Influence of hyperthermal regimes on experimental teratoma development in vitro

Abstract

SummaryWe screened for the impact of hyperthermal regimes varying in the cumulative equivalent minutes at 43°C (CEM43°C) and media composition on tumour development using an original teratoma in vitro model. Rat embryos (three germ layers) were microsurgically isolated and cultivated at the air‐liquid interface. During a two week period, ectodermal, mesodermal and endodermal derivatives developed within trilaminar teratomas. Controls were grown at 37°C. Overall growth was measured, and teratoma survival and differentiation were histologically assessed. Cell proliferation was stereologically quantified by the volume density of Proliferating Cell Nuclear Antigen. Hyperthermia of 42°C, applied for 15 minutes after plating (CEM43°C 3.75 minutes), diminished cell proliferation (P ˂ .0001) and enhanced differentiation of both myotubes (P ˂ .01) and cylindrical epithelium (P ˂ .05). Hyperthermia of 43°C applied each day for 30 minutes during the first week (CEM43°C 210 minutes) impaired overall growth (P ˂ .01) and diminished cell proliferation (P ˂ .0001). Long‐term hyperthermia of 40.5°C applied for two weeks (CEM43°C 630 minutes) significantly impaired survival (P ˂ .005). Long‐term hyperthermia of 40.5°C applied from the second day when differentiation of tissues begins (CEM43°C 585 minutes) impaired survival (P ˂ .0001), overall growth (P ˂ .01) and cartilage differentiation (P ˂ .05). No teratomas survived extreme regimes: 43°C for 24 hours (CEM43°C 1440 minutes), hyperthermia in the scant serum‐free medium (CEM43°C 630 minutes) or treatment with an anti‐HSP70 antibody before long‐term hyperthermia 40.5°C from the second day (CEM43°C 585 minutes). This in vitro research provided novel insights into the impact of hyperthermia on the development of experimental teratomas from their undifferentiated sources and are thus of potential interest for future therapeutic strategies in corresponding in vivo models.

Embryo‐derived teratoma in vitro biological system reveals antitumor and embryotoxic activity of valproate

“…Because both the overall growth and differentiation of embryo‐derived teratomas were severely affected with 2 m m VPA, we were interested in whether VPA also influenced the potential for cell proliferation that is known to remain in controls at the end of the culture period [28]. The proliferation marker [proliferating cell nuclear antigen (PCNA)] was found in both VPA‐treated and untreated embryo‐derived teratomas after the 14 days of culture (Fig.…”

“…Therefore, this model may be used for further screening of other HDACis or different dosages of VPA, as well as for other developmental hazards influencing the embryo itself. As the gastrulating human embryo is still not accessible for research [2016, 2016], only the rodent embryos may be investigated at this most critical phase of mammalian development in defined in vitro systems such as this [19,28].…”

“…Among drugs we tested so far was the DNA hypomethylating agent 5‐azacytidine that is used for the epigenetic therapy of myelodysplastic syndrome [23,24] and retinoic acid [25] used for the differentiation therapy of leukemia [26] or widespread acne vulgaris [27]. Moreover, we have recently described specific hyperthermal regimes able to destroy experimental teratoma tumors or enhance their differentiation in vitro [28].…”

“…Differentiation of tissues is possible only at the air–liquid interface in the optimal rat serum‐supplemented medium, while submerged embryos‐proper do not differentiate into a teratoma [29,30]. In the natural three‐dimensional (3D) system, where tissue interactions are not as disrupted as they are during the establishment of two‐dimensional (2D) cell cultures [31], a parallel assessment of the survival, overall growth, and differentiation is conducted [28].…”

“…Employment of in vitro biological systems tends to reduce the number of living animals in experiments according to the replacement, reduction, and refinement (3Rs) rule of Russel and Burch [36–39]. Because the embryo‐derived teratoma system starts with the critical developmental stage of gastrulation when the ‘all or none’ rule is lost from embryos and a therapy may result in congenital anomalies [12,40,41], this system also reveals the developmental toxicity of embryotoxic/teratogenic drugs or physical factors [24,25,28].…”