Introduction
Despite recent advances in cancer treatment, systemic chemotherapy still remains the only option for unresectable or metastatic pancreatic cancer. Folinic acid, fluorouracil, irinotecan and Oxaliplatin [
1] (FOLFORINOX) or Gemcitabine (GEM) in combination with Paclitaxel [
2] are the main chemotherapeutic options available however due to the toxicities associated with these drugs GEM alone has typically been prescribed in approximately 46% of patients [
3]. Even with greater first line options approximately 32% of patients receive gemcitabine monotherapy as first or second line treatment [
4]. Pancreatic cancer quickly develops resistance to these chemotherapeutic regimens, limiting their efficacy. The median survival after pancreatic cancer diagnosis is approximately 4.6 months [
5] which emphasises the urgent need for more effective treatment options. Thus, new chemotherapeutic regimens are required with improved efficacy [
6].
The clinical potential of immunotherapy is being realised through the use of checkpoint inhibitors (CPI) and engineered T-cells [
7]. However, with very few exceptions, these immunotherapeutic approaches have not been particularly successful when applied to pancreatic cancer [
8], likely due to the late presentation of the disease by which point the tumour is often locally advanced with a well-developed immune suppressive microenvironment [
9]. CPI in combination with single agent GEM or GEM plus Paclitaxcel has repeatedly failed to show significant efficacy in pancreatic cancer patients [
10‐
16] indicating that new chemotherapeutic approaches are needed for subsequent combination with immunotherapy.
Chemotherapeutic combinations for pancreatic cancer have typically been studied for their cytotoxic properties rather than their ability to increase the immunogenicity of pancreatic tumour cells. Our group has previously demonstrated immune modulatory properties of GEM [
17] indicating that it may be a suitable chemotherapeutic with which to develop novel combinations of immune modulating chemotherapeutic agents in the setting of pancreatic cancer. In this study chemotherapeutic agents Pomalidomide (POM), Oxaliplatin (OXP) and Zoledronic acid (ZA) were selected to be paired with GEM on the basis of their varied immune modulatory properties [
18‐
21]. In this study the effect of these GEM-based combinations was assessed in vitro on markers of immunogenicity in pancreatic tumour cell lines, dendritic cells and T-cells.
Methods
Reagents
Gemcitabine, Oxaliplatin, Pomalidomide and ZAedronic acid, Staphylococcus enterotoxin B (SEB) and WST-1 reagent were purchased from Sigma (Dorset, UK). Flow cytometry antibodies were purchased from Biolegend (London, UK) and R and D systems (Abingdon, UK). Cytomegalovirus, Epstein Barr Virus and Influenza virus (CEF) peptides were purchased from Sigma (Dorset, UK). CFSE reagent was purchased from Fisher Scientific—UK Ltd (Loughborough, UK).
Cell culture
Pancreatic tumour cell lines PANC-1, Miapaca-2 and BxCP-3 were cultured in Dulbecco’s modified eagles medium (DMEM) with 10% FBS and 5% Penicillin/Streptomycin at 37 °C, 5% CO
2. Leukocyte cones obtained from the NHS national blood service (London, UK). PBMC were isolated using Histopac purchased from Sigma (Dorset, UK) as described previously [
22].
Cytotoxicity assay
WST-1 viability assays were performed as detailed in the manufactures protocol. Briefly, pancreatic tumour cell lines were seeded into flat bottom 96 well plates at a concentration of 5 × 103 cells in 100 μl of DMEM. After overnight adherence the tumour cells were treated with the indicated concentration of chemotherapeutic agents and incubated for a further 72 h. Supernatant was removed prior to incubation of the cells in WST-1 reagent (Sigma, Dorset, UK) for 20 min. Colour change was assessed using a spectrometer at 490 nm.
Generation of MDDC and T-cells
MDDC were generated by isolating CD14+ cells using MACS CD14 isolation beads (Miltenyi, Bergisch Gladbach, Germany). The isolated cells were incubated with IL-4 and GMCSF for 6 days as previously described [
22]. T-cells were isolated by positive selection using MACS CD3 isolation beads according to the manufacturer’s instructions.
Flow cytometry
Pancreatic tumour cell lines, MDDC and T-cells were assessed for expression of different markers using the following antibodies: Anti-PDL-1 APC, anti-Galectin 9 PE, anti-CD39 FITC, anti-CD47 Percp-Cy5.5, anti-HLA-class I Alexa Flour 700, anti-MIC A/B PE, anti-ULBP1 FITC, anti-ULBP 2,5 6 Percp, anti-ULBP 3 APC,anti-CD86 PE, anti-CCR7 PE-CY7, anti-CD40 APC, anti-HLA-class II PE-CY5 anti-IFN-γ PE-CY7, anti-CD3 Alexa Flour 488, anti-CD4 APC-CY7, anti-CD8-PE and anti-CD69 APC (Biolegend, CA).
Tumour cell uptake assay
PANC-1 tumour cells were incubated with 1 µM of CFSE reagent overnight. Cells were subsequently washed in PBS and incubated with indicated concentration of chemotherapeutic agents for 24 h. The cells were washed twice and incubated with 1 × 105 MDDC for 4 h. The non-adherent MDDC were stained with an PE-CY5 anti-human HLA-class II antibody and assessed for uptake of CFSE labelled PANC-1 cells by flow cytometry (Biolegend, CA).
MDDC maturation
MDDC were incubated with supernatant from treated or untreated pancreatic tumour cell lines for 24 h prior to staining with antibodies specific for CD83, CD86, CCR7, PDL-1, HLA-class I, CD40 and HLA-class II (Biolegend, CA)..
T-cell activation
DC were matured as described above and loaded with CEF peptide or SEB antigen prior to co-culture with autologous CD14 negative PBMC. Brefeldin A was added after 18 h and the cells were incubated for a further 6 h prior to staining with antibodies for anti-human-CD3 FITC, anti-human, anti-human CD8-PE, anti-human IFN-γ-PE-CY7. Purified T-cells were also directly treated with combinations of chemotherapeutic agents for 24 h and stained for anti-human CD69–APC. To measure the onset of activation induced cell death (AICD) anti-PD-1 (1–10 μg/ml), anti-CD3 (5 μg/ml) and anti-CD28 (2.5 μg/ml) antibodies were used to stimulate T-cells prior to staining with anti-IFN-γ PE-CY7 and Annexin V APC (Biolegend, CA).
Discussion
Improving the immune modulatory properties of GEM-based therapy will benefit pancreatic cancer patients by providing more effective chemo-immunotherapy based treatments capable of killing tumour cells through direct cytotoxic effects and by supporting anti-tumour immune responses activated with immunotherapies such as checkpoint inhibition [
28,
29].
This study demonstrates the complexity of combining different agents with varied effects on diverse markers of the immune response. Of the agents studied here GEM was unique in its ability to upregulate markers of tumour recognition from pancreatic tumour cell lines including HLA-class I, MIC A/B and ULBP receptors (Figs.
1,
2). It would be interesting to ascertain whether GEM can enhance the targeting of pancreatic tumour cells by effector cells capable of recognising MIC A/B and ULBP such as NK and γδ-T-cells. GEM was the only agent studied which upregulated checkpoint molecules including PDL-1 and CD47. This has relevance for its potential as an immunotherapeutic agent in combination with anti-PD-1 or anti-PDL-1 inhibitors whilst implicating these checkpoints in blocking putative immune potentiating properties of GEM in vivo. Notably GEM could induce increases in HLA expression at concentrations inducing minimal cytotoxicity (Fig.
1) indicating that the immunogenic and cytotoxic properties of GEM may be independent. These data have implications for the use and dose of GEM in different therapeutic settings.
In contrast GEM was unable to induce expression of all three markers of ICD. Oxaliplatin, capable of ICD, increased the expression of CRT, ATP and HMGB1 from pancreatic tumour cell lines, in line with previous studies [
30]. GEM increased the expression of CRT on the surface of pancreatic tumour cells and enhanced the uptake of PANC-1 cells into DC but could not induce the expression of ATP or HMGB1 from the cell lines studied (Fig.
3). Given the ability of GEM to increase the expression of the checkpoint CD47 which is involved in blocking the CRT dependent uptake of tumour cells it would be interesting to ascertain whether blocking CD47 expression on PANC-1 cells further enhanced their GEM mediated uptake into APC’s.
Combination treatment of PANC-1 cells induced factors that could significantly increase the expression of markers of DC maturation compared to no treatment controls or single agent GEM (Fig.
4). These increases were lower compared to LPS or Poly IC but were associated with a significantly increased ability of these DC to activate antigen specific IFN-γ expression from CD8+ T-cells (Fig.
5). The increases in both DC maturation and T-cell activation were likely due in part to the presence of the iMiD POM in the combination treatment, consistent with our previous findings [
20]. GEM alone was unable to either induce DC maturation or activation of T-cell responses. GEM, but not OXP or ZA, was associated with inhibition of T-cell activation (Figs.
5,
6) suggesting a potential role for GEM in T-cell dysfunction.
Dysfunctional T-cell subsets, defined by expression of PD-1 and CD38 are associated with poor prognosis in GEM treated pancreatic patients, particularly on CD101+ expressing T-cells which represent an exhausted phenotype that cannot be salvaged by anti-PD-1 therapy [
26]. A recent study indicated that CD38
hi, PD-1
hi T-cells are susceptible to apoptosis upon interaction with anti-PD-1 antibody prior to T-cell priming [
27]. Incubation with GEM and/or POM did not significantly alter the expression of these markers upon stimulation of T-cell subsets from healthy donors. However, GEM inhibited IFN-γ expression from T-cells which could be partially restored with POM co stimulation (Fig.
6d). POM was also capable of reducing the onset of markers of apoptosis and increase the expression of IFN-γ from T-cells incubated with anti-PD-1 antibody prior to T-cell activation (Fig.
6b, e) consistent with the ability of POM to prime T-cell responses.
Its notable that GEM had the greatest effect on markers of tumour recognition, OXP on markers of ICD and POM on the priming of T-cell immune responses. Combinations of these agents rarely demonstrated additive or inhibitory properties, with the exception of GEM dependent inhibition of T-cell responses. Although several chemotherapeutics have well defined immunogenic effects their clinical efficacy has rarely been associated with the onset of immune responses, even for known inducers of ICD. A possible explanation for the limited observable immunotherapeutic effects of chemotherapeutic agents is that the promotion anti-tumour immune responses involves a multitude of checkpoints and effector cells whilst a single agent such as GEM mediates only a subset of these factors. The agents studied here demonstrated modest ability to induce markers of ICD (Fig.
3) or DC maturation (Fig.
4), suggesting that this component of chemo-immunotherapy needs to be addressed. Combinations involving chemotherapy and broad immune stimulants including TLR agonists such as Poly IC [
31,
32] or cytokines such as IFN-α [
33] have shown promise and the combination of GEM or POM with Poly IC studied here demonstrated increased DC maturation (Fig.
4g). The potential therapeutic benefit of combination of chemotherapy with TLR agonists is illustrated by our recent study demonstrating improved responses in PDAC patients with the addition of heat killed supported
Mycobacterium obuense to single agent GEM [
34].
A greater understanding of the immunological effects of combination chemotherapy, in addition to factors such as dose and sequence, is likely needed to improve immunotherapy in cancers such as pancreatic cancer. These data highlight that chemotherapeutics such as GEM can benefit by the addition immune modulators capable of inducing strong DC maturation and T-cell activation. It will be interesting to study the effect of these combinations in vivo during which the activation of tumour recognition and ICD, DC maturation and activation of T-cell responses may demonstrate cumulative anti-tumour effects which are not possible to study using the in vitro assays described here.
Checkpoint inhibition has demonstrated poor efficacy against pancreatic cancer. Priming with immune modulatory agents [
34] followed by immunogenic chemotherapy such as with GEM plus POM may promote greater effectiveness of checkpoint inhibition. We have previously reported on a complete response (>2 years) in a case study of metastatic pancreatic cancer involving treatment with enalidomide, GEM and a heat killed preparation of
Mycobacterium vaccae. In a more recent case study a complete response was observed involving GEM and
Mycobacterium obuense by the CPI Pembrolizumab (unpublished observation). In conclusion, this study has demonstrated distinct in vitro immune modulatory effects of GEM and POM on pancreatic tumour cell lines and T-cells respectively. This indicates that these agents are suitable for combination with immunotherapy such as checkpoint inhibition, particularly alongside innate immune agonists capable of promoting immunogenic cell death or DC maturation.
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