The development of a novel cancer immunotherapeutic platform using tumor-targeting mesenchymal stem cells and a protein vaccine.

An ideal anticancer strategy should target only the malignant cells but spare the normal ones. In this regard, we established a platform, consisting of an antigen-delivering vehicle and a protein vaccine, for developing an immunotherapeutic approach with the potential for eliminating various cancer types. Mesenchymal stem cells (MSCs) have been demonstrated capable of targeting tumors and integrating into the stroma. Moreover, we have developed a protein vaccine PE(ΔIII)-E7-KDEL3 which specifically recognized E7 antigen and elicited immunity against cervical cancer. Taking advantage of tumor-homing property of MSCs and PE(ΔIII)-E7-KDEL3, we used E6/E7-immortalized human MSCs (KP-hMSCs) as an E7 antigen-delivering vehicle to test if this protein vaccine could effectively eliminate non-E7-expressing tumor cells. Animals which received combined treatment of KP-hMSCs and PE(ΔIII)-E7-KDEL3 demonstrated a significant inhibition of tumor growth and lung-metastasis when compared to PE(ΔIII)-E7-KDEL3 only and KP-hMSCs only groups. The efficiency of tumor suppression correlated positively to the specific immune response induced by PE(ΔIII)-E7-KDEL3. In addition, this combined treatment inhibited tumor growth via inducing apoptosis. Our findings indicated that KP-hMSCs could be used as a tumor-targeting device and mediate antitumor effect of PE(ΔIII)-E7-KDEL3. We believe this strategy could serve as a platform for developing a universal vaccine for different cancer types.

The investigation of mitogen-activated protein kinase phosphatase-1 as a potential pharmacological target in non-small cell lung carcinomas, assisted by non-invasive molecular imaging.

BACKGROUND: Invasiveness and metastasis are the most common characteristics of non small cell lung cancer (NSCLC) and causes of tumour-related morbidity and mortality. Mitogen-activated protein kinases (MAPKs) signalling pathways have been shown to play critical roles in tumorigenesis. However, the precise pathological role(s) of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cancers has been controversial such that the up-regulation of MKP-1 in different cancers does not always correlate to a better prognosis. In this study, we showed that the induction of MKP-1 lead to a significant retardation of proliferation and metastasis in NSCLC cells. We also established that rosiglitazone (a PPARgamma agonist) elevated MKP-1 expression level in NSCLC cells and inhibited tumour metastasis. METHODS: Both wildtype and dominant negative forms of MKP-1 were constitutively expressed in NSCLC cell line H441GL. The migration and invasion abilities of these cells were examined in vitro. MKP-1 modulating agents such as rosiglitazone and triptolide were used to demonstrate MKP-1's role in tumorigenesis. Bioluminescent imaging was utilized to study tumorigenesis of MKP-1 over-expressing H441GL cells and anti-metastatic effect of rosiglitazone. RESULTS: Over-expression of MKP-1 reduced NSCLC cell proliferation rate as well as cell invasive and migratory abilities, evident by the reduced expression levels of MMP-2 and CXCR4. Mice inoculated with MKP-1 over-expressing H441 cells did not develop NSCLC while their control wildtype H441 inoculated littermates developed NSCLC and bone metastasis. Pharmacologically, rosiglitazone, a peroxisome proliferator activated receptor-gamma (PPARgamma) agonist appeared to induce MKP-1 expression while reduce MMP-2 and CXCR4 expression. H441GL-inoculated mice receiving daily oral rosiglitazone treatment demonstrated a significant inhibition of bone metastasis when compared to mice receiving sham treatment. We found that rosiglitazone treatment impeded the ability of cell migration and invasion in vitro. Cells pre-treated with triptolide (a MKP-1 inhibitor), reversed rosiglitazone-mediated cell invasion and migration. CONCLUSION: The induction of MKP-1 could significantly suppress the proliferative and metastatic abilities of NSCLC both in vitro and in vivo. Therefore, MKP-1 could be considered as a potential therapeutic target in NSCLC therapy and PPARgamma agonists could be explored for combined chemotherapy.

Molecular imaging with 123I-FIAU, 18F-FUdR, 18F-FET, and 18F-FDG for monitoring herpes simplex virus type 1 thymidine kinase and ganciclovir prodrug activation gene therapy of cancer.

UNLABELLED: The ability to monitor tumor responses during prodrug activation gene therapy and other anticancer gene therapies is critical for their translation into clinical practice. Previously, we demonstrated the feasibility of noninvasive in vivo imaging with 131I-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil (131I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here we tested the efficacy of SPECT with 123I-FIAU and PET with 5-18F-fluoro-2'-deoxyuridine (18F-FUdR), 2-18F-fluoroethyl-L-tyrosine (18F-FET), and 18F-FDG for monitoring tumor responses during prodrug activation gene therapy with HSV1-tk and ganciclovir (GCV). METHODS: In the flanks of FVB/N female mice, 4 tumors per animal were established by subcutaneous injection of 1 x 10(5) cells of NG4TL4 sarcoma cells, HSV1-tk-transduced NG4TL4-STK cells, or a mixture of these cells in different proportions to model different efficacies of transfection and HSV1-tk gene expression levels in tumors. Ten days later, the animals were treated with GCV (10 mg/kg/d intraperitoneally) for 7 d. Gamma-Imaging with 123I-FIAU and PET with 18F-FUdR, 18F-FET, and 18F-FDG were performed before and after initiation of therapy with GCV in the same animal. RESULTS: Before GCV treatment, no significant difference in weight and size was found in tumors that expressed different HSV1-tk levels, suggesting similar in vivo proliferation rates for NG4TL4 and NG4TL4-STK sarcomas. The accumulation of 123I-FIAU at 24 h after injection was directly proportional to the percentage of NG4TL4-STK cells in the tumors. The 123I-FIAU accumulation at 4 and 7 d of GCV therapy decreased significantly compared with pretreatment levels and was proportional to the percentage of HSV1-tk-positive tumor cells. Tumor uptake of 18F-FUdR in all HSV1-tk-expressing tumors also decreased significantly compared with pretreatment levels and was proportional to the percentage of HSV1-tk-positive tumor cells. The accumulation of 18F-FET decreased minimally (about 1.5-fold) and 18F-FDG decreased only 2-fold after 7 d of GCV therapy, and the degree of reduction was proportional to the percentage of HSV1-tk-positive tumor cells. CONCLUSION: We have shown that gamma-camera imaging with 123I-FIAU was the most reliable method for prediction of tumor response to GCV therapy, which was proportional to the magnitude of HSV1-tk expression in tumor tissue. 123I-FIAU imaging can be used to verify the efficacy of elimination of HSV1-tk-expressing cells by therapy with GCV. PET with 18F-FUdR reliably visualizes proliferating tumor tissue and is most suitable for the assessment of responses in tumors undergoing HSV1-tk plus GCV prodrug activation gene therapy. PET with 18F-FDG or 18F-FET can be used as additional "surrogate" biomarkers of the treatment response, although these radiotracers are less sensitive than 18F-FUdR for monitoring tumor responses to prodrug activation gene therapy with HSV1-tk and GCV in this sarcoma model.

Serial in vivo imaging of the lung metastases model and gene therapy using HSV1-tk and ganciclovir.

UNLABELLED: Noninvasive imaging in lung metastatic tumor models is used infrequently because of technical limitations in detecting metastases. We have previously used 2'-fluoro-2'-deoxy-5-iodo-1-beta-d-arabinofuranosyluracil labeled with (131)I ((131)I-FIAU) and demonstrated the applicability of noninvasive imaging for monitoring cancer gene therapy in an experimental animal model of herpes simplex virus type 1 thymidine kinase (HSV1-tk)-expressing tumor xenografts. We have now used the same animal model to effectively and noninvasively monitor the location, magnitude, and duration of therapeutic gene expression over time for the lung metastases model. METHODS: To improve the detectability of lung metastases, an experimental blood-borne lung metastases model in mice was established using intravenously administered HSV1-tk-expressing NG4TL4 fibrosarcoma cells (NG4TL4-TK) and simulated the clinical application of HSV1-tk plus ganciclovir (GCV) prodrug activation gene therapy. The efficacy of noninvasively monitoring the sites of development of lung metastatic lesions and their GCV-induced regression were assessed by SPECT with (131)I-FIAU. RESULTS: The results of this study showed that the lung metastases model of NG4TL4-TK cells could be successfully detected as early as 24 h after intravenous injection of tumor cells radiolabeled with (131)I-FIAU and also subsequently detected by extended monitoring with the intravenous injection of (131)I-FIAU on day 10. In mice treated with GCV, gamma-camera imaging demonstrated a significant growth inhibition of NG4TL4-TK cells of primary tumors and lung metastases on day 7 after initiating treatment. CONCLUSION: We conclude that this in vivo imaging approach will be useful for future studies of the lung metastases model and for the assessment of novel anticancer and antimetastatic therapies.

Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging.

The aim of this study was to assess the efficacy human mesenchymal stem cells (hMSC) for targeting microscopic tumors and suicide gene or cytokine gene therapy. Immunodeficient mice were transplanted s.c. with human colon cancer cells of HT-29 Inv2 or CCS line, and 3 to 4 days later, i.v. with "tracer" hMSCs expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) and enhanced green fluorescent protein (EGFP) reporter genes. Subsequently, these tumors were examined for specificity and magnitude of HSV1-TK(+), EGFP(+) stem cell engraftment and proliferation in tumor stroma by in vivo positron emission tomography (PET) with (18)F-labeled 9-(4-fluoro-3-hydroxymethylbutyl)-guanine ([(18)F]-FHBG). In vivo PET images of tumors growing for 4 weeks showed the presence of HSV1-TK(+) tumor stroma with an average of 0.36 +/- 0.24% ID/g [(18)F]-FHBG accumulation. In vivo imaging results were validated by in situ correlative histochemical, immunofluorescent, and cytometric analyses, which revealed EGFP expression in vWF(+) and CD31(+) endothelial cells of capillaries and larger blood vessels, in germinal layer of dermis and hair follicles proximal to the s.c. tumor site. These differentiated HSV1-TK(+), GFP(+) endothelial cells had limited proliferative capacity and a short life span of <2 weeks in tumor fragments transplanted into secondary hosts. We conclude that hMSCs can target microscopic tumors, subsequently proliferate and differentiate, and contribute to formation of a significant portion of tumor stroma. PET imaging should facilitate clinical translation of stem cell-based anticancer gene therapeutic approaches by providing the means for in vivo noninvasive whole-body monitoring of trafficking, tumor targeting, and proliferation of HSV1-tk-expressing "tracer" hMSCs in tumor stroma.