Tumor microenvironmental changes induced by the sulfamate carbonic anhydrase IX inhibitor S4 in a laryngeal tumor model

Abstract

Background and purpose: Carbonic anhydrase IX (CAIX) plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3'(3",5"-dimethylphenyl)-ureido)phenyl sulfamate (S4) on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding.

Methods: SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole) and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR.

Results: CAIX ectodomain shedding decreased after treatment with S4 (p<0.01). S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole) and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed.

Conclusion: As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.

Figure 1. Immunofluorescent images showing CAIX expression, pimonidazole, apoptosis and proliferation in SCCNij202.

CAIX is expressed in hypoxic regions as assessed by pimonidazole staining (A), but is also observed in non-pimonidazole areas (B). Yet, most of CAIX is expressed in pimonidazole positive regions (C). Proliferation (BrdUrd) and apoptosis (caspase-3) in relation to CAIX expression are shown in figure D and E respectively. Red, CAIX; Green, pimonidazole (A–B), BrdUrd (D) or caspase-3 (E); Yellow, overlap of CAIX (red) and pimonidazole (green); Light blue, vessels. Magnification 100×. Scale bars represent 100 µm. Closed circles represent CAIX expression in pimonidazole positive regions; open circles represent CAIX expression in pimonidazole negative regions. Abbrevations: 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.

Figure 2. CAIX ectodomain shedding after treatment with S4.

S4 decreases CAIX ectodomain shedding in the serum, especially 8 hours after the first injection. Abbreviations: CAIX, carbonic anhydrase IX; 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.

Figure 3. No cytotoxic effects of CAIX inhibition in SCCNij202.

S4 does not affect proliferation within CAIX positive areas (A), apoptosis within CAIX positive areas (B) or the amount of necrosis (C). Abbreviations: BrdUrd, bromodeoxyuridine; CAIX, carbonic anhydrase IX; LI, labeling index; NS, not significant, 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.

Figure 4. Intracellular pH and CAIX inhibition in SCCNij202.

S4 does not change CHOP mRNA (A) and MMP9 mRNA (B). Abbreviations: CHOP, C/EBP homologous protein; MMP9, matrix metallopeptidase 9; NS, not significant; 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.

Figure 5. Metabolism and CAIX inhibition in SCCNij202.

S4 does not influence the expression of several metabolic transporters and enzymes on the mRNA level. Abbreviations: ATP6V1C1, plasma membrane proton pump vacuolar ATPase (V-ATPase); CAIX, carbonic anhydrase IX; GLUT1, glucose transporter 1; MCT, monocarboxylate transporter; NHE1, sodium-hydrogen exchanger 1; NS, not significant; 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.