In vivo tumor models

Solid tumors of AT-1 cells were induced in vivo in male Copenhagen rats (body weight 145–332 g) and Walker-256 tumors in Wistar rats (body weight 213–284 g), housed in the animal care facility of the University of Halle. All experiments had previously been approved by the regional animal ethics committee and were conducted in accordance with the German Law for Animal Protection and the UKCCCR Guidelines [28].

Solid tumors were induced by heterotopic injection of cell suspension (6-8 × 10⁶ cells/0.4 ml isotonic saline) subcutaneously into the dorsum of the hind foot. Tumor volumes were determined by measuring the three orthogonal diameters with a caliper and with the formula: V = d₁·d₂·d₃·π/6. Tumors were investigated when they reached a volume of 0.35–1.50 ml.

In order to induce a more pronounced tumor acidosis in vivo, two different methods were used. Firstly, metabolic acidosis was induced by treating tumor-bearing animals with a combination of inspiratory hypoxia and meta-iodobenzylguanidine (MIBG) which forces glycolytic metabolism [29]. Therefore, animals received a MIBG injection (20 mg/kg b.w., i.p. dissolved in isotonic saline) and were then housed in a hypoxic atmosphere containing 10% O₂ and 90% N₂ for 24 h. This procedure reduces the extracellular pH in AT1 tumors from 7.02 ± 0.04 to 6.48 ± 0.08 and in Walker-256 tumors from 7.16 ± 0.03 to 6.65 ± 0.07 [30]. Animals housed in room air receiving only the solvent served as control. Secondly, tissue acidosis was intensified by direct intratumoral injection of lactic acid. Therefore, 50 μl of 0.222 mM lactic acid (in H₂O) were injected into the tumor tissue at a depth of 2–3 mm. Treatment of the contralateral tumor with 50 μl of 0.222 mM sodium lactate served as intra-individual control.

After 24 h animals were sacrificed, tumors were surgically removed, minced and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), while protein was isolated by using CST cell lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2,5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na₃VO₄, protease inhibitor cocktail).