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. 2021 Jan;23(1):110-121.
doi: 10.1007/s12094-020-02429-0. Epub 2020 Jul 13.

Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells

P L Smith  1 , Y Yogaratnam  2 , M Samad  2 , S Kasow  2 , A G Dalgleish  2
Affiliations

Effect of Gemcitabine based chemotherapy on the immunogenicity of pancreatic tumour cells and T-cells

P L Smith et al. Clin Transl Oncol. 2021 Jan.

Abstract

Purpose: Chemotherapy for advanced pancreatic cancer has limited efficacy due to the difficultly of treating established tumours and the evolution of tumour resistance. Chemotherapies for pancreatic cancer are typically studied for their cytotoxic properties rather than for their ability to increase the immunogenicity of pancreatic tumour cells. In this study Gemcitabine in combination with immune modulatory chemotherapies Oxaliplatin, zoledronic acid and pomalidomide was studied to determine how combination therapy alters the immunogenicity of pancreatic tumour cell lines and subsequent T-cell responses.

Methods: Pancreatic tumour cell lines were stimulated with the chemotherapeutic agents and markers of immune recognition were assessed. The effect of chemotherapeutic agents on DC function was measured using uptake of CFSE-stained PANC-1 cells, changes in markers of maturation and their ability to activate CD8+ T-cells. The effect of chemotherapeutic agents on T-cell priming prior to activation using anti-CD3 and anti-CD28 antibodies was determined by measuring IFN-γ expression and Annexin V staining using flow cytometry.

Results: These agents demonstrate both additive and inhibitory properties on a range of markers of immunogenicity. Gemcitabine was notable for its ability to induce the upregulation of human leukocyte antigen and checkpoints on pancreatic tumour cell lines whilst inhibiting T-cell activation. Pomalidomide demonstrated immune modulatory properties on dendritic cells and T-cells, even in the presence of gemcitabine.

Discussion: These data highlight the complex interactions of different agents in the modulation of tumour immunogenicity and immune cell activation and emphasise the complexity in rationally designing chemo immunogenic combinations for use with immunotherapy.

Keywords: Chemotherapy; Gemcitabine; Immunotherapy; Pancreatic cancer.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Effect of GEM on cytotoxicity and markers of tumour recognition. GEM (0.01–10 μM) was incubated with each pancreatic tumour cell line for 72 h prior to the addition of WST-1 reagent to measure cell viability (a). Expression of HLA (48 h) and cytotoxicity (72 h) of PANC-1 cells by different concentrations of GEM (b). Fold increase in the expression of HLA-class I, PDL-1 and CD47 by GEM in each cell line after 24 h incubation (c). N = a minimum of 3 experiments
Fig. 2
Fig. 2
GEM-based combination treatment and markers of tumour recognition. PANC-1 cells were treated for 48 h with different concentrations of GEM and changes in the expression of markers of tumour recognition HLA-class I, ULBP1, 3, 2/5/6 and MIC A/B (a) or immune checkpoints CD39, HVEM, PDL-1 (0.1 µM p = 0.007, 1 µM p = 0.006, 1 µM p = 0.014) Galectin 9 and CD47 (0.1 µM p = 0.007, 1 µM p = 0.006, 1 µM p = 0.05) were measured (b). N = 3. GEM (100 nM), OXP (100 nM), ZA (10 nM) and POM (100 nM) were used alone or in combination to stimulate PANC-1 cells for 48 h and markers of tumour recognition were measured by flow cytometry. N = 3. Paired t tests were performed to assess statistical significance
Fig. 3
Fig. 3
Induction of markers of immunogenic cell death from chemotherapeutic agents. Calreticulin (a) (0.1 µM GEM p = 0.04, 1 µM GEM p = 0.04; 1 µM OXP p = 0.016), ATP (b) (10 µM OXP p = 0.05) and HMGB1 (c) (0.1 µM GEM p = 0.0073, 1 µM GEM p = 0.013; 1 µM OXP p = 0.018) were measured in PANC-1 cells stimulated with different chemotherapeutic agents for 24–48 h. N = at least 3 experiments. A paired t test was used to determine statistical significance over unstimulated control. CFSE-stained PANC-1 cells treated with chemotherapeutic agents for 24 or 48 h were incubated for 4 h with MDDC and uptake was measured by flow cytometry. Incubation of CFSE + PANC-1 cells with chemotherapeutic agents promotes their internalisation by HLA-DR hi MDDC (d). Effect of uptake of PANC-1 cells at 24 and 48 h stimulation GEM (0.1 µM GEM p = 0.008, 1 µM GEM p = 0.027; 10 µM GEM p = 0.029) (e). GEM and OXP but not ZA promote PANC-1 uptake by DC (f)
Fig. 4
Fig. 4
GEM-based combinations enhance DC maturation: Immature MDDC were cultured with supernatant from pancreatic tumour cells treated with GEM-based combinations or left untreated. No supernatant (No stimulation) and LPS were utilised as controls. Flow cytometry was used to assess markers of maturation after 24 h HLA-class I (a), CD86 (b), CD40 (c), PDL-1 (d), CCR7 (e) and HLA-class II (f). N = experiments from 6 different donor PBMC. GEM (10 nM) or POM (10 nM) were paired with Poly IC (1 μg/ml) and incubated with MDDC for 24 h and markers of maturation were assessed (g). Paired t tests were used to assess statistical significance
Fig. 5
Fig. 5
Treatment with combinations of chemotherapeutic agents significantly increases antigen specific IFN-γ expression from CD8+ T-cells. IFN-γ expression from CD8+ T-cells stimulated with treated MDDC expressing CEF epitopes (a). CD8+ T-cells were stimulated with MDDC incubated with supernatant from PANC-1 cells treated with GEM, ZA or POM (b) or with MDDC directly stimulated with GEM, ZA, POM (c) and loaded with CEF peptide. CD8+ T-cells were stimulated with MDDC incubated with supernatant from PANC-1 cells treated with GEM, OXP or POM (d) or with MDDC directly stimulated with GEM, OXP, POM (e) and loaded with CEF peptide. N = at least 6 experiments. Isolated T-cells were stimulated with POM for 24 h and CD69 expression on CD8+ T-cells was measured by flow cytometry (f). CD69 expression from CD8+ T-cells stimulated for 24 h with GEM ± POM (g). N = 4
Fig. 6
Fig. 6
Pomalidomide modulates activation of CD8 + T-cells pre-incubated with anti-PD-1 antibody. T-cells were incubated with anti-PD-1 (10 μg/ml) ± GEM (1–100 nM) or POM (1–100 nM) or combinations of GEM (10 nM) and POM (10 nM) for 48 h prior to activation with anti-CD3 and anti-CD28 antibodies for a further 48 h (a). Effect of Anti-PD-1 pre-stimulation ± POM or GEM on IFN-γ expression (b), or Annexin V expression (c) from CD8+ T-cells. N = 4
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References

    1. Conroy T, Desseigne F, Ychou M, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 2011;364(19):1817–1825. doi: 10.1056/NEJMoa1011923. - DOI - PubMed
    1. Von Hoff DD, Ervin T, Arena FP, et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med. 2013;369(18):1691–1703. doi: 10.1056/NEJMoa1304369. - DOI - PMC - PubMed
    1. Siegel RL, Miller KD, Jemal A. Cancer statistics. CA Cancer J Clin. 2015;65:5–29. doi: 10.3322/caac.21254. - DOI - PubMed
    1. Abrams TA, Meyer G, Meyerhardt JA, et al. (2017) Patterns of chemotherapy use in a US-based cohort of patients with metastatic pancreatic cancer. Oncologist. 2017;22(8):925–933. doi: 10.1634/theoncologist.2016-0447. - DOI - PMC - PubMed
    1. Smyth EN, Bapat B, Ball DE, André T, Kaye JA. Metastatic pancreatic adenocarcinoma treatment patterns, health care resource use, and outcomes in France and the United Kingdom between 2009 and 2012: a retrospective study. Clin Ther. 2015;37:1301–1316. doi: 10.1016/j.clinthera.2015.03.016. - DOI - PubMed

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