Cell encapsulation technology as a novel strategy for human anti-tumor immunotherapy.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjuvant in autologous cell-based anti-tumor immunotherapy has recently been approved for clinical application. To avoid the need for individualized processing of autologous cells, we developed a novel strategy based on the encapsulation of GM-CSF-secreting human allogeneic cells. GM-CSF-producing K562 cells showed high, stable and reproducible cytokine secretion when enclosed into macrocapsules. For clinical development, the cryopreservation of these devices is critical. Thawing of capsules frozen at different time points displayed differences in GM-CSF release shortly after thawing. However, similar secretion values to those of non-frozen control capsules were obtained 8 days after thawing at a rate of >1000 ng GM-CSF per capsule every 24 h. For future human application, longer and reinforced capsules were designed. After irradiation and cryopreservation, these capsules produced >300 ng GM-CSF per capsule every 24 h 1 week after thawing. The in vivo implantation of encapsulated K562 cells was evaluated in mice and showed preserved cell survival. Finally, as a proof of principle of biological activity, capsules containing B16-GM-CSF allogeneic cells implanted in mice induced a prompt inflammatory reaction. The ability to reliably achieve high adjuvant release using a standardized procedure may lead to a new clinical application of GM-CSF in cell-based cancer immunization.

Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy.

Adenosine is an inhibitor of neuronal activity in the brain. The local release of adenosine from grafted cells was evaluated as an ex vivo gene therapy approach to suppress synchronous discharges and epileptic seizures. Fibroblasts were engineered to release adenosine by inactivating the adenosine-metabolizing enzymes adenosine kinase and adenosine deaminase. After encapsulation into semipermeable polymers, the cells were grafted into the brain ventricles of electrically kindled rats, a model of partial epilepsy. Grafted rats provided a nearly complete protection from behavioral seizures and a near-complete suppression of afterdischarges in electroencephalogram recordings, whereas the full tonic-clonic convulsions in control rats remained unaltered. Thus, the local release of adenosine resulting in adenosine concentrations <25 nM at the site of action is sufficient to suppress seizure activity and, therefore, provides a potential therapeutic principle for the treatment of drug-resistant partial epilepsies.

Growth inhibition of liver metastases by the anti-angiogenic drug TNP-470.

OBJECTIVE: This study was undertaken in order to assess the efficacy of a potent angiogenesis inhibitor, TNP-470, on tumor growth in a syngeneic rodent model of liver metastases from colorectal cancer. BACKGROUND: New blood vessel formation is a prerequisite for primary and metastatic tumor growth. TNP-470, a synthetic derivative of fumagillin when subcutaneously transplanted into nude mice, inhibits endothelial cell proliferation and migration, as well as the growth of various human cancers. However, the antitumor effect of this drug has not been studied in models reproducing a natural metastatic environment. Since the liver provides an extensive vascular bed for secondary tumor growth, an anti-angiogenic strategy may therefore be less efficient for treating hepatic metastases than primary tumors. METHODS: 10(7) DHD K12 colon carcinoma cells were injected intrasplenically into syngeneic BD IX rats to produce diffuse liver metastases. TNP-470 (30 mg/kg/day) was administered on alternate days starting 4 days after tumor implantation. The animals were sacrificed after 4 weeks and their livers were processed for histologic examination. In both the treatment and control groups (n=7), tumor volume was determined using a computerized analytical system, and tumor microvessel density was measured by immunostaining with anti-von Willebrand Factor monoclonal antibody. RESULTS: In vitro, TNP-470 demonstrated a direct toxicity towards the DHD K12 cell line with an IC50 of 0.1 microg/ml. Metastases were present in all animals from both groups. Liver weight (15.2 g vs 11.7 g, p=0.01), and tumor volume (1218 mm3 vs 406 mm3, p=0.03) were significantly reduced in the TNP-470 group compared to the control group. Tumor microvessel density was not statistically different between the two groups (67 vs 63 microvessels/x200 field, p=0.41). CONCLUSION: TNP-470 inhibits the growth of liver metastases in a syngeneic rat model of colorectal cancer. The mechanism responsible for this effect remains unclear, but may involve a combination of anti-angiogenic and direct cytotoxic effects.

Transplantation of encapsulated bovine chromaffin cells in the sheep subarachnoid space: a preclinical study for the treatment of cancer pain.

Chromaffin cells have been shown to release a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides. The allogeneic transplantation of chromaffin cells in the subarachnoid space has been shown to alleviate pain in various rodent models and possibly in terminal cancer patients. Because of the shortage of human cadaver donor tissue, we are investigating the possibility of transplanting xenogeneic cells in polymer capsules. In this technique, cells are surrounded by a permselective synthetic membrane whose pores are suitably sized to allow diffusion of nutrients, neurotransmitters and growth factors, but restrict the diffusion of the large molecules of the immune system and prevent contact with immunocompetent cells. The encapsulation technique therefore allows transplantation of xenogeneic tissue between species as well as retrieval of transplanted cells. Previously we have reported that encapsulated bovine chromaffin cells survive and alleviate pain in various rodent models. The purpose of the present study was to assess the feasibility of implanting a human sized device in a large animal model. Adrenals from 5 calves were surgically removed; chromaffin cells were isolated from these glands using a collagenase-based digestion-filtration technique. Cells were loaded into acrylic-based tubular (5 cm long, 920 microns wide) permselective capsules attached to silicone tethers. The capsules were maintained in vitro for at least 7 days following the encapsulation procedure. Nicotine evoked release was analyzed in a defined subgroup from each batch. One capsule was then implanted using a guiding cannula system in the lumbar subarachnoid space of each sheep for 4 (n = 5) and 8 (n = 1) wk. All capsules were retrieved intact by gentle pulling on the silicone tether. Except for one capsule, the evoked catecholamine release of the retrieved capsules was in the same range as that of other capsules from the same cohort that had been maintained in vitro. All retrieved capsules were devoid of host cell reaction. Clusters of viable cells dispersed in an alginate immobilizing matrix were observed throughout all the implanted capsules. This study demonstrates the feasibility of transplanting functional encapsulated xenogeneic chromaffin cells into the cerebrospinal fluid of a large animal model using a capsule of appropriate dimensions for human implants. We believe that these results suggest the appropriateness of human clinical trials in patients suffering from refractory terminal cancer pain.