Combination Therapy of a Host Defense-Like Lytic Peptide and Continuous Low-Dose Doxorubicin Inhibits Tumor Growth in a Syngeneic Immunocompetent Murine Fibrosarcoma (BFS-1) Model CURRENT STATUS: UNDER REVIEW

Background: The concept of a has been discussed with controversy. remain gold standard in STS therapy. It is still questionable whether a systemic therapy with chemotherapeutics has a positive impact on the overall survival rate especially in early stages of disease, because the therapeutic effect in the treatment of STS is limited by its toxicities and its low responding rates. Treatment options are rare. As a result the search for combination therapies by using low dose approaches is of high importance. Recent studies showed the therapeutic efficiency of a designer host defense-like lytic D,L- amino acid peptide [D]-K 3 H 3 L 9 . Therefore we tested a combination of this peptide with Doxorubicin on two different sarcoma cell lines in vitro and also in a syngeneic immunocompetent murine fibrosarcoma mouse model. Methods: In vitro the human synovial sarcoma cell line SW 982 and the murine fibrosarcoma cell line BFS-1 were exposed to the oncolytic peptide [D]-K 3 H 3 L 9 , to the Anthracycline Doxorubicin and to both agents simultaneously. In vivo the murine fibrosarcoma cell line BFS-1 was injected subcutaneously into the syngeneic mice. When the tumors engrafted the oncolytic designer peptide [D]-K 3 H 3 L 9 , Doxorubicin or a combination of both was administered thrice a week for a three weeks’ follow-up. Results: The combination treatment approach using an oncolytic designer host defense peptide and Doxorubicin inhibited the in vitro sarcoma cell proliferation significantly. The single therapies, either with local intratumoral application of [D]-K 3 H 3 L 9 or with intraperitoneal application of Doxorubicin in the syngeneic mouse model, inhibited at least the tumor progression. The combination of both substances revealed a significant inhibition of tumor growth and weight. Conclusion: The in vivo low dose combination schedule inhibited the tumor growth significantly. Histological analyses of the tumor sections revealed an antiproliferative and antiangiogenic effect. So, these results demonstrate the effectiveness of combined low-dose application forms with designer host defense-like lytic peptides and chemotherapeutics.

were already put in focus as oncolytic agents to potentially exceed the limits of current agents (25,26). At first they were seen as primarily antimicrobial active substances, but HDPs proved to have significant cytotoxic effects against malignant cells including bladder, breast and lung cancers as well as lymphomas, leukemia and sarcomas (25,26,(30)(31)(32)(33)(34). This oncolytic effect is caused via a membrane disrupting mechanism due to their molecular characteristics. HDPs are very short (10-40 amino acids) amphipathic and cationic proteins. Their positive charge leads to a higher affinity to the negatively charged membrane of tumor than to zwitterionic membranes of normal mammalian cells, based on the electrostatic interaction (26,31,(35)(36)(37). The limiting factor for their oncolytic potential in in vivo studies is their inactivation through enzymatic degradation or adhesion to serum components. In this context, research has focused on modifying the molecules´ structure to become first active in acidic tumor environments and therefore pass systemic degradation resulting in cationic peptides containing synthetic D-amino acids. This defines the difference between naturally occurring L-amino peptides to artificially synthesized D-amino peptides, resulting in higher adhesion to negatively charged membranes, the decrease in enzymatic degradation and the reduced negative side effects, as hemolysis, e.g. (38,39). The host defense-like lytic peptide [D]-K 3 H 3 L 9 used in this study consists of the D-and L-amino acids lysine, histidine and leucine. The activation of HDP in acidic environment in solid tumors is based on the changed metabolic and nutritional environment. The peptide becomes cationic by protonating histidine (pKa, 6.1) below pH 7.0 which leads to a selectivity against malignant tissue (22). The antiproliferative and antiangiogenic activity of the [D]-K 3 H 3 L 9 peptide could already be seen in preovious studies in a prostate cancer xenograft model and furthermore in suppression of soft tissue sarcoma in vitro and in vivo (22,37,40).
The aim of this study was to establish a syngeneic immunocompetent murine fibrosarcoma  model that would allow a study of host defense-like lytic peptide [D]-K 3 H 3 L 9 and Doxorubicin combination therapy, comparing the efficiency in vivo with the in vitro results.

Cell culture
The SW982 human synovial sarcoma cell line (Cell Line Service (CLS), Eppelheim, Germany), proved via nonaplex Polymerase Chain Reaction (PCR) in July, 2015 was cultivated in Dulbecco's Modified Eagle's Medium (DMEM) with 10 % Fetal Calf Serum (FCS) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1 % penicillin/streptomycin (PAA laboratories, Pasching, Austria). The murine fibrosarcoma cell line BFS-1wt (generously provided by Prof. T. Hehlgans, Department of Immunology, Regensburg, Germany) was hold in RPMI 1640 culture medium with 10 % FCS and antibiotics. Both cell lines were kept in a CO 2 (5%) 37°C incubator. The experiment started when the cells were grown confluent, medium changed twice a week.

Peptide and chemotherapeutics
The host defense-like lytic peptide [D]-K 3 H 3 L 9 , a short 15-mer D,L-amino acid peptide (LHLLHKLLKHLLKLL-NH2, D-amino acids underlined), was obtained by solid-phase synthesis and purified by RP-HPLC (NeoMPS S. A., Strasbourg, France) resulting in a concentration of 99.4 %. (The peptide's sequence was generously provided by Prof. Yechiel Shai, Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.). Doxorubicin was used as chemotherapeutic agent (Pharmacia Bergmannsheil).

Animals
All guidelines for the care and use of animals, stated by the Ministry for Environment, Agriculture, Conservation and Consumer Protection of the State of North Rhine-Westphalia, were followed. Immunocompetent C57/BL6 mice (Harlan Winkelmann GmbH, Borchen, Germany) (weighing 20-25 g, age 5 -6 weeks old) were used throughout this study. Their racks were ventilated and pathogen free.
They were held in equinox conditions (12 h light/12 h dark) with control of humidity and temperature (23 +/-2 °C). Sterile requirements were maintained throughout all surgical procedures.

Cell proliferation
Bromo Deoxyuridine (BrdU) cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics GmbH, Mannheim, Germany) was used for measurement of cell proliferation in accordance to the manufacturer's instructions. After seeding the cells in 96-well plates (3x10 4 cells/well), they grew for 24 hours. Followed by an 24-hour incubation with fresh FCS-free medium containing either the host defense-like lytic peptide [D]-K 3 H 3 L 9 or Doxorubicin or both agents and a 22-hour incubation after adding BrdU labeling solution. BrdU, a pyrimidine analogue, was incorporated into the DNA during cell proliferation and the rate of proliferation could be measured by the light emission detected via an Orion microplate luminometer (Berthold Detection Systems, Pforzheim, Germany). LC50 values for each medium were evaluated. Additionally, cell proliferation was measured using the concentration of the peptide and the chemotherapeutic which inhibited 15 -30 % of cell proliferation alone. The same concentration of each agent was used measuring the inhibition of cell proliferation with both substances combined and therefore the proliferation was measured in triplicate. The results show the cell viability in percentage compared to the negative control.

Solid tumor model
Murine fibrosarcoma BSF-1 cells in Matrigel (BD Biosciences, San Jose, CA, USA) were injected subcutaneously (1x10 6 cells) into the animal's flank using a laminar airflow cabinet. The mice weights were measured and the tumor volume was calculated with the formula of length x width x depth x 0.5 in mm 3 every second day. When the averaged volume of 130 mm 3 (BFS-1) was reached, the animals were randomly grouped following the rules of a randomized control trial. The different groups of mice were treated either with the [D]-K 3 H 3 L 9 peptide (n = 5), with Doxorubicin (n = 5), with both agents simultaneously (n = 5) or with Phosphate buffered saline (PBS) (pH 7.0 -7.5) as a control (n = 5) for three weeks from day 0 to day 19. A two-days-follow-up continued to day 21. [D]-K 3 H 3 L 9 was injected intratumorally thrice a week (single dose 5 mg/kg n = 5) and Doxorubicin was injected intraperitoneally twice a week (single dose 1.2 mg/kg, n = 5). The experiment was terminated early when a tumor length of over 20 mm was reached and the animals were euthanized by an overdose of isoflurane followed by cervical dislocation. The tumors were collected and analyzed. The evaluation of the data was based on a Kaplan-Meier graph.

Histologic and immunofluorescent staining
A fixation of the excised tumors in 5 % buffered formaldehyde was followed by a haematoxylin and eosin (H&E) staining of the paraffin-embedded 23 µm sections. The slides of treated tumors were microscopically examined (Zeiss Axioskop 2 Plus microscope) and photographed (AxioCam-HRc). For immunohistochemical examination slides needed to be deparaffinized and rehydrated. Citratemediated high temperature antigen retrieval (Vector Laboratories, Burlingame, CA, USA) and incubation with blocking buffers were performed to avoid nonspecific binding of antibodies. To examine the vascularization an incubation with monoclonal anti-human Ki-67 antibody followed (Acris Antibodies GmbH, Herford, Germany) which are specific to proliferating cells and CD31 antibody, known as the platelet endothelial cell adhesion molecule (PECAM-1).

Statistics
Data are reported as mean ± standard error of the mean (SEM) and all statistical analyses were performed with GraphPad Prism 6.0.1. Moreover, data were analyzed with student t-test or ANOVA if more than two groups were compared. Differences were considered as statistically significant with a p-value <0.05.

Results
Cell proliferation was determined after monotherapy with [D]-K 3 H 3 L 9 or Doxorubicin and comparison to the combination treatment ([D]-K 3 H 3 L 9 +Doxorubicin (DXR)). The human synovial sarcoma (SW982) and the murine Fibrosarcoma (BFS-1) cell line were simultaneously treated for 24 hours ( Figure 1).
Afterwards, the examination of the cell proliferation was detected using a BrdU detection system and normalized to carrier control. While single agents provoked a 15 -30 % inhibition of proliferation, we examined a possible synergistic effect of the combined use (>50% reduction).

Inhibition of solid soft tissue sarcoma growth by intratumoral administration of[D]-K 3 H 3 L 9 and
Doxorubicin in an immunocompetent mouse model Cells were implanted subcutaneously into the left flank of the immunocompetent C57BL/6 (BFS-1) mice. The oncolytic activity of [D]-K 3 H 3 L 9 was evaluated after reaching the defined tumor volume of approximately 130 mm 3 ( Figure 2). The injection of the peptide was adminstered intratumorally at a dose of 5 mg/kg, Doxorubicin was injected intraperitoneally at a dose of 1.2 mg/kg. As expected, there was no significant inhibition of the tumor volume after therapy with both agents in such low dose approaches. During the three weeks of therapy the subcutaneous tumors of the control group showed an exponential growth with a final mean tumor volume of 2 157 mm 3 . In the single-agent therapy they grew to a final mean tumor volume of 1 701 mm 3 (21% reduction) under the treatment with Doxorubicin and of 1 447 mm 3 (33% reduction) under the [D]-K 3 H 3 L 9 treatment. The administration of [D]-K 3 H 3 L 9 and Doxorubicin at the same time led to a significant (p<0.05) reduction of the tumor volume with 49% and a final volume of 1 099 mm 3 . These findings were reflected by a significant reduction (p<0.05) of tumor weight of the dissected primary tumors under combined treatment compared to the carrier control group (Figure 2).
[D]-K 3 H 3 L 9 +Doxorubicin treated mice signed for a significant reduced average tumor weight of 1.56 g which was about 38.58 % (p<0.05) lower than the weight in the control group (2.54 g). For the single agents neither Doxorubicin (mean weight 1.88 g, 26% reduction) nor [D]-K 3 H 3 L 9 (mean weight 2.12 g, 16.5% reduction) lead to significant tumor weight reduction. The tumor weight of the [D]-K 3 H 3 L 9 treated mice was 11.32 % higher in mean compared to the Doxorubicin group ( Figure 2). There were no acute toxicities or adverse events in the therapy groups, all mice could be included into the statistical analyses (5/5 mice in each group).

Immunostaining with CD 31 antibody revealed a highly significant reduction of tumors' vascularization treated with [D]-K 3 H 3 L 9 and Doxorubicin (p<0.01)
Additionally, a strong antiangiogenic effect of Doxorubicin in addition to the peptide was determined using a CD31 antibody (Figure 4). The immunohistochemical staining using anti-mouse CD31 antibody shows a significant reduction in vessel density within the combined treated tumors compared to the carrier control (PBS). The angiogenesis was quantified by high power field counting. Even in the mono therapy groups there was a significant effect on the tumors' vascularization (p<0.05). In comparison to the control group with 6.96 vessels/High-power field (HPF), vessel density was reduced to 4.8 vessels/HPF (31.03 % reduction) for the peptide group and down to 4.0 vessels/HPF in the Doxorubicin group (42.53 %). The results were highly significant (p<0.01) for the combined treated group with a vessel density of 2.46 vessels/HPF representing a 64.66 % reduction compared to the carrier control group (Figure 3). Cytotoxicity. The combination of both agents revealed an additive effect for the Synovialsarcoma (SW982) cell line (Fig.1a). The antiproliferative efficacy of the single agents with 30 % for Doxorubicin and 25 % for [D]-K3H3L9 led to an addition amount of 55 % under the combined application. The BFS-1 cells showed a synergistic effect underlining the potential of both substances (Fig.1b). A 17 % decreased cell proliferation for Doxorubicin (30 ng) and a 20 % decreased cell proliferation for the peptide (15 g) after single incubation, resulted in a 77 % synergistic inhibition. The BrdU assay displayed a highly significant (p<0.01) antiproliferative effect of the combined use of the peptide and Doxorubicin toward both cell lines.  Anti CD31-immunostaining. Comparison of the vessels' density in the murine fibrosarcoma model underlying the different therapy approaches shows the highly significant (64.66 %) reduction of the vessels' density after therapy with the HDP and Doxorubicin (p<0.01) using CD31 immunostaining. Comparison of vessels' density between different therapeutic regimens in murine fibrosarcoma. The combination therapy with the oncolytic peptide and Doxorubicin leads to a highly significant reduction (64.66%) of vessels' density in nonnecrotic areas (p<0.01).