HEAT RESPONSE OF HT29 CELLS DEPENDS STRONGLY ON PERFUSION - A P-31 NMR-SPECTROSCOPY, HPLC AND CELL-SURVIVAL ANALYSIS

A model system of perfused human colon adenocarcinoma cells (HT29) enc apsulated in alginate was used to examine metabolic response to heat t herapy with P-31 NMR spectroscopy, HPLC and cell survival analysis. Th e presented data show, that perfused (medium how during hyperthermia) and non-perfused (no medium flow during hyperthermia) cells are very d ifferent in their sensitivity to hyperthermia. Under equivalent experi mental conditions with respect to medium pH, oxygen and nutrient conce ntration, encapsulated perfused HT29 cells display a significantly low er thermal sensitivity than non-perfused cells. This reduced sensitivi ty of perfused cells is characterized by an increased cell survival an d relative ATP concentration, and reduced drop of the NTP/P-i ratio in the long-term follow up towards zero. The relative ATP concentration determined by HPLC after hyperthermia is correlated with the clonogeni c survival fraction. There is a direct relationship, depending on the specific experimental conditions (perfused, non-perfused). For perfuse d cells only a slight dependency of survival and relative ATP concentr ation on heat dose is observed. In consequence, the correlation betwee n survival and relative ATP concentration is weak, described by log(SF perf) = 0.7[ATP]-12.4, R(2) = 0.79, p < 0.04. For non-perfused cells the correlation is stronger resulting in a relationship of log(SFno pe rf) = 0.6[ATP]-9.0, R(2) = 0.98, P < 0.0002. Altogether, the presente d data suggest that the relative ATP concentration measured by HPLC af ter hyperthermia might be predictive for cell survival. On the other h and, a dependence between cell survival and long-term changes of NTP/P -i has been found. The results confirm the importance of tumour perfus ion for hyperthermia-induced metabolic changes and cytotoxicity and th erefore, for the therapeutic outcome.