Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jun;5(3):242-55.
doi: 10.1016/j.molonc.2011.03.007. Epub 2011 Apr 5.

Reversal of gastrointestinal carcinoma-induced immunosuppression and induction of antitumoural immunity by a combination of cyclophosphamide and gene transfer of IL-12

Mariana Malvicini  1 , Mariana Ingolotti, Flavia Piccioni, Mariana Garcia, Juan Bayo, Catalina Atorrasagasti, Laura Alaniz, Jorge B Aquino, Jaime A Espinoza, Manuel Gidekel, O Graciela Scharovsky, Pablo Matar, Guillermo Mazzolini
Affiliations

Reversal of gastrointestinal carcinoma-induced immunosuppression and induction of antitumoural immunity by a combination of cyclophosphamide and gene transfer of IL-12

Mariana Malvicini et al. Mol Oncol. 2011 Jun.

Abstract

Immunotherapy-based strategies for gastrointestinal carcinomas (GIC) have been exploited so far, but these approaches have to face strong mechanisms of immune escape induced by tumours. We previously demonstrated that sub-therapeutic doses of an adenovirus expressing IL-12 genes (AdIL-12) mediated a potent antitumour effect against subcutaneous (s.c.) colorectal carcinomas (CRC) in mice pre-treated with low doses of cyclophosphamide (Cy). In our study we used this combination to assess its impact on the immunosuppressive microenvironment. In s.c. CRC model we demonstrated that non-responder mice failed to decrease Tregs in tumour, spleen and peripheral blood. Reconstitution of Tregs into tumour-bearing mice treated with combined therapy abolished the antitumoural effect. In addition, Cy + AdIL-12 modified Tregs functionality by inhibiting the in vitro secretion of IL-10 and TGF-β and their ability to inhibit dendritic cells activation. Combined treatment decreased the number of myeloid-derived suppressor cells (MDSCs) in comparison to non-treated mice and, interestingly, administration of Tregs restored splenic MDSCs population. Furthermore, combined therapy potently generated specific cytotoxic IFN-γ-secreting CD4+ T cells able to eradicate established CRC tumours after adoptive transfer. Finally, we evaluated the combination on disseminated CRC and pancreatic carcinoma (PC). Cy + AdIL-12 were able to eradicate liver metastatic CRC (47%) and PC tumour nodules (40%) and to prolong animal survival. The results of this study support the hypothesis that Cy + AdIL-12 might be a valid immunotherapeutic strategy for advanced GIC.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Assessment of Tregs in mice R (responder) and NR (non‐responders) to combined treatment Cy + AdIL‐12 in s.c CT26 tumour model. (A) Tumour volume in R and NR mice. BALB/c mice (n = 35) were s.c. inoculated with 5 × 105 CT26 cells into the right flank (day 0) and tumours were allowed to reach 85 mm3 before the treatment began. Animals were treated with Cy (50 mg/kg i.p., day 8) + AdIL‐12 (109 TCID50 i.t., day 9) (n = 12) or with saline (n = 6). Tumour growth was assessed twice a week by calliper. Tumour volumes at days 7 and 14 after treatment are expressed as mean (bars, SEM). The experiment was performed four times. Mann Whitney test, Cy + AdIL‐12 NR vs. Cy + AdIL‐12 R: (##p < 0.01). Percentage of CD4+CD25+Foxp3+/CD4+ cells in peripheral blood (B) or spleen (C) of R and NR mice. Mann Whitney test, Peripheral blood, day 7 and day 14: Cy + AdIL‐12 R vs. saline (∗∗p < 0.01) and vs. Cy + AdIL‐12 NR (#p < 0.05). Spleen, day 7: Cy + AdIL‐12 R vs. saline and Cy + AdIL‐12 NR (#p < 0.05); Cy + AdIL‐12 NR vs. saline (∗p < 0.05); day 14: Cy + AdIL‐12 R vs. saline and Cy + AdIL‐12 NR (∗and #p < 0.05). (D) Percentage of tumour‐infiltrating CD4+ Tcells (left) and CD4+CD25+Foxp3+/CD4+ cells (right). Mann–Whitney test, CD4+ Tcells, day 7: Cy + AdIL‐12 R vs. saline, (∗p < 0.05); day 14: Cy + AdIL‐12 R vs. saline and Cy + AdIL‐12 NR (#p < 0.05); Cy + AdIL‐12 NR vs. saline (∗p < 0.05). CD4+CD25+Foxp3+/CD4+ cells, day 7: Cy + AdIL‐12 R vs. saline, (∗p < 0.05); day 14: Cy + AdIL‐12 R vs. saline and Cy + AdIL‐12 NR (#p < 0.05). The results shown represent four independent experiments.
Figure 2
Figure 2
Re‐infusion of Tregs in CT26‐bearing mice treated with the combination Cy + AdIL‐12. (A) Tumour growth: BALB/c mice were s.c. inoculated with 5 × 105 CT26 cells into the right flank (day 0) and tumours were allowed to reach 85 mm3 before the treatment began. Animals (n = 6/group) were treated with saline or Cy (50 mg/kg i.p., day 8) + AdIL‐12 (109 TCID50 i.t., day 9). In addition,saline or Cy + AdIL‐12‐treated mice were inoculated i.v. on days 11 and 13 with 1 × 106 CD4+CD25+ T cells (Tregs) isolated from untreated tumour‐bearing mice. Tumour volume was assessed twice a week by calliper. The experiment was performed four times. Data are expressed as mean (bars, SEM). Kruskal–Wallis test, day 28: Cy + AdIL‐12 vs. saline and Cy + AdIL‐12+Tregs (∗p < 0.05). B) Survival: Kaplan–Meier, log rank test, (∗∗p < 0.01). C) In vivo distribution pattern of re‐infused Tregs: Cy + AdIL‐12‐treated mice were inoculated with 1 × 106 fastDiO‐dyed CD4+CD25+ T cells (i.v; days 11 and 13) and biodistribution was analysed by flow cytometry. The results shown represent three independent experiments.
Figure 3
Figure 3
Effect of combined therapy Cy + AdIL‐12 on the interaction DC/Tregs. A) Quantification of IL‐12: Data are expressed as mean (bars, SEM). Kruskal–Wallis and Dunn's multiple comparisons test: DC vs. DC+Tregs (saline), ∗p < 0.05; DC+Tregs (saline) vs. DC+Tregs (Cy + AdIL‐12), ∗∗∗p < 0.001. B) Phenotype of DCs (CD11c+MHC‐II+CD40+). Mann Whitney test: DC vs. DC+Tregs (saline), ∗p < 0.05; DC+Tregs (saline) vs. DC+Tregs (Cy + AdIL‐12), ∗p < 0,05. The results shown represent three independent experiments. IL‐10 (C)and TGF‐β (D) secretion by Tregs: Mann Whitney test, saline vs. Cy, AdIL‐12, and Cy + AdIL‐12, ∗p < 0.05; Cy + AdIL‐12 vs. Cy and AdIL‐12; #p < 0.05. (E) Effect of Tregs supernatants (SN) on allogeneic spleen cell (SC) proliferation under stimulation with mitomycin C‐treated DC. (3H‐Thymidine). Mann Whitney test, SC+DC vs. SC+DC+Tregs SN (saline), ∗∗∗p < 0.001; SC+DC+Tregs SN (saline) vs. SC+DC+Tregs SN (Cy + AdIL‐12), ∗∗∗p < 0.001.
Figure 4
Figure 4
(A) Effect of combined treatment Cy + AdIL‐12 on splenic MDSCs (CD11b+Gr1+): Mann Whitney test, saline vs. Cy + Ad‐12 (∗∗p < 0.01). Data are representative of three independent experiments (n = 5/group per experiment) B) Effect of in vivo administration of Tregs on splenic MDSCs in mice treated with the combination Cy + AdIL‐12. Mann Whitney test, Cy + AdIL‐12 vs. Saline, ∗∗p < 0,01 and Cy + AdIL‐12 plus Tregs, #p < 0.05. Data are representative of two independent experiments (n = 6/group per experiment).
Figure 5
Figure 5
Antitumour effect of in vitro expanded CD4+ T‐lymphocytes from CT26‐bearing mice treated with Cy, AdIL‐12 or Cy + AdIL‐12. (A) Therapeutic model: BALB/c mice were s.c. inoculated with 5 × 105 CT26 cells into the right flank (day 0) and when tumours reached 65 mm3 in size (day 6) the mice were treated with vitro expanded CD4+ cells (2,5 × 106 i.v) from different experimental groups (n = 4/group) (left panel). Data are expressed as mean (bars, SEM). Mann Whitney test, days 23 and 28: CD4+CD25− (Cy + AdIL‐12) vs. CD4+CD25− (saline, Cy and AdIL‐12), ∗p < 0.05. Survival (right panel). Kaplan–Meier, log rank test, ∗∗p < 0.01. Data are representative of three independent experiments. B) Preventive model: BALB/c mice (n = 4/group) were simultaneously inoculated with 5 × 105 CT26 cells s.c. into the right flank and adoptive transfer of 2,5 × 106 CD4+ cells at the same time (day 0) (left panel). Data are expressed as mean (bars, SEM). Mann Whitney test, days 23 and 26:CD4+CD25− (Cy + AdIL‐12) vs. CD4+CD25− (saline, Cy and AdIL‐12), ∗p < 0.05. Survival (right panel). Kaplan–Meier, log rank test, ∗∗p < 0.01. Data are representative of three independent experiments. C) Specific CTL activity (leftt panel): CD4+ T cells from different experimental groups (n = 4/group) were isolated and stimulated in vitro with mitomycin C‐treated splenocytes and CT26 cells for 5 days. Specific CTL activity was evaluated against CT26 and BNL cells. The percentage of specific cytotoxicity was quantified whit the LDH Cytotoxicity Detection Kit and calculated according to the following formula: ([abs 492 nm experimental ‐ abs 492 nm background])/[abs 492 nm maximum ‐ abs492 nm background]) × 100. Mann Whitney test, Cy + AdIL‐12 vs. saline, ∗∗∗p < 0.001; Cy + AdIL‐12 vs. Cy and AdIL‐12, ###p < 0.01. Data represent the mean of triplicate cultures. Quantification of IFNγ (right panel): The amount of IFN‐γ secreted by CD4+T cells was evaluated after its isolation and in vitro stimulation as described above. Data are expressed as mean (bars, SEM). Kruskal–Wallis and Dunn's multiple comparisons test: Cy + AdIL‐12 vs. saline, ∗∗∗p < 0.001; Cy + AdIL‐12 vs. Cy and AdIL‐12, ##p < 0.01.
Figure 6
Figure 6
Effect of combined treatment Cy + AdIL‐12 on aggressive GIC models. A) Experimental liver metastases of CRC (CT26). Left panel, Liver metastases volume: BALB/c mice received an intra‐hepatic injection of 5 × 105 CT26 cells (day 0). Mice were distributed in different experimental groups and treated with saline (n = 6); Cy (50 mg/kg i.p; day 8, n = 6); AdIL‐12 (109 TCID50 i.t; day 9, n = 6); or Cy + AdIL‐12 (n = 8). Metastases volume was measured at day 20. Data are representative of three independent experiments. Mann Whitney test: Cy + AdIL‐12 vs. saline (∗∗∗p < 0.001); Cy + AdIL‐12 vs. AdIL‐12 or Cy (##p < 0.01). Right panel, Survival: Kaplan–Meier, log rank test, (∗∗∗p < 0.001). B) Pancreatic tumour model (Panc02). Left Panel, Tumour growth: C57BL/6 mice were inoculated s.c. in the right flank with 5 × 105 Panc02 cells. Treatments began when tumours reached 85 mm3 in size. Animals were treated with saline (n = 6), Cy (50 mg/kg i.p; day 8, n = 5), AdIL‐12 (109 TCID50 i.t; day 9, n = 5), or Cy + AdIL‐12 (n = 6). Tumour growth was assessed twice a week by calliper. Data are expressed as mean (bars, SEM). Data are representative of three independent experiments. Mann Whitney test: Cy + AdIL‐12 vs. Cy or AdIL‐12 (∗p < 0.05). Right panel, Survival: Kaplan–Meier, log rank test (∗∗∗p < 0.001). C) Analysis of the in vivo interaction between Cy and AdIL‐12 by the fractional product method (FTV) in Panc02 model. a FTV (experimental mean tumour volume)/(control mean tumour volume); b Day after treatment onset; c (Cy mean FTV) × (AdIL‐12 mean FTV); d R = Expected FTV/Observed FTV. A ratio >1 indicates a synergistic effect, and a ratio <1 indicates a less than additive effect.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alaniz, L. , Rizzo, M. , Malvicini, M. , Jaunarena, J. , Avella, D. , Atorrasagasti, C. , Aquino, J.B. , Garcia, M. , Matar, P. , Silva, M. , Mazzolini, G. , 2009. Low molecular weight hyaluronan inhibits colorectal carcinoma growth by decreasing tumor cell proliferation and stimulating immune response. Cancer Lett.. 278, 9–16. - PubMed
    1. Almand, B. , Clark, J.I. , Nikitina, E. , van Beynen, J. , English, N.R. , Knight, S.C. , Carbone, D.P. , Gabrilovich, D.I. , 2001. Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J. Immunol.. 166, 678–689. - PubMed
    1. Appay, V. , Zaunders, J.J. , Papagno, L. , Sutton, J. , Jaramillo, A. , Waters, A. , Easterbrook, P. , Grey, P. , Smith, D. , McMichael, A.J. , Cooper, D.A. , Rowland-Jones, S.L. , Kelleher, A.D. , 2002. Characterization of CD4(+) CTLs ex vivo. J. Immunol.. 168, 5954–5958. - PubMed
    1. Barajas, M. , Mazzolini, G. , Genove, G. , Bilbao, R. , Narvaiza, I. , Schmitz, V. , Sangro, B. , Melero, I. , Qian, C. , Prieto, J. , 2001. Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12. Hepatology. 33, 52–61. - PubMed
    1. Bell, D. , Chomarat, P. , Broyles, D. , Netto, G. , Harb, G.M. , Lebecque, S. , Valladeau, J. , Davoust, J. , Palucka, K.A. , Banchereau, J. , 1999. In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J. Exp. Med.. 190, 1417–1426. - PMC - PubMed

Publication types

MeSH terms