Subcutaneous Lung Cancer Xenograft Research Articles

Radiosensitization by microRNA30a-5p in a nude mouse model with subcutaneous lung-cancer xenograft*

Abstract Objective We aimed to observe the radiosensitization effect of mir-30a-5p in a nude mouse model with subcutaneous lung-cancer xenograft and to explore the underlying mechanism. Methods A549 cell lines with either stable upregulation or downregulation of mir-30a-5p, and their negative control, were transfected with lentivirus vectors. These cell lines were used to establish a nude mouse model with subcutaneous lung-cancer xenograft. Each group was randomly divided into irradiated and non-irradiated groups. The radiosensitization effect of mir-30a-5p in vivo was studied by observing xenograft growth trends and tumor weight. The mechanisms involved in this radiosensitization were investigated by detecting expressed radiosensitization-related proteins, using immunohistochemistry and Western blotting. Results The expression level of mir-30a-5p in the lenti-mir-30a-5p group was higher than that in the negative control (lenti-GFP) group and lower in the lenti-inhibitor group (P < 0.05). Subcutaneous lung-cancer xenografts in the irradiation group and lenti-mir-30a-5p increased in size slowly; tumors were lighter and tumor inhibition rates were higher than those in the non-irradiation and lenti-GFP groups. In contrast, the opposite of these effects was observed in the lenti-inhibitor group. Immunohistochemistry and Western blotting indicated that ATM protein expression level was lower in the lenti-mir-30a-5p group, with or without irradiation, compared to that in the lenti-GFP group. ATM protein levels were higher in the lenti-inhibitor groups. The phosphorylation level of ATM at residue 1981 was low in the groups without irradiation and increased significantly after irradiation (P < 0.05). Moreover, the phosphorylation level was lower in the lenti-mir-30a-5p group and higher in the lenti-inhibitor group than that in the lenti-GFP group after irradiation (P < 0.05). Conclusion Mir-30a-5p enhanced the radiosensitivity of nude mice with subcutaneous lung-cancer xenografts by inhibiting ATM phosphorylation.

Involvement of ER stress and reactive oxygen species generation in anti-cancer effect of CKD-516 for lung cancer.

CKD-516 (Valecobulin), a vascular-disrupting agent, inhibits microtubule elongation. We evaluated the effect of CKD-516 on lung cancer cells and the underlying molecular mechanisms. The effects of S516, an active metabolite of CKD-516, were evaluated in HUVECs and three lung cancer cell lines and by a microtubule polymerization assay. Tubulin cross-linking was used to identify the binding site of S516 on tubulin, and Western blotting was performed to identify the intracellular pathways leading to cell death. Subcutaneous lung cancer xenograft models were used to assess the in vivo effect of CKD-516 on tumor growth. S516 targeted the colchicine binding site on β-tubulin. In lung cancer cells, S516 increased endoplasmic reticulum (ER) stress and induced reactive oxygen species (ROS) generation by mitochondria and the ER. In addition, CKD-516 monotherapy strongly inhibited the growth of lung cancer xenograft tumors and exerted a synergistic effect with carboplatin. The findings suggest that CKD-516 exerts an anticancer effect in company with inducing ER stress and ROS production via microtubule disruption in lung cancer cells. CKD-516 may thus have therapeutic potential for lung cancer.

A novel role mediated by adenoviral E1A in suppressing cancer through modulating decorin.

Oncolytic adenovirus is an emerging alternative to current therapeutics. The adenoviral E1A, the first protein expressed upon oncolytic adenoviral infection, has been identified as an antitumor agent, but the mechanisms of its tumor inhibition ability are unclear enough. Decorin is ubiquitous in the extracellular matrix (ECM), which regulates multiple functions through interaction with ECM. Here, we intended to explore the effects of adenoviral E1A on the tumor extracellular matrix during gene therapy. We demonstrated that reduced decorin expression was found in patients with lung cancer. The adenoviral E1A or a mutant adenoviral E1A with Rb-binding ability absent (E1A 30-60aa, 120-127aa deletion) could increase the expression of decorin and down-regulate VEGF, two members of tumor ECM, involved in both vasculogenesis and angiogenesis. E1A/mE1A-mediated suppressing the migration and invasion ability of tumor cells was depended on decorin. E1A interacted with decorin directly and induced the proteasomal degradation of VEGF. In addition, E1A or mE1A can inhibit tumor growth in a subcutaneous lung cancer xenograft model. It suggested that decorin might be a crucial mediator among ECM components for adenoviral E1A-mediated antitumor activities. These studies on adenovirus E1A provide a new mechanism for the emerging therapies of tumor gene therapy.

FGFR1-ERK1/2-SOX2 axis promotes cell proliferation, epithelial-mesenchymal transition, and metastasis in FGFR1-amplified lung cancer.

Epithelial-mesenchymal transition (EMT) is an important process for cancer metastasis, drug resistance, and cancer stem cells. Activation of fibroblast growth factor receptor 1 (FGFR1) was found to promote EMT and metastasis in prostate and breast cancers, but the effects and mechanisms in lung cancer was unclear. In this study, we aimed to explore whether and how activation of FGFR1 promotes EMT and metastasis in FGFR1-amplified lung cancer. We show that activation of FGFR1 by its ligand fibroblast growth factor 2 (FGF2) promoted proliferation, EMT, migration, and invasion in FGFR1-amplified lung cancer cell lines H1581 and DMS114, whereas inhibition of FGFR1 suppressed these processes. FGFR1 activation upregulated expression of Sry-related HMG box 2 (SOX2) by downstream phosphorylated ERK1/2; moreover, the upregulation of SOX2 by autophosphorylation variant ERK2_R67S plasmid transfection was not suppressed by FGFR1 inhibitor AZD4547 or MEK/ERK inhibitor AZD6244 in vitro. And SOX2 expression was also significantly upregulated in ERK2_R67S lentivirus-transfected stable cell lines in vivo. Overexpression of SOX2 promoted cell proliferation, EMT, migration, and invasion. Importantly, activation of FGFR1 could not promote these processes in SOX2-silenced stable cell lines. In orthotopic and subcutaneous lung cancer xenograft models, inhibition of FGFR1 suppressed tumor growth, SOX2 expression, EMT, and metastasis in vivo; however, these processes caused by SOX2-overexpressing stable cell lines were not suppressed by FGFR1 inhibition. Higher expression of FGFR1 and SOX2 were positively correlated, and both were associated with shorter survival in lung cancer patients. In conclusion, our findings reveal that activation of FGFR1 promotes cell proliferation, EMT, and metastasis by the newly defined FGFR1-ERK1/2-SOX2 axis in FGFR1-amplified lung cancer.

A New Role for the Mitochondrial Pro-apoptotic Protein SMAC/Diablo in Phospholipid Synthesis Associated with Tumorigenesis.

The mitochondrial pro-apoptotic protein SMAC/Diablo participates in apoptosis by negatively regulating IAPs and activating caspases, thus encouraging apoptosis. Unexpectedly, we found that SMAC/Diablo is overexpressed in cancer. This paradox was addressed here by silencing SMAC/Diablo expression using specific siRNA (si-hSMAC). In cancer cell lines and subcutaneous lung cancer xenografts in mice, such silencing reduced cell and tumor growth. Immunohistochemistry and electron microscopy of the si-hSMAC-treated residual tumor demonstrated morphological changes, including cell differentiation and reorganization into glandular/alveoli-like structures and elimination of lamellar bodies, surfactant-producing organs. Next-generation sequencing of non-targeted or si-hSMAC-treated tumors revealed altered expression of genes associated with the cellular membrane and extracellular matrix, of genes found in the ER and Golgi lumen and in exosomal networks, of genes involved in lipid metabolism, and of lipid, metabolite, and ion transporters. SMAC/Diablo silencing decreased the levels of phospholipids, including phosphatidylcholine. These findings suggest that SMAC/Diablo possesses additional non-apoptotic functions related to regulating lipid synthesis essential for cancer growth and development and that this may explain SMAC/Diablo overexpression in cancer. The new lipid synthesis-related function of the pro-apoptotic protein SMAC/Diablo in cancer cells makes SMAC/Diablo a promising therapeutic target.

ImmunoPET for assessing the differential uptake of a CD146-specific monoclonal antibody in lung cancer.

Overexpression of CD146 in solid tumors has been linked to disease progression, invasion, and metastasis. We describe the generation of a 64Cu-labeled CD146-specific antibody and its use for quantitative immunoPET imaging of CD146 expression in six lung cancer models. The anti-CD146 antibody (YY146) was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA) and radiolabeled with 64Cu. CD146 expression was evaluated in six human lung cancer cell lines (A549, NCI-H358, NCI-H522, HCC4006, H23, and NCI-H460) by flow cytometry and quantitative western blot studies. The biodistribution and tumor uptake of 64Cu-NOTA-YY146 was assessed by sequential PET imaging in athymic nude mice bearing subcutaneous lung cancer xenografts. The correlation between CD146 expression and tumor uptake of 64Cu-NOTA-YY146 was evaluated by graphical software while ex vivo biodistribution and immunohistochemistry studies were performed to validate the accuracy of PET data and spatial expression of CD146. Flow cytometry and western blot studies showed similar findings with H460 and H23 cells showing high levels of expression of CD146. Small differences in CD146 expression levels were found among A549, H4006, H522, and H358 cells. Tumor uptake of 64Cu-NOTA-YY146 was highest in CD146-expressing H460 and H23 tumors, peaking at 20.1 ± 2.86 and 11.6 ± 2.34%ID/g at 48h after injection (n = 4). Tumor uptake was lowest in the H522 model (4.1 ± 0.98%ID/g at 48h after injection; n = 4), while H4006, A549 and H358 exhibited similar uptake of 64Cu-NOTA-YY146. A positive correlation was found between tumor uptake of 64Cu-NOTA-YY146 (%ID/g) and relative CD146 expression (r 2 = 0.98, p < 0.01). Ex vivo biodistribution confirmed the accuracy of the PET data. The strong correlation between tumor uptake of 64Cu-NOTA-YY146 and CD146 expression demonstrates the potential use of this radiotracer for imaging tumors that elicit varying levels of CD146. In the future, this tool may promote enhanced monitoring of therapeutic response and improved patient stratification.

Abstract 4557: Assessment of a humanized CXCR4 monoclonal antibody for therapeutic monitoring and intervention in experimental NSCLC and TNBC mouse models

Abstract The CXC chemokine receptor 4 (CXCR4) is over-expressed in lung and breast tumors, leading to increased metastasis, poor prognosis and survival rates. Considering the limited treatment options for triple negative breast (TNBC) and non-small cell lung (NSCLC) cancers, CXCR4-based therapy, guided by targeted imaging, will provide a more effective and optimal therapeutic strategy. Here, a humanized anti-CXCR4 antibody (mAb) and its 89Zr-labeled counterpart ([89Zr]mAb) were evaluated for therapeutic efficacy and CXCR4-specific tumor accumulation by positron emission tomography (PET) imaging with the aim to establish a correlation between imaging and therapy in NSCLC and TNBC xenografts in mice. The specificity of the [89Zr]mAb was evaluated in vitro using the NSCLC (CXCR4high H1155, CXCR4low A549) and TNBC (CXCR4high DU4775, CXCR4med MDA-MB-231) cancer cell lines. These cell lines were also used to establish subcutaneous and orthotopic breast and lung cancer xenografts in immunocompromised mice. The firefly luciferase-expressing MDA-MB-231 breast cancer cell line (231-Luc) was used to generate a spontaneous metastatic mouse model. PET imaging and ex vivo biodistribution studies were performed to evaluate [89Zr]mAb uptake in tumors and metastases. CXCR4 expression was confirmed by immunohistochemistry. For therapy, mice harboring lung and breast tumors were randomized (150mm3) and treated with vehicle (saline), control IgG4 mAb or targeted mAb (10mg/kg every third day, 5x, n = 7/group,). Weight of mice and tumor growth were monitored throughout the study. In vitro binding studies demonstrated CXCR4-specific radiotracer uptake and correlated with CXCR4 expression as determined by flow cytometry. PET imaging and biodistribution studies in subcutaneous lung cancer xenografts showed a%ID/g of 32.9 ± 6.2 in H1155 and 6.8 ± 0.9 in A549 tumors, 24 h p.i. Preferential H1155 tumor uptake was observed over 120 h with a consistent H1155/A549 ratio of 4.5-5. Similar results were observed in orthotopic lung and breast tumors. PET imaging of 231-Luc xenografted mice demonstrated a [89Zr]mAb uptake [percentage injected dose (%ID)/cc] of 15.7 ± 1.9 and 16.1 ± 2.9 in primary tumors and lymph node metastases, respectively, at 24 h post-injection (p.i.). Therapeutic doses significantly reduced tumor growth in CXCR4high H1155, but not in CXCR4low A549 xenografts. A similar but less pronounced effect was observed in TNBC xenografts that have comparatively lower CXCR4 expression. [89Zr]mAb-PET demonstrated CXCR4 uptake in primary tumors and metastases in experimental models of NSCLC and TNBC that correlated with therapeutic efficacy of the non-labeled mAb. [89Zr]mAb is a promising and translatable candidate for imaging of CXCR4 expression in various CXCR4 over-expressing cancers, which, in conjunction with the non-labeled antibody, can be utilized for therapeutic intervention and monitoring. Citation Format: Babak Behnam Azad, Samit Chatterjee, Ala Lisok, Mrudula Pullambhatla, Wojciech G. Lesniak, Zaver M. Bhujwalla, Martin Pomper, Sridhar Nimmagadda. Assessment of a humanized CXCR4 monoclonal antibody for therapeutic monitoring and intervention in experimental NSCLC and TNBC mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4557. doi:10.1158/1538-7445.AM2015-4557

Photothermal ablation of human lung cancer by low-power near-infrared laser and topical injection of indocyanine green.

The present study was designed to evaluate the efficacy of photothermal ablation therapy for lung cancer by low-power near-infrared laser and topical injection of indocyanine green (ICG). In vitro study 1: an 808 nm laser with 250 mW was irradiated for 10 minutes using different dilutions of ICG and the temporal thermal effect was monitored. ICG (1 mL of 0.5 g/L) was heated to a temperature of >30°C from the base temperature by laser irradiation. In vitro study 2: the cytotoxic effect of hyperthermia on human lung cancer cells was examined in different temperature and time settings. Cell viability was quantified by both an MTS assay and reculturing. Fatal conditions evaluated by reculturing were as follows: thermal treatment at 55°C for 5 minutes, 53°C for 10 minutes, and 51°C for 15 minutes. The MTS assay study suggested that thermal treatment at 59°C for 5 minutes and 57°C for 20 minutes showed a severe cytotoxic effect. In vivo study: nude mouse subcutaneous NCI-H460 human lung cancer xenograft models were used for the study. Saline or 0.5 g/L of ICG was injected topically into the tumor (n=3/group). Tumors were irradiated with a laser at 500 mW for 10 minutes. Although the tumor diameter reached 1 cm within 24 days after treatment in all 3 mice using saline/laser, tumor sizes were gradually reduced in all 3 mice using the ICG/laser. In 2 of the 3 mice using ICG/laser, tumors had disappeared macroscopically. The efficacy of the photothermal ablation therapy by low-power near-infrared laser and a topical injection of ICG was clarified using a mouse subcutaneous a lung cancer xenograft model.

Abstract 675: Jarid2 is a novel target for lung cancer therapy.

Abstract Increasing evidence indicates that aberrant expression/activity of polycomb repressive-complex-2 (PRC-2) contributes to the pathogenesis of a variety of human malignancies including lung cancers. In recent studies we have demonstrated that depletion of EZH2- a core component of PRC-2, either by biochemical or pharmacologic techniques inhibits the malignant phenotype of lung cancer cells, indicating that PRC-2 is a target for epigenetic therapy of lung cancer. In the present study, we sought to further examine the potential efficacy of targeting PRC-2 in lung cancers via depletion of Jarid2, which modulates gene expression in stem cells in part via targeting PRC-2 to DNA. Quantitative RT-PCR and immunoblot experiments demonstrated over-expression of Jarid2 in cultured lung cancer cells and primary lung cancers relative to normal respiratory epithelia or histologically normal lung tissues. In subsequent experiments, shRNA techniques were used to inhibit Jarid2 expression in lung cancer cells. Quantitative RT-PCR and immunoblot experiments demonstrated that knock-down of Jarid2 had relatively modest effects on global H3K27Me3 levels in lung cancer cells. These finding were in contrast to knock-down of EZH2, which markedly inhibited H3K27 trimethylation in these cancer cells. Interestingly, knock-down of Jarid2 inhibited in vitro proliferation of lung cancer cells by approximately 70% relative to respective controls, and significantly diminished growth of subcutaneous lung cancer xenografts in athymic nude mice. The anti-proliferative effects of Jarid2 knock-down were relatively comparable to those observed following depletion of EZH2 in lung cancer cells. Jarid2 knock- down only modestly enhanced cytotoxicity mediated by DZNep in lung cancer cells. Microarray experiments are currently in progress to compare gene expression profiles in lung cancer cells mediated by Jarid2 knock-down relative to EZH2 depletion or DZNep treatment, and to delineate the mechanisms by which Jarid2 enhances the malignant phenotype of lung cancer cells. Collectively, these preliminary findings warrant further analysis of Jarid2 expression during pulmonary carcinogenesis, and suggest that Jarid2 may be a novel target for lung cancer therapy. Citation Format: Young K. Hong, Mary Zhang, Scott Atay, Manish Raiji, Trevor Upham, Julie A. Hong, Mahadev Rao, David S. Schrump. Jarid2 is a novel target for lung cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2013-675