Regional Hyperthermia Enhances Mesenchymal Stem Cell Recruitment to Tumor Stroma: Implications for Mesenchymal Stem Cell-Based Tumor Therapy

Abstract

The tropism of mesenchymal stem cells (MSCs) for tumors forms the basis for their use as delivery vehicles for the tumor-specific transport of therapeutic genes, such as the theranostic sodium iodide symporter (NIS). Hyperthermia is used as an adjuvant for various tumor therapies and has been proposed to enhance leukocyte recruitment. Here, we describe the enhanced recruitment of adoptively applied NIS-expressing MSCs to tumors in response to regional hyperthermia. Hyperthermia (41°C, 1 h) of human hepatocellular carcinoma cells (HuH7) led to transiently increased production of immunomodulatory factors. MSCs showed enhanced chemotaxis to supernatants derived from heat-treated cells in a 3D live-cell tracking assay and was validated in vivo in subcutaneous HuH7 mouse xenografts. Cytomegalovirus (CMV)-NIS-MSCs were applied 6-48 h after or 24-48 h before hyperthermia treatment. Using ¹²³I-scintigraphy, thermo-stimulation (41°C, 1 h) 24 h after CMV-NIS-MSC injection resulted in a significantly increased uptake of ¹²³I in heat-treated tumors compared with controls. Immunohistochemical staining and real-time PCR confirmed tumor-selective, temperature-dependent MSC migration. Therapeutic efficacy was significantly enhanced by combining CMV-NIS-MSC-mediated ¹³¹I therapy with regional hyperthermia. We demonstrate here for the first time that hyperthermia can significantly boost tumoral MSC recruitment, thereby significantly enhancing therapeutic efficacy of MSC-mediated NIS gene therapy.

Keywords: gene therapy; hepatocellular carcinoma; mesenchymal stem cells; regional hyperthermia; sodium iodide symporter.

Graphical abstract

Figure 1

Chemokine and Cytokine Gene Expression Profile of HuH7 In Vitro Real-time PCR analysis of mRNA extracted from heat-treated HuH7 cells 0–48 h after hyperthermia compared with non-heated HuH7 using primers listed in Table 1. Results are normalized to the internal control β-actin (ACTB) and expressed as mean fold change ± SEM (n = 4; two-tailed Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ***p < 0.001).

Figure 2

Chemokine and Cytokine Protein Secretion Profile of HuH7 In Vitro Protein levels in culture supernatants derived from heat-treated HuH7 cells 0–48 h after hyperthermia were analyzed by ELISA. Results are presented as mean fold change ± SEM compared with non-heated control cells (n = 3; two-tailed Student’s t test; ∗p < 0.05).

Figure 3

CMV-NIS-MSCs Migration Assay Chemotaxis of MSCs in relation to a gradient of supernatants was tested using a live-cell tracking migration assay and monitored by time-lapse microscopy for 24 h. MSCs subjected to supernatants derived from untreated HuH7 cells in both chambers (A). Cells under the influence of a gradient between supernatants from untreated (top chamber) and thermo-stimulated HuH7 cells derived 0–48 h after hyperthermia (bottom chamber) (B–E). Quantification of chemotaxis parameters as mean forward migration index (yFMI) (F), mean directness (G), velocity (H), and the mean center of mass (yCoM; red dots in AE) (I) of MSCs toward supernatants from heat-treated compared with untreated HuH7 cells. One representative image each is shown from two independent experiments (mean ± SEM; two-tailed Student’s t test; ∗p < 0.05).

Figure 4

¹²³I-Scintigraphy following CMV-NIS-MSC Administration s.c. HuH7 tumor-bearing mice were injected with CMV-NIS-MSCs and subjected to hyperthermic treatment (1 h at 41°C or 37°C, as controls; n = 6) at different time points: 48 h (group A, n = 6), 24 h (group B, n = 6), 6 h (group C, n = 7) prior and 24 h (group D, n = 6, ∗∗p = 0.0055 [group D versus control], ∗p = 0.024 [group D versus group A], ∗p = 0.028 [group D versus group B], p = 0.086 [group D versus group C], p = 0.90 [group D versus group E]) and 48 h (group E, n = 6) after hyperthermia. Three days later, ¹²³I-scintigraphy was performed and tumoral iodine uptake and efflux were analyzed (A). Results are expressed as mean ± SEM, and significance was tested by ANOVA followed by post hoc Tukey (honestly significant difference) test. One representative image for the best performing hyperthermia treatment group and the control group, displaying besides the tumoral iodine accumulation a ¹²³I signal from the endogenously NIS expressing organs, thyroid, SGs, stomach, and the urinary bladder due to ¹²³I elimination. The competitive NIS inhibitor perchlorate was added 30 min prior to ¹²³I administration (B). mRNA isolated from frozen tumor sections was analyzed for NIS by real-time PCR (C) (n = 3; two-tailed Student’s t test; ∗∗p = 0.0033). On paraffin-embedded tumor section, NIS-specific immunohistochemistry (red) was performed on tumors of 37°C control animals, heat-treated tumors of mice, and control organs. One representative image is shown each at 20× magnification, scale bar, 50 μm (D). Quantification of NIS staining on tumor sections (dots represent counts in a single visual field, lines represent the median) (E).

Figure 5

Chemokine and Cytokine Secretion Profile of HuH7 In Vivo mRNA was isolated from frozen tumor sections from heat-treated tumors 8, 24, and 48 h later, including 8 h ¹²³I imaging (each n = 4) and controls (n = 4), and expression levels of different chemokines and cytokines were evaluated by real-time PCR. Results are normalized to the internal control (average of ACTB, r18s, and UBC) and expressed as mean ± SEM (two-tailed Student’s t test; ∗p < 0.05; ∗∗p < 0.01).

Figure 6

In Vivo¹³¹I Therapy Study In a s.c. HuH7 xenograft mouse model, the best treatment scheme identified in the imaging study was adapted for a therapy study using ¹³¹I (A). Tumor growth (B) and survival (C) were monitored for the treatment with CMV-NIS-MSCs, regional hyperthermia, and ¹³¹I (MSCs + 41°C + ¹³¹I; n = 8; red line), compared with the normothermic control group (MSCs + 37°C + ¹³¹I; n = 7; blue line) and the saline only group (NaCl + 37°C + NaCl; n = 5; gray line). Results are expressed as mean ± SEM (one-way ANOVA for tumor growth and log-rank test for Kaplan-Meier survival plots; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).