Hypoxic 3D in vitro culture models reveal distinct resistance processes to TKIs in renal cancer cells

Abstract

BackgroundThe aim of this study is to determine the effect of hypoxia on axitinib and sorafenib-treated renal cell carcinoma (RCC) cells. Hypoxia is a crucial factor influencing transcription process via protein modulation, which was shown i.e. in pancreatic cancer. Until now, hypoxia has been defined as associated with poorer outcome and inducing chemotherapy resistance in solid tumors. The unique phenomenon of pseudo-hypoxia connected with vhl mutation was observed in clear-cell, but not in papillary RCC, and the treatment of this subtype of cancer is still challenging. Despite the introduction of new antiangiogenic targeted therapies (inter alia tyrosine kinase inhibitors, TKIs), patients still develop both primary and acquired resistance. Overcoming resistance to TKIs, also in papillary RCC, may be possible by finding significantly modified protein expression. To do this, hypoxic 3D in vitro models must be developed to mimic both molecular pathways typical for low oxygen tension and cell–cell dynamics in tumor-like spatial structures.ResultsClear-cell and papillary renal cell carcinoma (cc and pRCC) cell lines were used in the study to determine the impact of hypoxia on primary drug resistance phenomenon previously observed in papillary, but not in ccRCC. Resistance was confirmed in monolayer culture and in 3D models in soft agar and suspension culture. Human papillary kidney cancer stem-like cells (HKCSCs) cultured in hypoxia developed resistance to sorafenib, while when cultured in normoxia resistance to axitinib has developed. Flow cytometry revealed that hypoxia decreased proliferation rates in all investigated RCC cells. In HKCSCs, there was an increase of quiescent cells (Ki67−) and percentage of cells arrested in S phase. It also appeared that map2k1 and eif4b protein expression is altered in papillary RCC resistant to tested drugs at different oxygen tensions. Also, HKCSCs did not express vegfr-1, braf nor c-kit, TKIs target receptors, which were present in ccRCC cells sensitive to TKI treatment.ConclusionsThe results confirm that low oxygen tension affects RCC cells. Hypoxia facilitates induction of sorafenib resistance in pRCC and induces map2k1 overexpression, while normoxic axitinib-resistant cells up-regulated eif4b. Further studies may determine if map2k1 or eif4b proteins play a role in pRCC resistance to TKIs. It is also of interest to establish if other than vegfr-1, braf, c-kit receptors can serve as potential molecular targets for more effective anti-RCC strategies.

Development of extracellular matrix supported 3D culture of renal cancer cells and renal cancer stem cells

“…Regarding 2D conditions, we have confirmed in stem cell promoting conditions, what has been shown in previous regular cell culture studies (Bielecka et al 2017 ; Gotink et al 2011 ), that kinase inhibitors effectively inhibit RCC cancer cell proliferation in higher concentrations (Fig. 6 , top; Figure S5), including both typical anti-RCC compounds sunitinib, and sorafenib (Figure S5, S6) and in typical normoxic (21% O2, as well as intra-tumoral like hypoxic conditions (Figure S7).…”

“…Such phenomenon has been already described in the literature (Paez-Ribes et al 2009 ). Although Temsirolimus used in this study for RCC cells treatment assessment is actually an antiangiogenic agent—an mTOR kinase inhibitor targeting endothelial cells, it should be noted that targeted therapy was already reported to also have direct cytostatic effect on RCC cells (Bielecka et al 2017 ; Gotink et al 2011 ). What is more, in the 3D model we can possibly observe other than cytostatic effect of kinase inhibitors on RCC cells.…”

“…No. 36117-44, Bielecka et al 2017 ) cell lines were used. During subsequent stages of the research, chosen cell lines were analyzed.…”

Stabilization of Hypoxia-Inducible Factors and BNIP3 Promoter Methylation Contribute to Acquired Sorafenib Resistance in Human Hepatocarcinoma Cells

“…Hypoxia contribution to chemoresistance development in HCC has been described after the administration of drugs, such as 5-fluorouracil (5-FU) [25], doxorubicin, and cisplatin [26]. Involvement of hypoxic environment in the acquired resistance to sorafenib has also been reported in renal cancer [27,28], gastric cancer [29], and acute myeloid leukaemia [30]. In samples of patients with untreated HCC sensitive and resistant to sorafenib, intratumoral hypoxia increased in patients resistant to sorafenib as opposed to sensitive ones [20].…”

Renal carcinoma CD105−/CD44− cells display stem-like properties in vitro and form aggressive tumors in vivo

“…In order to analyze appropriate RCC subtype specific cell lines must be selected based on their confirmed histology 9 . Data on the expression of markers should be interpreted along with the origin of RCC cells including tumor type and site of origin 8,20 . For initial characterization of RCC-CSCs candidates preselected cell surface were used and cells were isolated including CD105+ cells 13 and CD133+ cells 15 from nephrectomy specimens (primary tumor); CD44+ (ALDH1+) cells -from 293T human embryonic kidney cell line 10,11 ; CXCR4+ cells from RCC-26, RCC-53 cell lines 17 .…”