J Breast Cancer.  2016 Mar;19(1):34-44. 10.4048/jbc.2016.19.1.34.

Antitumor Effect of IP-10 by Using Two Different Approaches: Live Delivery System and Gene Therapy

• Taslimi Y
• Zahedifard F
• Habibzadeh S
• Taheri T
• Abbaspour
• Sadeghipour A
• Mohit E
• Rafati S

• KMID: 2176319
• DOI: http://doi.org/10.4048/jbc.2016.19.1.34

Abstract

PURPOSE
Immunotherapy is one of the treatment strategies for breast cancer, the most common cancer in women worldwide. In this approach, the patient's immune system is stimulated to attack microscopic tumors and control metastasis. Here, we used interferon γ-induced protein 10 (IP-10), which induces and strengthens antitumor immunity, as an immunotherapeutic agent. We employed Leishmania tarentolae, a nonpathogenic lizard parasite that lacks the ability to persist in mammalian macrophages, was used as a live delivery system for carrying the immunotherapeutic agent. It has been already shown that arginase activity, and consequently, polyamine production, are associated with tumor progression.
METHODS
A live delivery system was constructed by stable transfection of pLEXSY plasmid containing the IP-10-enhanced green fluorescent protein (IP-10-egfp) fusion gene into L. tarentolae. Then, the presence of the IP-10-egfp gene and the accurate integration location into the parasite genome were confirmed. The therapeutic efficacy of IP-10 delivered via L. tarentolae and recombinant pcDNA-(IP-10-egfp) plasmid was compared by determining the arginase activity in a mouse 4T1 breast cancer model.
RESULTS
The pcDNA-(IP-10-egfp) group showed a significant reduction in tumor weight and growth. Histological evaluation also revealed that only this group demonstrated inhibition of metastasis to the lung tissue. The arginase activity in the tissue of the pcDNA-(IP-10-egfp) mice significantly decreased in comparison with that in normal mice. No significant difference was observed in arginase activity in the sera of mice receiving other therapeutic strategies.
CONCLUSION
Our data indicates that IP-10 immunotherapy is a promising strategy for breast cancer treatment, as shown in the 4T1-implanted BALB/c mouse model. However, the L. tarentolae-(IP-10-EGFP) live delivery system requires dose modifications to achieve efficacy in the applied regimen (six injections in 3 weeks). Our results indicate that the arginase assay could be a good biomarker to differentiate tumoral tissues from the normal ones.

Keyword

Breast neoplasms; Chemokine CXCL10; Gene therapy; Immunotherapy

MeSH Terms

Animals
Arginase
Breast Neoplasms
Chemokine CXCL10
Female
Genetic Therapy*
Genome
Humans
Immune System
Immunotherapy
Interferons
Leishmania
Lizards
Lung
Macrophages
Mice
Neoplasm Metastasis
Parasites
Plasmids
Transfection
Tumor Burden
Arginase
Chemokine CXCL10
Interferons

Figure

• 

  Figure 1 Interferon γ-induced protein 10-enhanced green fluorescent protein (IP-10-EGFP) integration and expression in Leishmania tarentolae strain. (A) Schematic presentation of egfp gene into ssu ribosomal DNA lucus. (B) Polymerase chain reaction (PCR) results using L. tarentolae genomic DNA as template. Columns 1 and 2 demonstrate the IP-10-EGFP and EGFP amplification, respectively. Column 3 is the PCR amplification of the integrated fragment using integration primers (F3001 and A1715) primers. (C) Confirmation of IP-10-EGFP expression in mRNA level. Amplification of IP-10-EGFP and EGFP fragments were demonstrated in column 1 and 2 using recombinant L. tarentolae cDNA as template, respectively. (D) Fluorescence microscopic images of recombinant L. tarentolae stably expressing (IP-10-EGFP) 48 hours postinfection; both bright field and fluorescence (left) are shown.

• 

  Figure 2 Confirmation of interferon γ-induced protein 10 (IP-10) expression in COS-7 transfected cells. (A) GFP expression was assessed by fluorescence microscopy after 48 hours. (a) Untreated COS-7, (b) COS-7 treated with pEGFP, (c) COS-7 treated with pEGFP-(IP-10), and (d) COS-7 treated with pcDNA-(IP-10-egfp). (B) GFP positive percentage of transfected parasite by flowcytometer after 48 hours. (e) Untreated COS-7, (f) COS-7 treated with pEGFP, (g) COS-7 treated with pcDNA-(IP-10-egfp), and (h) COS-7 treated with pEGP-IP-10. (C) Western blot analysis using anti-GFP antibody. Column 1 and 2 show marker and untreated COS-7 as negative control, respectively. The ~27 kDa band determines the GFP protein expression in Leishmania tarentolae-EGFP as positive control (column 3) and pEGFP-transfected COS-7 cells (column 4). IP-10-EGFP expression was confirmed by detecting ~37 kDa in pcDNA-(IP-10-egfp) (column 5) and pEGFP-(IP-10-EGFP) treated COS-7 cells (column 6).GFP=green fluorescent protein; EGFP=enhanced green fluorescent protein; R1=COS-7 cells; RN1=GFP positive COS-7; FL1=fluorescein isothiocyanate (FITC) detector.

• 

  Figure 3 Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of interferon γ-induced protein 10 (IP-10) expression in pcDNA-(IP-10-egfp)-transfected COS-7 cells. qRT-PCR were performed 72 hours after transfection of pcDNA-egfp and pcDNA-(IP-10-egfp) into COS-7 cells. Data are presented as mean±SD (n=4).EGFP=enhanced green fluorescent protein.*p<0.05.

• 

  Figure 4 Effect of interferon γ-induced protein 10 (IP-10) on nitric oxide production. The supernatant of pcDNA-egfp and pcDNA-(IP-10-egfp)-transfected COS-7 cells were collected 6, 24, 48, 72, and 96 hours after transfection. Nitrate in the supernatant were measured with Griess reaction. Data are presented as mean±SD (n=4).EGFP=enhanced green fluorescent protein.*p<0.05.

• 

  Figure 5 The effect of different therapeutic strategy on 4T1 tumor growth, spleen growth and lung histological sections. (A) Schematic representation of the immunotherapy schedule. BALB/c mice (n=10) were injected subcutaneously with 1×105 4T1 cells. Different immunization regimes were given intratumorally twice a week for 3 weeks. (B) Tumor volumes were determined two times per week. (C) Tumors were sectioned at day 28 and weight was measured among the five groups. (D) Spleens also were sectioned and weighted. Data are presented as mean±SD (n=4). (E) At day 28 lungs were removed, sectioned and stained with H&E. (1) Normal alveolar cavity with normal respiratory bronchioles (black arrowhead) and no significant pathologic changes in pcDNA-(IP-10-egfp) (G1). Alveolar septa have normal thickness with no congestion, edema or inflammation. (2) Hemorrhagic lung tissue in L. tarentolae-(IP-10-GFP) (G3) with diffused alveolar edema fluid in alveolar spaces (arrow). Alveolar septa show congestion. (3) One foci of metastatic carcinoma cells in field (white arrowhead) in G5. Arrow head shows sheet of metastatic carcinoma which encircle a respiratory bronchiole. The tumoral cells have high nuclear/cytoplasmic ratio, vascular pleomorphic nuclei and prominent nucleoli (H&E stain, ×100).IP-10-GFP=interferon γ-induced protein 10-green fluorescent protein; L. tar=Leishmania tarentolae; PBS=phosphate buffered saline.*p<0.05; †p<0.001; ‡p<0.0001.

• 

  Figure 6 Arginase activity in tumor tissues and sera. Different immunization regimens were given to BALB/c mice (n=10) twice a week for 3 weeks, 3 days after 4T1 injection. (A) Arginase activity of different groups was measured in sera samples before 4T1 injection (N, normal), 3 days after injection 4T1 (G0, 4T1) and 23 days after injection of 4T1 (G1–G5). (B) Arginase activity in different groups was determined in tumor tissues 28 days after 4T1 injection. Data are representative of mean±SD (n=4).IP-10-GFP=interferon γ-induced protein 10-green fluorescent protein; L. tar=Leishmania tarentolae; PBS=phosphate buffered saline.*p<0.05.

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