Hypoxia-related proteins VEGF A and CA IX and resistance of soft tissue sarcoma cells to chemotherapy: ex vivo pilot study

Introduction. The identification of predictive factors is a cornerstone task of modern oncology. The development of new targeted drugs determines the need for prediction of chemosensitivity of each patient to the prescribed therapy, in this regard, the search for biomarkers of predictive response to therapy is actively conducted.

The study objective to investigate the relationship between tumor cell resistance and the expression levels of CA IX (carbonic anhydrase IX) and VEGF A (vascular endothelial growth factor А) in patient-derived cultures of soft tissue sarcomas.

Materials and methods: ex vivo soft tissue sarcoma cell culture, resazurin test, immunoblotting.

Results. We obtained 46 ex vivo samples of soft tissue sarcoma cultures for which chemosensitivity to doxorubicin, ifosfamide, docetaxel, gemcitabine, and their combinations was assessed by the resazurin cytotoxicity test. We analyzed the relationship between the expression of hypoxic proteins VEGF A and CA IX and the resistance to drugs. A correlation between the CA IX expression in hypoxia and cell resistance to ifosfamide and its combination with doxorubicin was found. Soft tissue sarcomas with high VEGF A index were resistant to doxorubicin, docetaxel, and its combination with gemcitabine (p <0.05).

Conclusion. The data obtained on patient-derived cultures indicate the relationship between hypoxic signaling and resistance of soft tissue sarcomas to chemotherapeutics.

1. Hoang N.T., Acevedo L.A., Mann M.J. et al. A review of soft-tissue sarcomas: translation of biological advances into treatment measures. Cancer Manag Res 2018;10:1089–114. DOI: 10.2147/CMAR.S159641.

2. Jimenez R.E., Zalupski M.M., Frank J.J. et al. Multidrug resistance phenotype in high grade soft tissue sarcoma: correlation of P-glycoprotein immunohistochemistry with pathologic response to chemotherapy. Cancer 1999;86(6):976–81. DOI: 10.1002/(sici)1097-0142(19990915)86:6<976::aidcncr12>3.0.co;2-3.

3. Hsu J.Y., Seligson N.D., Hays J.L. et al. Clinical utility of CDK4/6 Inhibitors in sarcoma: successes and future challenges. JCO Precis Oncol 2022;6:e2100211. DOI: 10.1200/po.21.00211.

4. Yuan J., Li X., Yu S. Molecular targeted therapy for advanced or metastatic soft tissue sarcoma. Cancer Control 2021;28:10732748211038424. DOI: 10.1177/10732748211038424.

5. Majidpoor J., Mortezaee K. Angiogenesis as a hallmark of solid tumors – clinical perspectives. Cell Oncol (Dordr) 2021;44(4):715–37. DOI: 10.1007/s13402-021-00602-3.

6. Ibrahim-Hashim A., Estrella V. Acidosis and cancer: from mechanism to neutralization. Cancer Metastasis Rev 2019;38(1–2):149–55. DOI: 10.1007/s10555-019-09787-4.

7. Swietach P. What is pH regulation, and why do cancer cells need it? Cancer Metastasis Rev 2019;38(1–2):5–15. DOI: 10.1007/s10555-018-09778-x.

8. Gatenby R.A., Gillies R.J. A microenvironmental model of carcinogenesis. Nat Rev Cancer 2008;8(1):56–61. DOI: 10.1038/nrc2255.

9. Becker H.M. Carbonic anhydrase IX and acid transport in cancer. Br J Cancer 2020;122(2):157–67. DOI: 10.1038/s41416-019-0642-z.

10. Chao C., Al-Saleem T., Brooks J.J. et al. Vascular endothelial growth factor and soft tissue sarcomas: tumor expression correlates with grade. Ann Surg Oncol 2001;8(3):260–7. DOI: 10.1007/s10434-001-0260-9.

11. Kilvaer T.K., Valkov A., Sorbye S. et al. Profiling of VEGFs and VEGFRs as prognostic factors in soft tissue sarcoma: VEGFR-3 is an independent predictor of poor prognosis. PloS One 2010;5(12):e15368. DOI: 10.1371/journal.pone.0015368.

12. Forker L., Gaunt P., Sioletic S. et al. The hypoxia marker CAIX is prognostic in the UK phase III VorteX-Biobank cohort: an important resource for translational research in soft tissue sarcoma. Br J Cancer 2018;118(5):698–704. DOI: 10.1038/bjc.2017.430.

13. Rodríguez-Corrales J., Josan J.S. Resazurin live cell assay: setup and finetuning for reliable cytotoxicity results. Methods Mol Biol 2017;1647:207–19. DOI: 10.1007/978-1-4939-7201-2_14.

14. Qi C.J., Ning Y.L., Zhu Y.L. et al. In vitro chemosensitivity in breast cancer using ATP-tumor chemosensitivity assay. Arch Pharm Res 2009;32(12):1737–42. DOI: 10.1007/s12272-009-2211-0.

15. Neubauer H., Stefanova M., Solomayer E. et al. Predicting resistance to platinumcontaining chemotherapy with the ATP tumor chemosensitivity assay in primary ovarian cancer. Anticancer Res 2008;28(2a):949–55.

16. Scherbakov A.M., Zhabinskii V.N., Khripach V.A. et al. Biological evaluation of a new brassinosteroid: antiproliferative effects and targeting estrogen receptor α pathways. Chem Biodivers 2019;16 (9):e1900332. DOI: 10.1002/cbdv.201900332.

17. Martin W.M., McNally N.J. Cytotoxicity of adriamycin to tumour cells in vivo and in vitro. Br J Cancer 1980;42(6):881–9. DOI: 10.1038/bjc.1980.336.

18. Yamauchi T., Raffin T.A., Yang P. et al. Differential protective effects of varying degrees of hypoxia on the cytotoxicities of etoposide and bleomycin. Cancer Chemother Pharmacol 1987;19(4):282–6. DOI: 10.1007/bf00261473.

19. Young S.D., Hill R.P. Effects of reoxygenation on cells from hypoxic regions of solid tumors: anticancer drug sensitivity and metastatic potential. J Nat Cancer Inst 1990;82(5):371–80. DOI: 10.1093/jnci/82.5.371.

20. Woods M.L., Koch C.J., Lord E.M. Detection of individual hypoxic cells in multicellular spheroids by flow cytometry using the 2-nitroimidazole, EF5, and monoclonal antibodies. Int J Radiat Oncol Biol Phys 1996;34(1):93–101. DOI: 10.1016/0360-3016(95)02006-3.

21. Harrison L., Blackwell K. Hypoxia and anemia: factors in decreased sensitivity to radiation therapy and chemotherapy? Oncologist 2004;9(Suppl. 5):31–40. DOI: 10.1634/theoncologist.9-90005-31.

22. Nordsmark M., Alsner J., Keller J. et al. Hypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutations. Br J Cancer 2001;84(8):1070–5. DOI: 10.1054/bjoc.2001.1728.

23. Måseide K., Kandel R.A., Bell R.S. et al. Carbonic anhydrase IX as a marker for poor prognosis in soft tissue sarcoma. Clin Cancer Res 2004;10(13):4464–71. DOI: 10.1158/1078-0432.ccr-03-0541.