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.

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.

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.

Is the novel SCKL3 at 14q23 the predominant Seckel locus?

Seckel syndrome (SCKL) is a rare disease with wide phenotypic heterogeneity. A locus (SCKL1) has been identified at 3q and another (SCKL2) at 18p, both in single kindreds afflicted with the syndrome. We report here a novel locus (SCKL3) at 14q by linkage analysis in 13 Turkish families. In total, 18 affected and 10 unaffected sibs were included in the study. Of the 10 informative families, nine with parental consanguinity and one reportedly nonconsanguineous but with two affected sibs, five were indicative of linkage to the novel locus. One of those families also linked to the SCKL1 locus. A consanguineous family with one affected sib was indicative of linkage to SCKL2. The novel gene locus SCKL3 is 1.18 cM and harbors ménage a trois 1, a gene with a role in DNA repair.

Highest heterogeneity for cystic fibrosis: 36 mutations account for 75% of all CF chromosomes in Turkish patients.

We analyzed the CFTR locus in 83 Turkish cystic fibrosis patients to identify mutations, haplotypes, and the carrier frequency in the population. We detected 36 different mutations in 125 (75%) of the total 166 CF chromosomes. Seven novel mutations were identified: four missense (K68E, Q493P, E608G, and V1147I), two splice-site (406 -3T > C and 3849 +5G > A), and one deletion (CFTRdele17b,18). The data showed that the Turkish population has the highest genetic heterogeneity at the CFTR locus reported so far. The results of this thorough molecular analysis at the CFTR locus of a population not of European descent shows that CF is not uncommon in all such populations. The large number of mutations present, as well as the high heterogeneity in haplotypes associated with the mutations suggests that most of the mutations have persisted for a long time in the population. Consistently, the carrier frequency is assessed to be high, indicating that the disease in the population is ancient.