Accepted Manuscript
IGF1-R inhibition and liposomal doxorubicin: progress in preclinical evaluation for the treatment of adrenocortical carcinoma
195H 0000-720P
Molecular and Cellular Endocrinology
Felix Beuschlein, Judith Jakoby, Susanne Mentz, Gerard Zambetti, Sara Jung, Martin Reincke, Regine Süss, Constanze Hantel
PII:
S0303-7207(16)30071-5
DOI:
10.1016/j.mce.2016.03.023
Reference:
MCE 9458
To appear in:
Molecular and Cellular Endocrinology
Received Date: 24 November 2015
Revised Date: 16 March 2016
Accepted Date: 16 March 2016
Please cite this article as: Beuschlein, F., Jakoby, J., Mentz, S., Zambetti, G., Jung, S., Reincke, M., Süss, R., Hantel, C., IGF1-R inhibition and liposomal doxorubicin: progress in preclinical evaluation for the treatment of adrenocortical carcinoma, Molecular and Cellular Endocrinology (2016), doi: 10.1016/ j.mce.2016.03.023.
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IGF1-R inhibition and liposomal doxorubicin: progress in preclinical evaluation for the treatment of adrenocortical carcinoma
3 4 5 6 *First 2 authors contributed equally and last 2 authors contributed equally as senior
7 scientists
8 Corr. author: Constanze Hantel
9 1Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany
10
11 2Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology 12 and Biopharmacy, Albert Ludwig University Freiburg, Freiburg, Germany
13 3Department of Pathology, St. Jude Children’s Research Hospital, Memphis,
14 Tennessee
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Felix Beuschlein*1, Judith Jakoby*2, Susanne Mentz1, Gerard Zambetti3, Sara Jung1, Martin Reincke1, Regine Süss*2, Constanze Hantel*1
ACCEPTED em Count, Rue
1 Abstract
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Adrenocortical carcinoma (ACC) is a tumor with poor prognosis and limited therapeutic options. Therefore, in addition to multi-chemotherapeutic regimens IGF-1 receptor (IGF-1R) targeting approaches have been evaluated including immunoliposomal (IL) preparations utilizing an IGF-1R inhibiting antibody. In the current study, we extended our experiments by long-term treatment regimens in the classical adrenocortical NCIH295R xenograft model as well as by short-term experiments in two novel xenograft models, which all displayed different levels of IGF-1R and IGF-2 expression. Interestingly, these experiments reveal sub-group dependent differences in therapeutic outcome, reflecting clinical observations and indicate, thus, that implementation of this panel of tumor models might be helpful for clinical translation of novel therapeutic regimens in the future.
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14 Keywords
Adrenocortical carcinoma, liposomal doxorubicin, immunoliposomes, Caelyx®, NCI- H295R; SJ-ACC3, MUC-1
17
18 Abbreviations
19 Ab antibody
20 ACC adrenocortical carcinoma
21 Chol cholesterol
22 DSPE-mPEG
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]
23 24 SPIT sterol-based post-insertion technique
25
1H7 anti IGF1-R ab
26 HBS HEPES buffered saline
27 SH sucrose histidine buffer
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1 1. Introduction
Adrenocortical carcinoma is a rare neoplasm with an annual incidence of 0.7-2.0 cases per million population which is characterized by a dismal 5-year survival rate of only 16-38% [1-3]. In a considerable proportion of patients diagnosis is made at stage when complete surgical resection is not achievable [4]. Even in case of RO resection between 40 and 80% of patients relapse depending on the proliferative potential of the tumor [5]. For patients with advanced disease systemic treatment options include mitotane in combination with cytotoxic drugs [6]. While the first 9 prospective randomized interventional trial for late stage ACC patients has recently demonstrated superiority of etoposide, doxorubicin, cisplatin and mitotane treatment over streptozotocin and mitotane [7], this trial also made evident the overall non- satisfactory response rates of available cytotoxic regimens. Thus, novel therapeutic strategies for patients with ACCs are urgently needed.
In the past, IGF1 receptor (IGF1-R) dependent signaling has been shown to promote tumorigenesis in ACC [8-11] and preclinical studies have provided for therapeutic applicability of IGF-1 receptor directed approaches [12, 13]. IGF-2, the ligand of IGF1-R is an important molecular marker for ACC as it is highly overexpressed in ACC tissue [14]. Moreover, strong evidence has pointed towards an autocrine influence of IGF-2 in adrenocortical tumor cell growth [8]. In line with this notion, an active role of IGF-2 has recently been confirmed by an RNA knock-down approach in NCI-H295R cells, which substantially impaired tumor cell growth, led to cell cycle arrest and induced apoptosis [15].
promSigaled these finci Quedado Which cancortical AC12 13) evidence
Based on findings, different IGF-1-R targeting approaches have been investigated in recent years for the treatment of ACC and some of them have led to promising results in preclinical and early clinical studies [12, 16-18]. However, subsequent clinical trials were disappointing and it remains uncertain whether inhibition of IGF1-R alone is sufficient to mediate sustained therapeutic effects [1, 16, 19]. Consequently, combinatory approaches with temsirolimus and mitotane have been initiated to complement effects of IGF-1-receptor directed therapies [6, 20, 21]. Currently, these combinations did not lead to the expected therapeutic benefits.
In 2010 our workgroup reported for the first time on the establishment of anti IGF1- receptor antibody (1H7) coupled, doxorubicin loaded, immunoliposomes and
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provided furthermore evidence for a putative therapeutic applicability against neuroendocrine tumors of the gastroenteropancreatic system (GEP-NETs) in vitro and in vivo. Moreover, in vitro experiments indicated that usage of this agent could also present a promising approach for other tumor entities as breast cancer, neuroblastoma and prostate cancer, as they showed very high cellular association and internalization rates into appropriate cell lines of human origin [22, 23]. In subsequent studies, we reported on an extraordinary uptake phenomenon of plain liposomes by adrenocortical tumor cells [24, 25]. While flow cytometry revealed again high cellular association and internalization of anti-IGF1-R immunoliposomes, also internalization of only pegylated liposomes and surprisingly an even higher uptake of plain liposomes by NCI-H295R cells were demonstrated in vitro. In vivo, single treatments were investigated on NCIh295 tumor xenografts in pharmacokinetic and therapeutic experiments. Accordingly, we detected after SSLD-PEG treatment a significantly higher accumulation of doxorubicin in the appropriate tumor tissue compared with SSLD-1H7 (Figure A below) while the highest therapeutic effects was still detectable in NCIh295 tumor-bearing mice after a single treatment with SSLD- 1H7. However, even though diminished, also a significant therapeutic efficacy for sterically stabilized liposomal doxorubicin (Caelyx®) alone was detectable for ACC. In the current study, we extend our findings by investigation of the immunoliposomal approach and versus a combinatory administration of the single substances in a long- term therapeutic study in the classical adrenocortical NCI-H295R xenograft model as well as in short-term experiments with the recently established new xenograft models for ACC SJ-ACC3 [26] and MUC-1 [27]. Our data provide further evidence, that a combination of IGF-1R inhibition and liposomal doxorubicin could represent a promising therapeutic approach for a subgroup of ACC patients. Moreover, our data indicate that the novel ACC xenograft models SJ-ACC3 and MUC-1 might be helpful for the identification of predictive and therapeutic biomarkers of IGF-1R inhibitory regimens in the future.
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1 2. Materials and methods
2.1. Surface modification of preformed liposomes
Chol-PEG-NHS was synthesized in our lab[24]. Caelyx® was obtained from Janssen- Cilag GmbH (Neuss, Germany). The monoclonal antibody to CD221 (IGF1-receptor alpha chain, 1H7 clone) was purchased from Acris Antibodies (Herford, Germany). Caelyx® was surface modified using the sterol-based post-insertion technique, the succinimide-activated Chol-PEG2000-NHS anchor and the 1H7 ab. For ab anchor 8 coupling, the ab solution in a concentration of 10 ug/ul was pipetted into an anchor coated vial resulting in an ab/anchor ratio of 1 to 50 (mol/mol). The mixture was vortexed and bath sonicated for 30 s following incubation at 17 ℃ under shaking (700 rpm). After 15 min the reaction was stopped by adding a 50 fold molar excess of histidine (in HBS, 20 g/l). After 10 min Caelyx® was added in an anchor to total lipid ratio of 1:80, vortexed and incubated for 60 min at 20 C. All steps were performed aseptically under laminar flow and no separation step of free ab-anchor conjugate was performed.
2.2. Therapeutic experiments
Female athymic NMRI nu/nu mice (6 - 8 weeks) were purchased from Harlan Winkelmann (Borchen, Germany) and housed under pathogen-free conditions. Cell line derived NCI-H295R and tissue-derived SJ-ACC3 and MUC-1 xenografts were induced as described elsewhere [25-27]. For therapeutic experiments sterically stabilized liposomal doxorubicin (Caelyx®) was used as basal formulation. As control treatment free ab-anchor conjugate was applied and with a time lag unmodified Caelyx® liposomes (designated as L + Ab). For ab-anchor conjugate preparation all steps were performed as described above unless liposome addition was omitted and for administration diluted with sterile HBS resulting in a final volume of about 100-200 ul. For short-term experiments single therapeutic treatments were administered intravenously to SJ-ACC3 (n=4-5) and MUC-1 (n=8) tumor bearing mice. 72 hours after a single treatment, mice were sacrificed, the tumors excised and subsequently analyzed. For long-term experiments on NCI-H295R-xenografts (n=7) treatments were given at days 11, 21, 29 and 37 after tumor induction. Dosages of 10 mg/kg body weight sterically stabilized liposomal doxorubicin (Caelyx®) and 3.9 mg/kg 1H7
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antibody or the respective immunoliposomal preparation were administered in short- as well as in long-term experiments. Mice were monitored daily and euthanized when the tumors reached a longest tumor diameter of 15 mm or in case of adverse effects as body weight loss, diarrhea, impaired phenotype, tumor ulceration or bleeding in tumor area. All experiments were carried out following protocols approved by the Regierung von Oberbayern and in accordance with the German guidelines for animal studies.
2.3. Real-time PCRs, Western Blots and immunohistochemistry
For Real-Time PCR analyses NCI-H295R (n=6), SW-13 (n=3), SJ-ACC3 (n=3) and MUC-1 tumors (n=3) were used for RNA extraction (SV Total RNA Isolation system, Promega) and reverse transcription (RevertAid™ H Minus First Strand cDNA Synthesis Kit, Fermentas). For Real-Time PCR analysis we utilized the EvaGreen® reaction mix (Bio-Rad, Munich, Germany) in the Stratagene Mx3000PTM Cycler (Agilent Technologies, Waldbronn, Germany). Human primer sequences or catalogue numbers were as follows: IGF-1R (forward 5’-AATAAGCCCCCAAAGGAATG, reverse 5’-TGGCAGCACTCATTGTTCTC) and IGF-2 primer (IGF-2 human Hs00171254_m1, TaqMan Gene Expression Assay) from Thermo Fischer Scientific (Karlsruhe, Germany).
For Western Blot analysis (n=3) 20µg protein preparations were run on Mini PROTEAN TGX Stain-Free Precast gels (Bio-Rad, Hercules, CA). ß-actin antibody (1:5000; Sigma-Aldrich, Saint Louis, MO), Akt (#9272 Cell Signaling, Danvers, MA) and phospho-Akt (#9271 Cell Signaling) were used as primary antibodies. As secondary antibodies Amersham ECL Rabbit IgG, HRP-linked F(ab’)2 fragment (from donkey), NA9340, and Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep), NA931, GE Healthcare, Freiburg, Germany, were used respectively. Quantification was achieved by the measurement of the pixels and adjacent analyses using the imaging program ImageJ (NIH, Bethesda, MD).
Paraffin-embedded sections were rehydrated and incubated with blocking buffer containing 3 % BSA (Roche Diagnostics, Mannheim, Germany), 5 % goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA), and 0.5 % Tween 20 for 15 min. For the specific stainings either monoclonal mouse anti-human Ki67
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1 (DakoCytomation, Glostrup, Denmark; 1:200 in BB), the monoclonal IGF-1R antibody 2 (IGF1-receptor alpha chain, 1H7 clone, Acris Antibodies, Herford, Germany) or the 3 4 5 6 7 DeadEnd colorimetric TUNEL system (Promega, Madison, WI; following the manufacturers protocol) were used and incubated overnight at 4 ℃. After rinsing for 15 min in PBS, secondary antibody (ImmPRESS™ HRP Anti-Mouse IgG (Peroxidase) Polymer Detection Kit, made in Horse (MP-7402), Vector Laboratories, Burlingame, CA, or biotinylated anti-rabbit (BA-1000), Vector Laboratories was 8 9 applied for 30 min at room temperature. Bound primary antibody was visualized using the Vectatstain ABC Kit (Vector Laboratories) according to the manufacturer’s 10 protocol with incubation for 30 min followed by 3,3’-diaminobenzidine (Sigma-Aldrich) staining. For quantification, 6 high power fields (HPF, 0,391 mm2, 400x magnification/ tumor) were investigated and quantified for Ki67 positive and negative cells.
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Furthermore, studies including patient biomaterial were approved by the local ethical committee.
2.4. Statistical Analyses
Statistical significance was determined by One-Way-ANOVA (with Dunnett post-test for direct comparison with the NaCI control group or normal adrenal glands) using Prism software (Houston, TX). Statistical significance is denoted as stars (*, p<0.05; ** , p<0.01; *** , p<0.001) in the figures if not stated otherwise.
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1 3. Results
3.1 Investigation of IGF-1 receptor and IGF-2 levels in four ACC xenograft models
IGF1-R mRNA levels were investigated by Real Time PCR (Figure 1A) in NCI- H295R, SW-13, SJ-ACC3 and MUC-1 xenografts. These experiments demonstrated high IGF-1R abundance in the NCI-H295R (100.0±17.4%) and SJ-ACC3 7 (108.3+8.2%) xenograft models, while the expression levels were significantly lower 8 9 10 11 in SW-13 and MUC-1 tumors (17.9±0.6 % and 29.2±1.3 %; both p<0.001 vs. NCI- H295R). In comparison, normal human adrenal glands displayed median IGF1- receptor expression levels compared with the different tumor models. (63.3±10.7%, p>0.05 vs. NCI-H295R and MUC-1 and p<0.05 vs SW-13 and SJ-ACC3). 12 Immunohistochemical investigation confirmed the obtained results for all four tumor models, showing again highest IGF-1R protein abundance in NCI-H295R and SJ- ACC3 tumors (Figure 1C). Moreover, Real Time PCR analyses revealed highly elevated IGF-2 expression for the pediatric SJ-ACC3 tumor model (1140.0±111.0%) compared with all other xenografts investigated (NCI-H295R: 100.0±11.6%, SW-13: 0.0±0% and MUC-1: 151.7±13.1%; all p<0.001 versus SJ-ACC3; Figure 1B).
3.2. Short-term therapeutic in vivo experiments in SJ-ACC3 and MUC-1 tumor 20 models
Following this initial expression analysis, we made usage of the recently established ACC xenograft models SJ-ACC3 and MUC-1 for the evaluation of IGF1-R targeted therapeutic approaches. Specifically, as treatment schemes L + Ab (liposomal Doxorubicin + free anti-IGF-1R antibody 1H7) and IL (anti-IGF1R targeted Doxorubicin loaded immunoliposomes) were studied. After a single administration of NaCI, L + Ab or IL, anti-tumoral effects were first investigated by immunohistochemistry (Figure 2). ☒
In a first step, tumor slides from xenografts of all treatment groups (SJ-ACC3: n=4-5 (Figure 2A and C) and MUC-1: n=8 (Figure 2B and D)) were analyzed by Ki67 immunohistochemistry including methylene-green counterstain and determined by quantification of six high power fields (HPF) per tumor to evaluate general anti- tumoral efficacy of the different approaches.
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1 The single administration of the therapeutic agents did not lead to significant anti-tumoral effects on any of the observed parameters. However, by trend a reduction in the number of proliferating cells upon L + Ab ( 79.1+24.5%, p=0.49 vs. controls) and IL treatments (66.±10.1%, p=0.12 vs. controls: 100.0±14.6%), overall areas of proliferation (L + Ab: 49.1±17.4%, p=0.26 and IL: 58.8±15.2%, 0.43 p vs. controls: 75.0±12.6%) and inhibition of IGF-1R-signaling (P-Akt/ Akt normalized to ß-actin in % of NaCI Akt; NaCl: 71.1±14.3%, L + Ab: 38.5±14.3%, IL: 45.8+7.2%) was indicated for SJ-ACC3 tumors , while no such tendencies were observed in the MUC-1 tumor model (Ki67 positive cells: NaCI:100.0±9.9%; L + Ab: 114.7.+15.8%, p=0.44; IL: 96.8.+6.6%, p=0.79 vs. NaCI; areas of proliferating cells: NaCl: 81.8+4.3%; L + Ab: 82.3±9.0%, p=1; IL: 97.2+2.4%, p=0.17; P-Akt/ Akt normalized to ß-actin in % of NaCI Akt; NaCl: 39.8±8.5%, L + Ab: 46.4±9.5%, IL: 73±3.7%).
3.3. Long-term therapeutic experiments on NCI-H295R xenografts in vivo
Recent experiments provided anti-tumoral effects of both, L + Ab and IL treatments, against NCI-H295R xenografts in short-term therapeutic experiments [24]. Primary endpoint of a long-term study on NCI-H295R xenografts was the investigation of tumor development during repeated treatments with NaCI, L + Ab and ILs. During this study mice were treated intravenously with four therapeutic cycles of each therapeutic treatment for all groups (n=7) at days 11, 21, 29 and 37. Although overall survival was not significantly different between the treatment groups (Figure 4A) this experiment demonstrated significant anti-tumoral effects of L + Ab and IL treatments compared with NaCI treated tumor bearing mice (Figure 4B). While tumor diameters [cm] were not significantly different at first day of therapies (NaCl: 0.3±0.1, L + Ab: 0.3±0.1, IL: 0.3+0.1; p>0.05), a significant reduction in tumor sizes was detectable at the last day of treatment with L + Ab (0.4+0.1; p<0.001) and IL (0.6±0.2; p<0.05), compared with the control group (1.3+0.2, Figure 4B). These discrepancies in reduction of tumor sizes is not reflected in prolonged overall survival as study termination in the specific treatment arms was mainly due to adverse effects such as body weight loss and diarrhea. In contrast, tumor diameter of 1.5 cm as the predefined endpoint was the reason for experiment termination mainly in NaCl treated animals (86%) while this was reached only in a small proportion of L + Ab (14%) and IL (28%) treated mice (Figure 5).
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1 Accordingly, while for L + Ab and IL treated tumor bearing mice response evaluation upon criteria in solid tumors (RECIST) revealed high rates of cures at the end of therapeutic intervention (L + Ab: complete remission: 43%, partial remission: 14%, stable disease: 14%, progressive disease: 29% ; IL: complete remission: 29%, partial remission: 0%, stable disease: 14%, progressive disease: 57%), no such effects were detectable for NaCI treated mice (complete remission: 0%, partial remission: 0%, stable disease: 14%, progressive disease: 86%).
ACCEPTED MANUS CRIPTem
1 4. Discussion
2 3 4 5 6 7 8 9 10 The aim of our current study was the investigation of a combination of liposomal doxorubicin and IGF-1R-inhibiting antibody (L + ab) as well as an immunoliposomal (IL) approach in a long-term therapeutic setting against NCI-H295R xenografts and in short-term-experiments on two newly established xenograft models for ACC. Integrated genomic characterization studies have recently demonstrated that ACC comprises a disease with distinct molecular subtypes[28]. Accordingly, recent clinical studies have supported the view that IGF-1R targeted therapies might be a useful strategy for subgroup of ACC patients with specific clinical and functional properties [18, 19, 21]. Overall, these findings and observations provide compelling evidence 11 that there is urgent need for ACC tumor models that reflect this heterogeneity also in 12 preclinical experiments. Herein, we have characterized the commonly available 13 human ACC tumor models NCI-H295R [29, 30] and SW-13 [31] as well as the two 14 novel xenograft models SJ-ACC3 [26] and MUC-1 [27] regarding their specific IGF- 15 1R and IGF2 expression levels. Thereby, we could demonstrate that these four tumor 16 models represent a wide spectrum of specific expression patterns that could be used for a more personalized approach in testing of IGF-1R inhibition. Furthermore, combined implementation of these models might be helpful for the identification of specific predictive and therapeutic biomarkers for different subgroups of patients. Moreover, as SW-13 derived from a small cell carcinoma of the adrenal gland and not from a primary ACC, the new human xenograft models might represent more ACC specific tools for future preclinical studies.
Our expression studies suggested NCI-H295R (with high levels of IGF-1R) and SJ- ACC3 (high IGF-1R and IGF2 levels) as tumor models with presumed good therapeutic responsiveness to IGF-1R inhibiting approaches, while for MUC-1 this therapeutic strategy was expected to be of less therapeutic benefit. Overall, our therapeutic experiments confirmed these predictions: Long-term therapy led to significantly reduced tumor sizes for L + Ab and IL treated NCI-H295R xenografts. Furthermore, in both specific treatment groups high rates of complete remissions were evident. Moreover, even though not reaching statistical significance after a single injection SJ-ACC displayed tendencies towards anti-tumoral activity for all three investigated endpoints, while no such trend was detectable in MUC-1 derived tumors in any of the observed parameters. The lack of statistical significance could
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1 be due to the administration of only a single dose of therapeutic agents. Notably, we had demonstrated in earlier studies that on cycle of etoposide, doxorubicin, cisplatin and mitotane has comparable tendencies towards therapeutic efficacy in a short-term setting of ACC xenografts, while resulting in a significant reduction of tumor sizes upon two therapeutic cycles [25].
Importantly, while 86% of NaCI treated xenografts were highly progressive and animals were sacrificed as they reached the critical tumor diameter of 1.5 cm, the tumors of specifically treated animals showed impressive response rates according to RECIST criteria. In fact, these therapeutic experiments were terminated due to 10 adverse effects as bodyweight loss and diarrhea as predefined in the animal study experimental protocol. From a clinical point of view the observed side effects represent mostly low grade toxicities. While the reason for body weight loss remains uncertain, major disturbances in glucose metabolism could be excluded (Supplemental Figure 1). NCI-H295R tumor bearing mice tended towards lower blood glucose levels after multiple treatments even though this was not statistically significant. From past clinical experience, such off target profiles of IGF-1R inhibiting approaches are manageable [19]. However, as an additional chemotherapeutic component is included, dosage and treatment cycle should be further evaluated to improve off-target profiles.
Interestingly, the combinatory administration of L + Ab was shown to be more effective against NCI-H295R compared to the immunoliposomal preparation. While the opposite effect, superiority of IL over L + Ab, was demonstrated in the past in a xenograft model for neuroendocrine tumors of the gastro-enteropancreatic system, recent findings on ACC had already indicated an extraordinary uptake phenomenon of plain liposomes by adrenocortical tumor cell s[24, 25]. As the utilized 1H7 antibody included in the L + Ab approach was furthermore optimized by steric stabilization (PEGylation) for our current experiments, at least comparable therapeutic efficacy to the immunoliposomal preparation could be expected. Of note, we utilized the clinically available formulation Caelyx® as basis for the establishment of ILs for in vivo experiments. Very recent analyses on our applied post-modification technique revealed an unexpected high impact of the Caelyx®-specific buffer system [23]. By changing the buffer composition on the bases of Caelyx® liposomes (from HBS to SH buffer) cellular association of our established immunoliposomes was significantly
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reduced, while replacement of the sucrose containing SH buffer by a NaCl containing (HBS) led to restoration of cellular association. These findings demonstrated, that buffer exchange and therefore further optimization of surface modification is possible and might further increase therapeutic efficacy of IL also against ACC.
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In summary, our study provides evidence for therapeutic efficacy of IGF-1R inhibiting approaches together with liposomal doxorubicin for the treatment of ACC. As for both, formulations are already clinically available, clinical evaluation of such a combination might be of interest for a sub-group of patients. Even though in our experiments the therapeutic outcome tended to correlate with IGF1-receptor levels in 10 the utilized preclinical tumor models for ACC, recent studies do not provide evidence 11 that IGF1R protein levels alone are able to predict responsiveness and that patient 12 selection is furthermore an important aspect with regard to IGF1R inhibitors. New biomarkers such as circulating IGF levels or activation status of the IGF1R axis are necessary to identify responding individuals [32]. Our experiments show, that preclinical progress in tumor model development might be helpful to identify those more specific and sub-type dependent markers for ACC in the future.
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Acknowledgements
The authors are indebted to Igor Shapiro for his excellent technical support. Furthermore, the authors thank Chris Morton (St. Jude Children’s Research Hospital, Memphis, Tennessee) and Dr. Cristina Ronchi (University of Würzburg, Germany).
Funding
This work was supported by the Mildred-Scheel-Stiftung to CH, FB and RS (grant number 110190). Moreover, the research leading to these results has received funding from the Seventh Framework Programme (FP7/2007-2013) under grant agreement nº259735.
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Figure 5: Causes for study termination (TD: tumor diameter, AE: adverse effects and LTS: long term survivors) and tumor response evaluation according to RECIST (Response Evaluation Criteria In Solid Tumors criteria: Progressive disease (PD, at least 20 % increase), partial remission (PR, at least 30% decrease), stable disease (SD, neither PR nor PD) and complete remission (CR, disappearance of target
6 7 lesions, no tumor measurable, palpable and resectable) for NCI-H295R tumor 8 bearing mice during long-term therapy with NaCI, L + Ab or IL.
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Short-term therapeutic experiments
Inoculation of SJ-ACC3 and MUC-1 xenografts
Implantation of tumor pieces (approximately 2 x 2 mm)
Single treatment NaCI, L+Ab, IL
End of experiment 72 hours after treatment
Long-term therapeutic experiments Inoculation of NCI-H295R xenografts
Treatments 1, 2, 3, 4 NaCI, L+Ab, IL at days 11, 21, 29, 37 after tumor induction
End of experiment by reaching critical endpoints as tumor diameter of 15 mm or in case of adverse effects
Injection of 15 x 106 tumor cells
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3 Supplemental Figure 2: Schematic illustration of the different approaches of xenograft induction and study designs for short and long-term therapeutic experiments
ACCEPTED MANUS CRIS OF
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ACCEPTED MANUSCRIPT
Highlights:
. We established novel combinatory approaches of IGF-1 receptor inhibition and liposomal doxorubicin for therapeutic treatment of adrenocortical carcinoma (ACC).
. We investigated therapeutic responsiveness in three different xenograft models for ACC (NCI-H295R, SJ-ACC3, MUC-1).
. Our experiments indicate, that implementation of this panel of tumor models might be helpful for clinical translation of novel therapeutic regimens in the future.
ACCEPTED MANUS CRIPelo, Die