Research activity is focused on a study of cancer diseases in various animal models and on their therapy by techniques that are capable to elicit anti-tumour immunity. Main emphasis is laid now on monitoring of effects of the devitalizaton the radiofrequency ablation and the field of pulse vector magnetic potential on tumour tissue and on analyses of biological processes induced by these treatments. For such experiments, we utilize mainly two own cancer models – the MeLiM strain of miniature pigs with hereditary melanoma and the Lewis laboratory rats with sarcoma that develops after inoculation of tumour cells cultured in vitro.

analyses utilized

Antibody Array Protein Microchips (multiplex cytokine detection)
Hematological analysis (detection of basic hematological parameters
Histological staining of paraffin sections
Imunocyto- a imunohistochemical staining (expression of various proteins at cellular and tissue level)
Cell cultivation in vitro
MACS technique (separation of various cell types on magnetic column for further characterization)
Flow cytometry (phenotypization of immunocompetent cells, Annexin Assay)
Sandwich – ELISA (cytokine and immunoglobuline quantification)
Western blotting (detection of various proteins e.g. heat shock and extracellular matrix proteins)

collaboration with

Czech University of Life Sciences in Prague, Faculty of Agrobiology, Food and Natural Resources (Department of Veterinary Sciences)
Charles University in Prague, 1st Medical School (Institute of Biochemistry and Experimental Oncology)
Mendel University of Agriculture and Forestry in Brno (Institute of Chemistry and Biochemistry)
Institute of Microbiology AS CR, v.v.i. in Prague
AREKO s.r.o. v Praze
ENJOY s.r.o. v Brně
INRA-CEA, Laboratoire de Radiobiologie et Etude du Génome, Jouy en Josas, Francie

what is the devitalization?

effect of devitalization on a healthy tissue in experimental animals

The first devitalization experiments were performed in ministure pigs and laboratory rats in the Institute of Animal Physiology and Genetics in Liběchov roughly in 1980 year. They dealt with study of devitalization in healthy animals with aim to ascertain eventual side-effects of such treatment. Various tissues were treated by devitalization, such as segments of the small and large intestine (including its content!), parts of the stomach, the kidney, the lung and the liver. We never observed any septic state followed by death of treated animals. They survived without any healthy complications. No substantial increase in body temperature and no changes of heamatological parameters were revealed that would suggest an extensive inflammatory process after devitalization. The devitalized tissues were progressively destroyed and reconstructed, so that a minute fibrous residue was found after several weeks.

effect of devitalization on tumour tissue in experimental animals

Using directed selection we established an original cancer model in the Institute of Animal Physiology and Genetics in Liběchov - a strain of miniature pigs that was designated with acronym MeLiM (Melanoma-bearing Libechov Minipig). Melanoma in this strain is heritable. Multiple skin nodular tumours (i.e. the most aggressive form of melanoma) appear on various parts of body in about a half of piglets. Their histological, immunohistochemical a biochemical characterization and a broad melanoma cell dissemination, which results in numerous organ metastases (mainly in the spleen, the lung, the liver and lymph nodes) followed with a death of affected animals, clearly document similarities with human melanoma and malignant behaviour of this porcine cancer. Melanoma is one of the most problematic cancers in present clinical oncology. Its incidence is continually increasing throughout the world by approximately 5% per year. Treatment of patients with regional or distal metastases is usually inefficient by standard procedures such as surgery, chemo- and radiotherapy. Thus, new more effective techniques are searched. The MeLiM animals are very suitable for this purpose. Minipigs with multiple nodular melanomas and metastases in lymph nodes and visceral organs (in the spleen and the liver) were used for devitalization experiments. One cutaneous melanoma was devitalized by the partially overlapping mattress sutures without any excision of tumour tissue. Such treatment caused a total destruction of melanoma cells in all cutaneous tumours as well as in all organ metastases observed before devitalization. Similar results were found if one whole metastatic lymph node or a part of metastatic spleen were devitalized. We did not observe any health complications after devitalization. Minipigs were fully healed of tumour cells during 4-6 months after devitalization and no tumour relapses were observed in the treated animals. Almost one hundred minipigs was treated by devitalization up to now with effectiveness almost 85%.

Results, which we found with therapy of porcine melanoma, confirmed our hypothesis about induction of anti-cancer immune reaction by devitalizaton. We ascertained a high expression of heat shock proteins HSP70 and gp96 by indirect immunofluorescence in the devitalized tumour. It is known that these proteins bind tumour-specific peptides and such complexes are capable to elicite the immune reaction though antigen-presenting cells. Using flow cytometry we observed thereafter a high concentration of the cytotoxic T lymphocytes in all melanomas. On the contrary, a negligible number of these immune cells was found in growing melanomas before devitalization. The cytotoxic T lymphocytes are effector immune cells that can specifically destroy tumour cells in all localities - in primary tumours as well as in organ metastases. Taken together, these results suggest that the devitalized tumour tissue induces cell-mediated anti-tumour immune reaction. Thus, the devitalization technique could be counted as one of immunotherapeutic procedures (specific active immunotherapy by autologous necrotic tumour tissue), which are in a centre of attention in many experimental and clinical oncological studies during last years. To clarify particularly biological processes that are induced by devitalization and that result in a general tumour tissue destruction we perform further experiments. To verify a general applicability, we carried out the devitalization also in about 90 dams of various age and breeds with histologically confirmed mammary gland carcinoma. Tumour destruction induced by devitalization was observed also in these animals. The effectiveness of this treatment was dependent on animal age (80% in younger dogs till 10 years of age, 40% in older animals). Inoculated sarcoma in the Lewis laboratory rat is another animal model in that we test effect of tumour devitalization. All ascertained results suggest that the unique anti-tumour effect of devitalization is neither species-specific nor tumour type-specific. Thus, the devitalization technique could be probably applicable with success also in therapy of some malignant tumours in clinical praxis as it was shown already in several patients with inoperable tumours of digestive tract (Fortýn et al. - Z. exp. Chir. Transplant. künstl. Organe 18:42-50, 1985; Fortýn et al. - Klin. onkol. (in Czech) 1: 7-10, 1989).

radiofrekvenční ablace jako potenciální imunoterapeutický postup pro léčbu nádorů?

Radiofrequency ablation (RFA) is a minimally invasive palliative technique utilized in clinical oncology for destruction of unresectable hepatic, pulmonary and renal malignancies. Using special devise with a heat electrode the tumours are destroyed very rapidly by high temperatures that vary from 60°C to 80°C. We test effects of RFA in both our models – the MeLiM miniature pigs and the Lewis rats with inoculated sarcoma. We search such conditions of RFA that would be sublethal for cancer cells. They would cause a slow destruction and heat stress in cancer cells. Such treatment could increase expression of heat shock proteins followed with induction of cell-mediated anti-tumour immunity (similarly as does the devitalization).

head of laboratory	Vratislav Horak, Dr., Ph.D.
researchers	Monika Burocziova, Dr., Ph.D. Jan Strnadel, Dr., Ph.D. Luca Vannucci, Prof., M.D., Ph.D.
PhD student	Daniela Planska, M.Sc. Monika Holubova, M.Sc. Michal Vure, DVM.
technician	Jaroslava Sestakova Jitka Klucinova