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Purpose:Mesenchymal stem cells (MSC) have emerged as cellular-based vehicles for the delivery of therapeutic genes in cancer therapy based on their inherent tumor-homing capability. As theranostic gene, the sodium iodide symporter (NIS) represents a successful target for noninvasive radionuclide-based imaging and therapy. In this study, we applied genetically engineered MSCs for tumor-targeted NIS gene transfer in experimental glioblastoma (GBM)—a tumor with an extremely poor prognosis.Experimental Design:A syngeneic, immunocompetent GL261 GBM mouse model was established by subcutaneous and orthotopic implantation. Furthermore, a subcutaneous xenograft U87 model was used. Bone marrow–derived MSCs were stably transfected with a NIS-expressing plasmid driven by the constitutively active cytomegalovirus promoter (NIS-MSC). After multiple or single intravenous injection of NIS-MSCs, tumoral iodide uptake was monitored in vivo using 123I-scintigraphy or 124I-PET. Following validation of functional NIS expression, a therapy trial with 131I was performed on the basis of the most optimal application regime as seen by 124I-PET imaging in the orthotopic approach.Results:A robust tumoral NIS-specific radionuclide accumulation was observed after NIS-MSC and radioiodide application by NIS-mediated in vivo imaging. NIS immunofluorescence staining of GBM and non-target tissues showed tumor-selective MSC homing along with NIS expression. Application of therapeutically effective 131I led to significantly delayed tumor growth and prolonged median survival after NIS-MSC treatment as compared with controls.Conclusions:A strong tumor-selective recruitment of systemically applied MSCs into GBM was found using NIS as reporter gene followed by successful therapeutic application of radioiodide demonstrating the potential use of NIS-based MSCs as therapy vehicles as a new GBM therapy approach.

PDF file - 68K, S5. Methylation of Sox9-promoter, Sox9 and VEGF expression in patient samples.

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PDF file - 70K, S2. A) Differential methylation of the Sox9-promoter in NMET and HMET cells. B) Methylation and hypoxia. NMET and HMET cells were cultivated under hypoxia or normoxia, respectively. (C) Effect of methylation inhibition on VEGF expression. Relative transcription of VEGF in LMET and HMET cells is demonstrated. D) Effect of methylation inhibition on Sox9 expression.

PDF file - 130K, Supplemental Table 1. Differential expression of the genes associated to cancer relevant pathways.

PDF file - 50K, S4. Sox9 does not influence stability of HIF1alpha.

Supplementary Figure 1: Radioiodide uptake of GL261 brain tumors after systemic MSC-mediated NIS reporter gene delivery in comparison to thyroid as endogenous NIS-expressing organ assessed by 124I PET/CT imaging. Results are expressed as mean value ± SEM.Supplementary Figure 2: Ex vivo analysis of non-target organs after NIS-MSC gene delivery. NIS (A) and Neomycin (B; selection marker of NIS-MSCs) mRNA expression was detected by qPCR in liver, spleen, kidney and lung after NIS-MSC application and showed no expression above the background level of untreated tumors (which was arbitrarily set to one; NIS mRNA levels of untreated ΔΔCt = 0.0003 and Neomycin mRNA levels of untreated ΔΔCt = 0.002). Data are represented as mean-fold change ± SEM (*p

Purpose:Mesenchymal stem cells (MSC) have emerged as cellular-based vehicles for the delivery of therapeutic genes in cancer therapy based on their inherent tumor-homing capability. As theranostic gene, the sodium iodide symporter (NIS) represents a successful target for noninvasive radionuclide-based imaging and therapy. In this study, we applied genetically engineered MSCs for tumor-targeted NIS gene transfer in experimental glioblastoma (GBM)—a tumor with an extremely poor prognosis.Experimental Design:A syngeneic, immunocompetent GL261 GBM mouse model was established by subcutaneous and orthotopic implantation. Furthermore, a subcutaneous xenograft U87 model was used. Bone marrow–derived MSCs were stably transfected with a NIS-expressing plasmid driven by the constitutively active cytomegalovirus promoter (NIS-MSC). After multiple or single intravenous injection of NIS-MSCs, tumoral iodide uptake was monitored in vivo using 123I-scintigraphy or 124I-PET. Following validation of functional NIS expression, a therapy trial with 131I was performed on the basis of the most optimal application regime as seen by 124I-PET imaging in the orthotopic approach.Results:A robust tumoral NIS-specific radionuclide accumulation was observed after NIS-MSC and radioiodide application by NIS-mediated in vivo imaging. NIS immunofluorescence staining of GBM and non-target tissues showed tumor-selective MSC homing along with NIS expression. Application of therapeutically effective 131I led to significantly delayed tumor growth and prolonged median survival after NIS-MSC treatment as compared with controls.Conclusions:A strong tumor-selective recruitment of systemically applied MSCs into GBM was found using NIS as reporter gene followed by successful therapeutic application of radioiodide demonstrating the potential use of NIS-based MSCs as therapy vehicles as a new GBM therapy approach.

Purpose:The innate tumor homing potential of mesenchymal stem cells (MSCs) has been used for a targeted delivery of the theranostic sodium iodide symporter (NIS) transgene into solid tumors. We have previously shown that external beam radiotherapy (EBRT) results in the enhanced recruitment of NIS-expressing MSCs into human hepatocellular carcinoma (HuH7). In parallel, the tumor-associated cytokine TGFB1 becomes strongly upregulated in HuH7 tumors in response to EBRT.Experimental Design:We therefore evaluated the effects of combining focused EBRT (5 Gy) with MSC-mediated systemic delivery of the theranostic NIS transgene under control of a synthetic TGFB1-inducible SMAD-responsive promoter (SMAD-NIS-MSCs) using 123I-scintigraphy followed by 131I therapy in CD1 nu/nu mice harboring subcutaneous human hepatocellular carcinoma (HuH7).Results:Following tumor irradiation and SMAD-NIS-MSC application, tumoral iodide uptake monitored in vivo by 123I-scintigraphy was enhanced as compared with nonirradiated tumors. Combination of EBRT and SMAD-NIS-MSC–mediated 131I therapy resulted in a significantly improved delay in tumor growth and prolonged survival in therapy mice as compared with the combined therapy using CMV-NIS-MSCs or to control groups receiving EBRT or saline only, or EBRT together with SMAD-NIS-MSCs and saline applications.Conclusions:MSC-based NIS-mediated 131I therapy after EBRT treatment dramatically enhanced therapeutic efficacy when a TGFB1-inducible SMAD-responsive promoter was used to drive NIS expression in adoptively applied MSCs. The remarkable therapeutic effect seen is thought to be linked in large part to the enhanced TGFB1 produced in this context, which leads to a highly selective and focused amplification of MSC-based NIS expression within the tumor milieu.

Supplementary Figure S2. Immunofluorescence staining of proliferating cells (Ki67) and blood vessels (CD31). Immunofluorescence analysis for Ki67 (green) and CD31 (red) showed no difference in proliferation as well as blood vessel density in resected tumors of mice treated with radiation and SMAD-NIS MSC followed by 131I treatment as compared to therapy mice receiving CMV-NIS-MSCs or irradiated tumors of mice injected with SMAD-NIS MSCs and NaCl or non-irradiated tumors receiving NaCl only (A-C). Slides of tumors were counterstained with Hoechst nuclear stain and one representative image is shown each (20Ã- magnification) (A).

constructs, murine HCC model, spheroid and imaging, RCC tissue pathology

Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577–90. ©2017 AACR.

Supplementary Table 2 from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

Supplementary Table 1 from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

Supplementary Figure 1 from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

Supplementary Figure 3 from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

Supplementary Figure 5 from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein that encodes metastasis-suppressor activity through the suppression of Src-mediated oncogenic signaling and vascular endothelial growth factor expression. SSeCKS expression is down-regulated in Src- and Ras-transformed fibroblasts, in human cancer cell lines and in several types of human cancer, including prostate. Normal human and mouse prostates express abundant SSeCKS in secretory epithelial cells and, to a lesser extent, in the surrounding mesenchyme. Here, we show that the loss of SSeCKS results in prostatic hyperplasia in the anterior and ventral lobes as well as increased levels of apoptosis throughout the prostate. Dysplastic foci were observed less frequently but were associated with the loss of E-cadherin staining and the loss of high molecular weight cytokeratin-positive basal epithelial cells. SSeCKS-null prostate tissues expressed significantly higher relative levels of AKTpoS473 compared with wild-type controls, suggesting that SSeCKS attenuates phosphatidylinositol-3-OH kinase signaling. The data suggest that SSeCKS-null mice have increased susceptibility for oncogenic transformation in the prostate. [Cancer Res 2008;68(13):5096–103]

Supplementary Figure 4 from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

Supplementary Figure 2 from Loss of the ssecks/gravin/akap12 Gene Results in Prostatic Hyperplasia

Cloning of the sodium iodide symporter (NIS) 25 years ago has opened an exciting chapter in molecular thyroidology with the characterization of NIS as one of the most powerful theranostic genes and the development of a promising gene therapy strategy based on image-guided selective NIS gene transfer in non-thyroidal tumors followed by application of 131I or alternative radionuclides, such as 188Re and 211At. Over the past two decades, significant progress has been made in the development of the NIS gene therapy concept, from local NIS gene delivery towards promising new applications in disseminated disease, in particular through the use of oncolytic viruses, non-viral polyplexes, and genetically engineered MSCs as highly effective, highly selective and flexible gene delivery vehicles. In addition to allowing the robust therapeutic application of radioiodine in non-thyroid cancer settings, these studies have also been able to take advantage of NIS as a sensitive reporter gene that allows temporal and spatial monitoring of vector biodistribution, replication, and elimination – critically important issues for preclinical development and clinical translation.

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 (123)I-scintigraphy, thermo-stimulation (41°C, 1 h) 24 h after CMV-NIS-MSC injection resulted in a significantly increased uptake of (123)I 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 (131)I 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.

The CytoSorb hemoadsorption device (CytoSorbents Inc, Monmouth Junction, NJ) is increasingly used in many critical disease states. The potential impact on the pharmacokinetic (PK) of concomitantly administered drugs must be considered in clinical practice. The current review summarizes relevant mechanistic principles, available preclinical and clinical data, and provides general guidance for the management of concomitant drug administration during CytoSorb therapy.Detailed search strategy using the PubMed and OVID MEDLINE databases, as well as presented congress abstracts for studies on drug removal by the CytoSorb device.Human, animal, and bench-top studies with PK or drug-removal data during CytoSorb therapy were selected for inclusion. Publications reporting on CytoSorb treatments for drug overdose were not considered.Relevant PK data were examined and synthesized for narrative review.To date, PK data during CytoSorb hemoadsorption are available for more than 50 drugs, including analgesics, antiarrhythmics, anticonvulsants, antidepressants, antihypertensives, antiinfectives, antithrombotics, anxiolytics, and immunosuppressants. Based on available PK data, drugs were categorized into low (30%), moderate (30-60%), or high rates of removal (60%), or, alternatively, according to clearance increase relative to endogenous clearance: negligible (25%), low (25-100%), moderate (100-400%), or high (400%). In most reports, additional impact of the extracorporeal platform where CytoSorb was integrated was not available. Based on available data and considering drug, patient, and setup-specific aspects, general dosing guidance for clinical practice was developed.CytoSorb therapy may increase drug elimination through active removal. However, the extent of removal is heterogeneous, and its clinical significance, if any, depends on the broader clinical context, including a patient's specific endogenous drug clearance and the underlying extracorporeal platform used. The available data, although not definitive, allow for general guidance on dosing adjustments during CytoSorb therapy; however, any treatment decisions should always be complemented by clinical judgment and therapeutic drug monitoring, when available.

Dysregulated lipid metabolism represents an important metabolic alteration in cancer. Fatty acids, cholesterol, and phospholipid are the three most prevalent lipids that act as energy producers, signaling molecules, and source material for the biogenesis of cell membranes. The enhanced synthesis, storage, and uptake of lipids contribute to cancer progression. The rewiring of lipid metabolism in cancer has been linked to the activation of oncogenic signaling pathways and cross talk with the tumor microenvironment. The resulting activity favors the survival and proliferation of tumor cells in the harsh conditions within the tumor. Lipid metabolism also plays a vital role in tumor immunogenicity via effects on the function of the noncancer cells within the tumor microenvironment, especially immune‐associated cells. Targeting altered lipid metabolism pathways has shown potential as a promising anticancer therapy. Here, we review recent evidence implicating the contribution of lipid metabolic reprogramming in cancer to cancer progression, and discuss the molecular mechanisms underlying lipid metabolism rewiring in cancer, and potential therapeutic strategies directed toward lipid metabolism in cancer. This review sheds new light to fully understanding of the role of lipid metabolic reprogramming in the context of cancer and provides valuable clues on therapeutic strategies targeting lipid metabolism in cancer., Lipid metabolic reprogramming‐mediated crosstalk between cancer cells and tumor microenvironment contributes to cancer progression. Stromal cells, such as adipocyte, CAF, epithelia cells contribute to lipid metabolic reprogramming in cancer cells. Cancer cells undergo lipid metabolism rewiring to generate building materials for membrane, lipid second messenger and energy supply. Cancer cells can also secret lipid metabolites to affect immune cell functions, creating a tumor‐favoring immune microenvironment.

Thyroid hormones are emerging as critical regulators of tumour growth and progression. To assess the contribution of thyroid hormone signalling via integrin αvβ3, expressed on many tumour cells, endothelial cells, and stromal cells, to tumour growth, we compared the effects of thyroid hormones vs tetrac, a specific inhibitor of thyroid hormone action at integrin αvβ3, in two murine xenograft tumour models with and without integrin αvβ3 expression. Integrin αvβ3-positive human anaplastic thyroid cancer cells SW1736 and integrin αvβ3-negative human hepatocellular carcinoma cells HuH7 were injected into the flanks of nude mice. Tumour growth was monitored in euthyroid, hyperthyroid, hypothyroid, and euthyroid tetrac-treated mice. In SW1736 xenografts, hyperthyroidism led to a significantly increased tumour growth resulting in a decreased survival compared to euthyroid mice, while tumour growth was significantly reduced and, hence, survival prolonged in hypothyroid and tetrac-treated mice. Both proliferation and vascularisation, as determined by Ki67 and CD31 immunofluorescence staining, respectively, were significantly increased in tumours from hyperthyroid mice as compared to hypothyroid and tetrac-treated mice. No differences in tumour growth, survival, or Ki67 staining were observed between the different groups in integrin αvβ3-negative HuH7 xenografts. Vascularisation, however, was significantly decreased in hypothyroid and tetrac-treated mice compared to euthyroid and hyperthyroid mice. Apoptosis was not affected in either tumour model, nor were cell proliferation or apoptosis in vitro. Tumour growth regulation by thyroid hormones in αvβ3-positive tumours has important implications for cancer patients, especially those with thyroid dysfunctions and thyroid cancer patients treated with thyrotropin-suppressive L-thyroxine doses.

Enolase 2 (ENO2) is a key glycolytic enzyme in the metabolic process of glycolysis, but its potential function in pancreatic ductal adenocarcinoma (PDAC) is unclear. In this study, we observed a significant overexpression of ENO2 in PDAC tissues, and its expression was correlated with metastasis and poor prognosis in PDAC patients. K394 was identified as a major acetylation site in ENO2 that regulates its enzymatic activity, cell metabolism and PDAC progression. Knockdown of ENO2 suppressed tumor growth and liver metastasis in PDAC. Re-expression of wild-type (WT) ENO2, but not the K394 acetylation mimetic mutant, could reverse the decreased tumor malignancy. We further characterized histone deacetylase 3 (HDAC3) and P300/CBP-associated factor (PCAF) as the potential deacetylase and acetyltransferase for ENO2, respectively. HDAC3-mediated deacetylation was shown to lead to ENO2 activation and enhancement of glycolysis. Importantly, insulin-like growth factor-1 (IGF-1) was found to decrease K394 acetylation and stimulate ENO2 activity in a dose- and time-dependent manner. The PI3K/AKT/mTOR pathway facilitated the phosphorylation of HDAC3 on S424, which promoted K394 deacetylation and activation of ENO2. Linsitinib, an oral small-molecule inhibitor of IGF-1R, could inhibit IGF-1-induced ENO2 deacetylation by HDAC3 and the PI3K/AKT/mTOR pathway. Furthermore, linsitinib showed a different effect on the growth and metastasis of PDAC depending on the overexpression of WT versus K394-mutant ENO2. Our results reveal a novel mechanism by which acetylation negatively regulates ENO2 activity in the metastasis of PDAC by modulating glycolysis. Blockade of IGF-1-induced ENO2 deacetylation represents a promising strategy to prevent the development of PDAC.

More than 70% of gastrointestinal (GI) cancers are diagnosed with metastases, leading to poor prognosis. For some cancer patients with limited sites of metastatic tumors, the term oligometastatic disease (OMD) has been coined as opposed to systemic polymetastasis (PMD) disease. Stephan Paget first described an organ-specific pattern of metastasis in 1889, now known as the "seed and soil" theory where distinct cancer types are found to metastasize to different tumor-specific sites. Our understanding of the biology of tumor metastasis and specifically the molecular mechanisms driving their formation are still limited, in particular, as it relates to the genesis of oligometastasis. In the following review, we discuss recent advances in general understanding of this metastatic behavior including the role of specific signaling pathways, various molecular features and biomarkers, as well as the interaction of carcinoma cells with their tissue microenvironments (both primary and metastatic niches). The unique features that underlie OMD provide potential targets for localized therapy. As it relates to clinical practice, OMD is emerging as treatable with surgical resection and/or other local therapy options. Strategies currently being applied in the clinical management of OMD will be discussed including surgical, radiation-based therapy, ablation procedures, and the results of emerging clinical trials involving immunotherapy.

Hyperfiltration is a state of high glomerular filtration rate (GFR) observed in early diabetes that damages glomeruli, resulting in an iterative process of increasing filtration load on fewer and fewer remaining functional glomeruli. To delineate underlying cellular mechanisms of damage associated with hyperfiltration, transcriptional profiles of kidney biopsies from Pima Indians with type 2 diabetes with or without early-stage diabetic kidney disease were grouped into two hyperfiltration categories based on annual iothalamate GFR measurements. Twenty-six participants with a peak GFR measurement within two years of biopsy were categorized as the hyperfiltration group, and 26 in whom biopsy preceded peak GFR by over two years were considered pre-hyperfiltration. The hyperfiltration group had higher hemoglobin A1c, higher urine albumin-to-creatinine ratio, increased glomerular basement membrane width and lower podocyte density compared to the pre-hyperfiltration group. A glomerular 1240-gene transcriptional signature identified in the hyperfiltration group was enriched for endothelial stress response signaling genes, including endothelin-1, tec-kinase and transforming growth factor-β1 pathways, with the majority of the transcripts mapped to endothelial and inflammatory cell clusters in kidney single cell transcriptional data. Thus, our analysis reveals molecular pathomechanisms associated with hyperfiltration in early diabetic kidney disease involving putative ligand-receptor pairs with downstream intracellular targets linked to cellular crosstalk between endothelial and mesangial cells.

Cloning of the sodium iodide symporter (NIS) in 1996 has provided an opportunity to use NIS as a powerful theranostic transgene. Novel gene therapy strategies rely on image-guided selective NIS gene transfer in non-thyroidal tumors followed by application of therapeutic radionuclides. This review highlights the remarkable progress during the last two decades in the development of the NIS gene therapy concept using selective non-viral gene delivery vehicles including synthetic polyplexes and genetically engineered mesenchymal stem cells. In addition, NIS is a sensitive reporter gene and can be monitored by high resolution PET imaging using the radiotracers sodium [124I]iodide ([124I]NaI) or [18F]tetrafluoroborate ([18F]TFB). We performed a small preclinical PET imaging study comparing sodium [124I]iodide and in-house synthesized [18F]TFB in an orthotopic NIS-expressing glioblastoma model. The results demonstrated an improved image quality using [18F]TFB. Building upon these results, we will be able to expand the NIS gene therapy approach using non-viral gene delivery vehicles to target orthotopic tumor models with low volume disease, such as glioblastoma.Trial registration not applicable.

Hyperfiltration (HF) is a state of high glomerular filtration rate (GFR) observed in early diabetes that damages glomeruli, resulting in an iterative process of increasing filtration load on fewer and fewer remaining functional glomeruli. To delineate underlying cellular mechanisms of damage induced by HF, transcriptional profiles of kidney biopsies from Pima Indians with type 2 diabetes with or without early-stage diabetic kidney disease (DKD) were grouped into two HF categories based on annual iothalamate GFR measurements. Twenty-six participants with a peak GFR measurement within two years of biopsy were categorized as the “HF group”, and 26 in whom biopsy preceded peak GFR by >2 years were considered “pre-HF”. The HF group had higher hemoglobin A1c, higher urine albumin-to-creatinine ratio, increased glomerular basement membrane width and lower podocyte density compared to the pre-HF group. A glomerular 1240-gene transcriptional signature identified in the HF group was enriched for endothelial stress response signaling genes, including from endothelin-1, tec-kinase and TGF-β1 pathways, with the majority of the transcripts mapped to endothelial and inflammatory cell clusters in kidney single cell transcriptional data. This analysis reveals molecular pathomechanisms contributing to development of HF and early DKD and involving putative ligand-receptor pairs and downstream intracellular targets linked to cellular crosstalk between endothelial and mesangial cells.

Non-classical thyroid hormone signalling via cell surface receptor integrin αvβ3, expressed on most cancer cells and proliferating endothelial cells, has been shown to drive tumour cell proliferation and survival, as well as angiogenesis. Tumours develop within a complex microenvironment that is composed of many different cell types, including mesenchymal stem cells. These multipotent progenitor cells actively home to growing tumours where they differentiate into cancer-associated fibroblast-like cells and blood vessel-stabilising pericytes and thus support the tumour’s fibrovascular network. Integrin αvβ3 expression on mesenchymal stem cells makes them susceptible to thyroid hormone stimulation. Indeed, our studies demonstrated – for the first time – that thyroid hormones stimulate the differentiation of mesenchymal stem cells towards a carcinoma-associated fibroblast-/pericyte-like and hypoxia-responsive, pro-angiogenic phenotype, characterised by the secretion of numerous paracrine pro-angiogenic factors, in addition to driving their migration, invasion, and recruitment to the tumour microenvironment in an experimental hepatocellular carcinoma model. The deaminated thyroid hormone metabolite tetrac, a specific inhibitor of thyroid hormone action at the integrin site, reverses these effects. The modulation of mesenchymal stem cell signalling and recruitment by thyroid hormones via integrin αvβ3 adds a further layer to the multifaceted effects of thyroid hormones on tumour progression, with important implications for the management of cancer patients and suggests a novel mechanism for the anti-tumour activity of tetrac.

Purpose: The innate tumor homing potential of mesenchymal stem cells (MSCs) has been used for a targeted delivery of the theranostic sodium iodide symporter (NIS) transgene into solid tumors. We have previously shown that external beam radiotherapy (EBRT) results in the enhanced recruitment of NIS-expressing MSCs into human hepatocellular carcinoma (HuH7). In parallel, the tumor-associated cytokine TGFB1 becomes strongly upregulated in HuH7 tumors in response to EBRT. Experimental Design: We therefore evaluated the effects of combining focused EBRT (5 Gy) with MSC-mediated systemic delivery of the theranostic NIS transgene under control of a synthetic TGFB1-inducible SMAD-responsive promoter (SMAD-NIS-MSCs) using 123I-scintigraphy followed by 131I therapy in CD1 nu/nu mice harboring subcutaneous human hepatocellular carcinoma (HuH7). Results: Following tumor irradiation and SMAD-NIS-MSC application, tumoral iodide uptake monitored in vivo by 123I-scintigraphy was enhanced as compared with nonirradiated tumors. Combination of EBRT and SMAD-NIS-MSC–mediated 131I therapy resulted in a significantly improved delay in tumor growth and prolonged survival in therapy mice as compared with the combined therapy using CMV-NIS-MSCs or to control groups receiving EBRT or saline only, or EBRT together with SMAD-NIS-MSCs and saline applications. Conclusions: MSC-based NIS-mediated 131I therapy after EBRT treatment dramatically enhanced therapeutic efficacy when a TGFB1-inducible SMAD-responsive promoter was used to drive NIS expression in adoptively applied MSCs. The remarkable therapeutic effect seen is thought to be linked in large part to the enhanced TGFB1 produced in this context, which leads to a highly selective and focused amplification of MSC-based NIS expression within the tumor milieu.

The thyroid hormones T3 and T4 have emerged as pro-angiogenic hormones with important implications for cancer management. Endogenous circulating hormone levels may help stimulate cancer progression and limit the effectiveness of anticancer therapy, though clinical data remain inconclusive. The capacity of thyroid hormones to modulate angiogenesis is mediated through non-canonical mechanisms initiated at the cell surface receptor integrin αvβ3. This integrin is predominantly expressed on tumour cells, proliferating endothelial cells and tumour stroma-associated cells, emphasising its potential relevance in angiogenesis and tumour biology. Thyroid hormone/integrin αvβ3 signalling results in the activation of intracellular pathways that are commonly associated with angiogenesis and are mediated through classical pro-angiogenic molecules such as vascular endothelial growth factor. The naturally occurring T4 analogue tetrac blocks the pro-angiogenic actions of thyroid hormones at the integrin receptor, in addition to agonist-independent anti-angiogenic effects. Tetrac reduces endothelial cell proliferation, migration and tube formation through a reduction in the transcription of vascular growth factors/growth factor receptors, hypoxia-inducible factor-1α, pro-angiogenic cytokines and a number of other pro-angiogenic genes, while at the same time stimulating the expression of endogenous angiogenesis inhibitors. It further modulates vascular growth factor activity by disrupting the crosstalk between integrin αvβ3 and adjacent growth factor receptors. Moreover, tetrac disrupts thyroid hormone-stimulated tumour recruitment, differentiation and the pro-angiogenic signalling of tumour stroma-associated mesenchymal stem cells. Tetrac affects tumour-associated angiogenesis via multiple mechanisms and interferes with other cancer cell survival pathways. In conjunction with its low toxicity and high tissue selectivity, tetrac is a promising candidate for clinical application.

Lipo-oligomers, post-functionalized with ligands to enhance targeting, represent promising new vehicles for the tumor-specific delivery of therapeutic genes such as the sodium iodide symporter (NIS). Due to its iodide trapping activity, NIS is a powerful theranostic tool for diagnostic imaging and the application of therapeutic radionuclides. 124I PET imaging allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression, and application of 131I enables cytoreduction. In our experimental design, we used epidermal growth factor receptor (EGFR)-targeted polyplexes (GE11) initially characterized in vitro using 125I uptake assays. Mice bearing an orthotopic glioblastoma were treated subsequently with mono-dibenzocyclooctyne (DBCO)-PEG24-GE11/NIS or bisDBCO-PEG24-GE11/NIS, and 24–48 h later, 124I uptake was assessed by positron emission tomography (PET) imaging. The best-performing polyplex in the imaging studies was then selected for 131I therapy studies. The in vitro studies showed EGFR-dependent and NIS-specific transfection efficiency of the polyplexes. The injection of monoDBCO-PEG24-GE11/NIS polyplexes 48 h before 124I application was characterized to be the optimal regime in the imaging studies and was therefore used for an 131I therapy study, showing a significant decrease in tumor growth and a significant extension of survival in the therapy group. These studies demonstrate the potential of EGFR-targeted polyplex-mediated NIS gene therapy as a new strategy for the therapy of glioblastoma., Graphical abstract, EGFR-targeted lipopolyplexes represent promising new vehicles for the tumor-specific delivery of therapeutic genes, such as the theranostic sodium iodide symporter (NIS). Spellerberg et al. established specific and highly efficient polyplexes for an effective NIS gene therapy concept of glioblastoma.

The tumor homing characteristics of mesenchymal stem cells (MSCs) make them attractive vehicles for the tumor-specific delivery of therapeutic agents, such as the sodium iodide symporter (NIS). NIS is a theranostic protein that allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression by radioiodine imaging as well as the therapeutic application of 131I. To enhance the actively recruitment of MSCs to growing tumor stroma and thereby trigger targeted delivery of the NIS gene to the tumor, we examined the combination with regional hyperthermia, as heat induces the secretion of immunomodulatory chemokines, cytokines and growth factors, well-known attractants of MSCs. Human hepatocellular carcinoma cells (HuH7) were heat-treated in a water bath at 41 °C for 1h, followed by incubation at 37 °C for 0-48h. mRNA and protein levels of chemokines involved in MSC migration was analyzed by RT-PCR and ELISA. Chemotaxis of MSCs in relation to a gradient of supernatants was tested in a 3D live cell tracking migration assay. In a subcutaneous HuH7 mouse xenograft tumor model, a single systemic injection of CMV-NIS-MSCs was applied 6h, 24h, 48h after or 24h, 48h before hyperthermia treatment and tumoral 123I accumulation was assessed by 123I-scintigraphy. Ex vivo NIS analysis of tumor sections was performed by RT-PCR and immunohistochemistry. The optimal imaging regime was then used for a 131I therapy study. Chemokine mRNA and protein analysis indicated a substantial increase in expression levels of chemokines and growth factors, involved in MSC tumor homing, after heat exposure. In addition, MSCs showed directed migration towards the supernatant of thermo-stimulated cancer cells. In vivo, with the optimal regime, we observed a significantly increased uptake of 123I in tumors of heat-treated animals (41 °C) when thermostimulated 24h after CMV-NIS-MSC injection compared to control animals (37 °C). Immunohistochemical staining of tumor sections showed strong tumoral NIS-specific immunoreactivity and RT-PCR an increased NIS mRNA expression in heat-treated tumors, thereby confirming tumor-selective, temperature-dependent MSC migration. CMV-NIS-MSC-mediated 131I therapy combined with regional hyperthermia resulted in a reduced tumor growth that was associated with prolonged survival of regional heat-treated animals compared to normothermic mice and to the saline control group. In summary, we have demonstrated a significantly increased, selective MSC migration towards the tumor stroma after regional hyperthermia in the 123I imaging study. The combination of MSC-mediated NIS gene therapy with mild regional hyperthermia resulting in stimulated therapeutic efficacy of NIS-mediated 131I therapy.