In vitro immunomodulatory properties of gemcitabine alone and in combination with interferon-alpha

Accepted Manuscript Title: In vitro immunomodulatory properties of gemcitabine alone and in combination with interferon-alpha Author: Jasmin Fritz Svetlana Karakhanova Ramona Brecht Ines Nachtigall Jens Werner Alexandr V. Bazhin PII: DOI: Reference:

S0165-2478(15)30041-9 http://dx.doi.org/doi:10.1016/j.imlet.2015.09.017 IMLET 5768

To appear in:

Immunology Letters

Received date: Revised date: Accepted date:

26-6-2015 28-9-2015 29-9-2015

Please cite this article as: Fritz Jasmin, Karakhanova Svetlana, Brecht Ramona, Nachtigall Ines, Werner Jens, Bazhin Alexandr V.In vitro immunomodulatory properties of gemcitabine alone and in combination with interferon-alpha.Immunology Letters http://dx.doi.org/10.1016/j.imlet.2015.09.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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In vitro immunomodulatory properties of gemcitabine alone and in combination with interferon-alpha

Jasmin Fritz1,§, Svetlana Karakhanova1,§, Ramona Brecht1, Ines Nachtigall2,Jens Werner2, Alexandr V. Bazhin2*

1

Department of General Surgery, University Hospital Heidelberg, Germany

2

Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Hospital

of the University of Munich, Germany

§

- equal contribution

Short title:Immunomodulatory effects of gemcitabine

*

Address correspondence to AVB: Department of General, Visceral, and Transplant Surgery,

Ludwig-Maximilians-University Munich, Germany, GERMANY Tel: +49-89-4400-0, Fax: +49-89-4400-76433 E-mail: [email protected]

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Highlights 

gemcitabine depletes the regulatory T cells in the splenocyte culture



gemcitabine in combination with interferon-alpha modulates composition of DC subpopulations and their activation state



combination of both therapeutics induces rather cumulative effects

Abbreviations 5FU:5-fluorouracil CTC:cytotoxic T cells DC:dendritic cells FACS:fluorescence activated cell sorting Gem:gemcitabine IFN:interferon-α LAK:lymphokine-activated killer MDSC:myeloid-derived suppressor cells NK cells:nature killer cells PDAC:pancreatic adenocarcinoma Tcon: conventional T cells Treg:regulatory T cells

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Abstract In general conventional chemotherapy is associated with significant toxicity leading to immunosuppression manifesting mainly in thelymphocyte depletion.This immunosuppression promotes tumor growth and elicits the tumor cell dissemination. However, chemotherapy can be immune stimulative especially in combination with an immunotherapy. In this work we investigated in vitro effects of gemcitabine alone and in combination with interferon-alpha on splenocytes obtained from healthy and pancreatic carcinoma bearing mice. We showed that gemcitabine alone depletes the regulatory T cells in the splenocyte culture. Gemcitabine in combination with interferon-alpha demonstrated some immunomodulatory features, but these effects were interferon-alpha dependent. We concluded that combination of both drugs induces rather cumulative effects, supposing that these therapeutic could be applied together for a chemo-immunotherapy.

Key Words: immunomodulation, chemotherapy,interferon-alpha, gemcitabine, pancreatic adenocarcinoma

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1. Introduction The best known immune effects of cancer chemotherapeutics are the myelo-,neutro- and lymphopaenie and therefore these drugs are also used as immunosuppressants. Besides these general effects, the influence of cancer drug on the immune system can be manifested in the blocking of T cell proliferation and/or in the affecting of their effector state [1]. Such undesirable immunosuppressive effects promote tumor growth and can also elicit the tumor cell dissemination [2]. The double-edge sword of the chemotherapy could be the reason that chemotherapeutic treatment leads to massive tumor-antigen release which from one side can induce the antigen specific immune response [3].But from another side chemotherapy may also trigger the immunosuppression due to the induction of different inflammatory mediators and the recruiting of innate immunosuppressive cells to tumor side [4]. In vivo support of this assumption has been found in studies with immunodeficient mice where it has been shown that chemotherapeutics are more effective in vanishing of tumor cells in the presence of the intact immune system [5,6]. Besides, anti-cancer drugs used at clinically efficient doses can mediate immunostimulatory effects (for review see [7]). The tumor-antigen releaseand certain immunostimulatory effects was a rationale for creating numerous clinical studies combining a conventional chemotherapy with immunotherapy. As immunotherapy, either unspecific immune stimulation by for example interferon-α (IFN) [810]or specific immunotherapy with DC or other cells[11] has been applied for treatment of different solid tumors with rather moderate clinical benefit. Pancreatic adenocarcinoma (PDAC) is namely rare but tremendous disease without any chance for healing in the advance stage [12]. In case of successful surgical treatment PDAC patientsgetusuallyadjuvant chemotherapy. The standard PDAC chemotherapy includes a combination of drugs with a chemotherapeutic backbone as gemcitabine (gem) or 5fluorouracil (5FU) [13]. A combined PDAC treatment with chemoradioimmunotherapy (5-FU

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plus IFN)was recently conducted(CapRI). While the combined therapy led to pronounce activation of immune system of the PDAC patients [14,15], no therapeutic benefit of the IFN adding

to

chemotherapy

was

registered

[16].

A

simultaneous

activation

of

immunosuppression due to IFN was proposed as a cause of the inefficacy of such drug combination [17]. It was shown that treatment with 5FU alone or in combination with IFN lead to an accumulation of the highly immunosuppressive myeloid-derived suppressor cells (MDSC) in the tumor of PDAC-bearing mice [14]. Thus, the chemotherapeutic (especially 5FU) with or without simultaneous application with IFN indeed can elicit immunoregulatory properties in PDAC. Immunological effects of gem are not deeply investigated yet. In a model of murine mesothelioma it was shown that gem inhibits proliferation of B cells [18]. One study in the gem treated PDAC patients demonstrated that this treatment led to a decrease in absolute number of CD3+ and CD20 + lymphocytes and to an increase in percentages of memory lymphocytes. An in vitro study on NK, CTL and lymphokine-activated killer (LAK) cells showed that gem inhibits generation of LAK or CTL [19]. Combination effects of gem with IFN have not been investigated so far. Therefore, in this work we examined immunological effects of treatment with gem, IFN or in combination of both drugs in ex vivo isolated splenocytes from PDAC-bearing and healthy mice.

2. Material andMethods 2.1 Antibodies and reagents Anti-mouse CD3-BD Horizon™ v500, CD3-PerCP-Cy5.5, CD4-APC-H7, CD8-APC BD Horizon™ v500, CD8-PerCP-Cy5.5, CD25-APC, Ly6C-BD Horizon™ v450, CD45-BD Horizon™ v500, and purified rat anti-mouse CD16/CD32 antibodies were purchased from BD Bioscience (Germany). FoxP3-FITC and FoxP3 fixation/permeabilization kit were from eBioscience (Germany). Gem was purchased from Fresenius Kabi (Germany).

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2.2 Mice C57BL/6 mice (six to eight weeks old) were purchased from Charles River (Sulzfeld, Germany) and kept under specific pathogen-free conditions in the animal facility of Heidelberg University (IBF, Heidelberg). Animal experiments were carried out following the approval of the German authorities (35-91585.81/G-184/11). 2.3 Orthotopic Mouse Model of Pancreatic Carcinoma Syngeneic pancreatic carcinoma cells (Panc02) were injected orthotopically in C57Bl/6 mice, as described elsewhere[20]. Mice were narcotized with isofluran/O2 inhalation. After achieving surgical tolerance (stage III2), mice were opened with an abdominal section. A volume of 5 µL of mycoplasma-free Panc02 cells in a concentration of 2 × 107 cells/mL PBS were injected into the pancreatic head with a 25 µL gastight syringe (Hamilton, Reno, NV, USA). The injection site was clamped for about 30 s after removal of the syringe. The tissue was carefully returned to its original position and all layers of the wound (peritoneum, muscles, and skin) were sutured with synthetic absorbable suture material (polysorb 6-0, (Tyco Healthcare, Neustadt, Germany)). Spleens were harvested four weeks after Panc02 cell implantations and used for the flow cell biology analysis. 3.4 Flow Cell Biology Analysis of Murine Splenocyte Samples Flow cytometry (FACS) analysis of murine splenocytes was performed as described elsewhere [21]. Briefly, single cell suspensions of spleens were prepared in the StainBuffer (PBS supplemented with 1% mouse serum and 1 mM EDTA). The cells were then counted and the density adjusted to 4 × 107/mL. The unspecific binding caused by the Fc receptors was blocked by incubating the cells with anti-mouse CD16/CD32 antibody (1 μL for 2 × 10 6 cells) at 4 °C in the dark for max 20 min. Then, a 50 μL cell suspension (2 × 106 cells) was incubated with 50 μL of the StainBuffer containing various monoclonal antibodies at 4 °C, also in dark conditions for max 20 min. A Foxp3 Staining Buffer set was used for intracellular staining according to the manufacturer’s instructions. Acquisition was performed by flow

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cytometry using FACS Canto II with FACSDiva Software (BD Biosciences, Heidelberg, Germany). Specifically, 300,000 events were collected in the mononuclear cell gate according to the FSC vs. SSC. FlowJo software (Tree Star) was used to analyze at least 500,000 events. 2.5 In Vitro Treatment of Murine Splenocytes Single cell suspensions of spleens were prepared as described elsewhere[22]. Spleens were homogenized, squeezed through a 100 µm and 40 µm cell strainer and flushed with 10 mL of PBS to collect as much cells as possible and finally suspended in PBS. The cells were then counted, the density adjusted to 2 × 10 6/mL and 5 mL of this cell suspension was cultivated in a RPMI-1640 medium with 10% fetal calf serum for 24 h, with or without IFN (5.000 U) and gem

(15μM). After that, the cells were gently scratched and stained with fluorescent-

labeledantibodies, as mentioned above. 2.6 Cell viability assays Cell viability after gem treatment was measured with a EZ4U Kit (Biomedica, Austria) in the whole medium as described elsewhere [23]. Briefly, for the EZ4U assay 20,000 cells per well were seeded in 96-well plates. After overnight growing (about 90% cells attached) gemat different concentrations was added without the medium exchange. After a 24 h incubation period a substrate compound from the kit was added, and the cells were incubated for 5 hours at 37°C to convert the yellow coloured tetrazolium to its red formazan derivate by living cells. Finally, the absorbance was measured at 450 nm. 2.7 Statistical analysis GraphPadPrism Version 5.01 software was used for statistical analyses. Distributions of continuous variables were described by means, SE, median, 25% and 75% percentiles, and were presented as box-and-whiskers plots or as column bar graphs. D’Agostino and Pearson omnibus normality tests were conducted to estimate the distribution of data. The null hypothesis (mean values were equal) versus the alternative hypothesis (mean values were not equal) was tested by repeated measures ANOVA with the Bonferroni’smultiple comparison

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post-hoc test for the normal distributed variants and by Mann-Whitney test for the data which did not pass the normality test.All statistical tests were two-tailed. The significance level was α=5%.

3. Results We first tested the cytotoxic effect of gem at different concentrations on the viability of splenocyte obtained from healthy or from tumor-bearing mice. No cytotoxicity effects have been found by all gem concentrations tested (Fig. S1). 3.1 Influence of chemotherapeutics on splenocytes from tumor-bearing mice To understand whether the gem alone or in combination with IFN has a direct effect on immune cells, the splenocytes from tumor-bearing mice have been isolated and cultivated with the therapeutics as described in Material and Methods. After 24 h cultivation the cells were analyzed by FACS (Fig. S2) using our established murine panels [21]. The IFN treatment of splenocytes led to an increase in the frequency of lymphocytes in the cultivated cells (Fig. 1A) and to a decrease in the CD4/CD8 ratio (Fig. 1D). The same shifting in the ratio has been observed after a combined treatment with IFN and gem, but not after a treatment with gem alone (Fig. 1D). IFN alone or in combination with gem reduced the frequency of effector cells from T cells (Fig. 1B and C). No effects of therapies on other lymphocyte subpopulations have been registered (data not shown). Regarding the frequencies of Treg and Tcon, it was shown that gem alone, but neither IFN nor both drugs, reduced the frequency of Treg after cultivation (Fig. 2A). IFN decreased the expression of the early activation marker CD69 on both – Treg and Tcon (Fig. 2B and C). Neither gem nor the drug combination had the influence on CD69. No immunological effects have been observed either on MDSC or macrophages (data not shown). While the therapeutics did not influence the frequency either of cDC or pDC, the number of CD80 +DC has been increased after treatment with IFN (Fig. 3A). Also the CD80

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expression on cDC has been stimulated by the drugs (Fig. 3B). Regarding CD86, only expression level of the co-stimulatory molecule, but not the frequency of CD86 + cells has been increased on cDC after the IFN treatment alone or in the combination with gem (Fig. 3C). It should be pointed that gem was able to decrease the frequency of CD80 +pDC (Fig. 3D). 3.2 Influence of chemotherapeutics on splenocytes from healthy mice As the next step we performed similar immunological analysis with the splenocytes from healthy mice. The same as in splenocytes from tumor-bearing animals, IFN increased a frequency of lymphocytes in the splenocyte culture (Fig. 4A) and diminished the CD4/CD8 ratio (Fig. 4D). While in contrast to tumor-bearing mice, no drug effects have been found on effector CD4+ T cells, the IFN treatment decreased the frequency of effector-memory CD4 + T cells (Fig. 4B). The negative effects of IFN treatment on effector CD8+ T cells have been also observed in the splenocytes from healthy mice (Fig. 4C).No effects of the drug on other lymphocyte subpopulations or Tcon or Tregfrom healthy mice have been found (data not shown). No influence of gem on macrophages or Gr1+CD11b+ Lys6C+/lowleukocytes was found(data not shown). In regard to DC, their co-stimulatory molecules have been also influenced by treatment with IFN alone or in combination with gem: CD80 and CD86 expression was upregulated on cDCand the frequency of CD86+pDC was increased (Fig. 5). 3.3 Frequency of Treg, CD80+ DC and expression of CD80/CD86 are tumor-dependent Comparing the data obtained from the both experimental models, we saw that the frequency of Treg was higher in the spleen from healthy mice compared to tumor-bearing ones independent on the treatment (Fig. 6A). The frequency of CD80+ DC was also constantly higher in healthy mice as in tumor-bearing animals (Fig. 6B and C). Inversely, intensity of the CD80 expression on DC was higher in tumor-bearing mice compared to healthy ones, independent on the drug applied (Fig. 6B and C). While no difference has been found in

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regard of CD86+ DC (data not shown), the CD86 expression was up-regulated on pDC from tumor-bearing mice compared to healthy animals (Fig. 6D).

4. Discussion As mentioned in Introduction chemotherapy in general is associated with significant toxicity manifesting in immunosuppression leading to increased risk of infection, and other side effects. The main immunosuppressive effect of chemotherapy is the depletion of lymphocytes due to the fact that chemotherapy targets highly proliferative cells, including activated lymphocytes. One of the main mechanisms accounting for Tregintratumoral accumulation is their recruitment[24]due tothe chemokines CCL22 and CCL17 that preferentially attract Treg through the CCR4 receptor [25].Therefore, it is not surprising that in our work the frequency of Treg was higher in the splenocytes obtained from healthy mice. Transient inhibition of Treg migration by CCR4 antagonists may play a therapeutic role in cancer immunotherapy generally, and particularly in PDAC [26], pointing towards the importance of Treg depletion by cancer patients. In this work we demonstrated that the chemotherapeutic gem applied alone in vitro to splenocytesobtained from PDAC-bearing mice reduced the frequency of Treg in the culture. This effect was not present in case of splenocytes from healthy mice and thus, seems to be tumor specificThis can be explained by the fact that gem showed its cytotoxic activity merely on the high proliferative Treg which is present in tumor bearing host [22,27].This result corroborates in vivo data showing that gem, applied conventionally or in low dose, successfully depletes Treg from tumors and spleens from-tumor bearing hosts including PDAC, and in turn enhances the anti-tumor immunity [22,28,29]).However, approaches aiming to deplete Treg in cancer should be recognized with caution, since these may cause some adverse effects [30]. An in vivo work of Suzuki et al demonstrated that the treatment of tumor-bearing mice with gem eliminates also MDSC. Treg und MDSC have been proposed to

11

influence each other and interplay between MDSC and Tregwas demonstrated in several systems[31,32]. In our in vitro experiments we did not see any effect of gem on MDSC, concluding that gem does not have a direct influence on these immunosuppressive cells. Our results demonstrated that IFN alone or in combination with gem contribute to the activation of cDC by the increasing expression of costimulatory molecules. This is in line with previous study from us and others showing the stimulating effect of IFN on DC [14]. Moreover in tumor-bearing mice, but not in healthy mice, even gem alone was able to increase the intensity of CD86 expression, speaking for the importance of tumor influence on DC for this effect. While in the in vivo studies cited the activation of anti-tumor response was well documented, in our work in in vitro setting we did not found any effect of gem on other immune cells, underlying the importance of the in vivo conditions with a specific immune cell constellation for immunological effectsof gem. In vivo as well in vitro effects of IFN on the immune cells are widely known[33], also in context of PDAC [14,15]. These effects are manifested by modulation of the immune system: improving differentiation, maturation, and function of DC, enhancing the survival of T cells by expression of anti-apoptotic genes inducing the generation of CD8 + memory cells, enhancing macrophage activities, and activating natural killer (NK) cells, and by enhancement of immunogenicity of tumors (increase of MHC class I expression)[33]. In concordance with these data, our in vitro study also demonstrated a positive effect of INF on immune cells in splenocytes both from healthy and tumor-bearing mice. It should be noted that combination of both drugs induces rather cumulative effects, supposing that these therapeutics could be applied together for a chemo-immunotherapy. However, while a combination of IFN with another chemotherapeutic –5FU, did highly activate the immune system of PDAC patients from the CapRI studies [14,15,34], this therapy did not improve survival of PDAC patients[29]. This negative result was attributed to the

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activation of immunosuppression induced merely by 5FU and/or INF [14,17]. Therefore, it is tempting to speculate that other combinations of IFN with chemotherapyshould be taken in consideration, making gem the “best friend” of IFN in the combination of immunotherapy with chemotherapy. Future preclinical and clinical study should be performed to elucidate this possibility.

Acknowledgements We thank Mr. Markus Herbst and Ms. Tina Maxelon for their excellent technical assistance.

Conflict of Interest No potential conflicts of interest were disclosed

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Figure Legends Figure 1.Immunomodulatory properties of gem and IFN alone or in combination on lymphocytesfrom tumor-bearing mice (A), effector CD4+ (B) and CD8+ (C) T cells, and CD4/CD8 ratio (D). Data are presented as box and whiskers plots (A-C) or column bar graph with SE (D) and analysed with the one-way ANOVA with the Dunnett’spost test, * p<0.05 and ** p<0.01.Right panel shows the representative FACS dot plots (A-C) for the IFN treatment.

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Figure 2.Immunomodulatory properties of gem and IFN alone or in combinationin regards toTreg and Tcon from tumor-bearing mice.Data are presented as column bar graph with SEand analysed with the one-way ANOVA with the Dunnett’spost test, * p<0.05, ** p<0.01and *** p<0.01.Right panel shows the representative FACS dot plots and histograms for gem (A) and IFN (B-C) treatment. Figure 3.Immunomodulatory properties of gem and IFN alone or in combinationin regards to DC from tumor-bearing mice.Data are presented as box and whiskers plots (A and D) or column bar graph with SE (B and C) and analysed with the one-way ANOVA with the Dunnett’spost test, * p<0.05, ** p<0.01and *** p<0.01.Right panel shows the representative FACS histograms for the IFN (A and B) and gem (C and D) treatment. Figure 4.Immunomodulatory properties of gem and IFN alone or in combinationin regards tolymphocytesfrom healthy mice (A), effector-memory CD4+ (B) and effector CD8+ (C) T cells, and CD4/CD8 ratio (D). Data are presented as box and whiskers plots (A-C) or column bar graph with SE (D) and analysed with the one-way ANOVA with the Dunnett’spost test, * p<0.05 and ** p<0.01.Right panel shows the representative FACS dot plots (A-C) for the IFN treatment. Figure 5.Immunomodulatory properties of gem and IFN alone or in combinationin regards to DC from healthy mice.Data are presented as column bar graph with SE (A and B)or box and whiskers plot (C) and analysed with the one-way ANOVA with the Dunnett’spost test, * p<0.05, ** p<0.01and *** p<0.01.Right panel shows the representative FACS histograms for the IFN treatment. Figure 6.Immunological effects of drugsin tumor-bearing mice compared with healthy animals. Data are presented as column bar graph with SEand analysed with thetwo-way ANOVA with the Bonferronipost test, * p<0.05, ** p<0.01and *** p<0.01.

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Supplemental Figures Figure S1. Gem does not affect the viability of murine splenocytes. Cultivated splenocytes obtained from healthy (A) or tumor-bearingmice (B) were treated with different gem concentration for 24 h. After that the cell viability was measure with a EZ4U Kit as described in the Material and methods.Data of three independent experiments are presented as column bar graph with SEand analysed with the one-way ANOVA with the Dunnett’spost test. Figure S2. Gating strategy for T lymphocytes and their subpopulations (A), for Treg (B), and

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0

Figure 4

D IF N ge m +I FN

3

N

ge m N

N

+I F

IF

ge m

m

n

+I FN

IF N

ge

co

50

IF

m

n

lymphocytes, % of leukocytes 60

ge m +I FN

1.5

ge

co

effector-memory cells, % of CD4 + T cells 10

ge m

co n

effector cells, % of CD8 + T cells

A

ge m

co n

CD4/CD8 ratio

19

*

co

**

40

30

20

B 15

*** **

5

0

C *** **

2

1

0

* *

1.0

0.5

0.0

IFN

20 A

B 50000

***

**

MFI of CD86+ cDC

MFI of CD80 + cDC

8000 6000 4000 2000

***

30000 20000 10000 0

0 co

N IF

m ge

m ge

+

IF

N

90

***

80

***

70 60 50 40 co

m ge

N IF ge

Figure 5

m

co

m ge

N IF ge

C CD86+ cells, % of pDC

40000

+

IF

N

m

+

N IF

21 D *** **

6

**

**

- tumor + tumor

4 2

40000 30000

*

**

*

*

- tumor + tumor

20000 10000

FN ge m

+I

co n

N IF

ge m +I FN

m

n

ge

co

IF N

0

0

ge m

Treg, % of CD4 + T cells

8

MFI of CD86 + pDC

A

B ***

***

***

***

80

- tumor + tumor

60 40 20

15000

MFI of CD80 + cDC

- tumor + tumor

***

10000

***

***

***

5000

FN ge m

+I

co n

FN

N IF

ge m +I

ge m

co n

IF N

0

0

ge m

CD80 + cells, % cDC

100

C **

**

15

*

*

10 5

- tumor + tumor

15000

MFI of CD80 + pDC

**

*

* 10000

*

5000

FN

IF N

ge m +I

N m +I F ge

IF N

m ge

n co

Figure 6

ge m

0

0

co n

CD80 + cells, % pDC

20

- tumor + tumor