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

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.

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.

Figure 2. Identification of Survivin expression in HCC specimens

(A) HCC specimens and paracancerous liver tissues collected from 10 patients with HCC were fixed in 10% formalin for 6 h to prepare the paraffin-embedded sections, and the expression of Survivin was detected by streptavidin-peroxidase (SP) immunohistochemistry; original magnification: ×200. (B) For each slice, the number of Survivin positive cells was counted within 5 medium-power magnification fields of view (20× objective lens) under microscope. The results were determined by scoring the stained cell ratio and the staining intensity, from 0 to 6 scores.

Figure 3. Anti-tumor effect of Hsp70 expression mediated by oncolytic adenovirus in HCC xenograft nude mouse models

(A) Fresh HCC tissues from 10 cases of clinical surgical specimens were cut to a depth of 2 mm in diameter and subcutaneously buried in the right axilla of eighty nude mice by a trocar puncture. Mice were assigned to 5 groups (AdSurp-Hsp70+CIK, AdSurp-Hsp70, CIK, AdSurp-EGFP, and the blank control group). After tumor xenografts were formed, mice in the AdSurp-Hsp70+CIK and CIK groups were infused with CIK cells through tail vein to a concentration of 10⁷ cells/mouse. Subsequently, the corresponding viral treatment was given based on the grouping at a total of 1×10⁹ pfu of adenoviruses. The blank control group was injected with the viral preservation solution instead of virus injection. Tumor size was measured regularly, and tumor volume was calculated to result in the tumor growth curves; **P<0.01, ***P<0.001. (B) Each experimental group was divided into a weakly positive Survivin subgroup and a strongly positive Survivin subgroup for further comparison of tumor volume. **P<0.01. (C) After 35 days of the first treatment, the observation was terminated. The tumors were collected and weighed; *P<0.05, **P<0.01, ***P<0.001. (D) Each experimental group was divided into a weakly positive Survivin subgroup and a strongly positive Survivin subgroup for further comparison of tumor weight; *P<0.05, **P<0.01.

Figure 4. Expression of E1a and Hsp70 mediated by oncolytic adenoviruses

(A) Tumor xenografts were fixed in 10% neutralized formalin for 6 h to prepare the paraffin-embedded sections. SP immunohistochemical staining was performed to locate the expressions of indicated proteins; original magnification: ×200. (B) For each slice, the percentages of positive cells were counted within 5 medium-power magnification fields of view (20× objective lens) under microscope. The results were determined by scoring the stained cell ratio and the staining intensity; ***P<0.001. (C) Mouse blood was collected to prepare sera. The expression of Hsp70 protein was detected using an ELISA; ***P<0.001.

Figure 5. CIK cell infiltration in tumor stroma and Ki67 expression in cancer cells

(A) Tumor xenografts were collected to prepare the paraffin-embedded sections as aforementioned. Immunohistochemical staining was performed to observe the number and distribution of the infiltrated CD3+ T cells in tumor stroma. For each slice, the number of positive cells was counted within 5 medium-power magnification fields of view (20× objective lens) under microscope. The results were determined by scoring the stained cell ratio and the staining intensity; original magnification: ×200; ***P<0.001. (B) Immunohistochemical staining was performed to observe the expression of the proliferating cell nuclear antigen Ki67 in tumor cells; original magnification: ×200. (C) The number of positive cells was counted within 5 medium-power magnification fields of view (20× objective lens) under microscope. Each experimental group was divided into a weakly positive Survivin subgroup and a strongly positive Survivin subgroup to further compare Ki67 expression in cancer cells; *P<0.05, **P<0.01, ***P<0.001.