Characterization and Propagation of Tumor Initiating Cells Derived from Colorectal Liver Metastases: Trials, Tribulations and a Cautionary Note

Abstract

Tumor initiating cells (TIC) are increasingly being put forward as a potential target for intervention within colorectal cancer. Whilst characterisation and outgrowth of these cells has been extensively undertaken in primary colorectal cancers, few data are available describing characteristics within the metastatic setting. Tissue was obtained from patients undergoing surgical resection for colorectal liver metastases, and processed into single cell suspension for assessment. Tumor initiating cells from liver metastases were characterised using combinations of EPCAM, Aldehyde dehydrogenase activity, CD133 and CD26. CD133 expression was significantly lower in patients who had received chemotherapy, but this was accounted for by a decrease observed in the male patient cohort only. ALDHhigh populations were rare (0.4 and 0.3% for EPCAM+/ALDHhigh/CD133- and EPCAM+/ALDHhigh/CD133+ populations respectively) and below the limits of detection in 28% of samples. Spheroid outgrowth of metastatic tumor cells across all samples could not be readily achieved using standard spheroid-formation techniques, thus requiring further method validation to reliably propagate cells from the majority of tissues. Spheroid formation was not enhanced using additional growth factors or fibroblast co-culture, but once cells were passaged through NOD-SCID mice, spheroid formation was observed in 82% samples, accompanied by a significant increase in CD26. Order of spheroid forming ability was ALDHhigh>CD133>CD26. Samples sorted by these markers each had the ability to reform ALDHhigh, CD133 and CD26 positive populations to a similar extent, suggestive of a high degree of plasticity for each population. Ex vivo TIC models are increasingly being utilised to assess efficacy of therapeutic interventions. It is therefore essential that such investigations use well-characterised models that are able to sustain TIC populations across a large patient cohort in order that the inherent heterogeneity observed in cancer populations is maintained.

Curcumin inhibits cancer stem cell phenotypes in ex vivo models of colorectal liver metastases, and is clinically safe and tolerable in combination with FOLFOX chemotherapy

“…In addition, curcumin alone decreased spheroid number to a greater extent than the 5-FU/oxaliplatin treatments. Previously we have shown that spheroid forming capacity in CRLM is reflected by the order of expression of ALDH high > CD133 > CD26 [26] . This order suggests that reduction in ALDH activity correlates with reduced spheroids and fewer CSCs [26] .…”

“…Previously we have shown that spheroid forming capacity in CRLM is reflected by the order of expression of ALDH high > CD133 > CD26 [26] . This order suggests that reduction in ALDH activity correlates with reduced spheroids and fewer CSCs [26] . Transcription factors associated with the ALDH high stem-like phenotype (Oct 3/4, Nanog, Otx2, AFP) were also downregulated by the combination of 5-FU/oxaliplatin + curcumin.…”

“…Tissues were processed as previously described [26] . Stem-like and epithelial components were characterised by flow cytometry (FACS Aria II, Becton Dickinson, Oxford, UK) using antibodies directed against epithelial cellular adhesion molecule (EPCAM), CD133, CD26 and aldehyde dehydrogenase activity.…”

“…Tumour tissues were maintained by serial passage through Non Obese Diabetic Severe Combined Immunodeficient Mice (NOD-SCID) mice [26] . All experiments were carried out under project license PPL 80/2167, granted to Leicester University by the United Kingdom Home Office, vetted by Leicester University Local Ethical Committee for Animal Experimentation and met the standards required by the UKCCR for animal welfare.…”

Aldehyde dehydrogenase as a marker and functional mediator of metastasis in solid tumors

“…Tumor cells displaying high ALDEFLUOR™ activity have been demonstrated to have enhanced motility and ability to invade through a 3D basement membrane in breast cancer [ 7 , 78 ], ovarian cancer [ 79 , 80 ], osteosarcoma [ 36 , 81 ], esophageal cancer [ 82 ], and prostate cancer [ 11 , 83 ]; as well as increased therapy resistance in breast cancer [ 28 , 84 ], ovarian cancer [ 79 ], osteosarcoma [ 85 ], and prostate cancer [ 86 ]. ALDEFLUOR™-positive cells have also been reported to exhibit increased capacity to form tumorspheres in breast cancer [ 8 , 15 , 52 , 87 ], human malignant fibrous histiocytoma (HMFH) [ 88 ], ovarian cancer [ 80 , 89 , 90 ], brain tumors [ 91 , 92 ], prostate cancer [ 11 ], head and neck squamous cell carcinoma (HNSCC) [ 93 ], colon cancer [ 94 , 95 ], non-squamous cell lung cancer (NSCLC) [ 96 – 98 ], esophageal cancer [ 82 ], and cervical cancer [ 99 ]. Interestingly, ALDH-positive cells display additional stem-like behaviors such as resistance to hypoxia in ovarian cancer [ 79 ] and the capacity for asymmetric division in brain tumor cells [ 91 ].…”