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Targeted Hsp70 expression combined with CIK-activated immune reconstruction synergistically exerts antitumor efficacy in patient-derived hepatocellular carcinoma xenograft mouse models.

Hu H, Qiu Y, Guo M, Huang Y, Fang L, Peng Z, Ji W, Xu Y, Shen S, Yan Y, Huang X, Zheng J, Su C - Oncotarget (2015)

Bottom Line: The results demonstrated that, either the immune anti-tumor effect caused by CIK cell infusion or the oncolytic effect generated by oncolytic adenovirus replication was very limited.However, the synergistic tumor inhibitory effect was significantly enhanced after treatments with oncolytic adenovirus expressing Hsp70 combined with CIK cells.This strategy can synergistically activate multiple anti-tumor mechanisms and exert effective anti-tumor activities that have a significant inhibitory effect against the growth of HCC xenografts.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Fuzhou General Hospital of Nanjing Military Area, Fuzhou, China.

ABSTRACT
The patient-derived tumor xenograft (PDTX) models can reproduce a similar natural genetic background and similar biological behaviors to tumor cells in patients, which is conducive to the assessment of personalized cancer treatment. In this study, to verify the targeting and effectiveness of the therapeutic strategy using a Survivin promoter-regulated oncolytic adenovirus expressing Hsp70, the PDTX models of hepatocellular carcinoma (HCC) were established in nude mice and the cytokine-induced killer (CIK) cells were intravenously infused into mice to partially reconstruct the mouse immune function. The results demonstrated that, either the immune anti-tumor effect caused by CIK cell infusion or the oncolytic effect generated by oncolytic adenovirus replication was very limited. However, the synergistic tumor inhibitory effect was significantly enhanced after treatments with oncolytic adenovirus expressing Hsp70 combined with CIK cells. Oncolytic adenovirus mediated the specific expression of Hsp70 in cancer tissues allowed the CIK chemotaxis, and induce the infiltration of CD3+ T cells in tumor stroma, thereby exhibiting anti-tumor activity. The anti-tumor effect was more effective for the highly malignant tumor xenografts with highly Survivin expression. This strategy can synergistically activate multiple anti-tumor mechanisms and exert effective anti-tumor activities that have a significant inhibitory effect against the growth of HCC xenografts.

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Related in: MedlinePlus

Identification of adenovirus vector in HCC cell lines(A) Schematic diagram of the oncolytic adenoviruses and amplification of the early replication gene E1a as well as the therapeutic gene Hsp70 was performed by PCR in adenoviral vectors AdSurp-Hsp70 and AdSurp-EGFP. The Survivin promoter was used to regulate the adenoviral E1a gene, and the expression cassettes of Hsp70 and EGFP were inserted into the upstream of E1a gene, then generated the recombinant oncolytic adenoviruses AdSurp-Hsp70 and AdSurp-EGFP. ITR: inverted terminal repeats; ψ: adenovirus 5 packaging signal; CMV: cytomegalovirus promoter; Surp: Survivin promoter. (B) The indicated cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-EGFP at an MOI of 1 pfu/cell, cultured for 48 h and observed the EGFP-positive cells under a fluorescence microscope, original magnification: ×200. (C) The cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-Hsp70 at an MOI of 1 pfu/cell, cultured for 48 h and the expression of Hsp70 was examined by Western blotting; ***P<0.001.
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Figure 1: Identification of adenovirus vector in HCC cell lines(A) Schematic diagram of the oncolytic adenoviruses and amplification of the early replication gene E1a as well as the therapeutic gene Hsp70 was performed by PCR in adenoviral vectors AdSurp-Hsp70 and AdSurp-EGFP. The Survivin promoter was used to regulate the adenoviral E1a gene, and the expression cassettes of Hsp70 and EGFP were inserted into the upstream of E1a gene, then generated the recombinant oncolytic adenoviruses AdSurp-Hsp70 and AdSurp-EGFP. ITR: inverted terminal repeats; ψ: adenovirus 5 packaging signal; CMV: cytomegalovirus promoter; Surp: Survivin promoter. (B) The indicated cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-EGFP at an MOI of 1 pfu/cell, cultured for 48 h and observed the EGFP-positive cells under a fluorescence microscope, original magnification: ×200. (C) The cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-Hsp70 at an MOI of 1 pfu/cell, cultured for 48 h and the expression of Hsp70 was examined by Western blotting; ***P<0.001.

Mentions: Two pre-constructed Survivin promoter-regulated oncolytic adenoviruses, AdSurp-Hsp70 and AdSurp-EGFP [1,3,4], were amplified in 293 cells and identified by polymerase chain reaction (PCR). The oncolytic adenoviruses AdSurp-Hsp70 and AdSurp-EGFP, regulated by the Survivin promoter, were E1a positive. The adenovirus AdSurp-Hsp70 carrying the Hsp70 gene was Hsp70 positive, while the control virus AdSurp-EGFP was Hsp70 negative (Fig. 1A), suggesting that the structure of the adenoviruses was consistent with expectations.


Targeted Hsp70 expression combined with CIK-activated immune reconstruction synergistically exerts antitumor efficacy in patient-derived hepatocellular carcinoma xenograft mouse models.

Hu H, Qiu Y, Guo M, Huang Y, Fang L, Peng Z, Ji W, Xu Y, Shen S, Yan Y, Huang X, Zheng J, Su C - Oncotarget (2015)

Identification of adenovirus vector in HCC cell lines(A) Schematic diagram of the oncolytic adenoviruses and amplification of the early replication gene E1a as well as the therapeutic gene Hsp70 was performed by PCR in adenoviral vectors AdSurp-Hsp70 and AdSurp-EGFP. The Survivin promoter was used to regulate the adenoviral E1a gene, and the expression cassettes of Hsp70 and EGFP were inserted into the upstream of E1a gene, then generated the recombinant oncolytic adenoviruses AdSurp-Hsp70 and AdSurp-EGFP. ITR: inverted terminal repeats; ψ: adenovirus 5 packaging signal; CMV: cytomegalovirus promoter; Surp: Survivin promoter. (B) The indicated cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-EGFP at an MOI of 1 pfu/cell, cultured for 48 h and observed the EGFP-positive cells under a fluorescence microscope, original magnification: ×200. (C) The cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-Hsp70 at an MOI of 1 pfu/cell, cultured for 48 h and the expression of Hsp70 was examined by Western blotting; ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359218&req=5

Figure 1: Identification of adenovirus vector in HCC cell lines(A) Schematic diagram of the oncolytic adenoviruses and amplification of the early replication gene E1a as well as the therapeutic gene Hsp70 was performed by PCR in adenoviral vectors AdSurp-Hsp70 and AdSurp-EGFP. The Survivin promoter was used to regulate the adenoviral E1a gene, and the expression cassettes of Hsp70 and EGFP were inserted into the upstream of E1a gene, then generated the recombinant oncolytic adenoviruses AdSurp-Hsp70 and AdSurp-EGFP. ITR: inverted terminal repeats; ψ: adenovirus 5 packaging signal; CMV: cytomegalovirus promoter; Surp: Survivin promoter. (B) The indicated cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-EGFP at an MOI of 1 pfu/cell, cultured for 48 h and observed the EGFP-positive cells under a fluorescence microscope, original magnification: ×200. (C) The cell lines were seeded into 24-well plates at a concentration of 1×105 cells/well, and infected with AdSurp-Hsp70 at an MOI of 1 pfu/cell, cultured for 48 h and the expression of Hsp70 was examined by Western blotting; ***P<0.001.
Mentions: Two pre-constructed Survivin promoter-regulated oncolytic adenoviruses, AdSurp-Hsp70 and AdSurp-EGFP [1,3,4], were amplified in 293 cells and identified by polymerase chain reaction (PCR). The oncolytic adenoviruses AdSurp-Hsp70 and AdSurp-EGFP, regulated by the Survivin promoter, were E1a positive. The adenovirus AdSurp-Hsp70 carrying the Hsp70 gene was Hsp70 positive, while the control virus AdSurp-EGFP was Hsp70 negative (Fig. 1A), suggesting that the structure of the adenoviruses was consistent with expectations.

Bottom Line: The results demonstrated that, either the immune anti-tumor effect caused by CIK cell infusion or the oncolytic effect generated by oncolytic adenovirus replication was very limited.However, the synergistic tumor inhibitory effect was significantly enhanced after treatments with oncolytic adenovirus expressing Hsp70 combined with CIK cells.This strategy can synergistically activate multiple anti-tumor mechanisms and exert effective anti-tumor activities that have a significant inhibitory effect against the growth of HCC xenografts.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Fuzhou General Hospital of Nanjing Military Area, Fuzhou, China.

ABSTRACT
The patient-derived tumor xenograft (PDTX) models can reproduce a similar natural genetic background and similar biological behaviors to tumor cells in patients, which is conducive to the assessment of personalized cancer treatment. In this study, to verify the targeting and effectiveness of the therapeutic strategy using a Survivin promoter-regulated oncolytic adenovirus expressing Hsp70, the PDTX models of hepatocellular carcinoma (HCC) were established in nude mice and the cytokine-induced killer (CIK) cells were intravenously infused into mice to partially reconstruct the mouse immune function. The results demonstrated that, either the immune anti-tumor effect caused by CIK cell infusion or the oncolytic effect generated by oncolytic adenovirus replication was very limited. However, the synergistic tumor inhibitory effect was significantly enhanced after treatments with oncolytic adenovirus expressing Hsp70 combined with CIK cells. Oncolytic adenovirus mediated the specific expression of Hsp70 in cancer tissues allowed the CIK chemotaxis, and induce the infiltration of CD3+ T cells in tumor stroma, thereby exhibiting anti-tumor activity. The anti-tumor effect was more effective for the highly malignant tumor xenografts with highly Survivin expression. This strategy can synergistically activate multiple anti-tumor mechanisms and exert effective anti-tumor activities that have a significant inhibitory effect against the growth of HCC xenografts.

Show MeSH
Related in: MedlinePlus