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Local bystander effect induces dormancy in human medullary thyroid carcinoma model in vivo
Cancer Letters 335 (2013) 299–305
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Local bystander effect induces dormancy in human medullary thyroid carcinoma model in vivo Lucia Kucerova a,⇑, Lucia Feketeova b, Miroslava Matuskova a, Zuzana Kozovska a, Pavol Janega b,c, Pavel Babal b, Martina Poturnajova a a b c
Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovakia Department of Pathology, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, 813 72 Bratislava, Slovakia Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Sienkiewiczova 1, 813 71 Bratislava, Slovakia
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Article history: Received 24 April 2012 Received in revised form 21 December 2012 Accepted 18 February 2013
Keywords: Human medullary thyroid carcinoma Gene-directed enzyme/prodrug therapy Fusion yeast cytosine deaminase 5-Fluorocytosine Bystander effect
a b s t r a c t The extent of local bystander effect induced by fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD) in combination with 5-fluorocytosine (5FC) was evaluated in xenogeneic model of human medullary thyroid carcinoma (MTC). This approach to gene-directed enzyme/prodrug therapy (GDEPT) induces strong bystander cytotoxicity. Effector yCD-TT mixed with target EGFP-TT cells in a ratio 2:9 could achieve significant tumor regression and 14-fold decrease in serum marker calcitonin upon 5FC administration. Histopathological analysis unraveled that antitumor effect resulted in tumor dormancy and proliferation arrest of remaining tumor cell clusters in vivo. yCD/5FC combination represents another GDEPT approach to achieve tumor growth control in MTC. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gene-directed enzyme/prodrug therapy (GDEPT) approaches were developed to limit host toxicity by introducing new catalytic functions into tumor cells in order to sensitize them to otherwise inert prodrugs [1–3]. Several suitable gene/prodrug combinations were proven efficient in preclinical antitumor testing. However, clinical trials have shown only limited extent of tumor control due to several reasons. Critical problem to overcome in cancer gene therapy remains to achieve gene delivery in sufficient cell numbers in order to target substantial proportion of tumor cells and mediate tumor regression [4]. Gene transfer efficiencies in clinical settings are unlikely to exceed more than 10% of target tumor cell population. Therefore an indirect effect of the treatment which causes cytotoxicity in neighboring non-transgenic cells within the tumor being called bystander effect is desirable in the context of enzyme/prodrug gene therapy. Bystander effect results in more cell deaths and higher cytotoxicity in comparison to the situation if only transgenic cells were killed by direct action of enzymatic conversion (suicide effect) [5]. GDEPT may elicit both local and distant bystander effect. The latter involves an intense systemic anti-tumor inflammatory ⇑ Corresponding author. Tel.: +421 2 59327425; fax: +421 2 59327250. E-mail address: [email protected] (L. Kucerova). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.040
infiltration and it is immune mediated. Distant bystander effect is considered to be one of the important factors for the clinical success of gene therapy in vivo [1]. On the other hand, local bystander effect is limited by the tissue penetration capacity of the prodrug to achieve sufficient concentrations and subsequently the ability of activated metabolites to spread into adjacent nontransduced cells [6]. Soluble toxic metabolites can be transferred by either passive diffusion or active transport, via apoptotic vesicles or gap junctions. Frequent deregulation in gap junction intercellular communication severely compromises the efficiency of the approaches using highly charged cytotoxic metabolites such as ganciclovir-triphosphate in GDEPT combination of herpes simplex virus thymidine kinase and ganciclovir [5]. Combination of bacterial or yeast enzyme cytosine deaminase with prodrug 5-fluorocytosine produces toxic effect via cytotoxic drug 5-fluorouracil, which can freely diffuse across the cell membranes, and thus induces significant local bystander effect [7]. Moreover, the fusion of the yeast cytosine deaminase with the uracil phosphoribosyltransferase has significantly improved the conversion efficiency of 5FC to 5FU and its metabolites further improving the antitumor effect in many preclinical models [7–10]. Although medullary thyroid carcinoma (MTC) constitutes only 2–4% of all thyroid cancer diagnosis, it has generally proven to be refractory to cytotoxic therapy [11]. Systemic therapy in patients with inoperable metastatic disease requires prolonged treatment
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with switching between different agents [12]. However, based on efficiency and toxicity not all patients can tolerate this therapy. Therefore novel multikinase inhibitors were tested in series of trials and have brought significant progress in treating patients with advanced progressive MTC during the past 5 years [13]. Messina and Robinson have recognized a potential role of gene therapy in MTC treatment summarizing also several studies using cytoreductive enzyme/prodrug combinations [14]. Our recently published data have extended the scale of effective GDEPT combinations against MTC to the therapeutic combination employing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD) in combination with 5-fluorocytosine (yCD/5FC) [9]. The absence of gap junction intercellular communication was observed in model MTC cells, and therefore the approach relying on passive metabolite diffusion was proposed to be employed to increase cytotoxicity. Potent cytotoxic effect was confirmed in vitro, and antitumor efficiency in vivo. Even though transgenic and nontransgenic tumor cells were mixed in a ratio 1:1 to induce tumor xenotransplant growth in immunodeficient mice, we have observed tumor inhibition of 92.5% indicative of strong suicide and local bystander effect in vivo. The aim of this study was to analyze the extent of local bystander effect induced by yCD/5FC with limited numbers of yCD-transduced cells present within the tumor mass. The proportion of effector-to-target tumor cells corresponded to the proportion necessary to achieve complete target tumor cell eradication in vitro. In order to track the target cells, these were modified to stably express the EGFP protein. Our xenogeneic model enables to evaluate local bystander effect in the absence of inflammatory anti-tumor responses in vivo. 5FC administration resulted in 71.4% tumor growth inhibition, 14-fold decrease in calcitonin serum levels and histopathologically confirmed tumor dormancy. These preclinical data provide evidence for potent local bystander toxicity and support the observation that yCD/5FC system is very efficient in tumor growth control in chemotherapy-resistant MTC model. We suggest that the GDEPT approach utilizing yCD/5FC combination might prove useful to overcome low antitumor efficiency and high systemic toxicity of 5FU.
2.3.1. GAPDH housekeeping gene protocol Quantitative PCR was performed with Master Mix containing: 1 MaximaÒ Probe qPCR Master Mix, final concentration of 5.5 mM MgCl2 (Thermo Fisher), 0.4 lM of GAPDH primers (sense 50 -GAA GGT GAA GGT CGG AGT C-30 and antisense 50 -GAA GAT GGT GAT GGG ATT TC-30 ), 0.1 lM GAPDH dual labeled probe (50 -HexCAA GCT TCC CGT TCT CAG CC-BHQ1-30 (Metabion, Martinsried, Germany) and 100 ng of cDNA. Thermal cycling conditions were as follows: DNA polymerase activation (50 °C for 2 min), initial denaturation step (95 °C for 10 min) followed by 40 cycles (95 °C for 15s, 60 °C for 60s and HEX fluorescence aquire). Correct amplification of 226 bp product was verified on 1% agarose gel. 2.3.2. Calcitonin gene protocol Calcitonin primers (sense 50 -TGC GGT AAT CTG AGT ACT TGC ATG C-30 and antisense 50 -CCA ACC CCA ATT GCA GTT TGG-30 ) were designed using Vector NTI software (Invitrogen) according to human calcitonin precursor mRNA calcitonin sequence (GenBank ID: X00356.1). The final reaction Master Mix contained: 1 MaximaÒ SybrGreen qPCR Master Mix (Fermentas) supplemented with 2.5 mM MgCl2, 0.18 lM of each primer and 100 ng of cDNA. Thermal cycling conditions: DNA polymerase activation (50 °C for 2 min), initial denaturation (95 °C for 10 min), followed by 35 cycles of (95 °C for 15 s, 63 °C for 60 s and acquire of SybrGreen fluorescence at 72 °C, 10 s). Correct amplification of 91 bp product was verified on 2.5% agarose gel and as a one sharp melting peak at 78 °C during melting in range 65–86 °C. 2.3.3. Statistical analysis and validation All samples were tested in parallels in four independent qPCR runs for GAPDH and qPCR runs for calcitonin expression. Data were normalized to housekeeper expression. Normalized expression for each sample relative was determined by DDCt method using CFX Manager software 1.5 (Bio-Rad). 2.4. Calcitonin EIA assay Calcitonin EIA Assay (Alpco Diagnostics) was used to quantify the protein levels. The immunoassay utilizes two mouse antibodies: biotinylated Ab against human Calcitonin 11-23 and peroxidase labeled Ab against Calcitonin 21-32. Quantification of calcitonin secretion was performed in two independent experiments, all samples in parallel according to manufacturer instruction. Briefly, 100 ll of calcitonin standards (calibrators), controls and samples in parallels were mixed with 50 ll of each antibody in streptavidin-coated microplate in a dark on an orbital rotator. It was incubated for 4 h at room temperature, washed away very carefully and incubated with TMB substrate for 30 min at room temperature in a dark with rotation. Reaction was stopped and optical density of each sample was measured at 450 and 405 nm. Concentration was derived from calcitonin standard curve. Calcitonin secretion was expressed as pg of calcitonin per 1 lg of total proteins in cell culture media as determined by Lowry method or pg of calcitonin per 1 ml of mouse serum. Standard cell culture medium or mouse serum from control mouse was used as background control.
2. Materials and methods 2.5. Proliferation analysis 2.1. Chemicals All chemicals were purchased form from Sigma–AldrichÒ, St. Louis, MO, if not stated otherwise.
2.2. Cells Epithelial adherent TT cell line (ATCC. No. CRL-1803™) derived from human medullary thyroid carcinoma was purchased from ATCC and cultured in RPMI medium (Roswell Park Memorial Institute, PAA Laboratories GmbH) supplemented with 10% fetal bovine serum (FBS, Biochrom AG), 10.000 IU/ml penicillin (Biotika, Part. Lupca, Slovakia), 2 mM glutamine and 5 lg/ml streptomycin (Sigma, St. Louis, MO). Transgenic TT cells expressing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase yCD or enhanced green fluorescent protein (EGFP) were prepared as described elsewhere in detail [9]. All cells were expanded according to standardized protocol and kept in humidified atmosphere and 5% CO2 at 37 °C. 2.3. Calcitonin expression analysis Parental TT and/or transduced TT cells were collected by accutase solution (PAA Laboratories GmbH), washed with PBS solution and pelleted (5–7 106 cells per pellet). These were frozen in RNAlater Stabilization Reagent (Qiagen, Hilden, Germany) and stored at 80 °C until use. RNA was isolated with NucleoSpinÒ RNA II Kit (Macherey–Nagel). Any traces of DNA were removed by RNase-free DNase I treatment (10 U/lg RNA) in the presence of 20 U RiboLock solution (Thermo Fisher). Total RNA was screened for complete DNA removal by PCR and then reverse transcribed using RevertAid™ H Minus First Strand cDNA Synthesis Kit and oligo-dT18 primer (Thermo Fisher).
One to 1.5 105 cells per well in 6-well plates were plated in duplicates, which ensured unrestricted exponential growth for the duration of the assay. Population doubling time (PDT) was calculated as an average of three measurements for the TT cells according to the formula G = t log 2/log Ntlog No, where t = time period, Nt = number of cells at time t, No = initial number of cells. In order to directly compare proliferation rates for EGFP-TT and yCD-TT cells, these cells were then labeled with 5 lL of Vybrant DiI (Invitrogen, Carlsbad, CA) diluted in 1 ml PBS per 1 106cells for 20 min at 37 °C. One part of these cells was proliferation-arrested by 10 lg/ml Mitomycin C (Mit-C, Kyowa, Hakko Ltd., UK) diluted in culture medium for 3 h and washed. Proliferating and arrested cells were kept in standard culture medium for 14 days. Cells were then detached by accutase solution, washed and fluorescence intensity was determined by flow cytometry. DiI fluorescent staining does not interfere with EGFP fluorescence, therefore this method is suitable for EGFP-TT stably expressing fluorescent reporter protein. 2.6. Xenotransplant growth and animal treatments in vivo A 6–8 weeks old athymic nude female mice (Balb/c nu/nu) were used in accordance to the institutional guidelines under the approved protocols. Tumors were induced with 1.1 107 EGFP-TT cells resuspended in serum-free RPMI diluted 1:1 with ECM gel (cat. No. #1270, Sigma–AldrichÒ, St. Louis, MO) s.c. Animals with growing xenotransplants (n = 5–6/group) were randomly divided into two groups for 5FU treatment, which was started on day 9. Treated animals received the dose of 10 mg 5FU/kg/day i.p. every other day for 22 days. Preliminary experiments have shown that this was the maximum tolerated dose in our experimental setting. In independent series of experiments, tumors were induced by 2 106 effector transgenic yCD-cells TT mixed with 9 106 target transgenic EGFP-TT (ratio effector-to-target cells 2:9) injected subcutaneously. Cells were resuspended in
L. Kucerova et al. / Cancer Letters 335 (2013) 299–305 serum-free RPMI diluted 1:1 with ECM gel (cat. No. #1270, Sigma–AldrichÒ, St. Louis, MO). Animals with growing xenotransplants (n = 5–6/group) were randomly divided into two groups for 5FC treatment, which was started on day 9. Treated animals received the dose of 500 mg 5FC/kg/day i.p. every day for 22 days. Animals were evaluated for tumor growth and size regularly. Tumor volume was calculated from caliper measurements according to formula volume = length width2/2. Results were evaluated as mean tumor volume ±SD. Untreated animals had to be sacrificed at the timepoint when they exhibited tumor ulceration, bleeding or any other signs of distress. Serum was harvested from blood samples taken from treated and untreated animals. Tumor xenotransplants or fibrotic remnants at the site of tumor growth were excised for further immunohistochemistry.
2.7. Immunohistochemistry Excised tumor xenotransplants were fixed in buffered formalin, embedded in paraffin, cut into 5 lm thick sections, stained with hematoxylin/eosin and evaluated by light microscopy (Leica DM2000, Wetzlar, Germany). Immunohistochemical staining was performed to detect proliferation markers Ki67 and PCNA (proliferating cell nuclear antigen). Tissue samples were also stained for EGFP to detect the presence of target EGFP-TT cells. Following reagents were used: monoclonal mouse anti-Ki67 antibody (Dako, Denmark), monoclonal mouse anti-PCNA antibody (Dako, Denmark) and polyclonal rabbit anti-EGFP antibody (Abcam, Cambridge, UK). Reaction was visualized by universal peroxidase polymer Histofine (anti-mouse, anti-rabbit) with diaminobenzidine (Dako, Denmark) as a substrate for color reaction. Marker expression was evaluated by semiquantitative method, positivity or negativity for nuclear markers was evaluated.
2.8. Statistical analysis Student’s two-sample t-test was used to compare the difference in means of two samples. Calcitonin secretion in sera of 5-FC treated vs. untreated mice were subjected to Mann–Whitney U test. The p-values with p < 0.05 were considered to be statistically significant.
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3. Results As previously reported two transgenic cell populations were prepared from human parental TT cells derived from MTC [9]. Retrovirally transduced yCD-TT cells express yeast fusion cytosine deaminase::phosphoribosyltransferase (yCD) and serve as effector cells for gene-directed enzyme/prodrug treatment. Enzymatic activity of yCD converts nontoxic 5-fluorocytosine (5FC) to metabolites of 5-fluorouracil, potent cytotoxic drug. Target transgenic TT cells were modified to express enhanced green fluorescent protein (EGFP) in order to track their presence in vivo. Both types of transgenic TT cells have retained calcitonin expression as an MTC characteristic (Fig. 1A). Calcitonin secretion was confirmed in cell culture supernatants, although the calcitonin production was significantly increased in supernatant from yCD-TT cells (Fig. 1B). Increased calcitonin secretion may be linked to the capability of fusion enzyme yCD to increase cell proliferation as previously reported in our work [15]. Introduction of yCD and its constitutive expression interferes with the cell-intrinsic nucleotide synthesis machinery and renders them with the capability to utilize the nucleotide precursors more efficiently. Indeed, yCD introduction shortened the population doubling time in transgenic yCD-TT cells which is not the case for retrovirally transduced EGFP-TT transgenic cells. EGFP-TT cell proliferation does not differ from the proliferation in parental TT cell population (Fig. 1C and D), therefore this efect is likely to depend on the yCD enzyme activity. MTC is generally reported to be refractory to chemotherapeutic treatment in clinical setting. Our previous data have shown moderate sensitivity of TT cells to the treatment with 5-fluorouracil with
Fig. 1. Transgenic yCD-TT cells have increased calcitonin secretion and proliferation. (A) Calcitonin expression was evaluated in parental, EGFP-TT and yCD-TT cells by quantitative RT-PCR. Effector yCD-TT cells exhibited increased calcitonin mRNA levels. (B) Cells were cultured in standard medium for 3 days and the concentration of calcitonin secreted into media was evaluated by EIA. Results were expressed as average concentration ±SD. p < 0.05. (C) Parental and transduced TT cells were plated at equal density to ensure the unrestricted exponential growth. Population doubling time was significantly lower for the effector yCD-TT cells. p < 0.05. (D) Vybrant DiI-dye retention assay to compare cell proliferation after prolonged culture was performed and evaluated by flow cytometry. Synchronized EGFP-TT or yCD-TT cells were stained with DiI, split into two parts, one portion was MitC-arrested and both aliquots were maintained in standard culture media for 14 days. Fluorescence intensity unraveled decreased fluorescence intensity in yCD-TT cells corresponding to increased proliferation. Parental TT cell displayed the analogous dye retention pattern to EGFP-TT and were omitted from figure for clarity.
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IC50(5FU) = 0.6 lg/ml in vitro. Our data also show that TT cells were responsive to vincristine and doxorubicin at high concentrations (data not shown). 5FU treatment of tumor xenotransplants derived from EGFP-TT cells could induce significant antitumor effect in vivo (Fig. 2A). 5FU administration resulted in 66.7% reduction in tumor volume as measured by caliper on day 22 of 5FU treatment (56.6 mm3 in the 5FU treated group in comparison to 170.3 mm3 in the untreated controls). Tumor volumes remained stable in the 5FU treated group with a tendency to increase after the termination of 5FU administration. Experiments had to be terminated due to cumulative 5FU toxicity and animals had to be sacrificed due to massive weight loss, diarrhea, and skin rash with open wounds. When tumor xenotransplants were grown from mixtures of transgenic yCD-TT effector cells and EGFP-TT target cells (ratio 2:9), rapid development and growth of tumor xenotransplant was observed as expected. Prodrug administration (5FC treatment) prevented further growth and resulted in tumor regression even though cells with enzymatic function yCD-TT represented only 18% of the tumor cell population (Fig. 2B). By the day 31 after experiment start the average tumor volume reached 229.4 mm3 in the untreated group and animals had to be sacrificed. Tumor volume in 5FC treated group was 65.5 mm3 by the same time point exhibiting tumor growth inhibition of 71.4%. In the absence of any bystander effect the theoretical extent of tumor growth inhibition corresponding to suicide action of enzymatically produced metabolites would have been less than 20% based on the starting effector-to-target cell ratio. Moreover, the tumor xenotransplants regressed further after treatment withdrawal and the average tumor volume decreased to 21.4 mm3 in this group by the day
51. Treated animals did not show any sign of systemic treatment toxicity. The bystander effect efficiency was reflected also by a decrease in serum calcitonin levels from 6027.1 pg/ml sera to 425.1 pg/ml sera, which is 14-fold decrease by the day 31 (Fig. 4C). In the EGFP-TT group calcitonin levels decreased from 4826 pg/ml sera in controls to 980 pg/ml sera in 5FU treated group, which is 4.9-fold decrease by the day 31. Taken together our data confirm the capability of gene directed enzyme/prodrug combination yCD/5FC to mediate local bystander effect accompanied by significant antitumor effect in the absence of any toxicity. The treatment efficiency of GDEPT approach is comparable to the effect of systemic 5FU administration. Immunohistochemical staining of untreated tumor xenotransplants unraveled retention of two populations of tumor cells within the tumor mass in the untreated control as expected (Fig. 3, left). In order to unravel whether the complete tumor cell eradication was achieved the fibrotic remnants of the tumor xenotransplants were analyzed at the experiment endpoint. Our data show small clusters of tumor cells within the fibrotic tissue at the site of tumor cell administration. Two populations of cells were visible: EGFPpositive and negative TT cells, which escaped the suicide and/or bystander cytotoxicity (Fig. 3 middle and right). More detailed analysis has shown that both effector and target TT cells were positive for proliferation markers Ki67 and PCNA in untreated tumors (Fig. 4 left). Contrastingly, clusters of EGFP-positive and negative cells which escaped the treatment and remained at the site of tumor growth had very rare Ki67 positivity (Fig. 4 right). PCNA staining was diffuse within the tumor clusters corresponding to the cells which were arrested in the S phase of cell
Fig. 2. Local bystander effect of yCD/5FC combination in vivo. (A) EGFP-TT cells diluted in ECM were inoculated in flank of immunodeficient females. Mice were randomly split into two groups. Starting on day 9 after inoculation one group was treated with 5FU for subsequent 21 days. Average tumor volume in 5FU treated group was significantly lower in comparison to untreated controls by day 31 (56.6 mm3 vs. 170.3 mm3). p < 0.01, Experimental animals had to be sacrificed due to ethical reasons, arrows depict the 5FU treatment timing. (B) Effector yCD-TT and target EGFP-TT cells (mixed in a ratio 2:9) diluted in ECM were inoculated in flank of immunodeficient females. One group of mice with tumor xenotransplants of average volume 75 mm3 (Day 9) started receiving 500 mg 5FC/kg/day for the next 22 days. By the day 31 there was significant difference in the median tumor volume when compared to 5FC untreated group. Treated animals were observed for another 20 days and exhibited further decrease in tumor volume from 65.5 mm3 to 21.4 mm3. p < 0.01, Experimental animals had to be sacrificed due to ethical reasons, arrows depict the 5FC treatment timing. (C) Mouse serum was collected from treated and untreated animals and the calcitonin concentration was determined by EIA. Calcitonin levels in serum of 5FC-treated and 5FU-treated mice was significantly lower in comparison to serum levels from untreated mice being in concordance with high tumor volumes in untreated mouse xenografts at the experiment endpoint. Background calcitonin level in the serum obtained from untreated healthy mouse was 100.5 pg/ml. p < 0.05.
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Fig. 3. Tumor xenotransplants derived from yCD-TT/EGFP-TT cells were analyzed by immunohistochemistry to detect transgenic EGFP-TT target tumor cell population. Left panel shows that untreated tumor tissue is composed of two tumor cell subpopulations both EGFP-positive (target EGFP-TT) and EGFP-negative (effector yCD-TT) cells which contribute to tumor growth. 5FC-treated tumor xenotransplants have shown regression with very small remnants palpable at the site of tumor. These fibrotic remnants (middle and right panel) are interspersed with clusters of tumor cells which escaped the suicide and bystander cytotoxicity. Both tumor cell populations of EGFP-positive and EGFP-negative cells were present within the clusters. Brown staining: EGFP-positivity, magnification 20. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. In order to determine the proliferation status in 5FC treated tumor xenotransplants, adjacent tumor sections were analyzed by immunohistochemistry to detect proliferation markers Ki67 and PCNA. Panels on the left confirm that both tumor cell populations yCD-TT and EGFP-TT are prominently positive for Ki67 and PCNA corresponding to cell proliferation and tumor growth. Right panel depicts very low Ki67 positivity in tumor cells within the clusters (red arrows). Diffuse staining for PCNA in combination with the absence of Ki67 marker is indicative of cell division arrest in the remaining cells being mostly in the S phase of the cell cycle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cycle. Very low Ki67 positivity corresponded to the arrest in cell division and these cells remained dormant. As both cell populations within the clusters in the treated tumors did not show any signs of proliferation activity, we concluded that even though these cells were retained at the site of tumor growth at the treatment endpoint they were incapable of proliferation and thus incapable of further tumor growth and spread. Taken together our data demonstrate the GDEPT approach to overcome the chemoresistance of MTC in vivo and suggest the
combination of gene directed enzyme/prodrug therapy approach which could induce strong local bystander effect even though only a small part of the tumor expressed the transgene.
4. Discussion Medullar thyroid carcinoma (MTC) represents a malignant disease which spreads quite early into adjacent lymph nodes and
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gives rise to multiple metastatic lesions in lungs, bones and liver. MTC is generally recognized as resistant to standard radiotherapy and chemotherapy [16,17]. Sporadic MTC grows usually slowly, but 50% of patients have tumor lesion within lymph nodes and 10% have already inoperable metastatic spread in distant organs at the time of diagnosis [18]. Heritable forms of MTC cover both familiar form (FMTC) and MTC as a major component of multiple endocrine neoplasia type 2 syndrome (MEN2). The latter MTC form exhibits quite rapid growth. Gain-of-function mutation in RET proto-oncogene is responsible for autosomal-dominantly inherited cancer syndrome MEN2. Cys634Trp mutation in RET proto-oncogene frequently occurs in patients with MEN2A and FMTC [18]. Model human TT cell line derived from medullary thyroid carcinoma has the above mentioned mutation and it was previously described as a relevant model for MTC [19]. In general, metastatic MTC is currently considered to be an incurable disease by standard treatment options. However it remains a subject to various experimental treatment strategies such as gene therapy in order to unravel the (combination of) approaches to achieve disease control. These include the efforts to correct the cellular defect by dominant-negative RET mutants, immunomodulatory gene therapy and cytotoxic gene directed enzyme/prodrug treatment approaches [14]. In our previous report we have shown so far unrecognized GDEPT strategy in MTC xenotransplant model by employing combination of fusion yeast cytosine deaminase with prodrug 5-fluorouracil [9]. This proofof-principle report highlighted its capability to overcome therapeutic refractoriness of MTC in vivo. In order to determine the extent of local bystander effect we have extended our previous study and induced tumor xenotransplant growth by lower proportion of transgenic effector yCD-TT to target EGFP-TT cells being more relevant for clinical application. Therapeutic yCD-TT cells expected to undergo cell suicide upon 5FC prodrug administration represented only 18% of the tumor cell population at the beginning. Our previous data have shown that this proportion of the effector cells could decrease the overall cell proliferation to less than 10% in vitro [9]. The adjacent yCD-TT cells spreading cytotoxic metabolites of 5FU could eradicate the majority of target TT cells within the 9 days in direct coculture. Correspondingly, 5FC administration to experimental animals with growing tumor xenotransplants in vivo resulted in the tumor size decrease correlating to expected antitumor effect. By the day 21 after treatment initiation a prominent decrease in the tumor volume could be observed based on caliper measurements, when tumors in the untreated group of animals increased their volume. However, based on our experience these measurements underestimate the extent of the antitumor effect. These outcomes cannot distinguish the contribution of the tumor cells from the necrotic tumor part and the fibrous remnants. Our analysis was therefore supplemented with the calcitonin level determination in order to evaluate the extent of antitumor effect by clinically relevant test. We could confirm 14-fold decrease in calcitonin concentration in 5FC treated animals. These data suggest the real extent of local bystander effect being higher than anticipated by volume comparison. 18% prodrug converting effector cells could achieve 92.9% inhibition of calcitonin secretion as determined in the serum of 5FC treated vs. untreated animals. In order to directly compare the efficiency of the approach, growing tumor xenotransplants were treated with 5FU at the same time schedule – treatment start on day 9 followed by 5FU administration for next 21 days. Significant decrease in tumor volume in 5FU treated animals could be detected in concordance with 79.7% decrease in serum calcitonin levels, although tumor volume remained stable and had tendency to increase after 5FU withdrawal. Contrastingly, no re-growth in any of the tumor xenotransplants could be observed after withdrawal of the 5FC administration. Treated tumors further decreased in volume. At the experiment endpoint the remaining
tissue at the site of tumor xenotransplant was analyzed and the presence of isolated tumor cell clusters was observed within the fibrous tissue. This finding further confirmed the higher extent of bystander effect in tumors than estimated by the caliper measurements. Moreover the remaining cells were found to lack Ki67 staining and PCNA detection which indicated their arrest in the S phase of the cell cycle. As we have performed our experiments in the immunodeficient mice we could directly evaluate the extent of local bystander effect in the absence of any immune-cell mediated reaction. We anticipate that the situation in the immunocompetent host would be even more prominent and distant bystander effect mediated by the immune cells could further increase the therapeutic efficiency [20,21]. Distant bystander effect might be important in the situations when the transduction efficiency within the tumor tissue remains low. Currently reported efficiencies from clinical setting do not exceed 10% transduction efficiencies in the target cells [4,22,23] and therefore we anticipate that distant bystander effect will substantially contribute to the final treatment outcome. Our data indicate comparable antitumor efficiency of the GDEPT approach in the absence of any systemic toxicity. Our experimental data indicated that TT cells exhibited limited chemosensitivity in vitro and in vivo. They responded to cytotoxic drugs such as vincristine and doxorubicin at high concentrations only (data not shown). The expression of drug transporters ABCB1 and ABCC1 which were previously linked to increased resistance to doxorubicin and vincristine is likely to contribute to their chemoresistance [9,24]. Although we determined that IC50 (5FU) = 0.6 lg/ ml for TT cells, we were not able to reach IC90 in vitro [9]. Even though TT cells do not express drug transporter ABCB5 previously linked to increased 5FU efflux and resistance [25], these cells have high expression of enzyme dihydropyrimidine dehydrogenase (DPD) that inactivates 5FU to non-toxic metabolites [26,27]. Tumor xenotransplant treatment with cytotoxic agent 5FU correlates to the clinical observations with chemotherapy efficiency in MTC. Maximum tolerated 5FU dose could achieve stabilization of tumor volume in our mouse model accompanied by high toxicity and tendency to re-grow after treatment termination (Fig. 2A). Immunohistochemical data showed that tumor xenotransplants had prominent Ki67 and PCNA staining indicating the proliferative status of tumor cells in vivo. This might support the hypothesis that the 5FU refractoriness was linked to the low intratumoral 5FU concentration. DPD expression in the liver contributed to rapid 5FU breakdown and decrease in plasma, DPD expression in the tumor cells further contributed to 5FU intratumoral breakdown and the absence of gap-junctional intracellular communication in these cells further eliminated the transmission of toxic metabolites throughout the tumor. Several previous reports have indicated potential ways to overcome the chemoresistance in MTC cells. TT cell chemoresistance could be abrogated by cyclooxygenase-2 (COX-2) inhibition with selective COX-2 inhibitor which sensitizes TT cells to the cytotoxic effects of doxorubicin, reduces ABCB1 expression and function [24]. Our data suggested that introduction of fusion enzyme cytosine deaminase::uracil phosphoribosyltransferase sensitized yCD-TT cells to 5FU (yCD-TT cells have IC90(5FU) 1 lg/ml corresponding to the achievable plasma concentrations) [9]. This effect is not unique to tumor cells as the similar phenomenon was reported for the nonmalignant transduced human mesenchymal stromal cells [15]. Chemosensitization by the expression of yCD transgene seems to represent a more general phenomenon: its constitutive expression interferes with the intrinsic nucleotide metabolism thus increasing the cell proliferation (Fig. 1C and D). It was previously shown that this effect cannot be attributed to the retroviral gene transfer procedure. The cells which were transduced with mock plasmid or marker EGFP vector did not have increased proliferation and chemosensitivity [15]. However, it is
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possible that increased proliferation of (tumor) cells mediated by yCD renders them more vulnerable to other cytotoxic agents. Thus the combination of the yCD/5FC antitumor gene therapy approach with other cytotoxic strategies might prove beneficial for therapeutic effect and tumor growth control. Another approach to chemosensitization and radiosensitization of tumor cells was reported by Wang et al. who expressed cytosine deaminase under the hypoxia responsive promoter. They reasoned that increased 5FC conversion to 5-fluorouracil could also increase radiosensitivity of the solid tumor [28]. In summary, yCD/5FC combination represents efficient GDEPT combination for MTC treatment. yCD/5FC induces strong local bystander effect in MTC model in vivo. yCD/5FC treatment decreased serum calcitonin by 92.9%, arrested tumor cell proliferation and induced tumor dormancy, thus representing potent antitumor strategy even in highly chemoresistant solid tumor model.
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Conflict of interest [16]
The authors declare no conflict of interest.
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Acknowledgements
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We thank M. Dubrovcakova, V. Frivalska, R. Bohovic and E. Klincova for the excellent technical assistance. Study was supported by the Slovak Research and Development Agency under the contract No. APVV-0230-11, the VEGA Grants Nos. 2/0088/11 (L.K.) and 2/0146/10 (M.M.). This work was supported by the Framework Program for Research and Technology Development, Project: Building of Centre of Excellency for Sudden Cerebral Vascular Events, Comenius University Faculty of Medicine in Bratislava (ITMS: 26240120023), cofinanced by European Regional Development Fund.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Local bystander effect induces dormancy in human medullary thyroid carcinoma model in vivo Lucia Kucerova a,⇑, Lucia Feketeova b, Miroslava Matuskova a, Zuzana Kozovska a, Pavol Janega b,c, Pavel Babal b, Martina Poturnajova a a b c
Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovakia Department of Pathology, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, 813 72 Bratislava, Slovakia Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Sienkiewiczova 1, 813 71 Bratislava, Slovakia
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Article history: Received 24 April 2012 Received in revised form 21 December 2012 Accepted 18 February 2013
Keywords: Human medullary thyroid carcinoma Gene-directed enzyme/prodrug therapy Fusion yeast cytosine deaminase 5-Fluorocytosine Bystander effect
a b s t r a c t The extent of local bystander effect induced by fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD) in combination with 5-fluorocytosine (5FC) was evaluated in xenogeneic model of human medullary thyroid carcinoma (MTC). This approach to gene-directed enzyme/prodrug therapy (GDEPT) induces strong bystander cytotoxicity. Effector yCD-TT mixed with target EGFP-TT cells in a ratio 2:9 could achieve significant tumor regression and 14-fold decrease in serum marker calcitonin upon 5FC administration. Histopathological analysis unraveled that antitumor effect resulted in tumor dormancy and proliferation arrest of remaining tumor cell clusters in vivo. yCD/5FC combination represents another GDEPT approach to achieve tumor growth control in MTC. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gene-directed enzyme/prodrug therapy (GDEPT) approaches were developed to limit host toxicity by introducing new catalytic functions into tumor cells in order to sensitize them to otherwise inert prodrugs [1–3]. Several suitable gene/prodrug combinations were proven efficient in preclinical antitumor testing. However, clinical trials have shown only limited extent of tumor control due to several reasons. Critical problem to overcome in cancer gene therapy remains to achieve gene delivery in sufficient cell numbers in order to target substantial proportion of tumor cells and mediate tumor regression [4]. Gene transfer efficiencies in clinical settings are unlikely to exceed more than 10% of target tumor cell population. Therefore an indirect effect of the treatment which causes cytotoxicity in neighboring non-transgenic cells within the tumor being called bystander effect is desirable in the context of enzyme/prodrug gene therapy. Bystander effect results in more cell deaths and higher cytotoxicity in comparison to the situation if only transgenic cells were killed by direct action of enzymatic conversion (suicide effect) [5]. GDEPT may elicit both local and distant bystander effect. The latter involves an intense systemic anti-tumor inflammatory ⇑ Corresponding author. Tel.: +421 2 59327425; fax: +421 2 59327250. E-mail address: [email protected] (L. Kucerova). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.040
infiltration and it is immune mediated. Distant bystander effect is considered to be one of the important factors for the clinical success of gene therapy in vivo [1]. On the other hand, local bystander effect is limited by the tissue penetration capacity of the prodrug to achieve sufficient concentrations and subsequently the ability of activated metabolites to spread into adjacent nontransduced cells [6]. Soluble toxic metabolites can be transferred by either passive diffusion or active transport, via apoptotic vesicles or gap junctions. Frequent deregulation in gap junction intercellular communication severely compromises the efficiency of the approaches using highly charged cytotoxic metabolites such as ganciclovir-triphosphate in GDEPT combination of herpes simplex virus thymidine kinase and ganciclovir [5]. Combination of bacterial or yeast enzyme cytosine deaminase with prodrug 5-fluorocytosine produces toxic effect via cytotoxic drug 5-fluorouracil, which can freely diffuse across the cell membranes, and thus induces significant local bystander effect [7]. Moreover, the fusion of the yeast cytosine deaminase with the uracil phosphoribosyltransferase has significantly improved the conversion efficiency of 5FC to 5FU and its metabolites further improving the antitumor effect in many preclinical models [7–10]. Although medullary thyroid carcinoma (MTC) constitutes only 2–4% of all thyroid cancer diagnosis, it has generally proven to be refractory to cytotoxic therapy [11]. Systemic therapy in patients with inoperable metastatic disease requires prolonged treatment
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with switching between different agents [12]. However, based on efficiency and toxicity not all patients can tolerate this therapy. Therefore novel multikinase inhibitors were tested in series of trials and have brought significant progress in treating patients with advanced progressive MTC during the past 5 years [13]. Messina and Robinson have recognized a potential role of gene therapy in MTC treatment summarizing also several studies using cytoreductive enzyme/prodrug combinations [14]. Our recently published data have extended the scale of effective GDEPT combinations against MTC to the therapeutic combination employing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD) in combination with 5-fluorocytosine (yCD/5FC) [9]. The absence of gap junction intercellular communication was observed in model MTC cells, and therefore the approach relying on passive metabolite diffusion was proposed to be employed to increase cytotoxicity. Potent cytotoxic effect was confirmed in vitro, and antitumor efficiency in vivo. Even though transgenic and nontransgenic tumor cells were mixed in a ratio 1:1 to induce tumor xenotransplant growth in immunodeficient mice, we have observed tumor inhibition of 92.5% indicative of strong suicide and local bystander effect in vivo. The aim of this study was to analyze the extent of local bystander effect induced by yCD/5FC with limited numbers of yCD-transduced cells present within the tumor mass. The proportion of effector-to-target tumor cells corresponded to the proportion necessary to achieve complete target tumor cell eradication in vitro. In order to track the target cells, these were modified to stably express the EGFP protein. Our xenogeneic model enables to evaluate local bystander effect in the absence of inflammatory anti-tumor responses in vivo. 5FC administration resulted in 71.4% tumor growth inhibition, 14-fold decrease in calcitonin serum levels and histopathologically confirmed tumor dormancy. These preclinical data provide evidence for potent local bystander toxicity and support the observation that yCD/5FC system is very efficient in tumor growth control in chemotherapy-resistant MTC model. We suggest that the GDEPT approach utilizing yCD/5FC combination might prove useful to overcome low antitumor efficiency and high systemic toxicity of 5FU.
2.3.1. GAPDH housekeeping gene protocol Quantitative PCR was performed with Master Mix containing: 1 MaximaÒ Probe qPCR Master Mix, final concentration of 5.5 mM MgCl2 (Thermo Fisher), 0.4 lM of GAPDH primers (sense 50 -GAA GGT GAA GGT CGG AGT C-30 and antisense 50 -GAA GAT GGT GAT GGG ATT TC-30 ), 0.1 lM GAPDH dual labeled probe (50 -HexCAA GCT TCC CGT TCT CAG CC-BHQ1-30 (Metabion, Martinsried, Germany) and 100 ng of cDNA. Thermal cycling conditions were as follows: DNA polymerase activation (50 °C for 2 min), initial denaturation step (95 °C for 10 min) followed by 40 cycles (95 °C for 15s, 60 °C for 60s and HEX fluorescence aquire). Correct amplification of 226 bp product was verified on 1% agarose gel. 2.3.2. Calcitonin gene protocol Calcitonin primers (sense 50 -TGC GGT AAT CTG AGT ACT TGC ATG C-30 and antisense 50 -CCA ACC CCA ATT GCA GTT TGG-30 ) were designed using Vector NTI software (Invitrogen) according to human calcitonin precursor mRNA calcitonin sequence (GenBank ID: X00356.1). The final reaction Master Mix contained: 1 MaximaÒ SybrGreen qPCR Master Mix (Fermentas) supplemented with 2.5 mM MgCl2, 0.18 lM of each primer and 100 ng of cDNA. Thermal cycling conditions: DNA polymerase activation (50 °C for 2 min), initial denaturation (95 °C for 10 min), followed by 35 cycles of (95 °C for 15 s, 63 °C for 60 s and acquire of SybrGreen fluorescence at 72 °C, 10 s). Correct amplification of 91 bp product was verified on 2.5% agarose gel and as a one sharp melting peak at 78 °C during melting in range 65–86 °C. 2.3.3. Statistical analysis and validation All samples were tested in parallels in four independent qPCR runs for GAPDH and qPCR runs for calcitonin expression. Data were normalized to housekeeper expression. Normalized expression for each sample relative was determined by DDCt method using CFX Manager software 1.5 (Bio-Rad). 2.4. Calcitonin EIA assay Calcitonin EIA Assay (Alpco Diagnostics) was used to quantify the protein levels. The immunoassay utilizes two mouse antibodies: biotinylated Ab against human Calcitonin 11-23 and peroxidase labeled Ab against Calcitonin 21-32. Quantification of calcitonin secretion was performed in two independent experiments, all samples in parallel according to manufacturer instruction. Briefly, 100 ll of calcitonin standards (calibrators), controls and samples in parallels were mixed with 50 ll of each antibody in streptavidin-coated microplate in a dark on an orbital rotator. It was incubated for 4 h at room temperature, washed away very carefully and incubated with TMB substrate for 30 min at room temperature in a dark with rotation. Reaction was stopped and optical density of each sample was measured at 450 and 405 nm. Concentration was derived from calcitonin standard curve. Calcitonin secretion was expressed as pg of calcitonin per 1 lg of total proteins in cell culture media as determined by Lowry method or pg of calcitonin per 1 ml of mouse serum. Standard cell culture medium or mouse serum from control mouse was used as background control.
2. Materials and methods 2.5. Proliferation analysis 2.1. Chemicals All chemicals were purchased form from Sigma–AldrichÒ, St. Louis, MO, if not stated otherwise.
2.2. Cells Epithelial adherent TT cell line (ATCC. No. CRL-1803™) derived from human medullary thyroid carcinoma was purchased from ATCC and cultured in RPMI medium (Roswell Park Memorial Institute, PAA Laboratories GmbH) supplemented with 10% fetal bovine serum (FBS, Biochrom AG), 10.000 IU/ml penicillin (Biotika, Part. Lupca, Slovakia), 2 mM glutamine and 5 lg/ml streptomycin (Sigma, St. Louis, MO). Transgenic TT cells expressing fusion yeast cytosine deaminase::uracil phosphoribosyltransferase yCD or enhanced green fluorescent protein (EGFP) were prepared as described elsewhere in detail [9]. All cells were expanded according to standardized protocol and kept in humidified atmosphere and 5% CO2 at 37 °C. 2.3. Calcitonin expression analysis Parental TT and/or transduced TT cells were collected by accutase solution (PAA Laboratories GmbH), washed with PBS solution and pelleted (5–7 106 cells per pellet). These were frozen in RNAlater Stabilization Reagent (Qiagen, Hilden, Germany) and stored at 80 °C until use. RNA was isolated with NucleoSpinÒ RNA II Kit (Macherey–Nagel). Any traces of DNA were removed by RNase-free DNase I treatment (10 U/lg RNA) in the presence of 20 U RiboLock solution (Thermo Fisher). Total RNA was screened for complete DNA removal by PCR and then reverse transcribed using RevertAid™ H Minus First Strand cDNA Synthesis Kit and oligo-dT18 primer (Thermo Fisher).
One to 1.5 105 cells per well in 6-well plates were plated in duplicates, which ensured unrestricted exponential growth for the duration of the assay. Population doubling time (PDT) was calculated as an average of three measurements for the TT cells according to the formula G = t log 2/log Ntlog No, where t = time period, Nt = number of cells at time t, No = initial number of cells. In order to directly compare proliferation rates for EGFP-TT and yCD-TT cells, these cells were then labeled with 5 lL of Vybrant DiI (Invitrogen, Carlsbad, CA) diluted in 1 ml PBS per 1 106cells for 20 min at 37 °C. One part of these cells was proliferation-arrested by 10 lg/ml Mitomycin C (Mit-C, Kyowa, Hakko Ltd., UK) diluted in culture medium for 3 h and washed. Proliferating and arrested cells were kept in standard culture medium for 14 days. Cells were then detached by accutase solution, washed and fluorescence intensity was determined by flow cytometry. DiI fluorescent staining does not interfere with EGFP fluorescence, therefore this method is suitable for EGFP-TT stably expressing fluorescent reporter protein. 2.6. Xenotransplant growth and animal treatments in vivo A 6–8 weeks old athymic nude female mice (Balb/c nu/nu) were used in accordance to the institutional guidelines under the approved protocols. Tumors were induced with 1.1 107 EGFP-TT cells resuspended in serum-free RPMI diluted 1:1 with ECM gel (cat. No. #1270, Sigma–AldrichÒ, St. Louis, MO) s.c. Animals with growing xenotransplants (n = 5–6/group) were randomly divided into two groups for 5FU treatment, which was started on day 9. Treated animals received the dose of 10 mg 5FU/kg/day i.p. every other day for 22 days. Preliminary experiments have shown that this was the maximum tolerated dose in our experimental setting. In independent series of experiments, tumors were induced by 2 106 effector transgenic yCD-cells TT mixed with 9 106 target transgenic EGFP-TT (ratio effector-to-target cells 2:9) injected subcutaneously. Cells were resuspended in
L. Kucerova et al. / Cancer Letters 335 (2013) 299–305 serum-free RPMI diluted 1:1 with ECM gel (cat. No. #1270, Sigma–AldrichÒ, St. Louis, MO). Animals with growing xenotransplants (n = 5–6/group) were randomly divided into two groups for 5FC treatment, which was started on day 9. Treated animals received the dose of 500 mg 5FC/kg/day i.p. every day for 22 days. Animals were evaluated for tumor growth and size regularly. Tumor volume was calculated from caliper measurements according to formula volume = length width2/2. Results were evaluated as mean tumor volume ±SD. Untreated animals had to be sacrificed at the timepoint when they exhibited tumor ulceration, bleeding or any other signs of distress. Serum was harvested from blood samples taken from treated and untreated animals. Tumor xenotransplants or fibrotic remnants at the site of tumor growth were excised for further immunohistochemistry.
2.7. Immunohistochemistry Excised tumor xenotransplants were fixed in buffered formalin, embedded in paraffin, cut into 5 lm thick sections, stained with hematoxylin/eosin and evaluated by light microscopy (Leica DM2000, Wetzlar, Germany). Immunohistochemical staining was performed to detect proliferation markers Ki67 and PCNA (proliferating cell nuclear antigen). Tissue samples were also stained for EGFP to detect the presence of target EGFP-TT cells. Following reagents were used: monoclonal mouse anti-Ki67 antibody (Dako, Denmark), monoclonal mouse anti-PCNA antibody (Dako, Denmark) and polyclonal rabbit anti-EGFP antibody (Abcam, Cambridge, UK). Reaction was visualized by universal peroxidase polymer Histofine (anti-mouse, anti-rabbit) with diaminobenzidine (Dako, Denmark) as a substrate for color reaction. Marker expression was evaluated by semiquantitative method, positivity or negativity for nuclear markers was evaluated.
2.8. Statistical analysis Student’s two-sample t-test was used to compare the difference in means of two samples. Calcitonin secretion in sera of 5-FC treated vs. untreated mice were subjected to Mann–Whitney U test. The p-values with p < 0.05 were considered to be statistically significant.
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3. Results As previously reported two transgenic cell populations were prepared from human parental TT cells derived from MTC [9]. Retrovirally transduced yCD-TT cells express yeast fusion cytosine deaminase::phosphoribosyltransferase (yCD) and serve as effector cells for gene-directed enzyme/prodrug treatment. Enzymatic activity of yCD converts nontoxic 5-fluorocytosine (5FC) to metabolites of 5-fluorouracil, potent cytotoxic drug. Target transgenic TT cells were modified to express enhanced green fluorescent protein (EGFP) in order to track their presence in vivo. Both types of transgenic TT cells have retained calcitonin expression as an MTC characteristic (Fig. 1A). Calcitonin secretion was confirmed in cell culture supernatants, although the calcitonin production was significantly increased in supernatant from yCD-TT cells (Fig. 1B). Increased calcitonin secretion may be linked to the capability of fusion enzyme yCD to increase cell proliferation as previously reported in our work [15]. Introduction of yCD and its constitutive expression interferes with the cell-intrinsic nucleotide synthesis machinery and renders them with the capability to utilize the nucleotide precursors more efficiently. Indeed, yCD introduction shortened the population doubling time in transgenic yCD-TT cells which is not the case for retrovirally transduced EGFP-TT transgenic cells. EGFP-TT cell proliferation does not differ from the proliferation in parental TT cell population (Fig. 1C and D), therefore this efect is likely to depend on the yCD enzyme activity. MTC is generally reported to be refractory to chemotherapeutic treatment in clinical setting. Our previous data have shown moderate sensitivity of TT cells to the treatment with 5-fluorouracil with
Fig. 1. Transgenic yCD-TT cells have increased calcitonin secretion and proliferation. (A) Calcitonin expression was evaluated in parental, EGFP-TT and yCD-TT cells by quantitative RT-PCR. Effector yCD-TT cells exhibited increased calcitonin mRNA levels. (B) Cells were cultured in standard medium for 3 days and the concentration of calcitonin secreted into media was evaluated by EIA. Results were expressed as average concentration ±SD. p < 0.05. (C) Parental and transduced TT cells were plated at equal density to ensure the unrestricted exponential growth. Population doubling time was significantly lower for the effector yCD-TT cells. p < 0.05. (D) Vybrant DiI-dye retention assay to compare cell proliferation after prolonged culture was performed and evaluated by flow cytometry. Synchronized EGFP-TT or yCD-TT cells were stained with DiI, split into two parts, one portion was MitC-arrested and both aliquots were maintained in standard culture media for 14 days. Fluorescence intensity unraveled decreased fluorescence intensity in yCD-TT cells corresponding to increased proliferation. Parental TT cell displayed the analogous dye retention pattern to EGFP-TT and were omitted from figure for clarity.
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IC50(5FU) = 0.6 lg/ml in vitro. Our data also show that TT cells were responsive to vincristine and doxorubicin at high concentrations (data not shown). 5FU treatment of tumor xenotransplants derived from EGFP-TT cells could induce significant antitumor effect in vivo (Fig. 2A). 5FU administration resulted in 66.7% reduction in tumor volume as measured by caliper on day 22 of 5FU treatment (56.6 mm3 in the 5FU treated group in comparison to 170.3 mm3 in the untreated controls). Tumor volumes remained stable in the 5FU treated group with a tendency to increase after the termination of 5FU administration. Experiments had to be terminated due to cumulative 5FU toxicity and animals had to be sacrificed due to massive weight loss, diarrhea, and skin rash with open wounds. When tumor xenotransplants were grown from mixtures of transgenic yCD-TT effector cells and EGFP-TT target cells (ratio 2:9), rapid development and growth of tumor xenotransplant was observed as expected. Prodrug administration (5FC treatment) prevented further growth and resulted in tumor regression even though cells with enzymatic function yCD-TT represented only 18% of the tumor cell population (Fig. 2B). By the day 31 after experiment start the average tumor volume reached 229.4 mm3 in the untreated group and animals had to be sacrificed. Tumor volume in 5FC treated group was 65.5 mm3 by the same time point exhibiting tumor growth inhibition of 71.4%. In the absence of any bystander effect the theoretical extent of tumor growth inhibition corresponding to suicide action of enzymatically produced metabolites would have been less than 20% based on the starting effector-to-target cell ratio. Moreover, the tumor xenotransplants regressed further after treatment withdrawal and the average tumor volume decreased to 21.4 mm3 in this group by the day
51. Treated animals did not show any sign of systemic treatment toxicity. The bystander effect efficiency was reflected also by a decrease in serum calcitonin levels from 6027.1 pg/ml sera to 425.1 pg/ml sera, which is 14-fold decrease by the day 31 (Fig. 4C). In the EGFP-TT group calcitonin levels decreased from 4826 pg/ml sera in controls to 980 pg/ml sera in 5FU treated group, which is 4.9-fold decrease by the day 31. Taken together our data confirm the capability of gene directed enzyme/prodrug combination yCD/5FC to mediate local bystander effect accompanied by significant antitumor effect in the absence of any toxicity. The treatment efficiency of GDEPT approach is comparable to the effect of systemic 5FU administration. Immunohistochemical staining of untreated tumor xenotransplants unraveled retention of two populations of tumor cells within the tumor mass in the untreated control as expected (Fig. 3, left). In order to unravel whether the complete tumor cell eradication was achieved the fibrotic remnants of the tumor xenotransplants were analyzed at the experiment endpoint. Our data show small clusters of tumor cells within the fibrotic tissue at the site of tumor cell administration. Two populations of cells were visible: EGFPpositive and negative TT cells, which escaped the suicide and/or bystander cytotoxicity (Fig. 3 middle and right). More detailed analysis has shown that both effector and target TT cells were positive for proliferation markers Ki67 and PCNA in untreated tumors (Fig. 4 left). Contrastingly, clusters of EGFP-positive and negative cells which escaped the treatment and remained at the site of tumor growth had very rare Ki67 positivity (Fig. 4 right). PCNA staining was diffuse within the tumor clusters corresponding to the cells which were arrested in the S phase of cell
Fig. 2. Local bystander effect of yCD/5FC combination in vivo. (A) EGFP-TT cells diluted in ECM were inoculated in flank of immunodeficient females. Mice were randomly split into two groups. Starting on day 9 after inoculation one group was treated with 5FU for subsequent 21 days. Average tumor volume in 5FU treated group was significantly lower in comparison to untreated controls by day 31 (56.6 mm3 vs. 170.3 mm3). p < 0.01, Experimental animals had to be sacrificed due to ethical reasons, arrows depict the 5FU treatment timing. (B) Effector yCD-TT and target EGFP-TT cells (mixed in a ratio 2:9) diluted in ECM were inoculated in flank of immunodeficient females. One group of mice with tumor xenotransplants of average volume 75 mm3 (Day 9) started receiving 500 mg 5FC/kg/day for the next 22 days. By the day 31 there was significant difference in the median tumor volume when compared to 5FC untreated group. Treated animals were observed for another 20 days and exhibited further decrease in tumor volume from 65.5 mm3 to 21.4 mm3. p < 0.01, Experimental animals had to be sacrificed due to ethical reasons, arrows depict the 5FC treatment timing. (C) Mouse serum was collected from treated and untreated animals and the calcitonin concentration was determined by EIA. Calcitonin levels in serum of 5FC-treated and 5FU-treated mice was significantly lower in comparison to serum levels from untreated mice being in concordance with high tumor volumes in untreated mouse xenografts at the experiment endpoint. Background calcitonin level in the serum obtained from untreated healthy mouse was 100.5 pg/ml. p < 0.05.
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Fig. 3. Tumor xenotransplants derived from yCD-TT/EGFP-TT cells were analyzed by immunohistochemistry to detect transgenic EGFP-TT target tumor cell population. Left panel shows that untreated tumor tissue is composed of two tumor cell subpopulations both EGFP-positive (target EGFP-TT) and EGFP-negative (effector yCD-TT) cells which contribute to tumor growth. 5FC-treated tumor xenotransplants have shown regression with very small remnants palpable at the site of tumor. These fibrotic remnants (middle and right panel) are interspersed with clusters of tumor cells which escaped the suicide and bystander cytotoxicity. Both tumor cell populations of EGFP-positive and EGFP-negative cells were present within the clusters. Brown staining: EGFP-positivity, magnification 20. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. In order to determine the proliferation status in 5FC treated tumor xenotransplants, adjacent tumor sections were analyzed by immunohistochemistry to detect proliferation markers Ki67 and PCNA. Panels on the left confirm that both tumor cell populations yCD-TT and EGFP-TT are prominently positive for Ki67 and PCNA corresponding to cell proliferation and tumor growth. Right panel depicts very low Ki67 positivity in tumor cells within the clusters (red arrows). Diffuse staining for PCNA in combination with the absence of Ki67 marker is indicative of cell division arrest in the remaining cells being mostly in the S phase of the cell cycle. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
cycle. Very low Ki67 positivity corresponded to the arrest in cell division and these cells remained dormant. As both cell populations within the clusters in the treated tumors did not show any signs of proliferation activity, we concluded that even though these cells were retained at the site of tumor growth at the treatment endpoint they were incapable of proliferation and thus incapable of further tumor growth and spread. Taken together our data demonstrate the GDEPT approach to overcome the chemoresistance of MTC in vivo and suggest the
combination of gene directed enzyme/prodrug therapy approach which could induce strong local bystander effect even though only a small part of the tumor expressed the transgene.
4. Discussion Medullar thyroid carcinoma (MTC) represents a malignant disease which spreads quite early into adjacent lymph nodes and
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gives rise to multiple metastatic lesions in lungs, bones and liver. MTC is generally recognized as resistant to standard radiotherapy and chemotherapy [16,17]. Sporadic MTC grows usually slowly, but 50% of patients have tumor lesion within lymph nodes and 10% have already inoperable metastatic spread in distant organs at the time of diagnosis [18]. Heritable forms of MTC cover both familiar form (FMTC) and MTC as a major component of multiple endocrine neoplasia type 2 syndrome (MEN2). The latter MTC form exhibits quite rapid growth. Gain-of-function mutation in RET proto-oncogene is responsible for autosomal-dominantly inherited cancer syndrome MEN2. Cys634Trp mutation in RET proto-oncogene frequently occurs in patients with MEN2A and FMTC [18]. Model human TT cell line derived from medullary thyroid carcinoma has the above mentioned mutation and it was previously described as a relevant model for MTC [19]. In general, metastatic MTC is currently considered to be an incurable disease by standard treatment options. However it remains a subject to various experimental treatment strategies such as gene therapy in order to unravel the (combination of) approaches to achieve disease control. These include the efforts to correct the cellular defect by dominant-negative RET mutants, immunomodulatory gene therapy and cytotoxic gene directed enzyme/prodrug treatment approaches [14]. In our previous report we have shown so far unrecognized GDEPT strategy in MTC xenotransplant model by employing combination of fusion yeast cytosine deaminase with prodrug 5-fluorouracil [9]. This proofof-principle report highlighted its capability to overcome therapeutic refractoriness of MTC in vivo. In order to determine the extent of local bystander effect we have extended our previous study and induced tumor xenotransplant growth by lower proportion of transgenic effector yCD-TT to target EGFP-TT cells being more relevant for clinical application. Therapeutic yCD-TT cells expected to undergo cell suicide upon 5FC prodrug administration represented only 18% of the tumor cell population at the beginning. Our previous data have shown that this proportion of the effector cells could decrease the overall cell proliferation to less than 10% in vitro [9]. The adjacent yCD-TT cells spreading cytotoxic metabolites of 5FU could eradicate the majority of target TT cells within the 9 days in direct coculture. Correspondingly, 5FC administration to experimental animals with growing tumor xenotransplants in vivo resulted in the tumor size decrease correlating to expected antitumor effect. By the day 21 after treatment initiation a prominent decrease in the tumor volume could be observed based on caliper measurements, when tumors in the untreated group of animals increased their volume. However, based on our experience these measurements underestimate the extent of the antitumor effect. These outcomes cannot distinguish the contribution of the tumor cells from the necrotic tumor part and the fibrous remnants. Our analysis was therefore supplemented with the calcitonin level determination in order to evaluate the extent of antitumor effect by clinically relevant test. We could confirm 14-fold decrease in calcitonin concentration in 5FC treated animals. These data suggest the real extent of local bystander effect being higher than anticipated by volume comparison. 18% prodrug converting effector cells could achieve 92.9% inhibition of calcitonin secretion as determined in the serum of 5FC treated vs. untreated animals. In order to directly compare the efficiency of the approach, growing tumor xenotransplants were treated with 5FU at the same time schedule – treatment start on day 9 followed by 5FU administration for next 21 days. Significant decrease in tumor volume in 5FU treated animals could be detected in concordance with 79.7% decrease in serum calcitonin levels, although tumor volume remained stable and had tendency to increase after 5FU withdrawal. Contrastingly, no re-growth in any of the tumor xenotransplants could be observed after withdrawal of the 5FC administration. Treated tumors further decreased in volume. At the experiment endpoint the remaining
tissue at the site of tumor xenotransplant was analyzed and the presence of isolated tumor cell clusters was observed within the fibrous tissue. This finding further confirmed the higher extent of bystander effect in tumors than estimated by the caliper measurements. Moreover the remaining cells were found to lack Ki67 staining and PCNA detection which indicated their arrest in the S phase of the cell cycle. As we have performed our experiments in the immunodeficient mice we could directly evaluate the extent of local bystander effect in the absence of any immune-cell mediated reaction. We anticipate that the situation in the immunocompetent host would be even more prominent and distant bystander effect mediated by the immune cells could further increase the therapeutic efficiency [20,21]. Distant bystander effect might be important in the situations when the transduction efficiency within the tumor tissue remains low. Currently reported efficiencies from clinical setting do not exceed 10% transduction efficiencies in the target cells [4,22,23] and therefore we anticipate that distant bystander effect will substantially contribute to the final treatment outcome. Our data indicate comparable antitumor efficiency of the GDEPT approach in the absence of any systemic toxicity. Our experimental data indicated that TT cells exhibited limited chemosensitivity in vitro and in vivo. They responded to cytotoxic drugs such as vincristine and doxorubicin at high concentrations only (data not shown). The expression of drug transporters ABCB1 and ABCC1 which were previously linked to increased resistance to doxorubicin and vincristine is likely to contribute to their chemoresistance [9,24]. Although we determined that IC50 (5FU) = 0.6 lg/ ml for TT cells, we were not able to reach IC90 in vitro [9]. Even though TT cells do not express drug transporter ABCB5 previously linked to increased 5FU efflux and resistance [25], these cells have high expression of enzyme dihydropyrimidine dehydrogenase (DPD) that inactivates 5FU to non-toxic metabolites [26,27]. Tumor xenotransplant treatment with cytotoxic agent 5FU correlates to the clinical observations with chemotherapy efficiency in MTC. Maximum tolerated 5FU dose could achieve stabilization of tumor volume in our mouse model accompanied by high toxicity and tendency to re-grow after treatment termination (Fig. 2A). Immunohistochemical data showed that tumor xenotransplants had prominent Ki67 and PCNA staining indicating the proliferative status of tumor cells in vivo. This might support the hypothesis that the 5FU refractoriness was linked to the low intratumoral 5FU concentration. DPD expression in the liver contributed to rapid 5FU breakdown and decrease in plasma, DPD expression in the tumor cells further contributed to 5FU intratumoral breakdown and the absence of gap-junctional intracellular communication in these cells further eliminated the transmission of toxic metabolites throughout the tumor. Several previous reports have indicated potential ways to overcome the chemoresistance in MTC cells. TT cell chemoresistance could be abrogated by cyclooxygenase-2 (COX-2) inhibition with selective COX-2 inhibitor which sensitizes TT cells to the cytotoxic effects of doxorubicin, reduces ABCB1 expression and function [24]. Our data suggested that introduction of fusion enzyme cytosine deaminase::uracil phosphoribosyltransferase sensitized yCD-TT cells to 5FU (yCD-TT cells have IC90(5FU) 1 lg/ml corresponding to the achievable plasma concentrations) [9]. This effect is not unique to tumor cells as the similar phenomenon was reported for the nonmalignant transduced human mesenchymal stromal cells [15]. Chemosensitization by the expression of yCD transgene seems to represent a more general phenomenon: its constitutive expression interferes with the intrinsic nucleotide metabolism thus increasing the cell proliferation (Fig. 1C and D). It was previously shown that this effect cannot be attributed to the retroviral gene transfer procedure. The cells which were transduced with mock plasmid or marker EGFP vector did not have increased proliferation and chemosensitivity [15]. However, it is
L. Kucerova et al. / Cancer Letters 335 (2013) 299–305
possible that increased proliferation of (tumor) cells mediated by yCD renders them more vulnerable to other cytotoxic agents. Thus the combination of the yCD/5FC antitumor gene therapy approach with other cytotoxic strategies might prove beneficial for therapeutic effect and tumor growth control. Another approach to chemosensitization and radiosensitization of tumor cells was reported by Wang et al. who expressed cytosine deaminase under the hypoxia responsive promoter. They reasoned that increased 5FC conversion to 5-fluorouracil could also increase radiosensitivity of the solid tumor [28]. In summary, yCD/5FC combination represents efficient GDEPT combination for MTC treatment. yCD/5FC induces strong local bystander effect in MTC model in vivo. yCD/5FC treatment decreased serum calcitonin by 92.9%, arrested tumor cell proliferation and induced tumor dormancy, thus representing potent antitumor strategy even in highly chemoresistant solid tumor model.
[8]
[9]
[10] [11]
[12] [13] [14]
[15]
Conflict of interest [16]
The authors declare no conflict of interest.
[17]
Acknowledgements
[18]
We thank M. Dubrovcakova, V. Frivalska, R. Bohovic and E. Klincova for the excellent technical assistance. Study was supported by the Slovak Research and Development Agency under the contract No. APVV-0230-11, the VEGA Grants Nos. 2/0088/11 (L.K.) and 2/0146/10 (M.M.). This work was supported by the Framework Program for Research and Technology Development, Project: Building of Centre of Excellency for Sudden Cerebral Vascular Events, Comenius University Faculty of Medicine in Bratislava (ITMS: 26240120023), cofinanced by European Regional Development Fund.
[19]
[20]
[21]
[22]
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