The functions of autophagy at the tumour-immune interface

Autophagy is frequently induced in the hypoxic tumour microenvironment. Accumulating evidence reveals important functions of autophagy at the tumour-immune interface. Herein, we propose an update on the roles of autophagy in modulating tumour immunity. Autophagy promotes adaptive resistance of established tumours to the cytotoxic effects of natural killer cells (NKs), macrophages and effector T cells. Increased autophagic flux in tumours dampen their immunogenicity and inhibits the expansion of cytotoxic T lymphocytes (CTLs) by suppressing the activation of STING type I interferon signalling (IFN-I) innate immune sensing pathway. Autophagy in suppressive tumour-infiltrating immune subsets maintains their survival through metabolic remodelling. On the other hand, autophagy is involved in the antigen processing and presentation process, which is essential for anti-tumour immune responses. Genetic deletion of autophagy induces spontaneous tumours in some models. Thus, the role of autophagy is context-dependent. In summary, our review has revealed the dichotomous roles of autophagy in modulating tumour immunity. Broad targeting of autophagy may not yield maximal benefits. The characterization of specific genes regulating tumour immunogenicity and innovation in targeted delivery of autophagy inhibitors into certain tumours are among the most urgent tasks to sensitize cold cancers to immunotherapy.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Radiotherapy Suppresses Angiogenesis in Mice through TGF-βRI/ALK5-Dependent Inhibition of Endothelial Cell Sprouting

Background

Radiotherapy is widely used to treat cancer. While rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence indicates that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity. In spite of its clinical relevance, such putative anti-angiogenic effect of radiotherapy has not been thoroughly characterized. We have investigated the effect of ionizing radiation on angiogenesis using in vivo, ex vivo and in vitro experimental models in combination with genetic and pharmacological interventions.

Principal Findings

Here we show that high doses ionizing radiation locally suppressed VEGF- and FGF-2-induced Matrigel plug angiogenesis in mice in vivo and prevented endothelial cell sprouting from mouse aortic rings following in vivo or ex vivo irradiation. Quiescent human endothelial cells exposed to ionizing radiation in vitro resisted apoptosis, demonstrated reduced sprouting, migration and proliferation capacities, showed enhanced adhesion to matrix proteins, and underwent premature senescence. Irradiation induced the expression of P53 and P21 proteins in endothelial cells, but p53 or p21 deficiency and P21 silencing did not prevent radiation-induced inhibition of sprouting or proliferation. Radiation induced Smad-2 phosphorylation in skin in vivo and in endothelial cells in vitro. Inhibition of the TGF-β type I receptor ALK5 rescued deficient endothelial cell sprouting and migration but not proliferation in vitro and restored defective Matrigel plug angiogenesis in irradiated mice in vivo. ALK5 inhibition, however, did not rescue deficient proliferation. Notch signaling, known to hinder angiogenesis, was activated by radiation but its inhibition, alone or in combination with ALK5 inhibition, did not rescue suppressed proliferation.

Conclusions

These results demonstrate that irradiation of quiescent endothelial cells suppresses subsequent angiogenesis and that ALK5 is a critical mediator of this suppression. These results extend our understanding of radiotherapy-induced endothelial dysfunctions, relevant to both therapeutic and unwanted effects of radiotherapy.     

p53 Stabilization Induces Cell Growth Inhibition and Affects IGF2 Pathway in Response to Radiotherapy in Adrenocortical Cancer Cells

Adrenocortical carcinoma (ACC) is a very rare endocrine tumour, with variable prognosis, depending on tumour stage and time of diagnosis. However, it is generally fatal, with an overall survival of 5 years from detection. Radiotherapy usefulness for ACC treatment has been widely debated and seems to be dependent on molecular alterations, which in turn lead to increased radio-resistance. Many studies have shown that p53 loss is an important risk factor for malignant adrenocortical tumour onset and it has been reported that somatic mutations in TP53 gene occur in 27 to 70% of adult sporadic ACCs. In this study, we investigated the role of somatic mutations of the TP53 gene in response to ionizing radiation (IR). We studied the status of p53 in two adrenocortical cell lines, H295R and SW-13, harbouring non-functioning forms of this protein, owing to the lack of exons 8 and 9 and a point mutation in exon 6, respectively. Moreover, these cell lines show high levels of p-Akt and IGF2, especially H295R. We noticed that restoration of p53 activity led to inhibition of growth after transient transfection of cells with wild type p53. Evaluation of their response to IR in terms of cell proliferation and viability was determined by means of cell count and TUNEL assay.^wtp53 over-expression also increased cell death by apoptosis following radiation in both cell lines. Moreover, RT-PCR and Western blotting analysis of some p53 target genes, such as BCL2, IGF2 and Akt demonstrated that p53 activation following IR led to a decrease in IGF2 expression. This was associated with a reduction in the active form of Akt. Taken together, these results highlight the role of p53 in response to radiation of ACC cell lines, suggesting its importance as a predictive factor for radiotherapy in malignant adrenocortical tumours cases.     

Purine nucleoside phosphorylase and fludarabine phosphate gene-directed enzyme prodrug therapy suppresses primary tumour growth and pseudo-metastases in a mouse model of prostate cancer

Gene-directed enzyme prodrug therapy based on the E. coli purine nucleoside phosphorylase (PNP) gene produces efficient tumour cell killing. PNP converts adenosine analogs into toxic metabolites that diffuse across cell membranes to kill neighbouring untransduced cells (PNP-GDEPT). Interference with DNA, RNA and protein synthesis kills dividing and non-dividing cells, an important consideration for slow-growing prostate tumours. This study examined the impact of administering PNP-GDEPT into orthotopically grown RM1 prostate cancers (PCas) on the growth of lung pseudo-metastases of immunocompetent mice. C57BL/6 mice bearing orthotopic RM1 PCas received a single intraprostatic injection of OAdV220 (10(10) particles), a recombinant ovine atadenovirus containing the PNP gene controlled by the Rous Sarcoma virus promoter, followed by fludarabine phosphate (approximately 600 mg/m(2)/day) administered intraperitoneally (ip) once daily for 5 days. Pseudo-metastases were induced 2 days after intraprostatic vector administration by tail-vein injection of untransduced RM1 cells. Mice given PNP-GDEPT showed a significant reduction both in prostate volume (approximately 50%) and in lung colony counts (approximately 60%). Apoptosis was increased two-fold in GDEPT-treated prostates compared with controls (P < 0.01), but was absent in the lungs. Staining for proliferating cell nuclear antigen (PCNA) indicated that proliferation of both RM1 prostate tumours (P < 0.01) and lung colonies (P < 0.01) was significantly suppressed after GDEPT. Although prostate tumour immune cell infiltration did not differ significantly between treatments, immunostaining for Thy-1.2 (CD90) showed that GDEPT promoted Thy-1.2(+) cell infiltration into the prostate tumour site. This study showed that a single course of PNP-GDEPT significantly suppressed local PCa growth and reduced lung colony formation in the aggressive RM1 tumour model.     

Differential radiosensitization of human tumour cells by 3-aminobenzamide and benzamide: inhibitors of poly(ADP-ribosylation)

The effect of 3-aminobenzamide (3AB) and benzamide (BZ) (inhibitors of poly(ADP-ribose) synthetase) on radiosensitivity was investigated in normal human fibroblasts and three human cell lines established from tumours with varying degrees of clinical radiocurability. The human tumour cell lines selected were: Ewing's sarcoma, a bone tumour usually considered radiocurable with moderate radiation doses; lung adenocarcinoma, a tumour considered radiocurable with high doses of radiotherapy; and osteosarcoma, a very resistant tumour which is rarely controlled by standard doses of radiotherapy. Poly(ADP-ribose) synthetase inhibitors were added to cultures 2 h prior to irradiation and removed 24 h after. Inhibitors were used at doses producing little or no toxicity in cells. In the presence of these inhibitors, a differential radiosensitization was observed. Ewing's sarcoma cells and normal human fibroblasts were sensitized to an equal extent by either 8 mM 3AB or 4 mM BZ. However, no sensitization was observed at these concentrations in the lung adenocarcinoma cells or osteosarcoma cells. The degree of radiosensitization in vitro by 3AB and BZ correlates well with the clinical radiocurability of these tumours in vivo.     

Immunotherapy success in prophylaxis cannot predict therapy: prime-boost vaccination against the 5T4 oncofoetal antigen

We have investigated the tumour therapeutic efficacy of homologous and heterologous prime-boost vaccine strategies against the 5T4 oncofoetal antigen, using both replication defective adenovirus expressing human 5T4 (Ad5T4), and retrovirally transduced DC lines (DCh5T4) in a subcutaneous B16 melanoma model (B16h5T4). In naïve mice we show that all vaccine combinations tested can provide significant tumour growth delay. While DCh5T4/Adh5T4 sequence is the best prophylactic regimen (P > 0.0001), it does not demonstrate any therapeutic efficacy in mice with established tumours. In active therapy the Adh5T4/DCh5T4 vaccination sequence is the best treatment regimen (P = 0.0045). In active therapy, we demonstrate that B16h5T4 tumour growth per se induces Th2 polarising immune responses against 5T4, and the success of subsequent vaccination is dependant on altering the polarizing immune responses from Th2 to Th1. We show that the first immunization with Adh5T4 can condition the mice to induce 5T4 specific Th1 immune responses, which can be sustained and subsequently boosted with DCh5T4. In contrast immunisation with DCh5T4 augments Th2 immune responses, such that a subsequent vaccination with Adh5T4 cannot rescue tumour growth. In this case the depletion of CD25⁺ regulatory cells after tumour challenge but before immunization can restore therapeutic efficacy. This study highlights that all vaccine vectors are not equal at generating TAA immune responses; in tumour bearing mice the capability of different vaccines to activate the most appropriate anti-tumour immune responses is greatly altered compared to what is found in naïve mice.     

A simplified tumour model established via Epstein-Barr virus-encoded, nasopharyngeal carcinoma-derived oncogene latent membrane protein 1 in immunocompetent mice

The expression and immune modulation of Epstein-Barr virus-encoded oncogene latent membrane protein 1 (N-LMP1) is essential in the pathogenesis of nasopharyngeal carcinoma. In previous studies, cell transformation has been induced by the expression of EBV-encoded N-LMP1 in non-tumour BALB/c-3T3 cells and these cells have then been used to form tumours in T-cell-deficient nude mice. However, studies using this model have been limited by the lack of a competent immune system. To facilitate the study of immune components in N-LMP1-driven oncogenesis, we herein developed a simplified N-LMP1-derived tumour model in immunocompetent mice. Cell transformation was induced by the expression of N-LMP1 in BALB/c-3T3 cells, and these transformants were used to induce oncogenesis in BALB/c mice. In contrast to the 100% successful tumour-induction rate in nude mice treated with monodispersed transformed cells, the tumour incidence in BALB/c mice was only 5-36%. However, the transplantation of tumour fragments into BALB/c mice yielded a reproducible tumour-induction rate of >85%, which is acceptable for most of the research needs. This novel model of N-LMP1-directed oncogenesis in an immunocompetent environment may serve as an important platform for the future assessment of N-LMP1-targeted tumour therapies.     

Sensitizing effect of 3-methyladenine on radiation-induced cytotoxicity in radio-resistant HepG2 cells in vitro and in tumor xenografts

Many recent efforts have focused on targeting cell death pathways for discovering new cancer therapies. The relative resistance of liver cancer cells to ionizing radiation (IR) and chemotherapeutic agents due to autophagic response limits the available treatment options for this type of cancer. In this study, 3-methyladenine (3-MA), an autophagy inhibitor, was investigated for its potential to enhance radio-sensitivity under radio-resistant conditions both in vitro and in vivo. Hep3B and HepG2 cells were used to examine the radio-resistance of liver cancer cells. The results show that Hep3B cells respond to irradiation with increased apoptotic cell death and that HepG2 is radio-resistant due to the IR-induced autophagy, as verified by DNA fragmentation, electron microscopy, acidic vesicular organelle formation, and Western blot analysis. Application of IR with 3-MA to inhibit autophagy simultaneously suppressed the expression of LC3 and enhanced cell death. The tumor xenograft model in nude mice verified the synergistic cytotoxic effect of 3-MA and IR, which resulted in significant repression of tumor growth. The results demonstrate that IR-induced autophagy provides a self-protective mechanism against radiotherapy in HepG2 cells. In addition, 3-MA enhances the cytotoxicity of IR in cell models and suppresses tumor growth in animal models. Based on the results, application of 3-MA, or other autophagy inhibitors, could be used as an adjuvant for radiotherapy when radio-resistance develops as a result of autophagy response.     

Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

High-grade gliomas, glioblastomas (GB), are refractory to conventional treatment combining surgery, chemotherapy, mainly temozolomide, and radiotherapy. This highlights an urgent need to develop novel therapies and increase the efficacy of radio/chemotherapy for these very aggressive and malignant brain tumors. Recently, tumor metabolism became an interesting potential therapeutic target in various cancers. Accordingly, combining drugs targeting cell metabolism with appropriate chemotherapeutic agents or radiotherapy has become attractive. In light of these perspectives, we were particularly interested in the anti-cancer properties of a biguanide molecule used for type 2 diabetes treatment, metformin. In our present work, we demonstrate that metformin decreases mitochondrial-dependent ATP production and oxygen consumption and increases lactate and glycolytic ATP production. We show that metformin induces decreased proliferation, cell cycle arrest, autophagy, apoptosis and cell death in vitro with a concomitant activation of AMPK, Redd1 and inhibition of the mTOR pathway. Cell sensitivity to metformin also depends on the genetic and mutational backgrounds of the different GB cells used in this study, particularly their PTEN status. Interestingly, knockdown of AMPK and Redd1 with siRNA partially, but incompletely, abrogates the induction of apoptosis by metformin suggesting both AMPK/Redd1-dependent and –independent effects. However, the primary determinant of the effect of metformin on cell growth is the genetic and mutational backgrounds of the glioma cells. We further demonstrate that metformin treatment in combination with temozolomide and/or irradiation induces a synergistic anti-tumoral response in glioma cell lines. Xenografts performed in nude mice demonstrate in vivo that metformin delays tumor growth. As current treatments for GB commonly fail to cure, the need for more effective therapeutic options is overwhelming. Based on these results, metformin could represent a potential enhancer of the cytotoxic effects of temozolomide and/or radiotherapy.     

Inhibitory effect of hydnocarpin D on T-cell acute lymphoblastic leukemia via induction of autophagy-dependent ferroptosis

Hydnocarpin D (HD) is a bioactive flavonolignan compound that possesses promising anti-tumor activity, although the mechanism is not fully understood. Using T cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat and Molt-4 as model system, we found that HD suppressed T-ALL proliferation in vitro, via induction of cell cycle arrest and subsequent apoptosis. Furthermore, HD increased the LC3-II levels and the formation of autophagolysosome vacuoles, both of which are markers for autophagy. The inhibition of autophagy by either knockdown of ATG5/7 or pre-treatment of 3-MA partially rescued HD-induced apoptosis, thus suggesting that autophagy enhanced the efficacy of HD. Interestingly, this cytotoxic autophagy triggered ferroptosis, as evidenced by the accumulation of lipid ROS and decrease of GSH and GPX4, while inhibition of autophagy impeded ferroptotic cell death. Our study suggests that HD triggers multiple cell death processes and is an interesting compound that should be evaluated in future preclinical studies.     

Mechanisms of "cytostasis" of tumours in vitro by syngeneic lymphoid cells of tumour bearers.

The cytostasis assay is an in vivo-in vitro radioactive technique which detects antitumour responses of the syngeneic tumour-bearing hosts. Examination and characterization of effector mechanisms at the cellular and humoral levels revealed that the cytostasis assay using Meth A (a 3-methylcholanthrene-induced) tumour was T cell independent. Furthermore, both B cells and macrophages were required. It was concluded that the mechanism involved complement-dependent antibody-mediated lysis of the tumour cells, with B cells producing antibody and macrophages producing the complement components during incubation. However, antibody-dependent cell-mediated cytotoxicity with or without complement could not be completely excluded. Although antibody was detected in vivo, specific antibody against Meth A tumour was produced in vitro by cultured lymphoid cells from the tumour-bearers. Antibody-coated Meth A cells caused regression of some tumours when inoculated into BALB/c mice. When these regressor mice were rechallenged with tumour, they were found to be permanently immune to the tumour. In the light of these findings, the role of antibody in the protection of tumours and its implications are discussed.     

TNFAIP8 regulates gastric cancer growth via mTOR-Akt-ULK1 pathway and autophagy signals

In this study, the purpose of this study was to investigate the role of TNFAIP8 in gastric cancer (GC). The expression of TNFAIP8 was detected by RT-PCR or western blot . TNFAIP8 was silenced or overexpressed in two cell lines. CCK-8 assay, transwell assay and flow cytometry were used to analyse cell viability, cell invasion capability and apoptosis, respectively. Nude mice were inoculated with TNFAIP8 silencing or overexpressing cells to form transplanted tumours. HE staining and immunohistochemistry assay were performed to assess histopathological changes in tumours. We found that the mRNA and protein expression of TNFAIP8 were significantly up-regulated in GC tumour tissues and cells compared with the normal counterparts. Overexpression of TNFAIP8 in GC cells increased cell viability, decreased apoptosis and promoted the cell migration ability. Meanwhile, increased expression of TNFAIP8 promoted autophagy, while inhibiting mTOR-Akt-ULK1 signal pathway. In conclusions, this study presents data that TNFAIP8 inhibits GC cells presumably by down-regulating mTOR-Akt-ULK1 signal pathway and activating autophagy signal.     

HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro

Background

We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions (e.g.G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo.

Methodology/Principal Findings

The effect of expression HYAL1 and HYAL2 was studied by colony formation inhibition, growth curve and cell proliferation tests in vitro and tumour growth assay in vivo. Very modest growth inhibition was detected in vitro in U2020 lung and KRC/Y renal carcinoma cell lines. In the in vivo experiment stably transfected KRC/Y cells expressing HYAL1 or HYAL2 were inoculated into SCID mice (10 and 12 mice respectively). Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60–67%).

Conclusions/Significance

The results suggest that the expression of either gene has led to inhibition of tumour growth in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment.     

PV1 down‐regulation via shRNA inhibits the growth of pancreatic adenocarcinoma xenografts

PV1 is an endothelial‐specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour‐bearing mice by single‐dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down‐regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC‐1 and BxPC‐3). The effect observed is because of down‐regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down‐regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.     

Characterization of fos-induced osteogenic tumours and tumour-derived murine cell lines

Osteogenic tumours from c-fos (MT-c-fos-LTR)-transgenic mice and from mice infected with the v-fos-bearing FBR murine osteosarcoma virus (FBR MSV) showed close morphological and neoplastic similarities. Fos mRNA expression was elevated in both types of tumours, and expression of several genes characteristic of differentiated bone cells was either lower, enhanced, or not detectable in comparison to that in normal bone. Tumour-derived cell lines showed variable levels of exogenous fos expression; bone-cell-specific genes were similarly expressed in both primary tumours and tumour-derived cell lines. Upon transplantation the tumour cells formed fibrosarcomas, some of which contained areas of focal osseochondrous differentiation. Non-tumorigenic cell lines established from bone tissue of normal and MT-c-fos-LTR transgenic mice showed osteoblastic characteristics, whereas no parathyroid hormone (PTH) response was observed in transgenic tumour cell lines in spite of high alkaline phosphatase activity. These data indicate that deregulated fos expression interferes with terminal osteogenic differentiation in v-fos- and c-fos-induced bone tumours.     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

Molecular Imaging of Induced Pluripotent Stem Cell Immunogenicity with In Vivo Development in Ischemic Myocardium

Whether differentiation of induced pluripotent stem cells (iPSCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection, is unclear. Here, we dynamically demonstrated the immunogenicity and rejection of iPSCs in ischemic myocardium using bioluminescent imaging (BLI). Murine iPSCs were transduced with a tri-fusion (TF) reporter gene consisting of firefly luciferase-red fluorescent protein-truncated thymidine kinase (fluc-mrfp-tTK). Ascorbic acid (Vc) were used to induce iPSCs to differentiate into cardiomyocytes (CM). iPSCs and iPS-CMs were intramyocardially injected into immunocompetent or immunosuppressed allogenic murine with myocardial infarction. BLI was performed to track transplanted cells. Immune cell infiltration was evaluated by immunohistochemistry. Syngeneic iPSCs were also injected and evaluated. The results demonstrated that undifferentiated iPSCs survived and proliferated in allogenic immunocompetent recipients early post-transplantation, accompanying with mild immune cell infiltration. With in vivo differentiation, a progressive immune cell infiltration could be detected. While transplantation of allogenic iPSC-CMs were observed an acute rejection from receipts. In immune-suppressed recipients, the proliferation of iPSCs could be maintained and intramyocardial teratomas were formed. Transplantation of syngeneic iPSCs and iPSC-CMs were also observed progressive immune cell infiltration. This study demonstrated that iPSC immunogenicity increases with in vivo differentiation, which will increase their chance for rejection in iPSC-based therapy.     

Resistance to Oncolytic Myxoma Virus Therapy in Nf1?/?/Trp53?/? Syngeneic Mouse Glioma Models Is Independent of Anti-Viral Type-I Interferon

Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 ^+/− Trp53 ^+/− mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection. To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/β is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.