Generation of magnetic biohybrid microrobots based on MSC.sTRAIL for targeted stem cell delivery and treatment of cancer.

Background: Combining the power of magnetic guidance and the biological activities of stem cells transformed into biohybrid microrobots holds great promise for the treatment of several diseases including cancer. Results: We found that human MSCs can be readily loaded with magnetic particles and that the resulting biohybrid microrobots could be guided by a rotating magnetic field. Rotating magnetic fields have the potential to be applied in the human setting and steer therapeutic stem cells to the desired sites of action in the body. We could demonstrate that the required loading of magnetic particles into stem cells is compatible with their biological activities. We examined this issue with a particular focus on the expression and functionality of therapeutic genes inside of human MSC-based biohybrid microrobots. The loading with magnetic particles did not cause a loss of viability or apoptosis in the human MSCs nor did it impact on the therapeutic gene expression from the cells. Furthermore, the therapeutic effect of the gene products was not affected, and the cells also did not lose their migration potential. Conclusion: These results demonstrate that the fabrication of guidable MSC-based biohybrid microrobots is compatible with their biological and therapeutic functions. Thus, MSC-based biohybrid microrobots represent a novel way of delivering gene therapies to tumours as well as in the context of other diseases.

IFN-γ combined with targeting of XIAP leads to increased apoptosis-sensitisation of TRAIL resistant pancreatic carcinoma cells.

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a specific and potent inducer of apoptosis in cancer cells, but the resistance of many tumour cells to TRAIL still represents a major hurdle for the clinical treatment of tumours with TRAIL. As apoptosis is regulated by the balance of activities of several anti-apoptotic factors and pro-apoptotic factors, we analysed the relative contribution of the two sides and found that down-regulation of Bcl-x(L) and in particular XIAP, but not c-Flip, sensitised the TRAIL resistant pancreatic cancer cell line Panc-1. A combination of both XIAP and Bcl-x(L) knock-downs showed no substantial added benefit indicating that both act in the same pathway. Notably, the degree of sensitisation by silencing of anti-apoptotic genes was further elevated by concomitantly increasing the pro-apoptotic potential in Panc-1 cells through over-expression of TRAIL-R1 or IFN-γ-mediated increases in caspase-8 levels. Similar sensitisation effects were obtained for another TRAIL-resistant pancreatic tumour cell line, AsPC-1. Our findings demonstrate that modulation of the balance between anti- and pro-apoptotic pathways from both sides by inhibition of apoptosis-antagonists and stimulation of pro-apoptotic factors provides the best way to enhance the anti-tumourigenic effect of TRAIL.

Both human and mouse mesenchymal stem cells promote breast cancer metastasis.

Cell therapy has the potential to offer novel treatment modalities for a number of diseases including cancer, and stem cells and in particular mesenchymal stem cells (MSCs) have been experimentally used to deliver therapeutic transgenes. However, conflicting reports have on the one side found that human MSCs can promote metastasis, while on the other hand other studies have shown that MSCs can stall the growth of metastatic lesions. In order to clarify the role of MSCs in metastasis development, we tested whether murine MSCs would behave similarly to human cells in mice. We found that the tissue distribution of human and mouse MSCs was nearly identical after intravenous injection. In mice with MDA-MB-231 mammary carcinoma xenografts we found that a fraction of MSCs infiltrated the primary tumor mass, but that the general tissue distribution of MSCs was unaffected by the tumor-burden. About half of the tumor-burdened animals that were treated with murine and human MSCs, respectively, harbored metastatic lesions with only 17% of controls showing metastatic nodules. Hence, both human and mouse MSCs possess metastasis-promoting activity raising concerns about the safe use of MSCs, but at the same time making the use of murine transgenic model systems feasible to study the role of MSCs in metastasis development and possibly finding ways of using them safely as cell therapeutic vehicles.

Targeting of XIAP combined with systemic mesenchymal stem cell-mediated delivery of sTRAIL ligand inhibits metastatic growth of pancreatic carcinoma cells.

Disseminating tumors are one of the gravest medical problems. Here, we combine the tumor-specific apoptosis-inducing activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with the ability of mesenchymal stem cells (MSCs) to infiltrate both tumor and lymphatic tissues to target primary tumors as well as disseminated cancer cells in a human pancreatic cancer mouse model. Furthermore, we targeted X-linked inhibitor of apoptosis protein (XIAP) by RNA interference (RNAi) inside the cancer cells to make use of the apoptosis sensitization as well the antimetastatic effect that is afforded by XIAP silencing. We generated MSCs, termed MSC.sTRAIL, that express and secrete a trimeric form of soluble TRAIL (sTRAIL). MSC.sTRAIL triggered limited apoptosis in human pancreatic carcinoma cells that were resistant to soluble recombinant TRAIL, which is most likely due to the enhanced effect of the direct, cell-mediated delivery of trimeric TRAIL. MSC.sTRAIL-mediated cell death was markedly increased by concomitant knockdown of XIAP by RNAi in the cancer cells. These findings were confirmed in xenograft models, in which tumors from the parental pancreatic carcinoma cells showed only growth retardation on treatment with MSC.sTRAIL, whereas tumors with silenced XIAP that were treated with MSC.sTRAIL went into remission. Moreover, animals with XIAP-negative xenografts treated with MSC.sTRAIL were almost free of lung metastasis, whereas animals treated with control MSCs showed substantial metastatic growth in the lungs. In summary, this is the first demonstration that a combined approach using systemic MSC-mediated delivery of sTRAIL together with XIAP inhibition suppresses metastatic growth of pancreatic carcinoma.

The TRAIL-receptor-1: TRAIL-receptor-3 and -4 ratio is a predictor for TRAIL sensitivity of cancer cells.

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in many cancer cells. However, a significant proportion of tumours are TRAIL-resistant erecting a major hurdle for a successful TRAIL-based treatment regimen in the future. In this context, it would be a major advantage to be able to identify the tumours that respond to TRAIL. The existence of two apoptosis-inducing receptors (TRAIL-R1 and TRAIL-R2) and two receptors that cannot transmit an apoptotic signal and have an inhibitory function (TRAIL-R3 and TRAIL-R4) make TRAIL signalling complicated. We analysed the surface expression of all four membrane-bound TRAIL receptors in cancer cell lines of various origin and primary cancer and normal cells and found a good correlation between TRAIL-sensitivity and the expression of TRAIL-R1 alone, but an even better correlation when a ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4 was analysed. Experimental overexpression of TRAIL-R1 alone or in combination with TRAIL-R4 in PANC-1 cells confirmed our correlation results. Similar to the surface expression-apoptosis correlation analysis we found a high correlation between TRAIL-sensitivity and the mRNA level ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4. A value of <0.85 for the ratio predicted TRAIL resistance in both protein and RNA analysis. Hence, TRAIL receptor RNA expression analysis by real-time PCR might be a feasible approach to predict possible TRAIL-responses in individual tumour samples.

Constitutively activated nuclear factor-kappaB, but not induced NF-kappaB, leads to TRAIL resistance by up-regulation of X-linked inhibitor of apoptosis protein in human cancer cells.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated, but its exact role is controversial. We studied different cell lines displaying varying responses to TRAIL and found that TRAIL can activate NF-kappaB in all our cancer cell lines regardless of their TRAIL sensitivity. Inhibition of NF-kappaB via adenoviral expression of the IkappaB-alpha super-repressor only sensitized the TRAIL-resistant pancreatic cancer cell line Panc-1. Panc-1 cells harbor constitutively activated NF-kappaB, pointing to a possible role of preactivated NF-kappaB in protection from TRAIL. Furthermore, we could reduce X-linked inhibitor of apoptosis protein (XIAP) levels in Panc-1 cells by inhibition of constitutively activated NF-kappaB and sensitize Panc-1 cells to TRAIL by RNA interference against XIAP. These results implicate elevated XIAP levels caused by high basal NF-kappaB activity in TRAIL resistance and suggest that therapeutic strategies involving TRAIL can be abetted by inhibition of NF-kappaB and/or XIAP only in tumor cells with constitutively activated NF-kappaB.

Betulinic acid as new activator of NF-kappaB: molecular mechanisms and implications for cancer therapy.

Recent evidence demonstrates that the anticancer activity of betulinic acid (BetA) can be markedly increased by combination protocols, for example with chemotherapy, ionizing radiation or TRAIL. Since nuclear factor-kappaB (NF-kappaB), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-kappaB in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-kappaB in a variety of tumor cell lines. NF-kappaB DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluorescence microscopy. BetA-induced NF-kappaB activation involved increased IKK activity and phosphorylation of IkappaB-alpha at serine 32/36 followed by degradation of IkappaB-alpha. Reporter assays revealed that NF-kappaB activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-kappaB activation by different chemical inhibitors (proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-kappaB inhibition by transient or stable expression of IkappaB-alpha super-repressor inhibited BetA-induced apoptosis in SH-EP neuroblastoma cells, while transient expression of IkappaB-alpha super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our findings that activation of NF-kappaB by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-kappaB inhibitors in combination with BetA would have no therapeutic benefit or could even be contraproductive in certain tumors, which has important implications for the design of BetA-based combination protocols.

Caspase-8L expression protects CD34+ hematopoietic progenitor cells and leukemic cells from CD95-mediated apoptosis.

Regulation of sensitivity or resistance for apoptosis by death receptor ligand systems is a key control mechanism in the hematopoietic system. Dysfunctional or deregulated apoptosis can potentially contribute to the development of immune deficiencies, autoimmune diseases, and leukemia. Control of homeostasis starts at the level of hematopoietic stem cells (HSC). To this end, we found that CD34+ hematopoietic progenitor cells are constitutively resistant to CD95-mediated apoptosis and cannot be sensitized during short-term culture to death receptor-mediated apoptosis by cytokines. Detailed analysis of the death machinery revealed that CD34+ cells do not express caspase-8a/b, a crucial constituent of the death-inducing signaling complex (DISC) of death receptors. Instead, we found a smaller splice variant termed caspase-8L to be present in HSC. Forced expression of caspase-8L using a recombinant lentiviral vector was able to protect hematopoietic cells from death receptor-induced apoptosis even in the presence of caspase-8a/b. Furthermore, we found that caspase-8L is recruited to the DISC after CD95 triggering, thereby preventing CD95 from connecting to the caspase cascade. These results demonstrate an antiapoptotic function of caspase-8L and suggest a critical role as apoptosis regulator in HSC. Similar to CD34+ HSC, stem cell-derived leukemic blasts from AML(M0) patients only expressed caspase-8L. Additionally we found, caspase-8L expression in several AML and ALL samples. Thus, caspase-8L expression might explain constitutive resistance to CD95-mediated apoptosis in CD34+ progenitor cells and might participate in the development of stem cell-derived and other leukemias by providing protection from regulatory apoptosis.