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. Author manuscript; available in PMC: 2014 Aug 5.
Published in final edited form as: Cancer Biol Ther. 2005 Sep 18;4(9):937–942. doi: 10.4161/cbt.4.9.2182

Non-Invasive Fluorescence Imaging of Cell Death in Fresh Human Colon Epithelia Treated with 5-Fluorouracil, CPT-11 and/or TRAIL

Niklas Finnberg 1, Seok-Hyun Kim 1, Emma E Furth 1, Jue Judy Liu 1, Pierre Russo 2, David A Piccoli 2, Adda Grimberg 1,2, Wafik S El-Deiry 1,*
PMCID: PMC4121850  NIHMSID: NIHMS617728  PMID: 16251801

Abstract

Apoptosis is instrumental in several physiological/pathophysiological processes and is a frequently used end-point in the development of anti-neoplastic compounds. Despite ample data on several colon cancer cell lines, little is known about the susceptibility of human colon to apoptosis following treatment with established chemotherapeutics. By treating fresh human colonic explants with 5-Fluorouracil (200 μg/ml), CPT-11 (100 μg/ml) and/or TRAIL (100 ng/ml) we readily detected a signal in situ using FITC-VAD-FMK at different time points, whereas labeling of colonic explants with EGFP-conjugated Annexin V proved less specific. Although TRAIL treatment alone appeared to cause little apoptosis in human colonic epithelia versus the control, we observed a greater number of cells undergoing apoptosis when a combination of CPT-11 and TRAIL was used as compared to either agent alone. This is the initial demonstration of TRAIL-induced apoptosis with or without a chemotherapeutic agent in fresh primary human colon epithelia explants. Thus, human colonic explants may provide a valuable reference point when candidate therapeutic compounds triggering apoptosis in colon cancer cell lines, xenografts or mouse models are developed. The results support the feasibility of developing non-invasive optical imaging strategies to detect apoptosis through direct visualization of injury to human colonic epithelia in vivo.

Keywords: non-invasive imaging, fluorescence imaging, apoptosis, cancer therapy, drug toxicity, colon epithelia, FLICA, TRAIL

INTRODUCTION

Anti-neoplastic compounds frequently engage a physiologically important process described as programmed cell death, or apoptosis, to eliminate cancer cells.1 Apoptosis, an energy-dependent process that is characterized by several molecular and morphological changes to the cell, is crucial to physiological processes including embryogenesis, tissue homeostasis and the prevention of transformation of cells damaged beyond repair. Apoptosis constitutes a fundamental component of cell removal because it lacks the severe bystander effects and inflammation that are observed with tissue damage and subsequent passive cell death, i.e., necrosis.1 A wealth of data on the regulation of apoptosis in the context of chemotherapy has been generated from experiments on tissues, tissue culture and cells from experimental animals. However, studies of the process in human cells are typically restricted to the use of continuously growing human (cancer) cell lines that may or may not accurately replicate the magnitude of cell death in human tumor or normal tissues in vivo.

Although apoptosis was first described over thirty years ago, there is still a lack of consensus about how to monitor the process in patients receiving chemotherapy.2,3 Due to the heterogeneous nature of cancer, the individual variation among patients and hence the need to individually tailor anti-cancer therapies, apoptosis can be a useful pharmaco-kinetic marker for response to chemotherapy. Thus several efforts have been made to identify reliable biomarkers of apoptosis.3 Traditionally morphological and biochemical changes have been monitored in preclinical settings, e.g., by staining with DNA binding fluorochromes and subsequent analysis by flow cytometry.4 A number of soluble surrogate markers for tumor apoptosis present in peripheral blood, more easily utilized in clinical settings, have also been identified.5,6

Although the colon is considered resistant to the effects of DNA damage in comparison to the hematopoietic organs or the small bowel, treatment with chemotherapeutic agents and localized treatment with ionizing radiation has shown loss of colonic epithelium, presumably due to the ablation of colonic stem cells, subsequent inflammation and fibrosis.7,8 Animal experiments have shown an increased expression of pro-apoptotic proteins and an increased rate of apoptosis in the proliferating crypts of the colon following DNA damage.9-12

In an attempt to monitor apoptosis in the acute response to anti-neoplastic agents in the human colon, we isolated explants from a surgically resected human colon and treated the explants with two well-established DNA-damaging agents frequently used in the treatment of colorectal cancer [5-Fluorouracil (5-FU) and CPT-11 (Camptosar™)] and/or the cytokine Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL). 5-FU functions as an anti-metabolite that blocks the enzyme thymidylate synthase, leading to a disturbed synthesis of thymine and eventually a block in DNA-synthesis and repair,13 whereas CPT-11 functions as a Topoisomerase I poison, stalling replication forks and introducing lethal double-strand breaks in DNA.14 TRAIL is known to trigger apoptosis independent of DNA damage through two different receptors, KILLER/DR5 and DR4; DR5 is inducible by p53 following DNA damage.15 TRAIL can kill several different types of cancer cells in vitro, but shows little or no toxicity towards normal tissues, and monoclonal antibodies towards DR4 and DR5 as well as TRAIL are currently undergoing phase-1 evaluation.16

By using a Fluorochrome-Labeled Inhibitor of Caspase Activation (FLICA)17 on viable colonic explants, we readily detected apoptosis following treatment with 5-FU or CPT-11. To our surprise, we found an increase in TRAIL-induced apoptosis following 30 hours of incubation and an additive effect between TRAIL and CPT-11. Although additional data is warranted, this may indicate that apoptosis can be triggered and readily observed in human colon by 5-FU, CPT-11 and/or TRAIL. Thus when studying the apoptosis induced by TRAIL alone or in combination with other anti-cancer therapies in cancer cells, primary colonic explants or colonocytes may serve as a reference point in order to further evaluate a potential therapeutic dose.

MATERIAL AND METHODS

Isolation of primary colonic explants

Parts of the large intestine from a 16-year old female APC-patient who underwent prophylactic colectomy was obtained following informed patient consent and approval from the Institutional Review Boards at both the Children's Hospital of Philadelphia and the University of Pennsylvania. The colon was cut open, the mucosa was separated from the serosa and placed in Base Medium consisting of four parts MCDB 201 and one part L15 (Sigma), supplemented with 2 ng/ml human recombinant EGF (Sigma), 5 μg/ml insulin (Sigma), 5 μg/ml transferrin, 50 μg/ml streptomycin and gentamycin and 2% FCS.18 Tissues were further dissected into 3-7 mm slices, cultured in 100 mm dishes containing DMEM-10 consisting of Dulbecco's Minimum Essential Medium (Invitrogen), 10 % FBS and 1% Penicillin/Streptomycin/ Fungizone, and incubated in a humidified chamber at 37°C and 5% CO2. The colonic tissue isolated for these studies was from non-polypoid mucosa. The colon had small polyps that were not isolated for this study.

In vitro treatments

Healthy colonic explants were treated with 200 μg/ml of 5-Fluorouracil (American Pharmaceutical Partners Inc. IL), 100 μg/ml of CPT-11 (Camptosar®, Pharmacia Upjohn, MI) or 100 ng/ml of recombinant murine TRAIL (BioMol, CA) or a combination of 5-Fluorouracil or CPT-11 plus TRAIL for 18–48 hours. Recombinant murine TRAIL has been shown to kill Jurkat cells effectively.19 Experiments in our laboratory have also shown that recombinant murine TRAIL effectively kills HCT116, a human colon cancer cell line that has been shown in several studies to be sensitive to recombinant human TRAIL.20,21

Propidium iodide (PI) staining

To assess viability, colonic explants were incubated with 25 μg/ml of Propidium Iodide (PI) for five minutes, washed in PBS and analyzed using an Axiovert 100 inverted fluorescence microscope (Zeiss). Pictures were captured using the NIH software Scion Image 1.62.

Apoptosis assays

Annexin V-staining

Primary colonic explants were transferred to Binding Buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 1 mM MgCl2, 5 mM KCl) and 1 μl of Annexin V-EGFP (BioVision, CA), and 25 μg/ml (final concentration) of PI was added to each sample. The colonic explants were washed in PBS and transferred to a glass slide, covered with a glass cover slip and analyzed using an Axiovert 100 inverted fluorescent microscope (Zeiss).

Fluorochrome-labeled inhibitor of caspase activity (FLICA)-assay in situ

The PCE's were incubated with 10[.proportional]M of FITC-VAD-FMK (CaspACE™, Promega, WI) and 25 μg/ml of propidium iodide for 20 minutes at 37°C protected from light. This was followed by washing in PBS and then transfer to glass slides, cover slip placement and analysis using an Axiovert 100 inverted fluorescence microscope (Zeiss).

RESULTS

To investigate the role of cell death in viable primary colonic tissues following treatment with established chemotherapy (CPT-11, 5-Fluorouracil) and the cytokine TRAIL, we isolated colonic explants from a surgically resected colon from a juvenile APC-patient and cultured them in vitro.

Viability assessment of colon explants

Viability of the isolated explants was determined by the amount of PI that was taken up by the explants upon addition to the medium (see Fig. 1). PI up-take in colonic explants increased over time following isolation of the explants, suggesting that viability decreased with increased culturing time. Due to low viability, the colonic explants were cultured for no more than six days (data not shown). At early time points (four hours, see Fig. 1), only parts of the explants stained for PI suggesting that the reminder of the colonic explants, at least partially, contained viable cells. Thus, in order to minimize background death, we limited experiments to the first 3 days following isolation of the explants.

Figure 1.

Figure 1

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Viability of colonic explants as assessed by PI-staining. PI was added to the medium of the colonic explants at different time-points following isolation. Increased staining intensity (red) was observed over time following isolation.

Treatment with 5-Fluorouracil (5-FU) and/or TRAIL

5-FU has been shown to kill several human colon cell lines at doses that are significantly lower than the one used in this study,22 whereas primary human hepatocytes are resistant to doses of 5-FU up to 200 μg/ml.23 TRAIL has been shown to kill primary human hepatocytes and primary human esophageal cells at doses as low as 10 ng/ml.24-26 However, the TRAIL-induced cell death observed in primary hepatocytes could be related to the preparation of the cells, as toxicity has not been replicated by some investigators even in the presence of proteasomal inhibitors.23

The colonic explants were treated for 24 and 48 hours with 200 μg/ml of 5-FU. At 48 hours, a large number of treated cells labeled positive using the FLICA-assay (FITC-VAD- FMK) (Fig. 2A). We suspected that the high levels of cell death in the tissue at this time point could overshadow a potential additive effect of 5-FU and TRAIL. At 24 hours following treatment with 5-FU alone, there was a noticeable elevation of FLICA-positive cells in the tissue (Fig. 2B). TRAIL alone did not result in a significant increase of FLICA-positive cells, and the combination of 5-FU and TRAIL did not give an increase in the numbers of FLICA-positive cells relative to 5-FU treatment alone. PI-uptake was increased following treatment with 5-FU alone for 24 hours whereas no increase was observed when the colonic explants were treated with TRAIL alone for 18 hours (Fig. 2B). Combination treatment with 5-FU and TRAIL only very modestly increased the PI-positivity in the tissue.

Figure 2.

Figure 2

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Cell death as assessed by the FLICA assay following treatment with 5-FU and/or TRAIL. (A) Cell death was assessed by PI up-take (red) and the cellular binding of FITC-conjugated inhibitors of caspase activity (green) in control colonic explants and colonic explants treated with 5-FU (200 μg/ml) for 48 hours. (B) Cell death was also assessed following combination treatment with 5-FU and TRAIL. Colonic explants were treated with either 5-FU (200 μg/ml) for 24 hours alone or with TRAIL (100 ng/ml) for 18 hours.

Although we were able to document cell death by FLICA/PI, we were not able to address which cell type in the explants is primarily affected by treatment with CPT-11, 5-FU and TRAIL. Upon resection of the colon, only the mucosa was isolated from the organ. Thus, it is likely, considering the frequency of cell death, that this involves primarily the colonic mucosa.

Cells that undergo apoptosis translocate phosphatidylserine groups from the inner monolayer to the outer monolayer of their plasma membrane. Phosphatidylserine groups can be detected using fluorochrome-conjugated annex-in V, a molecule that binds phosphatidylserine groups with high affinity.27 Adding EGFP-conjugated annexin to colonic explants treated with 5-FU and TRAIL in an identical manner as described above did not convincingly show specific labeling of apoptotic cells (Fig. 3). Thus it seems that this method may not be optimal to detect cells undergoing apoptosis in colonic explants following treatment with 5-FU and/or TRAIL.

Figure 3.

Figure 3

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Cell death as assessed by Annexin V binding following treatment with 5-FU and TRAIL. Colonic explants were treated with either 5-FU (200 μg/ml) for 24 hours alone or with TRAIL (100 ng/ml) for 18 hours.

In conclusion, 5-FU can trigger cell death and caspase activation within 24-48 hours in primary colonic explants whereas no TRAIL-induced apoptosis could be detected at 18 hours. The combination 5-FU and TRAIL does not enhance 5-FU mediated caspase activation as assessed by FLICA and only modestly enhances PI-positivity. Thus, in combination with 5-FU, TRAIL may trigger cell death independently of caspase activation.

Treatment with CPT-11 and TRAIL

Colonic explants were treated with CPT-11, a member of the family of topotecans and an inducer of lethal DNA-strand breaks. Colonic explants were treated for 36 hours with CPT-11 (100 μg/ml) and 30 hours with TRAIL (100 ng/ml) or a combination of CPT-11 and TRAIL, following six hours of pretreatment with CPT-11. The pro-apoptotic response was monitored using the FLICA-assay.

An increase in FLICA labeling and PI-staining was observed following treatment with CPT-11 for 36 hours as compared to the control (Fig. 4). Interestingly, increased labeling by FLICA and PI of the colonic explanted tissues was observed following 30 hours of incubation with TRAIL (Fig. 4), which stands in sharp contrast to the results obtained when the colonic explants were treated with TRAIL for 18 hours (Fig. 2). Also, the combination of both CPT-11 and TRAIL yields a more frequent labeling by both the FLICA-assay and PI as compared to either TRAIL or CPT-11 alone (Fig. 4).

Figure 4.

Figure 4

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FLICA-assay following treatment with CPT-11 and TRAIL. Colonic explants were treated with CPT-11 (100 μg/ml) for 36 hours and/or TRAIL (100 ng/ml) for 30 hours.

Thus, exposure of primary human colonic epithelia for 36 hours to CPT-11 (100 ng/ml) yield a readily detectable apoptotic response as measured by FLICA and PI assays. This is also the case for 30 hours of TRAIL treatment. An additive effect of TRAIL and CPT-11 is also observed.

DISCUSSION

Relatively little is known about the relevance or molecular nature of apoptosis in the acute phase following chemotherapy in the human colon. In this communication we evaluate the utility of two methods, Annexin V labeling and an in situ FLICA-assay (using FITC-VAD-FMK), that are commonly used on viable cells grown as continuous cell lines in vitro to assess apoptosis following a diverse set of stimuli. We show that the FLICA-assay in combination with PI-staining is the most robust method for detecting cell death in human colonic explants in vitro following treatment with 5-FU, CPT-11 and/or TRAIL, whereas the specificity of the Annexin V assay remains to be proven in this context.

Treatment of the colonic explants with 5-FU for 24 or 48 hours resulted in an easily detectable labeling by both the FLICA-assay and PI. One important note is that although there was partial overlap in the FLICA/PI labeling, large areas of the colonic explants were only positive for PI-uptake. Thus, a population of cells in the tissue might be dying due to a caspase-independent mechanism, and cells that show uptake of FITC-VAD-FMK but not PI maybe the result of an early onset of caspase activation that has not yet reached the point of compromised membrane integrity to allow PI influx.

Combination treatment with 5-FU and TRAIL did not lead to an increased labeling in the FLICA-assay, whereas combination treatment with CPT-11 and TRAIL did. One possible explanation may be the discrepancy in duration of the TRAIL treatments; i.e., TRAIL treatment for 18 hours showed no increase in FLICA/ PI-labeling relative to control (Fig. 2), whereas TRAIL treatment for 30 hours resulted in a clearly detectable response (Fig. 4). Thus, the time point chosen for treatment with TRAIL needs to be carefully considered since apoptosis may occur later. Indeed, one study of the colon carcinoma cell line HCT116 has shown a bax-dependent synergistic effect of 5-FU and TRAIL with lower doses of both compounds.22 On the other hand, CPT-11 has been shown previously to act in synergy with TRAIL in triggering apoptosis, presumably by down-regulating anti-apoptotic members of the Bcl-2 family and activating p53.28,29 Thus CPT-11 may utilize alternative, more effective molecular mechanisms to sensitize both tumor and normal cells to the pro-apoptotic function of TRAIL. Additional experiments are needed to further clarify this.

Little is known about the effect chemotherapeutics have on non-transformed primary human colonocytes with regard to apoptosis. Our study which used APC+/− human colon suggests that apoptosis can be triggered in the colon not only by 5-FU or CPT-11, but also by TRAIL. Although the APC+/− colon used here was not transformed and grossly had small polyps that were not included in the tissue used here, the samples used came from a premalignant tissue that is predisposed to developing hyperplasia, polyps and eventually cancer. Several studies have previously reported that TRAIL readily kills several tumor cells but fails to kill primary human cells.30 Although several cancer cell lines isolated from patients with colon cancer show sensitivity towards TRAIL, our data suggest that at least some cells in the colon can be sensitive to doses of TRAIL that are equivalent to those killing colon cancer cells in vitro. This may affect anti-cancer therapies that employ the ligand or antibodies towards the death receptors, especially when combined with conventional cytotoxic agents.

In conclusion, the use of tumor cell lines has resulted in a lot of data on the triggering of apoptosis by several chemotherapeutics and TRAIL, but little is known about the process in primary human epithelia. In sharp contrast to continuously growing cell lines, human colonic tissue exhibits a number of different cell types at various stages of differentiation and thus may differ in proliferation rates from that of a continuously dividing cultured cell line. We report that we are able to monitor apoptosis, or programmed cell death, following acute exposure to 5-FU, CPT-11 and/or TRAIL in primary human colonic explants. We report readily detectable levels of cell death following all treatments. Thus, further investigation of human primary tissues might prove useful as a reference point when new pro-apoptotic anti-cancer agents are being evaluated. Moreover, the studies described here support the feasibility to further develop noninvasive techniques to both qualitatively and quantitatively monitor cell death or injury in vivo, i.e., endoscopically or in biopsies. However, once imaging techniques have been developed that allow monitoring of cell death non-invasively and quantitatively, there is a need to verify the correlation between apoptosis in a tissue and the resulting tissue toxicity following anti-cancer therapy in vivo.

ACKNOWLEDGEMENTS

We are grateful to our patient and her family for supporting our research and allowing us to study her colon; without their generosity, this research would not have been possible. This work was presented in part by W.S.E-D. at the 13th SPORE Investigators’ Workshop in July, 2005 in Washington D.C. A.G. was supported by NIDDK grant, 5K08 DK64352. This work was supported by funds from the NCI Network for Translational Research in Optical Imaging grant CA105008 to W.S.E-D.

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