Tumor-induced myeloid cells are reduced by gemcitabine-loaded

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Tumor-induced myeloid cells are reduced by gemcitabineloaded PAMAM dendrimers decorated with anti-Flt1 antibody Digdem Yoyen-Ermis, Kivilcim Ozturk-Atar, Muammer Alper Kursunel, Cisel Aydin, Didem Ozkazanc, mustafa gürbüz, Aysegul Uner, Metin Tulu, Sema Calis, and Gunes Esendagli Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01075 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Molecular Pharmaceutics

Tumor-induced myeloid cells are reduced by gemcitabine-loaded PAMAM dendrimers decorated with anti-Flt1 antibody Digdem Yoyen-Ermisa,#, Kivilcim Ozturk-Atarb,#, M. Alper Kursunela,#, Cisel Aydinc, Didem Ozkazanca, Mustafa Ulvi Gurbuzd, Aysegul Unerc, Metin Tulud, Sema Calisb, Gunes Esendaglia

a

Hacettepe University Cancer Institute, Department of Basic Oncology, Ankara, Turkey

b

Hacettepe University Faculty of Pharmacy, Department of Pharmaceutical Technology,

Ankara, Turkey c

Hacettepe University Medical Faculty, Department of Pathology, Ankara, Turkey

d

Yıldız Technical University, Faculty of Arts and Sciences, Department of Chemistry,

Istanbul, Turkey

#

These authors contributed equally to the study.

Correspondence to: Gunes Esendagli, PhD., Department of Basic Oncology, Hacettepe University Cancer Institute, 06100, Sihhiye, Ankara - Turkey Fax: +90 312 324 20 09, e-mail: [email protected]

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract While reshaping its microenvironment, tumors are also capable of influencing systemic processes such as myeloid cell production. Therefore, the tumor-induced myeloid cells, such as myeloid-derived suppressor cells (MDSCs) which are characterized with pro-cancer properties, became another target in order to increase success of the therapy. This study evaluated the capacity of a novel dendrimeric drug delivery platform to eliminate tumorinduced myeloid cells. As described in a previous study by our research group, the anti-Flt1 antibody-conjugated

polyethylene

glycol (PEG)-cored poly(amidoamine) (PAMAM)

dendrimers improved the efficacy of gemcitabine against pancreatic cancer. Here, the biodistribution studies showed that these dendrimeric structures accumulated into the compartments that became rich in myeloid cells in the pancreatic tumor-bearing mice. When gemcitabine was loaded into the dendrimer complexes, the number of myeloid cells were significantly reduced while the percentage distribution of granulocytic and monocytic myeloid cells was not always significantly altered. The CD11b+Ly6G-Ly6C+ monocytes were more severely affected from the treatments than CD11b+Ly6G+Ly6C+ granulocytes. Immune infiltration levels in the tumor tissue was also altered. Myeloid cells in the spleen and F4/80+ macrophages of the liver were protected. The compartments such as the liver and the bone marrow, which are known with high vascular endothelial growth factor (VEGF) - Flt1 pathway activity, were particularly targeted by gemcitabine when delivered through anti-Flt1 antibody-conjugated

PAMAM

dendrimers. In

conclusion,

chemotherapeutic agents

complexed with dendrimers not only improve anti-cancer efficacy but also assist elimination of the tumor-induced myeloid cells.

Key words: PAMAM, dendrimer; drug delivery; myeloid cells; cancer.


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Introduction Immune cells of myeloid origin, i.e. neutrophils and monocytes, are one of the most affected cell types upon tumor development. Factors derived from the tumor microenvironment recruits the myeloid cells into the tumor mass and augment the production of myeloid cells in the hematopoietic organs1. Not only the bone marrow but also the spleen and the liver become hubs for myelopoiesis2. Nevertheless, these cells cannot fully mature and serve as supporters for tumorigenesis and can suppress anti-tumor immune responses. The tumor-induced myeloid cells are acknowledged as myeloid-derived suppressor cells (MDSCs)3. Therefore, elimination of these immature myeloid cells can diminish the progression of the disease. In addition to in vivo administration of monoclonal antibodies against myeloid cells, conventional chemotherapeutics such as gemcitabine, doxorubicin, and 5-fluorouracil have been shown to reduce MDSCs4-8. Pancreatic cancer is one of the deadliest of all types of cancer which can be characterized with upregulation of myeloid cells even in the pre-malignant stages9-12. Gemcitabine is a standard chemotherapy for advanced pancreatic cancer patients; it targets DNA replication and telomerase activity, therefore, myeloid cells are also non-specifically affected by this systemically administered drug6, 13. Nevertheless, drug delivery systems are intended to be developed to enhance solubility, stability and biocompatibility of the drugs, and to lower their cytotoxic side effects14, 15. A delivery system endowed with tumor-homing agents of guidance such as antibodies aiming the molecules that are enriched in the malignant microenvironment is preferred to increase the local concentration of the drug16. In a previous study, our laboratory group designed and synthesized polyethylene glycol (PEG)-cored poly(amidoamine) (PAMAM) dendrimer structures that contained anionic



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carboxylic acid groups modified with PEG chains. Conjugation of these chains with antibodies against Flt1 was effective as a targeted drug delivery system for cancer chemotherapy17. When gemcitabine was loaded into these nano-sized dendrimeric structures, its efficacy in the reduction of tumor burden was increased in vitro and in a xenograft pancreatic cancer model17. Flt1 is also known as vascular endothelial growth factor (VEGF) receptor 1 that contributes to angiogenesis18. Flt1 can be expressed in various types of cancers. Additionally, VEGF is a pivotal mediator for mobilization and recruitment of bone marrow-derived leukocytes and hampers the differentiation of these myeloid cells19, 20. It is widely expressed by hypoxic regions in the tumor microenvironment dare shown to recruit MDSCs21,

22

. As a positive feed-back mechanism, VEGF expression can upregulate Flt1

expression on myeloid cells22. Signaling through Flt1, VEGF facilitates acquisition of an immune regulatory character by the myeloid cells and formation of pre-metastatic niche20, 23, 24

. Accordingly, it was hypothesized that dendrimers decorated with anti-Flt1 antibodies

can possess the capacity to hamper accumulation of tumor-induced myeloid cells in hematopoietic organs. Here, gemcitabine delivered via anti-Flt1 antibody-conjugated dendrimers precisely altered the myeloid cell dynamics and reduced their numbers especially in the bone marrow which is the generator and reservoir for myeloid cells.

Materials and Methods Preparation of dendrimeric structures and complex formation with gemcitabine The synthesis methods and in vitro properties of PAMAM dendrimers were previously described in detail and published15, 17. Briefly, PAMAM dendrimers with peripheral amine (NH2) groups were synthesized starting from poly(ethylene glycol) tetra amine (4-arm-PEG) cores. The dendrimers were grown up to 4.5 generations through reactions with methyl


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acrylate and ethylenediamine. PEGylation, with 10% Poly(ethylene glycol) bis(amine) was performed for surface modification. Esteric surfaces were converted to carboxylic acid groups to increase the solubility. The resulting products were characterized with nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy. Following the conjugation with anti-Flt1 antibody, unconjugated PAMAM was removed with dialysis15. The resulting product was confirmed by NMR as well15, 17. Change in the dendrimers’ properties and gemcitabine loading and releasing capacity of the dendrimers was determined by highperformance liquid chromatography (HPLC), zeta potential, differential scanning calorimetry analyses. Accordingly, encapsulation efficiency, particle size and charge, physical status of the drug and release kinetics of gemcitabine from the dendrimers were previously described and published15, 17. Schematic diagram of anti-Flt1 antibody-conjugated PEG-cored PAMAM dendrimers loaded with gemcitabine (D-αFlt1-Gem) is given in Figure 1A. The dendrimer structures and their abbreviations used in this study is given in Table-1.

In vivo experiments with tumor-bearing mice CFPAC-1 human pancreatic cancer cell line (American Type Culture Collection, LGC Promochem, Rockville, MD, USA) was cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Biowest, Nuaillé, France) containing 100 U/ml of penicillin and 100 mg/ml of streptomycin (Biological Industries, Beit-Haemek, Israel), and 10% FBS (Biological Industries, Beit-Haemek, Israel) under humidified atmosphere conditions with 5% CO2 at 37°C. Subcutaneous tumors were established by subcutaneous inoculation of 1.5x107 CFPAC1 cells in right flank of 6-to-8-week-old male CD-1 Nude mice (Kobay As., Ankara, Turkey). The tumor-bearing animals (approximate tumor diameter 0.5 cm) were administered intraperitoneally (2x/week) with 0.09% NaCl in dH2O (control group, n=6), gemcitabine HCl solution (100 mg/kg, n=6) prepared in 0.09% NaCl, gemcitabine loaded into dendrimers D-



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Gem (100 mg/kg, n=6) or D-αFlt1-Gem (100 mg/kg, n=7). The (i.p.) injections were performed. The change in tumor growth and weight of animals were followed and the organs were dissected and macroscopically evaluated after termination of the experiments. The procedures were approved by the Local Animal Care and Use Committee. The ARRIVE guidelines were strictly adhered in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.

Flow cytometry Cell suspensions from the spleen, the liver and the tumor were obtained with mechanical agitation in phosphate buffered saline (1xPBS), and passed through 40 µm pore-sized filters. Peripheral blood was collected in EDTA-containing tubes. Bone marrow cells were purged by passing PBS by using a syringe. Following separation of the cells by density gradient centrifugation (Histopaque®-1119; Sigma, Steinheim, Germany), the cells were labeled with monoclonal anti-bodies against CD11b (M1/70), Gr-1 (RB6-8C5), Ly6C (HK1.4), F4/80 (BM8), and Ly6G (1A8) (Biolegend, San Diego, CA, USA). Gating strategy is given in Supplementary Figure 1. Percentage of positive cells was taken by comparison with the samples incubated with appropriate isotype-matched antibodies. All analyses were performed on a FACSAria II flow cytometer (Becton Dickinson, San Jose, CA, USA).

Biodistribution studies In vivo distribution in tumor-bearing mice were performed upon i.p. injection of the dendrimers (antibody conjugated or not) loaded with Rhodamine123 fluorescent tracer (0.07 mg Rhodamine123/10 mg dendrimer). The animals were killed after 12 h and cell suspensions were freshly prepared from the tissues for evaluation of in vivo cellular uptake. These freshly isolated cells were read on flow cytometer against autofluorescence of the cells obtained from


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untreated control animals. Median fluorescence intensity (MFI) values were also obtained. In addition, cryosections from the liver were prepared in order to visualize dendrimer inclusions and documentation were done by fluorescent microscopy (Leica, Wetzlar/Germany).

Histopathology Tumor samples were fixed in 10% formalin and embedded in paraffin, then, thin tissue sections were taken. After staining with hematoxylin and eosin, histopathological evaluation was performed under conventional light microscopy. The amount of lymphohistiocytes in and around the tumor tissue was scored in every specimen. An average of four non-overlapping fields was analyzed and a semi-quantitative scoring (high, medium, low, no) based on a ratio of cells with specific morphology over total cells was determined.

Statistical analysis Number of animals used in each group was decided according to power analysis obtained from anticipated results or preliminary data obtained. For the statistical analysis, each group was tested for normality distribution and homogeneity of variance according to normality tests. Statistical evaluations were done with the analysis of variance (ANOVA) followed by post-hoc Tukey’s test for pairwise diﬀerence testing. Differences were regarded as significant when a P value smaller than 0.05 was obtained. Data are shown as arithmetic mean ± standard deviation (SD).

Results PEG-cored PAMAM dendrimers conjugated with anti-Flt1 antibody accumulate into the compartments rich in myeloid cells in tumor-bearing mice.



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Conjugation of anti-Flt1 antibody to PEGylated surface carboxylic acid groups of PEG-cored PAMAM dendrimers (abbreviated as D-αFlt1, Table-1) was previously determined as a novel drug delivery system improving the therapeutic efficacy of gemcitabine (Fig. 1A) in a xenograft pancreatic tumor model17. Since Flt1 is implicated in many biological processes affecting myeloid cell kinetics; here, biodistribution of D-αFlt1 complexes through the organs where myelopoiesis takes place was assessed. The capacity of D-αFlt1 to deliver a fluorescent tracer (Rhodamine123) into these compartments was tested upon intraperitoneal administration into the mice which bear human CFPAC-1 pancreatic tumors. The results were compared with that of the dendrimers without antibody conjugation (Fig. 1B and C). Typically, the dendrimers primarily stayed in the peritoneal cavity while conjugating them with anti-Flt1 antibody increased their distribution to the tissues where myeloid cells are located25 such as the liver, the spleen, and the bone marrow (Fig. 1B and C). Rhodamine123loaded D-αFlt1 dendrimers were found to be associated with CD11b+ cells which are of basically myeloid origin. In addition to subpopulations identified with low or high CD11b expression, these structures were also identified on CD11b-negative cells (Suppl. Fig. 2A). A significant accumulation was observed in the liver, surrounding sinusoids and canaliculi (Suppl. Fig. 2B). Accordingly, following the establishment of human xenograft pancreatic tumors in immune deficient mice, the effect of tumor progression on myeloid cells in the blood, spleen, and liver was monitored. Percentage of CD11b+Gr-1+ myeloid cells was elevated with tumor progression, specifically in the blood and the spleen (Fig. 2A). In the blood, this increase was documented as early as day-8, following the formation of palpable (approximate diameter 0.25 cm) tumors. On the other hand, on day-16, CD11b+Gr-1+ cells were substantially gathered in the spleen and, to a minimal extend, in the liver (Fig. 2A). With the progression of the

pancreatic

tumors,

the

fraction

of

granulocytic

myeloid

cells,

which

is


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CD11b+Ly6C+Ly6G+ subpopulation, was largely elevated; as a result, the ratio of monocytic CD11b+Ly6C+Ly6G- cells was decreased in all compartments studied (Fig. 2B and C). Collectively, based on these observations, anti-Flt1-conjugated PEG-cored PAMAM dendrimers, D-αFlt1, were thought to be useful drug delivery agents for targeting the compartments where tumor-induced myeloid cells were located.

Effect of gemcitabine-loaded dendrimers on the percentage distribution of granulocytic and monocytic myeloid cells Amongst the organs studied, the liver hosted only a limited amount of CD11b+Gr-1+ myeloid cells (2.52 ± 0.45%) whereas the spleen was more rich in the myeloid cells (31.05 ± 6.5%) than the blood (17.5 ± 3.53%) (Fig. 3A and C). Gemcitabine treatment, either with or without dendrimer structures, resulted in a slight decrease in the proportion of hepatic myeloid cells but it did not have a significant impact on the percentage of myeloid cells in the spleen (Fig. 3B and C). On the other hand, the amount of circulating myeloid cells was significantly reduced to half in the mice groups administered with gemcitabine containing agents (Fig. 3B and C). When gemcitabine was loaded into the dendrimers, the percentage of CD11b+Ly6C+Ly6G- monocytic myeloid cells was increased which was due to the decrease in the percentage of CD11b+Ly6C+Ly6G- granulocytes in the liver (an increase by 3.8% and 2.77% when compared to gemcitabine treatment alone). However, an opposite situation was observed in the blood (a decrease by 12.12% and 12.35% when compared to gemcitabine treatment alone) (Fig. 3B and C). The amount of F4/80+ liver macrophages was not significantly modulated upon treatments (Suppl. Fig. 3). Therefore, the decrement in the circulating myeloid cells was basically due to the negative impact of gemcitabine on monocytes when it was administered with the dendrimer complexes.



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The bone marrow is the main center for myelopoiesis and a reservoir for immature myeloid cells26. Next, it was checked whether the decreased ratio of peripheral blood monocytic cells in the mice administered with gemcitabine-loaded dendrimers was because of a dysregulation in the bone marrow. Only a small reduction (range 4.5-9.97% when compared to the untreated control group) was observed in the percentage of myeloid cells (mainly the CD11b+Ly6C+Ly6G+ granulocytic subset) in the animals that received any of the formulations containing gemcitabine (Fig. 4A and B).

Bone marrow becomes a particular target for gemcitabine when delivered through antiFlt1 antibody-conjugated PAMAM dendrimers Chemotherapeutics have been acknowledged with their capacities to target proliferating cells; therefore, leukocytes especially that reside in the hematopoietic compartments are also severely affected25. Nevertheless, as observed in our study, the subset distribution of leukocytes is not always significantly altered27. When the absolute number of myeloid cells was calculated a severe myelopenia (29.78% to 74.55% reduction compared to the tumorbearing control mice) was observed in the mice administered either with the gemcitabine alone or with the gemcitabine-loaded dendrimers (Fig. 5A). The spleen was the minimally affected organ where an insignificant reduction in the amount of Ly6G+Ly6C+ granulocytes was determined only after the treatment with gemcitabine-loaded D-αFlt1 (Fig. 5A and B). Myeloid cells in the liver and the bone marrow were significantly decreased with all preparations containing gemcitabine however the mice in the D-αFlt1-Gem group displayed severe myelopenia especially in the bone marrow (CD11b+Gr-1+, 995.277 ± 76,61 (x104) cells vs. 4.594 ± 0.84 (x104) cells in the tumor-bearing SF vs D-αFlt1-Gem-treated group, respectively) (Fig. 5A). These observations were also evidenced in the peripheral blood; nevertheless, dendrimers without antibody conjugation did not significantly hamper the


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number of circulating myeloid cells (Fig. 5A). Intriguingly, in the blood of the mice treated with gemcitabine-loaded dendrimers (D-Gem) Ly6G-Ly6C+ monocytes were primarily eliminated (~85.6 % reduction compared to the tumor-bearing control mice). On the other hand, in the liver and the bone marrow, both granulocytes and monocytes were significantly decreased. In the D-αFlt1-Gem group, they were almost absent (Ly6G-Ly6C+ monocytes in the tumor-bearing SF vs D-αFlt1-Gem-treated group, in the liver 53.36 ± 19.44 cells/mg vs 21.29 ± 7.74 cells/mg; in the bone marrow, 217.72 ± 19.05 (x104) cells vs 1.24 ± 0.33 (x104) cells, respectively) (Fig. 5B and C). Collectively, loading gemcitabine into the D-αFlt1 enhanced its efficacy on cancer-induced myeloid cells, especially on the monocytic subset. Positive and significant correlations between the tumor size and the absolute myeloid cell counts from the bone marrow (r=0.7, P=0.003) and the liver (r=0.56, P=0.025) were determined (Fig. 5D). Expectedly, the tumor size tended to decrease as the amount of myeloid cells was decreased indicating the success of the therapeutic application (Suppl. Fig. 4). Both the tumor size and the amount of myeloid cells were significantly reduced following gemcitabine delivery through D-αFlt1 dendrimers (Fig. 5D and Suppl. Fig. 4). Treatment with D-αFlt1-Gem was more successful in reducing the pancreatic tumor burden in mice17. Accordingly, when histopathological analyses were performed on tumor tissue, the mice in the D-αFlt1-Gem group had lower leukocyte infiltration than that of in the D-Gem group (Fig. 6A and B). In general, peri-tumoral regions displayed higher lymphohistiocytic cells than intra-tumoral regions (Fig. 6A and B). Even though not reaching to a level of statistical significance, an increased trend of high immune infiltration in the tumor mass was observed in the animals that received D-Gem preparations (Fig. 6A and B). Therefore, myeloid cell numbers were diminished not only in the hematopoietic organs but also in the tumor microenvironment following the delivery of gemcitabine via anti-Flt1conjugated PEG-cored PAMAM dendrimers. 11 ACS Paragon Plus Environment


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Discussion The polyethylene glycol (PEG)-cored PAMAM dendrimers bearing surface PEG-modified anionic carboxylic acid groups that are conjugated with anti-Flt1 antibody have been shown to be an efficient delivery vehicle for gemcitabine to target pancreatic cancer17. These dendrimers not only increased the access of the cargo into the tumor mass17, but also accumulated in various compartments where the amount of myeloid cells was augmented under the influence of the tumor. Here, we report that the tumor-induced myeloid cells, especially in the organs known to have an active VEGF pathway, become a preferred target for gemcitabine when complexed with PAMAM dendrimers conjugated with anti-Flt1 antibodies (Suppl. Fig. 5). According to the fact that the tumor-induced myeloid cells, i.e. the myeloid-derived suppressor cells (MDSCs), assist progression of the disease; therefore, efficient elimination of these cells by the anti-Flt1-conjugated dendrimers can be an additional mechanism that augments anti-cancer efficacy. Dendrimers are nano-sized biocompatible and water-soluble carriers that contain modifiable charged surface groups14. PAMAM dendrimers are hyper-branched polymeric nanostructures generally having an ethylene diamine (EDA) core and amidoamine monomer groups together with active functional groups outside its surface28, 29. A newly synthesized and characterized PEG-cored PAMAM dendrimers that had surface modifications such as PEGylation to enhance biocompatibility and avoid quick removal by the reticuloendothelial system15, 17, 28, 30, 31 were chosen for gemcitabine delivery. Moreover, to neutralize the surface charge and improve distribution of the cargo through the target tissues, antibodies and several ligand molecules can be conjugated onto PEG groups as ‘homing agents’ 15, 28. VEGF has an important role in the myelopoiesis-associated organs, the axis between myeloid cells and this growth factor may play an active role in the progression of pancreatic cancer32, 33. Therefore,


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in this study, an antibody reactive with Flt1 molecule (the receptor for VEGF) was chosen to be conjugated with PEG-cored PEGylated PAMAM dendrimers. Accumulation of immature myeloid cells due to chronic inflammation is wellestablished in cancer34. In tumor-bearing mice, CD11b+Gr-1+ phenotype is defined as MDSCs with two major subsets, monocytic MDSCs (CD11b+Ly6C+Ly6G-) and granulocytic MDSCs (CD11b+Ly6C-Ly6G+)34. In the mice with CFPAC-1 pancreatic tumors, the amount of these MDSC-like monocytic and granulocytic cells was significantly increased. Nevertheless, since we did not perform any assays to test their suppressive activity, in the current study, these cells were designated as myeloid cells instead of MDSCs. Recently, elimination of MDSCs in tumor-bearing mice by pluronic-stabilized poly(propylene sulfide) nanoparticles35 and dextran (C-dextran) and polyethyleneimine (PEI) cationic polymers36 has been introduced as promising anti-cancer approaches. In our study, the peritoneal cavity was the primary compartment where dendrimers brought the cargo (i.e. Rhodamine123 as the tracer) in line with the intraperitoneal route which was used for drug administration. Conjugation with anti-Flt1 antibody decreased the retaining of dendrimers in the peritoneal cavity. On the other hand, anti-Flt1-conjugated dendrimers reached to the liver, the blood and the spleen as successful as their unconjugated counterparts. Flt1 pathways are prominent in the bone marrow and the liver37, 38. Our results indicate the capacity of dendrimers with anti-Flt1 antibodies to target the bone marrow which is one of the most inaccessible compartments, in vivo39. Flt1 is expressed on human monocytes and macrophages40. Murine monocytes and macrophages can also express receptors for VEGF since they are implicated in lymphangiogenesis and indirect angiogenesis41. Accordingly, monocytes were very much sensitive to gemcitabine delivered by anti-Flt1-conjugated dendrimers. The liver macrophages were not altered following the treatments since these cells can be resistant to chemotherapeutics42. As a basic rule, the so-



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called selectivity of chemotherapy depends on the cells’ proliferation25. Here, the monocytes and bone marrow-derived cells that were not in terminal maturation were significantly targeted. Cancer alters the myelopoiesis in the bone marrow and can induce secondary hematopoiesis in the liver and the spleen; thus, increases the number of immature myeloid cells2. As a consequence of tumor progression, monocytic cells can differentiate into granulocytic cells43. Therefore, the decrease in monocytic cells may have eventually leaded to the decrement in granulocytes as well. Considering the therapeutic approaches tested here, the ratio of granulocytes and monocytes was not usually altered however the number of cells were significantly reduced. Therefore, an even toxic effect or production block on myeloid cells is achieved with gemcitabine even when loaded into the dendrimers. Flt1 targeting significantly potentiated the effect of gemcitabine on the bone marrow. Although there are reports declaring early gemcitabine treatments targets splenic MDSCs in tumor-bearing mice6, 44

, in all our groups studied, the myeloid cells in the spleen were only slightly affected from

the chemotherapy. Thus, this is an open question for further analyses testing the influence of tumor model and drug delivery method used. Considering the substantial disorder in the bone marrow upon gemcitabine treatment via Flt1-targeting dendrimers, not underscoring its potential effects directly on circulating leukocytes, the drastic reduction of myeloid cells in the peripheral blood and the liver could have been anticipated. It can be speculated that monocytes were more vulnerable than granulocytes to direct and indirect (through alteration of hematopoiesis and myeloid cell reservoir) effects of gemcitabine-loaded dendrimers, in vivo. Moreover, the success of the treatment in reducing the pancreatic tumor burden in mice can also decrease the stimuli on myeloid cell production. In accordance with our data, this statement may be correct especially for the myeloid cells in the bone marrow and the liver; and, even for the tumor infiltrating


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histiocytic cells. The impact of tumor size reduction on myeloid cell counts can also be valid for the blood when only monocytes were considered. In conclusion, the gemcitabine-loaded anti-Flt1 antibody-conjugated PEG-cored PAMAM dendrimers’ success in the reduction of tumor mass17 were accompanied by the elimination of tumor-induced myeloid cells in various compartments (Suppl. Fig. 5). Therefore, this novel approach can not only be regarded as a promising strategy to diminish pancreatic cancer growth but also to target myeloid cells propagated under the influence the tumor mass.

Acknowledgements This research is partially supported by The Scientific and Technological Research Council of Turkey (Project no.:112S205). It is covered under the European Cooperation in Science and Technology (COST-EU) Action BM1404 (Mye-EUNITER). COST Action BM1404 MyeEUNITER (http://www.mye-euniter.eu). COST is supported by the EU Framework Program Horizon 2020.

Conflict of interest None declared.

Supporting Information Supporting Information. The data auxiliary to the main content of the Materials and Methods and the Results sections supplied as Supporting Information.



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Figure legends Figure 1. Biodistribution of anti-Flt1 antibody-conjugated PEG-cored PAMAM dendrimers. As shown in schematic drawing of the dendrimers (A), dendrimer arms were propagated from a PEG core and their surface was PEGylated where up to four anti-FLt1 antibody molecules were attached. Instead of gemcitabine, Rhodamine123 (Rho123) was loaded as a tracer into the space through the dendrimer arms for the biodistribution analyses. B) The delivery of Rho was evaluated by flow cytometric analysis of median fluorescence intensity (MFI) values of cell suspensions obtained from peritoneal cavity (Pt. cavity), liver, spleen, blood, and bone marrow (B. marrow). C) Representative flow cytometry overlay histograms for Rho staining. (*P

Tumor-induced myeloid cells are reduced by gemcitabine-loaded

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