Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells

Abstract

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.

Figure 1

Colon cancer stem cells (CSCs) are resistant toward chemotherapy. (a) Spheroid primary cultures were derived from primary human colorectal tumors and transduced with TOP-GFP construct. In these cultures, Wnt pathway activity drives the expression of GFP (TOP-GFP). These spheroid cultures with TOP-GFP (Co100) were treated with chemotherapy, for example, oxaliplatin for 24 h. Subsequently, spheroid cultures were stained with CaspGlow to measure caspase-3 activity. Cells are first gated on TOP-GFP^low (blue bar) and TOP-GFP^high (red bar), differentiated tumor cells and CSCs, respectively. Caspase-3 activity is then separately analyzed for the differentiated tumor cells and CSCs. (b) This method was used to test various chemotherapeutic therapies. Spheroid cultures treated for 24 h with oxaliplatin, cisplatin, 5-FU, etoposide, FOLFIRI and FOLFOX or 4 h with TRAIL are shown. Caspase-3 activity shows more cell death in differentiated cells compared with CSCs. Significance comparing differentiated versus CSCs is indicated (*P<0.05, **P<0.01, ***P<0.001)

Figure 2

Colon cancer stem cell resistance can be observed with multiple techniques in various patient-derived spheroid cultures. (a) Spheroid cultures were treated with oxaliplatin for 24 h and (a) phosphatidylserine (PS) exposure or (b) loss of mitochondrial activity was measured. (c) Different spheroid cultures derived from different patients (Co108 and Co123) were treated with oxaliplatin and PS exposure was measured in differentiated tumor cells (TOP-GFP^low) and CSCs (TOP-GFP^high). All spheroid cultures showed cell death in the differentiated tumor cells and not in the CSCs. Significance comparing differentiated versus CSCs is indicated (*P<0.05, **P<0.01)

Figure 3

Colon CSCs and differentiated cells are differentially mitochondrial primed. (a) Spheroid culture (Co100) ectopically overexpressing BCLXL protein was generated, as shown by western blot analysis of BCLXL (upper panel). Lower panel shows a control western blot for ERK1/2. (b) After oxaliplatin treatment, differentiated tumor cells (TOP-GFP^low) ectopically overexpressing BCLXL are no longer sensitive and show significantly reduced cell death by caspase-3 measurement. (c) Figure showing specificity of BH3 peptides for binding to anti-apoptotic BCL2 family protein members. Red indicates high affinity and green indicates low affinity. (d) Spheroid culture (Co100) was subjected to BH3 profiling using various BH3 peptides. Cells were stained for CD133, treated with indicated BH3 peptides and mitochondrial depolarization was measured with JC1. Mitochondrial depolarization in differentiated tumor cells (CD133^low) and CSCs (CD133^high) is shown relative to complete depolarization obtained with CCCP. Significance comparing oxaliplatin-treated control versus BCLXL overexpressing differentiated tumor cells is indicated in b and comparing differentiated versus CSCs in d (*P<0.05, **P<0.01, ***P<0.001)

Figure 4

ABT-737 and WEHI-539 sensitize colon CSCs toward chemotherapy. (a) Untransduced or (b) BCLXL-transduced spheroid cultures were treated with 1 μM of ABT-737, ABT-199 or WEHI-539 for 24 h and limiting dilution assay was performed on CSCs (TOP-GFP^high). ABT-737 and WEHI-539 decreased clonogenic capacity in CSCs (a), but not when BCLXL is overexpressed (b). Significance is indicated (***P<0.001) and is related to vehicle (DMSO) alone treatment. (c) Western blot analysis on the different spheroid cultures for BCL2 (upper panel), BCLXL (middle panel) and BCLW (lower panel) proteins. Control western blots for the kinase ERK1/2, which is stably expressed in the spheroids, are shown. (d) Spheroid cultures were treated with 100 nM ABT-737 or WEHI-539 in combination with 50 μM oxaliplatin for 24 h and subsequently analyzed for caspase-3 activity or (e) clonogenic capacity using limiting dilution on CSCs (TOP-GFP^high). Significance is indicated (**P<0.01 and ***P<0.001) and is related to oxaliplatin alone treatment. (f) Spheroid cultures were treated with 100 nM ABT-737, ABT-199 or WEHI-539 in combination with 0.5 μM oxaliplatin for 24 h and cell numbers were measured at various time points using cell titer blue