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. 2012 Nov;61(11):2113-23.
doi: 10.1007/s00262-012-1279-4. Epub 2012 May 11.

Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment

Sebastian Dempe  1 , Muriel Lavie, Sofie Struyf, Rauf Bhat, Hannelien Verbeke, Stephanie Paschek, Nele Berghmans, Renate Geibig, Jean Rommelaere, Jo Van Damme, Christiane Dinsart
Affiliations

Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment

Sebastian Dempe et al. Cancer Immunol Immunother. 2012 Nov.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related death in western countries. The patients are often diagnosed in advanced metastatic stages, and the prognosis remains extremely poor with an overall 5-year survival rate less than 5 %. Currently, novel therapeutic strategies are being pursued to combat PDAC, including oncolytic viruses, either in their natural forms or armed with immunostimulatory molecules. Natural killer cells are critical players against tumours and infected cells. Recently, we showed that IL-2-activated human NK cells displayed killing activity against PDAC cells, which could further be enhanced through the infection of PDAC cells with the rodent parvovirus H-1PV. In this study, the therapeutic efficacy of parvovirus-mediated delivery of three distinct cyto/chemokines (Il-2, MCP-3/CCL7 and IP-10/CXCL10) was evaluated in xenograft models of human PDAC. We show here that activated NK and monocytic cells were found to be recruited by PDAC tumours upon infection with parvoviruses armed with IL-2 or the chemokine MCP-3/CCL7, resulting in a strong anti-tumour response.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Transgene production from recombinant H-1PV-transduced Panc-1 cells. 2 × 105 Panc-1 cells were infected with MCP-3-, IP-10- or IL-2-transducing Chi-H1/-based vectors (MOI = 3 RU/c). Cell-free conditioned media were analysed at the indicated time-points p.i. for cytokine accumulation using specific ELISAs and expressed in ng/2 × 105 cells. Data represent means ± SD of three measurements of a representative infection
Fig. 2
Fig. 2
IL-2-transduced Panc-1 cells induce human NK cell activation and chemotaxis. a 1 × 106 NK cells were stimulated for 5 days with recombinant (rec) IL-2 at 100 IU/mL or conditioned media harvested at day 2 p.i. from 1 × 106 Panc-1 cells buffer-treated (Mock) or infected (MOI = 3 RU/c) with control (∆800), MCP-3 or IL-2-encoding recombinant H-1PV vectors. Stimulated effector NK cells were then co-cultured with NK cell-susceptible Cr51-labelled target K562 cells for 4 h at the indicated effector to target (E/T) cell ratios, and the percentage of specific lysis was calculated as described in the M&M section. Data represent means ± SD of a representative experiment. b 5 × 103 NK cells were placed onto the porous membrane of a 96-well chemotaxis plate and allowed to transmigrate for 3 h into the lower chamber, filled with the conditioned media of (infected) Panc-1 cells, as described in a. NK cells present in the lower chamber were microscopically counted. Data represent the means ± SD of the chemotactic index above buffer-treated cells obtained from three independent experiments
Fig. 3
Fig. 3
Secretion of Gaussia luciferase in Panc-1 xenografts infected with Chi-H1/GLuc. 5 × 106 Panc-1 cells were infected with Chi-H1/GLuc or control Chi-H1/∆800 (MOI = 3 RU/c), s.c. implanted into nude Balb/c recipient mice and 100 μg of coelenterazine was intraperitoneally injected at the indicated time-points p.imp. GLuc activity was monitored 7 min thereafter for 5 min using a CCD camera (left panel), photon emission quantified and expressed as photons × 105 released per second ± SD (right panel)
Fig. 4
Fig. 4
IL-2- and MCP-3-encoding H-1PV vectors suppress human PDAC tumour development. a 5 × 106 Panc-1 cells were buffer-treated (Mock), infected at an MOI = 1.5 RU/c with wild-type H-1PV (H-1PV) or Chi-H1/-based vectors, empty (∆800) or transducing IP-10, MCP-3 or IL-2, and s.c. implanted into female Balb/c mice (n = 8, wild-type H-1PV: n = 5). The two-dimensional protrusions of the developing tumours were measured twice a week using an electronic calliper and tumour volumes calculated using the following formula: V = ½ × L × B2. Data represent mean tumour volumes ± SEM. b 2.5 × 106 MiaPaCa-2 cells were buffer-treated (Mock) or infected (MOI = 2 RU/c) with Chi-H1/∆800, Chi-H1/MCP-3 or Chi-H1/IL-2, s.c. implanted into nude Balb/c mice (n = 10, Chi-H1/IL-2: n = 6), and tumours were monitored as described in a. c 1.5 × 105 Panc-1 and MiaPaCa-2 cells were infected with Chi-H1/MCP-3 (MOI = 3 RU/c), and the daily secretion of MCP-3 was analysed in cell supernatants using specific ELISA. Data represent means ± SD of three measurements of a representative infection
Fig. 5
Fig. 5
Leucocyte infiltration into IL-2 and MCP-3-transduced PDAC tumours. Left panel 5 × 106 Panc-1 cells were buffer-treated (Mock) and infected (MOI = 1.5 RU/c) with wild-type (H-1PV) or recombinant vectors (Chi-H1/): control empty vector (∆800) and vector transducing MCP-3, IP-10 and IL-2. Right panel 2.5 × 106 MiaPaCa-2 were infected (MOI = 2 RU/c) with Chi-H1/Δ800 or Chi-H1/MCP-3. PDAC cells were s.c. implanted into female Balb/c mice (n = 3), and developing tumours were excised at day 5 (Panc-1) or 11 (MiaPaCa-2) p.imp. Three micrograms of total RNA were reverse-transcribed and cDNAs amplified using primers specific for control- (Ctrl), virus-, NK cell-, monocyte- (Mcyte) or dendritic cell (DC)-related transcripts and visualized on 1 % ethidium bromide-stained agarose gels
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