Jordi Martinez-Quintanilla

Therapeutic Efficacy and Fate of Bimodal Engineered Stem Cells in Malignant Brain Tumors

Therapeutically engineered stem cells (SC) are emerging as an effective tumor‐targeted approach for different cancer types. However, the assessment of the long‐term fate of therapeutic SC post‐tumor treatment is critical if such promising therapies are to be translated into clinical practice. In this study, we have developed an efficient SC‐based therapeutic strategy that simultaneously allows killing of tumor cells and assessment and eradication of SC after treatment of highly malignant glioblastoma multiforme (GBM). Mesenchymal stem cells (MSC) engineered to co‐express the prodrug converting enzyme, herpes simplex virus thymidine kinase (HSV‐TK) and a potent and secretable variant of tumor necrosis factor apoptosis‐inducing ligand (S‐TRAIL) induced caspase‐mediated GBM cell death and showed selective MSC sensitization to the prodrug ganciclovir (GCV). A significant decrease in tumor growth and a subsequent increase in survival were observed when mice bearing highly aggressive GBM were treated with MSC coexpressing S‐TRAIL and HSV‐TK. Furthermore, the systemic administration of GCV post‐tumor treatment selectively eliminated therapeutic MSC expressing HSV‐TK in vitro and in vivo, which was monitored in real time by positron emission‐computed tomography imaging using 18F‐FHBG, a substrate for HSV‐TK. These findings demonstrate the development and validation of a novel therapeutic strategy that has implications in translating SC‐based therapies in cancer patients. STEM Cells 2013;31:1706–1714

Bioselection of a Gain of Function Mutation that Enhances Adenovirus 5 Release and Improves Its Antitumoral Potency

Genetic bioselection of a mutagenized Ad5wt stock in human tumor xenografts led us to isolate AdT1, a mutant displaying a large-plaque phenotype in vitro and an enhanced systemic antitumor activity in vivo. AdT1 phenotype correlates with an increased progeny release without affecting total viral yield in different human tumors and cancer-associated fibroblasts. An approach combining hybrid Ad5/AdT1 recombinants and sequencing identified a truncating insertion in the endoplasmic reticulum retention domain of the E3/19K protein (445A mutation) which relocates the protein to the plasma membrane and is responsible for AdT1s enhanced release. E3/19K-445A phenotype does not correlate with the proteins ability to interact with MHC-I or induce apoptosis. Intracellular calcium measurement revealed that the 445A mutation induces extracellular Ca(2+) influx, deregulating intracellular Ca(2+) homeostasis and inducing membrane permeabilization, a viroporin-like function. E3/19K-445A mutants also display enhanced antitumoral activity when injected both intratumorally and systemically in different models in vivo. Our results indicate that the inclusion of mutation 445A in tumor-selective adenoviruses would be a very powerful tool to enhance their antitumor efficacy.

Minimal RB-responsive E1A Promoter Modification to Attain Potency, Selectivity, and Transgene-arming Capacity in Oncolytic Adenoviruses

Oncolytic adenoviruses are promising anticancer agents due to their ability to self-amplify at the tumor mass. However, tumor stroma imposes barriers difficult to overcome by these agents. Transgene expression is a valuable strategy to counteract these limitations and to enhance antitumor activity. For this purpose, the genetic backbone in which the transgene is inserted should be optimized to render transgene expression compatible with the adenovirus replication cycle and to keep genome size within the encapsidation size limit. In order to design a potent and selective oncolytic adenovirus that keeps intact all the viral functions with minimal increase in genome size, we inserted palindromic E2F-binding sites into the endogenous E1A promoter. The insertion of these sites controlling E1A-Δ24 results in a low systemic toxicity profile in mice. Importantly, the E2F-binding sites also increased the cytotoxicity and the systemic antitumor activity relative to wild-type adenovirus in all cancer models tested. The low toxicity and the increased potency results in improved antitumor efficacy after systemic injection and increased survival of mice carrying tumors. Furthermore, the constrained genome size of this backbone allows an efficient and potent expression of transgenes, indicating that this virus holds promise for overcoming the limitations of oncolytic adenoviral therapy.

Stem Cells Loaded With Multimechanistic Oncolytic Herpes Simplex Virus Variants for Brain Tumor Therapy

BACKGROUND The current treatment regimen for malignant glioblastoma multiforme (GBM) is tumor resection followed by chemotherapy and radiation therapy. Despite the proven safety of oncolytic herpes simplex virus (oHSV) in clinical trials for GBMs, its efficacy is suboptimal mainly because of insufficient viral spread after tumor resection. METHODS Human mesenchymal stem cells (MSC) were loaded with oHSV (MSC-oHSV), and their fate was explored by real-time imaging in vitro and in vivo. Using novel diagnostic and armed oHSV mutants and real-time multimodality imaging, the efficacy of MSC-oHSV and its proapoptotic variant, oHSV-TRAIL encapsulated in biocompatible synthetic extracellular matrix (sECM), was tested in different mouse GBM models, which more accurately reflect the current clinical settings of malignant, resistant, and resected tumors. All statistical tests were two-sided. RESULTS MSC-oHSVs effectively produce oHSV progeny, which results in killing of GBMs in vitro and in vivo mediated by a dynamic process of oHSV infection and tumor destruction. sECM-encapsulated MSC-oHSVs result in statistically significant increased anti-GBM efficacy compared with direct injection of purified oHSV in a preclinical model of GBM resection, resulting in prolonged median survival in mice (P < .001 with Gehan-Breslow-Wilcoxin test). To supersede resistant tumors, MSC loaded with oHSV-TRAIL effectively induce apoptosis-mediated killing and prolonged median survival in mice bearing oHSV- and TRAIL-resistant GBM in vitro (P < .001 with χ(2) contingency test). CONCLUSIONS Human MSC loaded with different oHSV variants provide a platform to translate oncolytic virus therapies to clinics in a broad spectrum of GBMs after resection and could also have direct implications in different cancer types.

Encapsulated Stem Cells Loaded With Hyaluronidase-expressing Oncolytic Virus for Brain Tumor Therapy

Despite the proven safety of oncolytic viruses (OV) in clinical trials for glioblastoma (GBM), their efficacy has been hindered by suboptimal spreading within the tumor. We show that hyaluronan or hyaluronic acid (HA), an important component of extracellular matrix (ECM), is highly expressed in a majority of tumor xenografts established from patient-derived GBM lines that present both invasive and nodular phenotypes. Intratumoral injection of a conditionally replicating adenovirus expressing soluble hyaluronidase (ICOVIR17) into nodular GBM, mediated HA degradation and enhanced viral spread, resulting in a significant antitumor effect and mice survival. In an effort to translate OV-based therapeutics into clinical settings, we encapsulated human adipose-derived mesenchymal stem cells (MSC) loaded with ICOVIR17 in biocompatible synthetic extracellular matrix (sECM) and tested their efficacy in a clinically relevant mouse model of GBM resection. Compared with direct injection of ICOVIR17, sECM-MSC loaded with ICOVIR17 resulted in a significant decrease in tumor regrowth and increased mice survival. This is the first report of its kind revealing the expression of HA in GBM and the role of OV-mediated HA targeting in clinically relevant mouse model of GBM resection and thus has clinical implications.

A modified E2F-1 promoter improves the efficacy to toxicity ratio of oncolytic adenoviruses.

The E2F-1 promoter has been used to confer tumor-selective E1A expression in oncolytic adenoviruses. Tumor specificity is mainly conferred by a unique structure of E2F-responsive sites organized in palindromes. Binding of the E2F–pRb complex to these palindromes results in repression of transcription in normal cells. Owing to deregulation of the Rb/p16 pathway in tumor cells, binding of free E2F activates transcription and initiates an autoactivation loop involving E1A and E4-6/7. ICOVIR-7 is a new oncolytic adenovirus designed to increase the E2F dependency of E1A gene expression. It incorporates additional palindromes of E2F-responsive sites in an insulated E2F-1 promoter controlling E1A-Δ24. The E2F palindromes inhibited replication in normal cells, resulting in a low systemic toxicity at high doses in immunocompetent mice. The Δ24 deletion avoids a loop of E2F-mediated self-activation in nontumor cells. Importantly, the additional E2F-binding hairpins boost the positive feedback loop on the basis of E1A-mediated transcriptional regulation of E4-6/7 turned on in cancer cells and increased antitumoral potency as shown in murine subcutaneous xenograft models treated by intravenous injection. These results suggest that the unique genetic combination featured in ICOVIR-7 may be promising for treating disseminated neoplasias.

Targeting breast to brain metastatic tumours with death receptor ligand expressing therapeutic stem cells

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study, we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging, we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next, we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain, we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective, potent and secretable variant of a TRAIL, S-TRAIL, and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore, the incorporation of pro-drug converting enzyme, herpes simplex virus thymidine kinase, into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications.

Positive selection of gene-modified cells increases the efficacy of pancreatic cancer suicide gene therapy

Thymidine kinase (TK)-mediated suicide gene therapy has been considered for the treatment of pancreatic cancer. However, despite a bystander effect, the proportion of transduced tumor cells has proven too low to result in efficacy. We propose the use of a drug-selectable marker (MDR1) to enrich TK-expressing cells using chemotherapy. This enrichment or positive selection phase may increase the efficacy of suicide gene therapy. To test this strategy, we generated stable NP18MDR/TK-GFP transfectants and showed docetaxel resistance in vivo. Mixed tumors of MDR/TK-expressing cells and parental NP18 cells were established and docetaxel was used to increase the proportion of TK-expressing cells. After this positive selection phase, suicide gene therapy with ganciclovir was applied. Upon positive selection, the proportion of TK-expressing cells increased from 4% to 22%. Subsequent suicide gene therapy was more effective compared with a control group without positive selection. Starting with 10% of TK-expressing cells the positive-negative selection strategy completely inhibited tumor growth. Taken together, these results suggest that a positive-negative selection strategy based on MDR and TK genes represents an efficient way to increase the proportion of TK-expressing cells in the tumor and the efficacy of TK-mediated suicide gene therapy. [Mol Cancer Ther 2009;8(11):3098–107]

Antitumor therapy based on cellular competition.

A major obstacle for the efficacy of cancer gene therapy is the need to transduce a high proportion of tumor cells with genes that directly or indirectly cause their death. During the formation of certain organs, cells compete among themselves to colonize the whole tissue. We reasoned that cell competition could be used to increase the proportion of cells that become transfected in a tumor. For this, a transgene that provides a selective advantage to the transfected cells should be used. If the same gene conferred a suicide mechanism the tumor could be eradicated after a period of selection. Bystander effect of transfected cells over neighboring nonmodified cells may eliminate tumors even with incomplete replacement of tumor cells. To test this strategy a competitive advantage was provided to colon cancer cells, using a gene encoding a fusion protein of dihydrofolate reductase (DHFR) and thymidine kinase (TK). DHFR confers resistance to methotrexate (MTX) and TK confers sensitivity to ganciclovir (GCV). Modified cells were also transduced with green fluorescent protein and parental cells with red fluorescent protein. In vitro and in vivo experiments were performed, using various proportions of modified cells and applying positive selection with MTX followed by negative selection with GCV. In vitro, cell competition was evident. Under MTX treatment, tumor cells transfected with the DHFR-TK fusion gene efficiently replaced the parental cells (from 0.1 to 90% in 35 days). After this positive selection period, negative selection with GCV eliminated the transfected cells. In vivo, positive selection was also achieved and resulted in a statistically significant therapeutic effect.

Somatostatin receptor type 2 as a radiotheranostic PET reporter gene for oncologic interventions

Reporter gene systems can serve as therapy targets. However, the therapeutic use of reporters has been limited by the challenges of transgene delivery to a majority of cancer cells. This study specifically assesses the efficacy of targeting human somatostatin receptor subtype 2 (hSSTR2) with peptide receptor radionuclide therapy (PRRT) when a small subpopulation of cells bears the transgene. Methods: The hSSTR2 transgene was delivered to A549 and Panc-1tumors using the lentiviral vector, LV-hSSTR2-IRES-GFP or murine mesenchymal stem cells (mMSC)s using a retroviral vector. SSTR2 expression was assessed using Western blot and correlated to GFP fluorescence and 68Ga-DOTATOC uptake. Wild type (WT), transduced (TD), and mixed population A549 or Panc-1 xenografts were implanted in nude mice. Separate groups with A549WT and Panc-1WT tumors received intratumoral injection of SSTR2-expressing mMSCs. Tumor-bearing mice were treated with 90Y-DOTATOC or saline and evaluated with 68Ga-DOTATOC PET before and after treatment. Results: Cell studies showed a strong correlation between 68Ga-DOTATOC uptake and SSTR2 expression in A549 (p < 0.004) and Panc-1 cells (p < 0.01). 68Ga-DOTATOC PET SUVmean was 8- and 5-fold higher in TD compared to WT A549 and Panc-1 tumors, respectively (p < 0.001). After 90Y-DOTATOC treatment, 100% TD and mixed population TD xenografts showed growth cessation while the WT xenografts did not. A549WT and Panc-1WT tumors with SSTR2-expressing mMSCs treated with 90Y-DOTATOC showed significantly lower tumor volumes compared to controls (p < 0.05). 68Ga-DOTATOC PET SUVmean of treated TD tumors monotonically declined and was significantly lower than that of non-treated xenografts. Conclusions: We showed that SSTR2 delivery to a small population of cells in tumor in conjunction with PRRT is effective in tumor growth cessation. The availability of various transgene delivery methods for hSSTR2 and radiotherpaeutic somatostatin analogs highlights the direct translational potential of this paradigm in the treatment of various cancers.