A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development

Abstract

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor, cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first, and randomized, after tumor regression to continuing treatment with gemcitabine, a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24, CD44, ALDH, nestin, and the hedgehog pathway. After induction with gemcitabine, treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently, a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer.

Figure 1

After 4 wk of induction with gemcitabine, the cohort 2 showed a plateau in effect. After re-randomization, the group released from gemcitabine (3) rapidly regrew, as did the group receiving cyclopamine (5). The group that continued gemcitabine (4) remained in an activity plateau. Only the combination therapy group (6) showed incremental effect, and these tumors homogeneously regressed to 50% of the initial size after 4 wk. Bars, SD.

Figure 2

A, immunohistochemical assessment of CD24, CD44, and ALDH by immunohistochemistry. CD44 presented a flat staining that was not different between groups. However, both CD24 and ALDH had a pattern consisting in inversely intense staining with tumor size, with maximal staining in gemcitabine-treated and gemcitabine + cyclopamine-treated groups. B, tumor growth, mouse nestin expression, cytoplasmic CD24, ALDH by reverse transcription-PCR and immunohistochemistry, and GLI1 mRNA expression. ALDH by immunohistochemistry was particularly differentiating as the low-level expression followed an all-or-nothing pattern. ALDH was only positive in 2% of the cells in the gemcitabine-treated groups. In the group where gemcitabine is ceased, the TSC markers (both CD24 and ALDH) decreased back to the levels of the untreated group, suggesting a repopulation of proliferating cells and a dilution of the TSC-enriching effect.