Tumor Growth Delay Assay Research Articles

Establishment and Application of Novel Hypoxia-driven Dual-reporter Model to Investigate Hypoxic Impact on Radiation Sensitivity in Human Nasopharyngeal Carcinoma Xenografts.

Background: Tumor hypoxia has been frequently detected in nasopharyngeal carcinoma (NPC) and is intently associated with therapeutic resistance. The aim of the study is to establish a clonogenically stable hypoxia-inducible dual reporter model and apply it to investigate the effect of tumor hypoxia on DNA double strand break (DSB) and synergistic effect of irradiation in combination with chemotherapy or targeted therapy. Methods: The plasmid vector consisting of hypoxia response elements to regulate HSV1-TK and GFP genes, was constructed and stably transfected into human NPC cells. The expected clone was identified and validated by in vivo and in vitro assay. DSB repair was measured by γH2AX foci formation. Tumor growth delay assay and spatial biodistribution of various biomarkers was designed to investigate the anti-tumor effect. Results: The system has the propensity of high expression of reporter genes under hypoxia and low to no expression under normoxia. Intratumoral biodistributions of GFP and classic hypoxic biomarkers were identical in poor-perfused region. Upon equilibration with 10% O2, the xenografts showed higher expression of hypoxic biomarkers. Cisplatin radiosensitized SUNE-1/HRE cells under hypoxia by suppressing DSB repair while the addition of PI3K/mTOR inhibitor further enhanced the anti-tumoral therapeutic efficacy. Combination of IR, DDP and NVP-BEZ235 exhibited most effective anti-tumor response in vivo. These observations underline the importance of dual reporter model for imaging tumor hypoxia in therapeutic study. Conclusions: Our preclinical model enables the investigation of heterogeneous tumor hypoxic regions in xenograft tissues and explores the treatment efficacy of combinations of various therapeutic approaches to overcome hypoxia.

Biological Effects of Prostagrandin E2-EP4 Antagonist (AAT-008): Enhancement of Immunoresponse to Radiotherapy and a Potential as a Radiosensitizer

The cyclooxygenase-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four G protein-coupled receptors (EP1-4). Recently, an antagonist of PGE2-EP4 (AAT-008) has been newly developed for clinical use. The purpose of this study is to investigate the biological effects of AAT-008 as a radiosensitizer for the treatment of murine colon cancer (CT26WT) in vivo and explore the mechanism using flow cytometry.CT26WT tumor cells transplanted into the hind legs of BALB/c mice were used. Treatments were carried out when the mean diameter of the tumors reached approximately 10 mm for tumor growth delay assay and 5 mm for flow cytometry (day 0). AAT-008 (0, 3, 10, and 30 mg/kg/day) was administered orally twice a day from day 0 to day 18. The tumor was irradiated at 9 Gy on day 3 for the radiation treatment (RT) group. In the tumor growth assay, twelve mice were used for each group. The three dimensions of each tumor were measured every other day using a caliper and the tumor doubling time was calculated. For the first flow cytometry, the dose of AAT-008 was fixed to 10 mg/kg/day. Administration of AAT-008 and RT dose were the same as in the tumor growth assay. The population of cytotoxic T-cells (CD45+CD8+CD69+: CTL) was investigated using the tumor cells on day 19. For the second flow cytometry, the dose of AAT was fixed to 30 mg/kg/day. AAT-008 was administered from day 0 to day 12. The population of CTL and the ratio of CTL / regulatory T-cells (Foxp3+: Treg) were investigated using the tumor cells on day 13.In the tumor growth delay assay, the median tumor doubling times in the 0-, 3-, 10-, and 30-mg group without RT were 3.2, 4.4, 4.4, and 5.7 days, respectively. The times in those dose groups with RT were 6.1, 6.6, 17.3, and 19.8 days, respectively (P < 0.001 between the 0- and 10-mg/30-mg groups with RT). There was no significant difference between the 10- and 30-mg groups with RT (P = 0.56). In the first flow cytometry, the mean CTL proportion of the irradiated tumors in the 10 mg + RT (n = 4) and 0 mg + RT groups (n = 5) was 43% and 31%, respectively. Of note, 67% of CTL were observed in responded mice (n = 2) in the 10 mg + RT group. In the second flow cytometry, the mean Treg proportion and the CTL/ Treg ratio of the irradiated tumors in the 30 mg + RT (n = 5) and 0 mg + RT groups (n = 5) were 4.0% and 1.5%, respectively (P = 0.02) and 10 and 22, respectively (P = 0.02).AAT-008 had a potential of enhancing radiosensitivity in the treatment of colon cancer cells. It was assumed that the optimal dose of AAT-008 with RT was 10 mg/kg/day. It was suggested that AAT-008 stimulated the immune system against the cancer cells.

Enhancement of antitumor effect of radiotherapy via combination with Au@SiO2 nanoparticles targeted to tumor-associated macrophages

Tumor-associated macrophages (TAMs) exhibit the M2 phenotype and serve as critical tumor-promoting immune cells in the tumor microenvironment. As TAMs are an important target, we examined the effect of gold nanoparticles (AuNPs) with radiotherapy (RT) on M2 TAMs in tumors. We synthesized CD163 antibody-conjugated, silica-coated AuNPs (CD163-GNPs) that were specifically recognized by M2 TAMs. Bone marrow-derived macrophages and Raw 264.7 macrophages were polarized into M1 and M2 phenotypes. The effect of CD163-GNPs combined with RT was evaluated in a CT26 syngeneic mouse model (BALB/c mice). Immunostaining, flow cytometry, microscopic analyses, enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and tumor growth delay assay were performed following irradiation combined with CD163-GNP treatment. We observed selective phagocytosis of CD163-GNPs by Raw 264.7 macrophages following M1/M2 polarization. Immunostaining analyses revealed higher numbers of CD163-GNPs taken up by M2 macrophages than M0 or M1 type. CD163-GNPs combined with RT significantly reduced tumor growth in the CT26 syngeneic mouse model. Macrophages subjected to the combination treatment showed increased expression of M1 markers. The depletion of M2 TAMs in tumors upon combination treatment with CD163-GNPs enhances the efficiency of RT.

Whole Genome CRISPR-Cas9 Screen Identifies STING as a Determinant of Intrinsic Radiosensitivity in Head and Neck Squamous Cell Carcinoma

Radiation therapy is a critical component of curative therapy for HNSCC. However, the mechanisms of radioresistance in HPV-Negative HNSCC, a molecularly distinct disease with poor prognosis relative to HPV-Positive HNSCC, are incompletely understood. Although RNA interference based screening methods have been used to understand resistance to radiotherapy, they are hampered by off-target effects and partial target suppression. The recent introduction of CRISPR technology has provided a more efficient method for gene targeting and suppression. As such we hypothesized that a whole genome CRISPR-Cas9 screen in HPV-Negative HNSCC would identify novel components of the cellular response to ionizing radiation. A whole genome CRISPR-Cas9 library containing 123,411 guide RNAs (gRNAs) targeting 19,050 genes was amplified, sequenced to ensure diversity, and packaged into lentivirus. HPV-Negative HNSCC cells (160 million) were transduced with lentivirus in a pooled format and irradiated. Following irradiation, 50 million cells per condition were collected (400x coverage) and gRNA enrichment or depletion determined via MAGeCK analysis of next-generation sequencing (NGS) results. Clonogenic assay (CA) was used to determine the effects of gene knockout (KO) on radiation and cisplatin sensitivity. Immunoblotting was performed to determine effects of knockout on protein expression. Tumor growth delay assays were performed using tumor cells implanted subcutaneously in athymic nude mice. Cas9 expression alone showed no effect on radiosensitivity by CA indicating feasibility to proceed with genetic screening. Analysis of NGS results show the top-ranked gRNAs enriched after radiation target STING (stimulator of interferon genes; p < 0.001; FDR <0.01), suggesting that STING KO imparts a radioresistant phenotype. Isogenic STING KO cell lines were established by CRISPR-Cas9 KO. CA shows that STING KO significantly enhanced radiation survival in multiple KO lines by a factor of 5-10. STING KO also conferred resistance to cisplatin treatment (3-7 fold) in CAs suggesting that STING regulates an intrinsic response to DNA damage. To determine the effects of STING KO on in vivo radiosensitivity, tumor growth delay assays were performed in immunodeficient mice. STING KO tumors were significantly more radioresistant when compared to isogenic STING proficient tumors (p< 0.05) with a dose modifying factor of 0.35. Whereas STING has previously been shown to regulate the extrinsic (immune-based) response of tumors to radiation, our data suggest that STING, as identified by a whole genome CRISPR-Cas9 screen, regulates the intrinsic response to radiation in HNSCC. This supports future investigation of strategies activating the STING pathway in combination with radiotherapy to enhance therapeutic response.

Ro 90-7501 Is a Novel Radiosensitizer for Cervical Cancer Cells that Inhibits ATM Phosphorylation.

Ro 90-7501 has been reported as an inhibitor of the amyloid β42 fibril assembly that is associated with Alzheimer's disease. The present study aimed to elucidate the radiosensitizing effects of Ro 90-7501 and focused on ATM signaling after irradiation. Clonogenic survival, apoptosis, and cell-cycle assays as well as western blotting were performed in HeLa cells treated with irradiation and Ro 90-7501. Tumor growth delay assay was also performed using BALB/c-nu mice. The combination of irradiation with Ro 90-7501 showed significant radiosensitizing effects in clonogenic survival and tumor growth delay assays. Ro 90-7501 significantly increased apoptosis and impaired cell cycle after irradiation. Western blotting showed that Ro 90-7501 suppressed the phosphorylation of ATM and its downstream proteins, such as H2AX, Chk1, and Chk2, after irradiation. Ro 90-7501 inhibits DNA damage response by inhibiting ATM and has significant radiosensitizing effects on cervical cancer cells.

Radiosensitization of Glioblastoma Cells by a Novel DNA Methyltransferase-inhibiting Phthalimido-Alkanamide Derivative.

Strategies to enhance the therapeutic ratio of radiotherapy in glioblastoma are warranted. Our aim was to report a novel DNA methyltransferase inhibitor as a potential radiosensitizing agent in glioblastoma. Four glioblastoma cell lines and one normal astrocyte cell line were incubated with a newly-synthetized phthalimido-alkanamide derivative, MA17, and its radiosensitizing effects were assessed. We performed a tumor growth delay assay in two glioblastoma lines: U87MG and U138MG. We evaluated DNA methyltransferase (DNMT) inhibition, apoptosis, autophagy, DNA damage repair, and FANCA expression. MA17 radiosensitized all glioblastoma cells (all p<0.05), but it did not affect normal astrocytes (p=0.193). MA17 significantly prolonged the mean tumor doubling time in vivo, in cells treated in addition with radiotherapy, compared to radiotherapy alone (p<0.05). DNMT activity was down-regulated, and apoptosis and autophagy were induced by MA17. Double-stranded DNA break foci were observed for prolonged periods in cells treated with MA17. FANCA expression was also inhibited. A novel phthalimido-alkanamide derivative demonstrated significant radiosensitization in glioblastoma cells in vitro and in vivo. Further investigation is needed to translate these results to the clinic.

BAP1 Is a Novel Target in HPV-Negative Head and Neck Cancer.

Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC).Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)-negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients.Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy.Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600-7. ©2017 AACR.

Metformin Inhibits Growth and Metastasis With Enhancement of Radiation Response in Hepatocellulcar Carcinoma Xenograft Model

This study aimed to investigate the pntential of metformin to inhibits the growth and metastasis with enhancement of radiation response in hepatocellular carcinoma (HCC) xenograft mice model. Huh-7 HCC cells were injected to the right thigh in Balb/c nude mice. Huh-7 bearing mice were treated with local tumor irradiation with a single 15 Gy of X-ray (RT), intraperitoneal administration of metformin with 150 mg/kg/day until the day of sacrifice (MET), or combination of both (administration of metformin 6 hours prior irradiation) (RT-MET). Tumor response to treatment was determined by a tumor growth delay assay. Metastatic potential was evaluated by gross qualitative observation of spontaneous pulmonary metastasis in a lung metastasis model on day 45 and 60. RT or MET resulted in a significant reduction of tumor growth and RT-MET enhanced it further than each treatment alone (55% reduction compared with RT and 67% reduction compared with MET). More importantly, RT or MET inhibited in the growth of metastatic nodules in lung and RT-MET enhanced it further than each treatment alone (93% reduction compared with RT and 76% reduction compared with MET). RT-MET decreased the expression of vascular endothelial growth factor and matrix metallopeptidase 9 by immunohistochemistry. Metformin inhibits the growth and metastasis of HCC with enhancement of radiation response in vivo. Our data suggest that metformin can be a clinically useful adjunct to radiotherapy in HCC.

In Vitro and in vivo Evaluation of Electrochemotherapy with trans-platinum Analogue trans-[PtCl2(3-Hmpy)2

BackgroundCisplatin is used in cancer therapy, but its side effects and acquired resistance to cisplatin have led to the synthesis and evaluation of new platinum compounds. Recently, the synthesized platinum compound trans-[PtCl2(3-Hmpy)2] (3-Hmpy = 3-hydroxymethylpyridine) (compound 2) showed a considerable cytotoxic and antitumour effectiveness. To improve compound 2 cytotoxicity in vitro and antitumour effectiveness in vivo, electroporation was used as drug delivery approach to increase membrane permeability (electrochemotherapy).Materials and methodsIn vitro, survival of sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensitive, TBLCl2Pt resistant and SA-1 moderately sensitive) was determined using a clonogenic assay after treatment with compound 2 or cisplatin electrochemotherapy. In vivo, the antitumour effectiveness of electrochemotherapy with compound 2 or cisplatin was evaluated using a tumour growth delay assay. In addition, platinum in the serum, tumours and platinum bound to the DNA in the cells were performed using inductively coupled plasma mass spectrometry.ResultsIn vitro, cell survival after treatment with compound 2 electrochemotherapy was significantly decreased in all tested sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensitive, TBLCl2Pt resistant and SA-1 moderately sensitive). However, this effect was less pronounced compared to cisplatin. Interestingly, the enhancement factor (5-fold) of compound 2 cytotoxicity was equal in cisplatin-sensitive TBLCl2 and cisplatin-resistant TBLCl2Pt cells. In vivo, the growth delay of subcutaneous tumours after treatment with compound 2 electrochemotherapy was lower compared to cisplatin. The highest antitumour effectiveness after cisplatin or compound 2 electrochemotherapy was obtained in TBLCl2 tumours, resulting in 67% and 11% of tumour cures, respectively. Compound 2 induced significantly smaller loss of animal body weight compared to cisplatin. Furthermore, platinum amounts in tumours after compound 2 or cisplatin electrochemotherapy were approximately 2-fold higher compared to the drug treatment only, and the same increase of platinum bound to DNA was observed.ConclusionsThe obtained results in vitro and in vivo suggest compound 2 as a potential antitumour agent in electrochemotherapy.

TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects.

Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. p16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. HNSCC cells and xenografts (HPV/p16-positive and -negative) were used. p16-overexpressing and small hairpin RNA-knockdown cells were generated, and the effect of p16 on radiosensitivity was determined by clonogenic cell survival and tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction; and data from The Cancer Genome Atlas HNSCC project were analyzed. p16 overexpression led to downregulation of TRIP12, which in turn led to increased RNF168 levels, repressed DNA damage repair (DDR), increased 53BP1 foci and enhanced radioresponsiveness. Inhibition of TRIP12 expression further led to radiosensitization, and overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. These findings reveal that p16 participates in radiosensitization through influencing DDR and support the rationale of blocking TRIP12 to improve radiotherapy outcomes.

Abstract 1661: TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects

Abstract Objective: Patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) have better responses to radiotherapy and higher overall survival rates than do patients with HPV-negative HNSCC, but the mechanisms underlying this phenomenon are unknown. P16 is used as a surrogate marker for HPV infection. Our goal was to examine the role of p16 in HPV-related favorable treatment outcomes and to investigate the mechanisms by which p16 may regulate radiosensitivity. Methods: HNSCC cells and xenografts were used. P16-overexpressing (HPV/p16-negative HN5 and UMSCC-1) and p16 shRNA knockdown (HPV/p16-positive UMSCC-47 and UCPI-SCC154) cells, TRIP12 shRNA or siRNA knockdown (HPV/p16-negative HN5 and Fadu) cells were generated. The effects of p16 or TRIP12 on HNSCC cell radiosensitivity were determined by clonogenic cell survival. The effects of p16 on tumor xenografts (HPV/p16-negative HN5 and HPV/p16-positive UMSCC-47) radioresponse were evaluated by tumor growth delay assays. DNA double-strand breaks (DSBs) were assessed by immunofluorescence analysis of 53BP1 foci; DSB levels were determined by neutral comet assay; western blotting was used to evaluate protein changes; changes in protein half-life were tested with a cycloheximide assay; gene expression was examined by real-time polymerase chain reaction (PCR); and data from The Cancer Genome Atlas HNSCC project were analyzed. Results: P16 expression led to downregulation of TRIP12 protein both in vitro and in vivo via decreasing TRIP12 protein's half life, which in turn led to increased RNF168 levels and subsequently repressed DNA damage repair, represented by increased 53BP1 foci and neutral comet moment tails at 24 hours after irradiation. As a result, p16 expression enhanced radioresponsiveness both in vitro and in vivo. Inhibition of TRIP12 expression led to radiosensitization through repressed DNA damage repair, represented by decreased BRCA1 foci at 1 and 5 hours after irradiation; and increased neutral comet moment tails at 24 hours after irradiation. Furthermore, overexpression of TRIP12 was associated with poor survival in patients with HPV-positive HNSCC. Conclusions: The findings of our study reveal that p16 participates in radiosensitization through influencing DNA damage repair. P16 downregulates TRIP12 protein expression via post-translational regulation. Inhibition of TRIP12 sensitized HNSCC cells via prohibition of DNA damage repair. These findings support the rationale of blocking TRIP12 signaling to improve radiotherapy outcomes. Citation Format: Li Wang, Peijing Zhang, David Molkentine, Jessica Molkentine, Uma Raju, David Valdecanas, Ramesh Tailor, Howard Thames, Thomas Buchholz, Junjie Chen, Li Ma, Kathy Mason, Raymond Meyn, Heath D. Skinner. TRIP12 as a mediator of human papillomavirus/p16-related radiation enhancement effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1661.

Electrochemotherapy by pulsed electromagnetic field treatment (PEMF) in mouse melanoma B16F10 in vivo.

IntroductionPulsed electromagnetic field (PEMF) induces pulsed electric field, which presumably increases membrane permeabilization of the exposed cells, similar to the conventional electroporation. Thus, contactless PEMF could represent a promising approach for drug delivery.Materials and methodsNoninvasive electroporation was performed by magnetic field pulse generator connected to an applicator consisting of round coil. Subcutaneous mouse B16F10 melanoma tumors were treated with intravenously injection of cisplatin (CDDP) (4 mg/kg), PEMF (480 bipolar pulses, at frequency of 80 Hz, pulse duration of 340 μs) or with the combination of both therapies (electrochemotherapy − PEMF + CDDP). Antitumor effectiveness of treatments was evaluated by tumor growth delay assay. In addition, the platinum (Pt) uptake in tumors and serum, as well as Pt bound to the DNA in the cells and Pt in the extracellular fraction were measured by inductively coupled plasma mass spectrometry.ResultsThe antitumor effectiveness of electrochemotherapy with CDDP mediated by PEMF was comparable to the conventional electrochemotherapy with CDDP, with the induction of 2.3 days and 3.0 days tumor growth delay, respectively. The exposure of tumors to PEMF only, had no effect on tumor growth, as well as the injection of CDDP only. The antitumor effect in combined treatment was related to increased drug uptake into the electroporated tumor cells, demonstrated by increased amount of Pt bound to the DNA. Approximately 2-fold increase in cellular uptake of Pt was measured.ConclusionsThe obtained results in mouse melanoma model in vivo demonstrate the possible use of PEMF induced electroporation for biomedical applications, such as electrochemotherapy. The main advantages of electroporation mediated by PEMF are contactless and painless application, as well as effective electroporation compared to conventional electroporation.

Abstract C145: Targeted radiosensitization of non-small cell lung cancer (NSCLC) through ADAM17 inhibition

Abstract Introduction: Radiotherapy is a mainstay for the treatment of locally advanced NSCLC. However, the response rates and clinical outcomes are still disappointing, with the 5-year survival rate being only approximately 15%. Growth and survival of NSCLC cells are often dependent on ectodomain cleavage of membrane-anchored proteins, which is primarily mediated by metalloproteases such as ADAMs (a disintegrin and metalloproteinase). Among all metalloproteases, ADAM17 is actively associated with this process of proteolytic ‘shedding’ and hence the release of growth factors and cytokines regulating cell proliferation and migration. In the current study, we provide novel insights into activation of ADAM17 and subsequent ligand shedding in response to irradiation. We show for the first time that direct targeting of ADAM17 with a clinically relevant inhibitor radiosensitizes lung carcinoma in vitro and in vivo. Material and methods: Large scale secretome profiling (&amp;gt;300 factors) was performed using antibody arrays for a wide range of secretory factors. Secretion kinetics of selected ADAM17 substrates were determined using ELISA across different established tumor cells and in murine blood serum, derived from irradiated A549 tumor xenograft-carrying mice. Clonogenic survival and xenograft tumor growth delay assays were performed in response to IR in siRNA-targeted tumor cell lines or in combination with small molecular agents. Tumor proliferation and ADAM17 downstream was evaluated employing immunohistochemistry and ex vivo blood serum analysis. Results: Based on a large scale secretome screening, we investigated secretion of auto- or paracrine factors in non–small cell lung cancer in response to irradiation and discovered the ADAM17 network as crucial mediator of resistance to IR. Irradiation triggered secretion of multiple ADAM17 substrates as ALCAM and amphiregulin in a similar IR-induced time- and dose-dependent manner across a panel of NSCLC cell lines and from A549-derived tumor xenografts. Irradiation also induced a dose-dependent increase of furin-mediated cleavage of the proform of ADAM17 to active ADAM17, which resulted in enhanced ADAM17 activity in vitro and in vivo. Genetic or pharmacologic targeting of ADAM17 suppressed IR-induced shedding of secreted factors, downregulated ErbB-signaling in target cells and enhanced IR-induced cytotoxicity in vitro. Furthermore, the combined treatment modality of IR with the ADAM17 inhibitor TMI-005 resulted in a supra-additive antitumor response in A549 tumor xenografts. Inhibition of ADAM17 with TMI-005 also strongly reduced immunohistochemical staining for ADAM17 substrates and MIB1, demonstrating an enduring antiproliferative effect and potency of ADAM17 targeting in combination with radiotherapy to overcome treatment resistance. Conclusions: These findings implicate that radiotherapy significantly activates ADAM17 in non–small cell lung cancer (NSCLC) cells, which results in shedding of multiple survival factors, growth factor pathway activation and contributes to treatment resistance. We provide a sound rationale for repositioning ADAM17 inhibitors as short-term adjuvants to improve the radiotherapy outcome of NSCLC. Citation Format: Ashish Sharma, Sabine Bender, Oliver Riesterer, Angela Broggini-Tenzer, Martin Pruschy. Targeted radiosensitization of non-small cell lung cancer (NSCLC) through ADAM17 inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C145.

PLK1 blockade enhances therapeutic effects of radiation by inducing cell cycle arrest at the mitotic phase

The cytotoxicity of ionizing radiation depends on the cell cycle phase; therefore, its pharmacological manipulation, especially the induction of cell cycle arrest at the radiosensitive mitotic-phase (M-phase), has been attempted for effective radiation therapy. Polo-like kinase 1 (PLK1) is a serine/threonine kinase that functions in mitotic progression, and is now recognized as a potential target for radiosensitization. We herein investigated whether PLK1 blockade enhanced the cytotoxic effects of radiation by modulating cell cycle phases of cancer cells using the novel small molecule inhibitor of PLK1, TAK-960. The TAK-960 treatment exhibited radiosensitizing effects in vitro, especially when it increased the proportion of M-phase cells. TAK-960 did not sensitize cancer cells to radiation when an insufficient amount of time was provided to induce mitotic arrest. The overexpression of a PLK1 mutant, PLK1-R136G&T210D, which was confirmed to cancel the TAK-960-mediated increase in the proportion of mitotic cells, abrogated the radiosensitizing effects of TAK-960. A tumor growth delay assay also demonstrated that the radiosensitizing effects of TAK-960 depended on an increase in the proportion of M-phase cells. These results provide a rational basis for targeting PLK1 for radiosensitization when considering the therapeutic time window for M-phase arrest as the best timing for radiation treatments.

Evaluation of therapeutic gain for fractionated carbon-ion radiotherapy using the tumor growth delay and crypt survival assays

Background and purposeThe aim of the study was to evaluate the therapeutic gain of carbon ion (C-ion) radiotherapy using a mouse model. Materials and methodsTransplanted fibrosarcoma (NFSa) growing in C3H/He mice and murine small intestine were irradiated with 290MeV/nucleon C-ion beams (C-ions) in 1–12 fractions separated by 4h. The cell killing efficiencies of C-ions were measured using jejunum crypt survival and tumor growth delay (TGD) assays. ResultsThe equieffect dose for crypt survival and TGD increased with increasing number of fractions after X-rays and 20keV/μm C-ions, whereas TGD after 77keV/μm C-ions rather decreased. Crypts showed stronger LET-dependent increase in α terms than the tumor while β terms less depended on LET irrespective of tissues. Therapeutic gain factor, i.e., a ratio of tumor RBE over crypt RBE, of 77keV/μm C-ions was more than unity at any doses while that of 20keV/μm C-ions increased with an increase in dose per fraction. ConclusionsThese specific data imply that use of large dose per fraction would be suitable for C-ion radiotherapy irrespective of LET from the point of view of therapeutic gain, though small dose per fraction by high-LET radiation decreases total dose for tumor.

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model

ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Western blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.

Inhibition of EGFR or IGF-1R signaling enhances radiation response in head and neck cancer models but concurrent inhibition has no added benefit.

Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established in many cancer types. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (RT). The effects of cetuximab and IMC-A12 on cell viability and radiosensitivity were determined by clonogenic cell survival assay. Formation of nuclear γ-H2AX and 53BP1 foci was monitored by immunofluorescence. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of triple therapy with IMC-A12, cetuximab, and RT. In vitro data showed that cetuximab differentially affected the survival and the radiosensitivity of HNSCC cells. Cetuximab suppressed DNA repair that was evident by the prolonged presence of nuclear γ-H2AX and 53BP1 foci. IMC-A12 did not have any effect on the cell survival. However, it increased the radiosensitivity of one of the cell lines. EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. Addition of IMC-A12 to cetuximab did not increase the radiosensitivity of these cells. Tumor xenografts exhibited enhanced response to RT in the presence of either cetuximab or IMC-A12. Concurrent treatment regimen failed to further enhance the tumor response to cetuximab and/or RT. Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways did not yield additional therapeutic benefit in overcoming resistance to RT.

Abstract 857: Preclinical evaluation: efficacy of DI17E6, a therapeutic antibody against alpha v integrins, compared to cilengitide in sensitizing human cancers to radiation therapy

Abstract Purpose: Integrins are implicated in resistance of solid tumors to radiation therapy, suggesting that their inhibition would enhance therapeutic efficacy. We investigated the efficacy of DI17E6, a therapeutic antibody targeting alpha v (αv) integrins, in enhancing the radiosensitivity of non-small cell lung carcinoma (NSCLC) and head &amp; neck cancer (HNSCC) cells using in vitro and in vivo models, and compared to the previously reported effects of the cyclic peptide cilengitide (inhibits αvβ3 and αvβ5 integrins). Methods: NSCLC lines (A549 and H460) and HNSCC lines (FaDu and HN-5) were examined. Basal level of αv integrin was assessed by viable cell flow-cytometry and by Western blot. The effects of DI17E6 on cell viability were determined by MTS assay, and on cellular radiosensitivity by clonogenic cell survival assay after exposing the cells to γ-radiation with or without DI17E6. In vivo effect of DI17E6 on radioresponse of xenografts was evaluated by tumor growth delay assay. DI17E6 was given i.p.; as a single loading dose of 180 mg/kg followed by weekly doses of 10, 30 or 100 mg/kg given for 6 weeks; fractionated doses of local tumor irradiation was delivered using 137Cs γ-ray source (2 Gy daily for 7 days). When combined, the loading dose of DI17E6 was given either 4 h before or after the first radiation dose. Results: DI17E6 (0.01-10 µg/ml, 24 h) had a minimal effect on the cell viability. However, DI17E6 reduced the plating efficiency of A549, H460 and FaDu with no effect on HN-5 cells. When combined with radiation, DI17E6 enhanced the radiosensitivity of NSCLC cells but not the HNSCC cells. These data are consistent with previously observed effects of cilengitide in vitro. In vivo experiments with A549 xenografts showed that DI17E6 as a single agent had no antitumor activity, but in combination with radiation it suppressed the tumor growth in a schedule- and dose-dependent manner. These data were compared to the previously reported effect of cilengitide, which did not enhance the response of A549 xenografts to radiation therapy. DI17E6 has an extended pharmacokinetics (with a half-life of &amp;gt;7 days) and blocks more αv integrins than cilengitide. The results imply that when αvβ3 and αvβ5 integrins were inhibited by cilengitide either the duration of blockade was insufficient, or that other αv integrins may have provided tumor growth advantage. In vivo studies on other tumor types are underway. Conclusions: Inhibition of αv integrins by DI17E6 in combination with radiation enhanced radiosensitivity of NSCLC cell lines in vitro and in vivo enhanced A549 tumor xenograft response to radiation in mice. These results suggest that DI17E6 may improve the treatment outcome of patients with NSCLC when combined with radiotherapy in a clinical setting. Supported by EMD Serono Merck KGaA, Germany Citation Format: Amit A. Deorukhkar, David P. Molkentine, David R. Valdecanas, Kathryn A. Mason, Uma Raju. Preclinical evaluation: efficacy of DI17E6, a therapeutic antibody against alpha v integrins, compared to cilengitide in sensitizing human cancers to radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 857. doi:10.1158/1538-7445.AM2014-857

Combination of Metformin and Statins Significantly Enhanced the Local Radio therapeutic Effect and Attenuated the Lung Metastasis of Lewis Lung Carcinoma (LLC) Animal Model

Purpose/Objective(s): Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established inmany cancer types. Ligands of these receptors are capable of activating both pathways. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (IR). Materials/Methods: SixHNSCCmodels expressing different levels of EGFR and IGF-1R were studied. The effects of cetuximab and IMC-A12 on cell viability (MTS assay) and radiosensitivity (clonogenic survival assay) were determined. Formation of nuclear g-H2AX and 53BP1 foci was monitored by immunofluorescence to assess the IR-induced DNA damage and repair kinetics. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of IMCA12 in enhancing tumor response to cetuximab and radiation using two HNSCC tumor xenograft models generated in mice. Results: In vitro studies in six HNSCC lines showed that exposure to cetuximab resulted in increased cell radiosensitivity in two cell lines. EGFR inhibition suppressed DNA repair that was evident by the prolonged presence of nuclear g-H2AX and 53BP1 foci. Western blot analyses showed that EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. When IMC-A12 was added concurrently to the treatment regimen along with cetuximab, a further increase in radiosensitivity was observed in one of the three cell lines tested. In vivo tumor growth delay data in two of the HNSCC tumor models showed that tumor xenografts exhibited enhanced tumor response to RT in the presence of either cetuximab or IMC-A12 depending on the tumor type. Addition of IMC-A12 to cetuximab and IR treatment regimen failed to further enhance the tumor response to cetuximab as well as to IR. Conclusions: Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways using their respective antibodies did not yield additional therapeutic benefit in overcoming resistance to RT. Author Disclosure: U. Raju: None. D.P. Molkentine: None. D.R. Valdecanas: None. A.A. Deorukhkar: None. K.A. Mason: None. T.A. Buchholz: None. R.E. Meyn: None. K.K. Ang: None. H. Skinner: None.

Inhibition of IGF-1R Signaling Enhances the Head and Neck Tumor Radioresponse but Fails to Further Improve Tumor Response to Cetuximab and Radiation Therapy In Vivo

Purpose/Objective(s): Interaction between the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGF-1R) has been well established inmany cancer types. Ligands of these receptors are capable of activating both pathways. We investigated the effects of cetuximab (EGFR antibody) and IMC-A12 (IGF-1R antibody) on the response of head and neck squamous cell carcinoma (HNSCC) to radiation therapy (IR). Materials/Methods: SixHNSCCmodels expressing different levels of EGFR and IGF-1R were studied. The effects of cetuximab and IMC-A12 on cell viability (MTS assay) and radiosensitivity (clonogenic survival assay) were determined. Formation of nuclear g-H2AX and 53BP1 foci was monitored by immunofluorescence to assess the IR-induced DNA damage and repair kinetics. Alterations in target signaling were analyzed by Western blots. In vivo tumor growth delay assay was performed to determine the efficacy of IMCA12 in enhancing tumor response to cetuximab and radiation using two HNSCC tumor xenograft models generated in mice. Results: In vitro studies in six HNSCC lines showed that exposure to cetuximab resulted in increased cell radiosensitivity in two cell lines. EGFR inhibition suppressed DNA repair that was evident by the prolonged presence of nuclear g-H2AX and 53BP1 foci. Western blot analyses showed that EGFR inhibition increased IGF-1R expression levels and also the association between EGFR and IGF-1R. When IMC-A12 was added concurrently to the treatment regimen along with cetuximab, a further increase in radiosensitivity was observed in one of the three cell lines tested. In vivo tumor growth delay data in two of the HNSCC tumor models showed that tumor xenografts exhibited enhanced tumor response to RT in the presence of either cetuximab or IMC-A12 depending on the tumor type. Addition of IMC-A12 to cetuximab and IR treatment regimen failed to further enhance the tumor response to cetuximab as well as to IR. Conclusions: Taken together our data suggest that concomitant inhibition of both EGFR and IGF-1R pathways using their respective antibodies did not yield additional therapeutic benefit in overcoming resistance to RT. Author Disclosure: U. Raju: None. D.P. Molkentine: None. D.R. Valdecanas: None. A.A. Deorukhkar: None. K.A. Mason: None. T.A. Buchholz: None. R.E. Meyn: None. K.K. Ang: None. H. Skinner: None.