor cells.

The hepatitis B virus X protein (HBx) has pleiotropic biological effects, which underlies its potential role in cell transformation. However, its involvement in hepatic fibrosis remains unclear. In this study, we wanted to clarify, in vivo, the role of HBx protein in the development of liver fibrosis. Mice transgenic for the full-length HBx (FL-HBx) were used. To create liver fibrosis, FL-HBx transgenic and control mice were chronically exposed to carbon tetrachloride (CCl4). Modulation of the expression of proteins involved in matrix remodeling, hepatic metabolism and epithelial-mesenchymal transition (EMT) were investigated. In transgenic mice, FL-HBx expression potentiates CCl4-induced liver fibrosis with increased expression of proteins involved in matrix remodeling (Collagen1a, α-Sma, PdgfR-ß, MMP-13). In FL-HBx transgenic mice, an increase in EMT was observed with a higher transcription of two inflammatory cytokines (TNF-α and TGF-ß) and a decrease of glutamine synthetase expression level. This was associated with a sustained cell cycle and hepatocyte polyploidy alteration consistent with p38 and ERK1/2 overactivation, increase of PLK1 transcription, accumulation of SQSTM1/p62 protein and increase expression of Beclin-1. This correlates with a higher expression of tumor progenitor cell markers (AFP, Ly6D and EpCam), indicating a higher risk of progression from fibrosis to hepatocellular carcinoma (HCC) in the presence of FL-HBx protein. In conclusion, our results show that FL-HBx protein enhances the development of liver fibrosis and contributes to the progression of liver disease from chronic hepatitis to HCC.

Evaluation of the cancer stem cell marker DCLK1 in patients with lymph node metastases of head and neck cancer.

BACKGROUND: Lymph node metastases are frequently detected in head and neck squamous cell carcinoma (HNSCC) patients. Little is known about biomarkers expressed in lymph node metastases or their influence on clinical outcome. Doublecortin-like kinase 1 (DCLK1) is one marker that might be associated with outcome, owing to its correlation with stem cell-like characteristics. METHODS: We assessed the expression of DCLK1 in 74 postoperatively irradiated patients in histologically confirmed HNSCC lymph node metastases. Statistical analysis of the association with DCLK1 on clinical outcomes was performed. RESULTS: DCLK1 was expressed in 63.5% of our patient cohort. DCLK1(+) HNSCC patients, compared with those without DCLK1 expression, showed a significantly poorer time to recurrence. Moreover, we observed a significantly poorer time to recurrence in HPV(-) HNSCC patients, and significantly shorter overall and disease-free survival rates in HPV(-) oropharyngeal cancer patients, compared with HPV(+) patients with these cancers. HPV(+) patients showed no significant differences in survival time according to DCLK1 expression. However, recurrent disease occurred in only DCLK1(+) patients. Mulitivariate analysis showed that DCLK1 expression in lymph node metastases is an independent marker for recurrence. CONCLUSION: DCLK1 expression might be associated with poorer clinical outcomes in HNSCC patients, specifically in HPV(-) move patients. However, larger studies are required to verify our results.

Oncolytic Effect of Adenoviruses Serotypes 5 and 6 Against U87 Glioblastoma Cancer Stem Cells.

BACKGROUND/AIM: Oncolytic adenoviruses are promising therapeutic agents against both the bulk of tumor cells and cancer stem cells. The present study intended to test the oncolytic capability of adenovirus serotype 6 (Ad6), which has a lower seroprevalence and hepatotoxicity relatively to adenovirus 5 (Ad5), against the glioblastoma and its cancer stem cells. MATERIALS AND METHODS: Oncolytic efficacy of Ad6 was compared to widespread Ad5 both in vitro and in vivo, using the U87 and U251 human glioblastoma cell lines and subcutaneously transplanted U87 cells in SCID mice, respectively. RESULTS: Ad6 had a dose-dependent cytotoxicity toward glioblastoma cells in vitro and its intratumoral injections lead to a significant (p<0.05) decrease in volume of U87 xenografts, similarly to Ad5. Based on the innate capability of glioblastoma cancer stem cells to internalize a fluorescent-labeled double-stranded DNA probe, the spatial localization of these cells was estimated and it was shown that the number of cancer stem cells tended to decrease under adenovirus therapy as compared to the control group. CONCLUSION: Ad6 was shown to be a promising agent for treating glioblastomas.

SPLUNC1 and MLL3 regulate cancer stem cells in nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is one of the common types of cancer that originate from the nasopharyngeal region. Recurrence and early metastasis represent major problems associated with NPC mortality. These are mainly caused by various molecular changes that take place during the conversion of normal stem cells into treatment-resistant stem cells. The aim of our study was to investigate the proliferative behavior of cancer stem cells in different stages of NPC and to identify the functional roles of SPLUNC1 and MLL3 associated with cancer stem cells. METHODS: We successfully developed a NPC mouse model using C666-1 cells. Immunohistochemistry and Western blotting were used to analyze the expression of SOX2, SPLUNC1 and MLL3. RESULTS: Null BALB/c mice developed initial and aggressive stages of NPC in 3 and 10 weeks, respectively. Histological results showed that the proliferative ability of cells increased as the tumor progressed to the next level. The SOX2 protein showed a peculiar pattern of upregulation in aggressive NPC when compared with control tissues and initial NPC. Remarkably, our study found that SPLUNC1 and MLL3 expression showed upregulation in initial NPC, which indicates their role in the tumor resistance mechanism even if their expression was downregulated in aggressive NPC. CONCLUSIONS: Our results conclude that SPLUNC1 and MLL3 expression control the resistance mechanism of cancer stem cells in initial NPC, but their downregulation in aggressive stages contributes to developing resistance in nasopharyngeal cancer stem cells.

Diversity of cancer stem cells in head and neck carcinomas: The role of HPV in cancer stem cell heterogeneity, plasticity and treatment response.

Head and neck squamous cell carcinomas (HNSCC) resulting from oncogenic transformations following human papillomavirus (HPV) infection consistently demonstrate better treatment outcomes than HNSCC from other aetiologies. Squamous cell carcinoma of the oropharynx (OPSCC) shows the highest prevalence of HPV involvement at around 70-80%. While strongly prognostic, HPV status alone is not sufficient to predict therapy response or any potential dose de-escalation. Cancer stem cell (CSC) populations within these tumour types represent the most therapy-resistant cells and are the source of recurrence and metastases, setting a benchmark for tumour control. This review examines clinical and preclinical evidence of differences in response to treatment by the HPV statuses of HNSCC and the role played by CSCs in treatment resistance and their repopulation from non-CSCs. Evidence was collated from literature searches of PubMed, Scopus and Ovid for differential treatment response by HPV status and contribution by critical biomarkers including CSC fractions and chemo-radiosensitivity. While HPV and CSC are yet to fulfil promise as biomarkers of treatment response, understanding how HPV positive and negative aetiologies affect CSC response to treatment and tumour plasticity will facilitate their use for greater treatment individualisation.

Zika virus infection induces MiR34c expression in glioblastoma stem cells: new perspectives for brain tumor treatments.

Zika virus (ZIKV) is a flavivirus with a marked effect on fetal nervous system development. ZIKV treatment has recently been found to also have a benefit against glioblastoma, a highly aggressive brain tumor with a poor prognosis. The reported data do not completely explain the mechanism beyond this effect. Nevertheless, in the majority of the cases no adverse effect has been found in healthy adult humans. In this study, we characterized the ZIKV infection mechanism on glioblastoma stem cells, which are considered responsible for the tumor progression and resistance to conventional therapies. Moreover, we explain why the action of this virus is directed to the stem cells in the nervous system counterpart. Our results confirm the effectiveness of ZIKV treatment against glioblastoma, indicating novel molecular targets that can be introduced for more powerful therapies.

Concise Review: Targeting Cancer Stem Cells and Their Supporting Niche Using Oncolytic Viruses.

Cancer stem cells (CSCs) have the capacity to self-renew and differentiate to give rise to heterogenous cancer cell lineages in solid tumors. These CSC populations are associated with metastasis, tumor relapse, and resistance to conventional anticancer therapies. Here, we focus on the use of oncolytic viruses (OVs) to target CSCs as well as the OV-driven interferon production in the tumor microenvironment (TME) that can repress CSC properties. We explore the ability of OVs to deliver combinations of immune-modulating therapeutic transgenes, such as immune checkpoint inhibitor antibodies. In particular, we highlight the advantages of virally encoded bi-specific T cell engagers (BiTEs) to not only target cell-surface markers on CSCs, but also tumor-associated antigens on contributing components of the surrounding TME and other cancer cells. We also highlight the crucial role of combination anticancer treatments, evidenced by synergy of OV-delivered BiTEs and chimeric-antigen receptor T cell therapy. Stem Cells 2019;37:716-723.

The osteopontin-CD44 axis in hepatic cancer stem cells regulates IFN signaling and HCV replication.

Osteopontin (OPN) is involved in cell proliferation, migration, inflammation, and tumor progression in various tissues. OPN induces stemness by interacting with CD44, but the functional relevance of OPN-mediated interferon (IFN) signaling and hepatitis C virus (HCV) replication in stem cell populations remains unclear. In this study, we investigated the effect of OPN on HCV replication and IFN signaling in cancer stem cells (CSCs) positive for epithelial cell adhesion molecule (EpCAM) and CD44. We show that the EpCAM+/CD44+ CSCs show marked HCV replication when compared to EpCAM-/CD44- cells. In addition, OPN significantly enhances this HCV replication in EpCAM+/CD44+ CSCs and markedly suppresses IFN-stimulated gene expression. The GSK-3ß inhibitor BIO increases the EpCAM+/CD44+ CSC population and OPN expression and impairs IFN signaling via STAT1 degradation. Taken together, our data suggest that OPN enhances HCV replication in the EpCAM+/CD44+ CSCs, while it also negatively regulates the IFN signaling pathway via inhibition of STAT1 phosphorylation and degradation. Therefore, OPN may represent a novel therapeutic target for treating HCV-related hepatocellular carcinoma.

Treatment of Human Glioblastoma with a Live Attenuated Zika Virus Vaccine Candidate.

Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy.IMPORTANCE Glioblastoma (GBM), the deadliest type of brain tumor, is currently incurable because of its high recurrence rate after traditional treatments, including surgery to remove the main part of the tumor and radiation and chemotherapy to target residual tumor cells. These treatments fail mainly due to the presence of a cell subpopulation called glioma stem cells (GSCs), which are resistant to radiation and chemotherapy and capable of self-renewal and tumorigenicity. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we tested a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a human GSC-derived orthotopic model. Our results showed that ZIKV-LAV retained good efficacy against glioblastoma by selectively killing GSCs within the tumor. In addition, ZIKV-LAV exhibited an excellent safety profile upon intracerebral injection into the treated animals. The good balance between the safety of ZIKV-LAV and its efficacy against human GSCs suggests that it is a potential candidate for combination with the current treatment regimen for GBM therapy.

Restriction of Replication of Oncolytic Herpes Simplex Virus with a Deletion of γ34.5 in Glioblastoma Stem-Like Cells.

Oncolytic viruses, including herpes simplex viruses (HSVs), are a new class of cancer therapeutic engineered to infect and kill cancer cells while sparing normal tissue. To ensure that oncolytic HSV (oHSV) is safe in the brain, all oHSVs in clinical trial for glioma lack the γ34.5 genes responsible for neurovirulence. However, loss of γ34.5 attenuates growth in cancer cells. Glioblastoma (GBM) is a lethal brain tumor that is heterogeneous and contains a subpopulation of cancer stem cells, termed GBM stem-like cells (GSCs), that likely promote tumor progression and recurrence. GSCs and matched serum-cultured GBM cells (ScGCs), representative of bulk or differentiated tumor cells, were isolated from the same patient tumor specimens. ScGCs are permissive to replication and cell killing by oHSV with deletion of the γ34.5 genes (γ34.5- oHSV), while patient-matched GSCs were not, implying an underlying biological difference between stem and bulk cancer cells. GSCs specifically restrict the synthesis of HSV-1 true late (TL) proteins, without affecting viral DNA replication or transcription of TL genes. A global shutoff of cellular protein synthesis also occurs late after γ34.5- oHSV infection of GSCs but does not affect the synthesis of early and leaky late viral proteins. Levels of phosphorylated eIF2α and eIF4E do not correlate with cell permissivity. Expression of Us11 in GSCs rescues replication of γ34.5- oHSV. The difference in degrees of permissivity between GSCs and ScGCs to γ34.5- oHSV illustrates a selective translational regulatory pathway in GSCs that may be operative in other stem-like cells and has implications for creating oHSVs.IMPORTANCE Herpes simplex virus (HSV) can be genetically engineered to endow cancer-selective replication and oncolytic activity. γ34.5, a key neurovirulence gene, has been deleted in all oncolytic HSVs in clinical trial for glioma. Glioblastoma stem-like cells (GSCs) are a subpopulation of tumor cells thought to drive tumor heterogeneity and therapeutic resistance. GSCs are nonpermissive for γ34.5- HSV, while non-stem-like cancer cells from the same patient tumors are permissive. GSCs restrict true late protein synthesis, despite normal viral DNA replication and transcription of all kinetic classes. This is specific for true late translation as early and leaky late transcripts are translated late in infection, notwithstanding shutoff of cellular protein synthesis. Expression of Us11 in GSCs rescues the replication of γ34.5- HSV. We have identified a cell type-specific innate response to HSV-1 that limits oncolytic activity in glioblastoma.

HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer.

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV+ OPSCC metastasize faster than HPV- tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV+ OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44high /EpCAMhigh ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44high /EpCAMlow ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV+ OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44high /EpCAMlow CSCs were validated by RT-qPCR and in situ hybridization on HPV16+ OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16+ OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV+ and HPV- metastasis. As inhibition of this miRNA led to a markedly reduction of CD44high /EpCAMlow cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

GOLPH2-regulated oncolytic adenovirus, GD55, exerts strong killing effect on human prostate cancer stem-like cells in vitro and in vivo.

GOLPH2 (also called GP73) is a Golgi glycoprotein, which has been identified as a novel tumor marker upregulated in various cancers, including prostate cancer (PCa). GD55 is a novel GOLPH2-regulated oncolytic adenovirus that exhibits a strong killing effect on hepatoma cells. Here, we investigate the antitumor effect of GD55 on prostate cancer stem cell (CSC)-like cells in vitro and in vivo. Prostate CSC-like sphere cells were acquired and enriched by culturing DU145, LNCap or P3 prostate cancer cells in suspension. The prostate CSC-like sphere cells were capable of self-renewal, differentiation and quiescence, displaying tumorigenic feature and chemo-resistance to 5-FU, doxorubicin and DDP. Treatment with GD55 (1, 5, 10 MOI) dose-dependently suppressed the viability of DU145 sphere cells, which was a more pronounced compared to its cytotoxic action on the parental DU145 cells. In a mouse xenograft prostate CSC-like model, intratumoral injection of GD55 markedly suppressed the growth rate of xenograft tumors and induced higher levels of cell death and necrosis within the tumor tissues. Our results demonstrate that GD55 infection exerts strong anticancer effects on prostate CSC-like cells in vitro and in vivo, and has a potential to be used in the clinical therapy of PCa.