Days For Tumors Research Articles

Evaluating the immunologically “cold” tumor microenvironment after treatment with immune checkpoint inhibitors utilizing PET imaging of CD4 + and CD8 + T cells in breast cancer mouse models

BackgroundImmune-positron emission tomography (PET) imaging with tracers that target CD8 and granzyme B has shown promise in predicting the therapeutic response following immune checkpoint blockade (ICB) in immunologically “hot” tumors. However, immune dynamics in the low T-cell infiltrating “cold” tumor immune microenvironment during ICB remain poorly understood. This study uses molecular imaging to evaluate changes in CD4 + T cells and CD8 + T cells during ICB in breast cancer models and examines biomarkers of response.Methods[89Zr]Zr-DFO-CD4 and [89Zr]Zr-DFO-CD8 radiotracers were used to quantify changes in intratumoral and splenic CD4 T cells and CD8 T cells in response to ICB treatment in 4T1 and MMTV-HER2 mouse models, which represent immunologically “cold” tumors. A correlation between PET quantification metrics and long-term anti-tumor response was observed. Further biological validation was obtained by autoradiography and immunofluorescence.ResultsFollowing ICB treatment, an increase in the CD8-specific PET signal was observed within 6 days, and an increase in the CD4-specific PET signal was observed within 2 days in tumors that eventually responded to immunotherapy, while no significant differences in CD4 or CD8 were found at the baseline of treatment that differentiated responders from nonresponders. Furthermore, mice whose tumors responded to ICB had a lower CD8 PET signal in the spleen and a higher CD4 PET signal in the spleen compared to non-responders. Intratumoral spatial heterogeneity of the CD8 and CD4-specific PET signals was lower in responders compared to non-responders. Finally, PET imaging, autoradiography, and immunofluorescence signals were correlated when comparing in vivo imaging to ex vivo validations.ConclusionsCD4- and CD8-specific immuno-PET imaging can be used to characterize the in vivo distribution of CD4 + and CD8 + T cells in response to immune checkpoint blockade. Imaging metrics that describe the overall levels and distribution of CD8 + T cells and CD4 + T cells can provide insight into immunological alterations, predict biomarkers of response to immunotherapy, and guide clinical decision-making in those tumors where the kinetics of the response differ.

Peripheral insulin resistance is early, progressive, and correlated with cachexia in Walker-256 tumor-bearing rats.

Insulin (INS) resistance is often found in cancer-bearing, but its correlation with cachexia development is not completely established. This study investigated the temporal sequence of the development of INS resistance and cachexia to establish the relationship between these factors in Walker-256 tumor-bearing rats (TBrats). INS hepatic sensitivity and INS resistance-inducing factors, such as free fatty acids (FFA) and tumor necrosis factor-α (TNF-α), were also evaluated. Studies were carried out on Days 2, 5, 8, and/or 12 after inoculation of tumor cells in rats. The peripheral INS sensitivity was assessed by theINS tolerance testand the INS hepatic sensitivity in in situ liver perfusion. TBrats with 5, 8, and 12 days of tumor, but not 2 days, showed decreased peripheral INS sensitivity (INS resistance), retroperitoneal fat, and body weight, compared to healthy rats, which were more pronounced on Day 12. Gastrocnemius muscle wasting was observed only on Day 12 of tumor. The peripheral INS resistance was significantly correlated (r = -.81) with weight loss. Liver INS sensitivity of TBrats with 2 and 5 days of tumor was unchanged, compared to healthy rats. TBrats with 12 days of tumor showed increased plasma FFA and increased TNF-α in retroperitoneal fat and liver, but not in the gastrocnemius, compared to healthy rats. In conclusion, peripheral INS resistance is early, starts along with fat and weight loss and before muscle wasting, progressive, and correlated with cachexia, suggesting that it may play an important role in the pathogenesis of the cachectic process in TBrats. Therefore, early correction of INS resistance may be a therapeutic approach to prevent and treat cancer cachexia.

Estimating Distributions of Breast Cancer Onset and Growth in a Swedish Mammography Screening Cohort.

In recent years, biologically motivated continuous tumor growth models have been introduced for breast cancer screening data. These provide a novel framework from which mammography screening effectiveness can be studied. We use a newly developed natural history model, which is unique in that it includes a carcinogenesis model for tumor onset, to analyze data from a large Swedish mammography cohort consisting of 65,536 participants, followed for periods of up to 6.5 years. Using patient data on age at diagnosis, tumor size, and mode of detection, as well as screening histories, we estimate distributions of patient's age at onset, (inverse) tumor growth rates, symptomatic detection rates, and screening sensitivities. We also allow the growth rate distribution to depend on the age at onset. We estimate that by the age of 75, 13.4% of women have experienced onset. On the basis of a model that accounts for the role of mammographic density in screening sensitivity, we estimated median tumor doubling times of 167 days for tumors with onset occurring at age 40, and 207 days for tumors with onset occurring at age 60. With breast cancer natural history models and population screening data, we can estimate latent processes of tumor onset, tumor growth, and mammography screening sensitivity. We can also study the relationship between the age at onset and tumor growth rates. Quantifying the underlying processes of breast cancer progression is important in the era of individualized screening.

Insulin secretion decline in Walker-256 tumor-bearing rats is early, follows the course of cachexia, and is not improved by lixisenatide.

Lixisenatide, a glucagon-like peptide-1 receptor agonist, is used to stimulate insulin secretion in patients with type 2 diabetes mellitus. However, its effect on insulin secretion in cancer patients, particularly during the cachexia course, has not yet been evaluated. The purpose of this study was to investigate the lixisenatide effect on INS secretion decline during the cachexia course (2, 6, and 12 days of tumor) in pancreatic islets isolated from Walker-256 tumor-bearing rats. Pancreatic islets of healthy and tumor-bearing rats were incubated in the presence or absence of lixisenatide (10 nM). Tumor-bearing rats showed reduction of body weight and fat and muscle mass, characterizing the development of cachexia, as well as reduction of insulinemia and INS secretion stimulated by glucose (5.6, 8.3, 11.1, 16.7, and 20 mM) on days 2, 6, and/or 12 of tumor. Lixisenatide increased the 16.7 mM glucose-stimulated insulin secretion, but not by 5.6 mM glucose, in the islets of healthy rats, without changing the insulin intracellular content. However, lixisenatide did not prevent the decreased 16.7 mM glucose-stimulated insulin secretion in the pancreatic islets of rats with 2, 6, and 12 days of tumor and neither the decreased insulin intracellular content of rats with 12 days of tumor. In consistency, in vivo treatment with lixisenatide (50 μg kg-1, SC, once daily, for 6 days) visually increased insulinemia of healthy fasted rats, but did not prevent hypoinsulinemia of tumor-bearing rats. In conclusion, Walker-256 tumor-bearing rats showed early decline (2 days of tumor) of insulin secretion, which followed the cachexia course (6 and 12 days of tumor) and was not improved by lixisenatide, evidencing that this insulin secretagogue, used to treat type 2 diabetes, does not have beneficial effect in cancer bearing-rats.

Moderate Heat Application Enhances the Efficacy of Nanosecond Pulse Stimulation for the Treatment of Squamous Cell Carcinoma

Nanosecond pulse stimulation as a tumor ablation therapy has been studied for the treatment of various carcinomas in animal models and has shown a significant survival benefit. In the current study, we found that moderate heating at 43°C for 2 minutes significantly enhanced in vitro nanosecond pulse stimulation-induced cell death of KLN205 murine squamous cell carcinoma cells by 2.43-fold at 600 V and by 2.32-fold at 900 V, as evidenced by propidium iodide uptake. Furthermore, the ablation zone in KLN205 cells placed in a 3-dimensional cell-culture model and pulsed at a voltage of 900 V at 43°C was 3 times larger than in cells exposed to nanosecond pulse stimulation at room temperature. Application of moderate heating alone did not cause cell death. A nanosecond pulse stimulation electrode with integrated controllable laser heating was developed to treat murine ectopic squamous cell carcinoma. With this innovative system, we were able to quickly heat and maintain the temperature of the target tumor at 43°C during nanosecond pulse stimulation. Nanosecond pulse stimulation with moderate heating was shown to significantly extend overall survival, delay tumor growth, and achieve a high rate of complete tumor regression. Moderate heating extended survival nearly 3-fold where median overall survival was 22 days for 9.8 kV without moderate heating and over 63 days for tumors pulsed with 600, 100 ns pulses at 5 Hz, at voltage of 9.8 kV with moderate heating. Median overall survival in the control groups was 24 and 31 days for mice with untreated tumors and tumors receiving moderate heat alone, respectively. Nearly 69% (11 of 16) of tumor-bearing mice treated with nanosecond pulse stimulation with moderate heating were tumor free at the completion of the study, whereas complete tumor regression was not observed in the control groups and in 9.8 kV without moderate heating. These results suggest moderate heating can reduce the necessary applied voltage for tumor ablation with nanosecond pulse stimulation.

Abstract 4900: Cysteamine suppresses tumor metastasis by inhibiting activity of matrix metalloproteases without inducing toxicity in mouse models of human ovarian cancer

Abstract Previously, we demonstrated that cysteamine, a small molecule inhibitor of matrixmetalloproteases (MMPs), inhibited tumor invasion in vitro and metastasis in vivo in mouse models of human pancreatic and ovarian cancer. We have also shown that subcutaneous cysteamine administration improved the survival of mice with orthotopically transplanted pancreatic tumors. Herein, we examined the effect of cysteamine on total MMP activity, MMP isoforms and activity by substrate gel zymography in tumors obtained from orthotopic mouse model of human ovarian cancer. We also investigated any gastro-intestinal and general toxicity in vital organs of mice treated with the highest dose of cysteamine (250 mg/kg). We developed orthotopic tumors by injecting two human ovarian cancer cell lines in the ovary of female athymic nude mice. After 4 days of tumor implant, the mice were treated with different doses of cysteamine and followed for 5 weeks. Total MMP activity by fluorogenic substrate, MMP-2, 9 and 14 by ELISA and MMP activity by substrate gel zymography were measured in tumor lysates and compared with untreated tumors. We performed H&E staining in vital organs such as heart, lung, liver, kidney, spleen and brain for evaluating general toxicity of cysteamine. In addition, we examined stomach and duodenum from cysteamine treated and vehicle treated mice if they developed any ulcerations. In two orthotopic murine models of human ovarian cancer, consistent with our previous observations, tumor metastasis was significantly decreased compared to controls in cysteamine treated mice. Similarly, total MMP activity was also significantly decreased in primary tumors treated with cysteamine in a dose dependent manner (P≤0.05). Zymographic MMP activity was also decreased significantly in cysteamine treated tumors compared to control corroborating with total MMP activity (P≤0.05). Interestingly, ELISA did not show any changes in MMP expression in cysteamine treated and untreated tumors. Furthermore, we did not observe any symptoms of general toxicity in animals treated at the highest dose of cysteamine and major vital organs and gastro-intestinal organs from treated animals showed no evidence of histological changes as evident by H&E staining. In conclusion, our results suggest that cysteamine is a potent inhibitor of MMP activity without affecting expression. Cysteamine does not cause any gastro-intestinal or vital organ toxicity and may be useful therapeutic agent for ovarian cancer either alone or in combination therapy with known anti-cancer agents. Citation Format: Akiko Suzuki, Rukmini Bhardwaj, Pamela Leland, Bharat H. Joshi, Raj K. Puri. Cysteamine suppresses tumor metastasis by inhibiting activity of matrix metalloproteases without inducing toxicity in mouse models of human ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4900. doi:10.1158/1538-7445.AM2017-4900

Diethyldithiophosphate (DEDTP) Role in Proportion of Macrophages and Natural Killer Cells in Lymph Vessel and 4T1 Murine Breast Tumor Model

The diethyldithiophosphate (DEDTP) is a metabolite produced by the degradation of organophosphorus pesticides (POFs), and a dialkylphosphate (DAP) which is also chemically synthesized with a widespread commercial use and so the world population is chronically exposed to this compound. It is suggested that organophosphorus compounds produce deleterious effects on the immune system of the exposed population, since it has been demonstrated that DAPs affect peripheral blood lymphocytes activation and proliferation of T lymphocytes, furthermore, in vivo assays using C57 mice, the exposure to DEDTP enlarges skin tumors of BMK (Baby Mouse Kidney) cells, these results suggest that DEDTP exposure in vivo may modify the immune response and enhance the growth of tumor cells. Therefore, our objective is to determine the effect of DEDTP in the proportion of macrophages and natural killer cells (NKs) in lymph nodes and 4T1 breast cancer tumors in mice. To this end, we used Balb/c female mice of 6–8 weeks old, exposed for 8 days to 0.01 g/kg i.p. of DEDTP and inoculated with 1×105 4T1 cells. Tumors and lymph nodes were excised and measured. The percentage of total (F4/80+) and M2 (CD206+) macrophages and natural killer (NK) cells (CD355+) were assessed by flow cytometry. Exposure to DEDTP for 8 days increased the proportion of M2 macrophages. After 15 days of exposure to DEDTP, NK cells proportion was reduced but increased after 20 days. In mice challenged with 4T1 cells during DEDTP exposure, the number of total macrophages increased while the proportion of CD355+ cells in the lymph node proximal to the tumor decreased after 5 days; 10 days after, the number of M2 macrophages increased. Finally, after 20 days the number of M2 macrophages and NK cells in the proximal lymph node increased. In tumors, the number of infiltrating NK cells and total macrophages were reduced, followed by a reduction of M2 macrophages. Subsequently, after 15 days we observed an increase of infiltrating NK cells and M2 macrophages; after 20 days of tumor, M2 macrophages were increased. None of these changes had a significant impact on the establishment and development of tumors. With these results we conclude that exposure to DEDTP, in the absence of a particular antigen challenge, exerts immunomodulatory effects on individuals, apparently by inducing alternative activation of macrophages, however, this effect had no significant effect in the growth of 4T1 tumor cells. This could probably be because the more aggressive behavior of 4T1 tumor cells than BMK cells (previously tested on C57 mice) and the pronounced Th2 immune response profile of Balb/c mice (contrary to C57 animals). These results suggest that the effect of DEDTP on the immune system is not decisive against an aggressive tumor challenge, but may have relevance against a bacterial or parasitic infectious challenge.Support or Funding InformationCONACyT grant 153468

Pharmacokinetics and Biodistribution of Locked Nucleic Acid (LNA)-Mir-221 Inhibitor: A New Promising Anti-Myeloma Agent

miR-221/222 are highly homologous microRNAs (miRNAs) whose upregulation has been found in several malignancies, including multiple myeloma (MM). Both miRNAs are thought to promote cell proliferation via down-regulation of p27 and/or p57, two negative regulators of G1 to S phase cell cycle progression.We proved that silencing of both miRNAs results in significant anti-MM activity and in downregulation of canonical targets both in vitro and in vivo. In the aim to progress to clinical translation, we designed an original 13mer synthetic inhibitor, specific for systemic delivery, named LNA-i-miR-221, which took advantage from both locked nucleic acid (LNA) technology and phosphorothioate backbone, for increasing the seed sequence binding and nuclease resistance in vivo, respectively. Since no data are presently available, we evaluated the specificity of LNA-i-miR-221 to inhibit endogenous miR-221 and the pharmacokinetic properties (i.e. tissue distribution) in mice and Cynomolgus monkeys.We demonstrated that LNA-i-miR-221 inhibit growth and survival of MM cells bearing high miR-221 levels. Moreover, LNA-i-miR-221-mediated silencing of miR-221 triggered upregulation of p27Kip1 mRNA and protein, evaluated by q-RT-PCR and western blotting, respectively. In vivo, i.p.treatment with 25 mg/kg of LNA-i-miR-221 reduced tumor growth in SCID/NOD mice bearing MM xenografts. Tumors and vital organs (including liver, kidney, bone marrow and heart) were harvested from treated animals and evaluated for pharmacokinetics purposes using in situ hybridization (ISH) assay. After a single i.p. dose of 25 mg/kg, we detected the presence of LNA-i-miR-221 from 2 up to 7 days in tumors and mouse tissues. Interestingly, no toxicity was observed even after long-lasting presence of the inhibitors in vital organs. We also evaluated the LNA-i-miR-221 half-life in mouse plasma using HPLC-MS/MS, which confirmed the rapid uptake of LNA-i-miR-221 in tissues. To evaluate the maximum tolerated doses, in vivo dose escalation treatments was performed using doses from 10 to 100 mg/kg delivered at days 1,4,8,15,22. All treatments were well tolerated. No changes in mice behavior or organ toxicity were observed in treated mice.Finally, a pilot toxicity study was performed in Cynomolgus monkeys. PK results demonstrated that LNA-i-miR-221 has a short serum half-life with a rapid tissue uptake and minimum urinary escretion.In conclusion, our results suggest that LNA-i-miR-221 is a promising anti-MM agent associated with a safety profile in mice and in monkeys, supporting the rationale for development of this novel miR-221 inhibitor in early clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Abstract 69: In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128.

Abstract Radiation-induced translational control of gene expression has been shown to be a component of the cellular radioresponse. Furthermore, recent data suggest strategies targeting eIF4E may be effective for tumor cell radiosensitization. The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of eIF4E and mRNA translation. The newly developed ATP-competitive mTOR inhibitor, INK128, inhibits both mTORC1 and mTORC2 activity, and is in currently undergoing evaluation in Phase 1 clinical trials. In the study described here we evaluated the effects of INK128 treatment on tumor cell radiosensitivity in vitro using a clonogenic survival assay and in vivo according to tumor growth delay. For tumor cell lines initiated from different histologic origins in vitro exposure to INK128 resulted in the complete inhibition of both mTORC1 and 2. Treatment with the same concentration of INK128 immediately after irradiation increased tumor cell radiosensitivity with DEFs greater than 1.3. To determine whether the INK128 induced radiosensitization could be extended to an in vivo model we used subcutaneous xenografts initiated from U251 or PSN1 tumor cells. INK128 treatment (oral gavage) inhibited mTOR activity in tumors established from both cell lines as defined by loss of 4E-BP1 phosphorylation. Furthermore, m7-GTP batch chromatography showed that INK128 treatment decreased cap-binding activity in xenografts. To evaluate the effects of INK128 on in vivo tumor radiosensitivity a tumor growth delay assay was performed using mice bearing U251 tumor xenografts. When tumors reached approximately 210mm3 mice were randomized into 4 groups: vehicle, radiation, INK128, and INK128 plus radiation. INK128 was delivered once a day (3 mg/kg, oral gavage) for four days with the tumor locally irradiated (2 Gy) 2h after each of the four drug treatments. Absolute growth delays were calculated as the time in days for tumors in treated mice to grow from 210 to 1000mm3 minus the time in days for tumors to reach the same size in vehicle treated mice. The absolute growth delays for INK128 alone and radiation alone groups were 1.7 and 12.7 days respectively. The growth delay in mice treated with the combination of INK128 and radiation was 22.2 days, which is greater than the sum of the growth delays caused by INK128 and radiation alone. The DEF, obtained by dividing the normalized tumor growth delay in mice treated with the radiation/PP242 combination (20.5) by the absolute growth delay in mice treated with radiation only (12.7), was 1.6. These results suggest that mTOR inhibition by the clinically relevant ATP-competitive mTOR inhibitor, INK128, enhances tumor cell radiosensitivity both in vitro and in vivo. Citation Format: Thomas J. Hayman, Tamalee Kramp, Kevin Camphausen, Philip J. Tofilon. In vitro and in vivo tumor cell radiosensitization by the competitive mTOR inhibitor INK128. [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 69. doi:10.1158/1538-7445.AM2013-69

Abstract 1461: MK-4827, a PARP-1/-2 inhibitor, strongly enhances response of human lung and breast cancer xenografts to radiation

Abstract The poly-(ADP-ribose) polymerase (PARP) inhibitor, MK-4827, is a novel potent, orally bioavailable PARP-1 and PARP-2 inhibitor currently in phase I clinical trials for cancer treatment. No preclinical data currently exist on the combination of MK-4827 with radiotherapy (XRT). We examined whether MK-4827 enhances radiosensitivity of a variety of human tumor xenografts of differing p53 status. Human lung cancer xenografts, Calu-6 (p53 null), A549 (p53 wild-type [wt]) and H-460 (p53 wt) and triple negative MDA-MB-231 human breast carcinoma xenografts were used. Tumor xenografts growing in the hind leg of nude mice were exposed to fractionated XRT at 8 mm (diameter). Fractionated XRT (2 Gy per fraction) was delivered for 7 (twice daily, 6 h in between) or 14 (once daily) consecutive days, total dose 28 Gy. MK-4827 was given by gavage once (50 mg/kg, 1 h before first XRT of the day) or twice daily (25 mg/kg, 1 h before each XRT of the day) starting when tumors were 6 mm diameter. Mice received (a) no treatment, (b) MK-4827, (c) XRT or (d) MK-4827 plus XRT. Tumor growth delay (TGD, days for tumors to grow from 8.0 mm to 12.0 mm) was the endpoint. Radiation enhancement factor (EF) was then calculated. MK-4827 alone had no effect on tumor growth for any of the tumors types. For p53 mutant Calu-6 xenografts TGD for untreated tumors was 13.2 ± 0.5 and for twice daily XRT was 30.9 ± 2.4 days. Combination of XRT for 7 days with MK-4827, 25mg/kg twice daily for 21 consecutive days, further extended TGD to 39.7 ± 2.0 days (EF 1.4) and 47.8 ± 4.0 days (EF 2.03) for MK-4827, 50mg/kg once daily. Maximum radiation enhancement was observed when MK-4827 was given at a dose of 50 mg/kg once daily. For XRT and MK-4827 given once daily, MK-4827 was continued until 2 days after completion of XRT, MK-4827 was found to be similarly effective and the EF was 1.7. For p53 wt H460and A459 xenografts, twice daily XRT and once daily MK-4827 was used. MK-4827 was continued until 2 days after completion of XRT. TGD for untreated H460 and A459 xenografts was 7.8 ± 0.5 and 33.2 ± 1.5 days respectively; whereas it was 15.6 ± 0.5 and 53.2 ± 3.1 days respectively for irradiated tumors. TGDs were further extended to 26.0 ± 3.5 days for H460 (EF 2.2) and 64.1 ± 4.2 days (EF 1.9) for A549 xenografts in MK-4827 plus XRT groups. Similarly, using MDA-MB-231 human breast carcinoma xenografts, the potency of combination treatment was greater than XRT alone showing essentially no local tumor regrowth until the occurrence of metastases. When MK-4827 was given as a single dose of 50 mg/kg and tumors were assayed for PAR, MK-4827 reduced PAR levels in tumors by 1 h after administration which persisted for up to 24 h. In conclusion, MK-4827 strongly enhanced the effect of radiation on a variety of human tumor xenografts irrespective of p53 status. The long lasting PARP inhibition (1-24h) potentially adds flexibility to future clinical trial design. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1461. doi:1538-7445.AM2012-1461

Combination radiofrequency (RF) ablation and IV liposomal heat shock protein suppression: Reduced tumor growth and increased animal endpoint survival in a small animal tumor model

BackgroundTo investigate the effect of IV liposomal quercetin (a known down-regulator of heat shock proteins) alone and with liposomal doxorubicin on tumor growth and end-point survival when combined with radiofrequency (RF) tumor ablation in a rat tumor model. MethodsSolitary subcutaneous R3230 mammary adenocarcinoma tumors (1.3–1.5cm) were implanted in 48 female Fischer rats. Initially, 32 tumors (n=8, each group) were randomized into four experimental groups: (a) conventional monopolar RF alone (70°C for 5min), (b) IV liposomal quercetin alone (1mg/kg), (c) IV liposomal quercetin followed 24hr later with RF, and (d) no treatment. Next, 16 additional tumors were randomized into two groups (n=8, each) that received a combined RF and liposomal doxorubicin (15min post-RF, 8mg/kg) either with or without liposomal quercetin. Kaplan-Meier survival analysis was performed using a tumor diameter of 3.0cm as the defined survival endpoint. ResultsDifferences in endpoint survival and tumor doubling time among the groups were highly significant (P<0.001). Endpoint survivals were 12.5±2.2days for the control group, 16.6±2.9days for tumors treated with RF alone, 15.5±2.1days for tumors treated with liposomal quercetin alone, and 22.0±3.9days with combined RF and quercetin. Additionally, combination quercetin/RF/doxorubicin therapy resulted in the longest survival (48.3±20.4days), followed by RF/doxorubicin (29.9±3.8days). ConclusionsIV liposomal quercetin in combination with RF ablation reduces tumor growth rates and improves animal endpoint survival. Further increases in endpoint survival can be seen by adding an additional anti-tumor adjuvant agent liposomal doxorubicin. This suggests that targeting several post-ablation processes with multi-drug nanotherapies can increase overall ablation efficacy.

Antiangiogenic Cancer Therapy: Monitoring with Molecular US and a Clinically Translatable Contrast Agent (BR55)

To develop and test human kinase insert domain receptor (KDR)-targeted microbubbles (MBs) (MB(KDR)) for imaging KDR at the molecular level and for monitoring antiangiogenic therapy in a human colon cancer xenograft tumor model in mice. Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. A heterodimeric peptide that binds to human KDR with low nanomolar affinity (K(D) = 0.5 nmol/L) was coupled onto the surface of perfluorobutane-containing lipid-shelled MBs (MB(KDR)). Binding specificity of MB(KDR) to human KDR and cross-reactivity with murine vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) were tested in cell culture under flow shear stress conditions (at 100 sec(-1)). In vivo binding specificity of MB(KDR) to VEGFR2 was tested in human LS174T colon cancer xenografts in mice with a 40-MHz ultrasonographic (US) transducer. Targeted contrast material-enhanced US imaging signal by using MB(KDR) was longitudinally measured during 6 days in tumors with (n = 6) and without (n = 6) antiangiogenic treatment (anti-VEGF antibody). Ex vivo VEGFR2 staining and microvessel density analysis were performed. Significant differences were evaluated (t, Mann-Whitney, or Wilcoxon test). Cell culture experiments showed four times greater binding specificity of MB(KDR) to human KDR and cross-reactivity to murine VEGFR2 (P < or = .01). In vivo imaging signal was more than three times higher (P = .01) with MB(KDR) compared with control MBs and decreased significantly (approximately fourfold lower, P = .03) following in vivo receptor blocking with anti-VEGFR2 antibody. One day after initiation of antiangiogenic therapy, imaging signal was significantly decreased (approximately 46% lower, P = .02) in treated versus untreated tumors; it remained significantly lower (range, 46%-84% decreased; P = .038) during the following 5 days. Microvessel density was significantly reduced (P = .04) in treated (mean, 7.3 microvessels per square millimeter +/- 4.7 [standard deviation]) versus untreated tumors (mean, 22.0 microvessels per square millimeter +/- 9.4); VEGFR2 expression was significantly decreased (>50% lower, P = .03) in treated tumors. Human MB(KDR) allow in vivo imaging and longitudinal monitoring of VEGFR2 expression in human colon cancer xenografts.

Abstract 4420: ABI-013: A novel nanoparticle albumin-bound (nab) docetaxel analog with superior antitumor activity over docetaxel

Abstract Background: Docetaxel-based chemotherapy is currently used to treat patients with a variety of tumors. A critical challenge has been to improve its chemotherapeutic levels without systemic toxicity. We developed ABI-013, a novel analog of docetaxel (Taxotere®) as a solvent (polysorbate 80/ethanol)-free, nanoparticle albumin-bound 70-nm (nab) form with improved stability in vivo as a tumor-targeting chemotherapeutic agent. In this study, we evaluated the comparative antitumor activity of ABI-013 versus Taxotere® in human tumor xenografts. Methods: Subcutaneous human breast (MDA-MB-231), colon (HT29 and HCT-116), and lung (H358) tumors were grown in athymic nude mice and treated intravenously (IV) with ABI-013 (15, 30, 60, 90, 120 or 150 mg/kg) or Taxotere (MTD of 15 mg/kg) on a q4dx3 schedule. Evaluations of anti-tumor activity were based on the number of partial and complete regressions, the median number of days for tumors in each group to reach one tumor mass doubling (T-C) or the percentage tumor growth inhibition (%TGI). Results: Significant dose-dependent TGI was observed with complete or partial tumor regressions in all xenograft models treated with ABI-013. In the MDA-MB-231 breast tumor model, ABI-013 effectively delayed the tumor growth with T-C values of 83.1, 80.7, and &amp;gt;84.8 days at dosages of 120, 90, and 60 mg/kg, respectively, with &amp;gt;96% TGI compared to vehicle control (P &amp;lt; 0.0001 vs. saline). In contrast, Taxotere delayed the tumor growth with a T-C value of 49.5 days and decreased tumor growth by 88% (P &amp;lt; 0.0001 vs. saline). In the HT 29 colon tumor model, Taxotere revealed dose-limiting toxicity with a 27% body weight loss (BWL) despite 86 to 91% TGI (P &amp;lt; 0.01 vs. saline). In contrast, ABI-013 (90 mg/kg) caused complete tumor regression with a maximum BWL of 19% (P &amp;lt; 0.0001 vs. saline). Treatment with Taxotere delayed the growth of HCT-116 colon tumor with a T-C value of 14.9 days with a maximum BWL of 21%. In contrast, complete tumor regression was achieved in 40% of ABI-013-treated mice. In the H358 human non-small cell lung cancer model, unlike Taxotere, ABI-013 (60 mg/kg) caused partial tumor regression with minimal toxicity. Conclusion: ABI-013, a solvent-free nanoparticle albumin-bound 70-nm particle form of a novel docetaxel analog, had superior antitumor activity in human breast, colorectal, and lung cancer xenograft models compared with equitoxic doses of solvent-based formulation of docetaxel. The nab form of the novel docetaxel analog shows promise in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4420.

Abstract 1765: Preclinical optimization of the Chk1/2 inhibitor AZD7762 in combination with gemcitabine in pancreatic cancer

Abstract Pancreatic cancer remains among the most lethal cancers with an overall 5-year survival of less than 5%. Chk1 inhibition has emerged as a promising approach to sensitize pancreatic cancer to gemcitabine (Gem), the current standard therapy for both local control and metastatic disease. Here, we characterized the ability of AZD7762, a small molecule Chk1/2 inhibitor currently in Phase I trials, to sensitize pancreatic cancer cells to Gem both in vitro and in vivo. We first evaluated the in vitro schedule-dependence of AZD7762-mediated Gem sensitization by clonogenic assay. We found that AZD7762 effectively sensitized pancreatic cancer cell lines to Gem-induced loss of clonogenicity, with shifts in IC50 from 3- to 38-fold, compared to cells treated with Gem alone. At low concentrations of Gem (≤IC50), AZD7762 was most effective when present during Gem treatment. At higher concentrations of Gem, however, giving AZD7762 24 h after Gem resulted in the greatest sensitization. We next assessed the molecular mechanisms of AZD7762-mediated chemosensitization in one of these cell lines, MiaPaCa2, by immunoblot, flow cytometry and immunofluorescent staining. Gemcitabine sensitization was associated with inhibition of Chk1, rather than Chk2 and abrogation of the S-M and G2-M checkpoints. Despite checkpoint abrogation, we found that Gem/AZD7762-treated cells were unable to exit S-phase and, over time, AZD7762 inhibited DNA synthesis, as measured by BrdUrd incorporation. This inhibition was associated with pan γ-H2AX staining in Gem-treated cells, in addition to disruption of Rad51 foci and inhibition of homologous recombination repair activity. We next evaluated the effects of AZD7762 in two different human pancreatic cancer xenograft models; MiaPaCa2, and a patient-derived early passage pancreatic cancer xenograft, EPX-J. AZD7762 had anti-tumor activity both as a single agent, and in combination with Gem in both MiaPaCa2 and EPX-J, with average times to tumor doubling of 37 and 49 days, respectively, in tumors treated with both Gem/AZD7762, compared to 18 and 29 days for tumors treated with Gem alone (*p≤0.05). Chk1 inhibition, as determined by S296- and S345-Chk1 phosphorylation status, correlated with in vivo response. Finally, we found increased S345-Chk1 phosphorylation in normal tissues from mice treated with Gem/AZD7762, suggesting this may be a useful surrogate marker for AZD7762 activity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1765.

Prolonged Hypercalcemia Following Resection of Dysgerminoma: A Case Report

Background. Hypercalcemia is a rare but potentially dangerous complication of pediatric cancer. Of the dysgerminoma cases reported to date, associated hypercalcemia is corrected within 2–7 days of tumor resection. Case. A 13-year-old female with an ovarian dysgerminoma was found to be hypercalcemic on presentation. Following dysgerminoma resection, moderate hypercaclemia persisted for 7 days and calcium remained mildly elevated for an additional 7 days. PTHrP was undetectable. Immunolocalization studies indicated that 1α-hydroxylase was expressed in dysgerminoma tissue but 1,25(OH)2D3 was not elevated. Conclusion. Persistently elevated calcium levels following tumor resection suggests that this case involves a previously undescribed mechanism. Elucidation of this mechanism may offer new insights into tumor biology and opportunities for therapeutic correction of hypercalcemia in this patient population.

Synergistic antitumoral effects of human telomerase reverse transcriptase‐mediated dual‐apoptosis‐related gene vector delivered by orally attenuated Salmonella enterica Serovar Typhimurium in murine tumor models

Salmonella enterica Serovar Typhimurium (S. typhimurium), a facultative anaerobe, has been reported to be capable of both selectively amplifying and expressing exogenous genes within tumors. In addition, previous studies have demonstrated that the human telomerase reverse transcriptase (hTERT) promoter could confine gene expression strictly to the telomerase-positive cancer cells. In the present study, we used attenuated S. typhimurium as a carrier to deliver a eukaryotic expression vector (pSNhTS) that contains the second mitochondria-derived activator of caspases (Smac) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genes under the control of the hTERT promoter. The results of in vitro experiments showed that Smac could enhance TRAIL-induced apoptosis in tumor cells and the hTERT promoter could drive specific gene expression in tumor cells, but not in normal cells. In vivo data in mice indicated that salmonella-mediated exogenous gene expression could persist for at least 14 days in tumors. Furthermore, S.L./SNhTS (S. typhimurium carrying the pSNhTS) was orally administered into mice bearing tumors, and its antitumoral effect was evaluated. It was observed that S.L./SNhTS significantly inhibited tumor growth by 70-90% and prolonged the survival of mice. This strong antitumoral activity achieved by S.L./SNhTS was due to the synergistic antitumoral properties of Smac and TRAIL-recombinant proteins. Our data also support the idea that the hTERT promoter could be an excellent candidate for cancer-specific therapy and S. typhimurium appeared to be a promising strategy for the treatment of solid tumors.

Optimum Overall Times II: Extended Modelling for Head and Neck Radiotherapy

Aims A previous paper in this journal (part I) concluded that there was no pronounced optimum overall time, at least up to 70 fractions of 1.15 Gy at two fractions/day in 50 days. The maximum tolerable tumour doses increased only 2% from the best short schedules of 21 or 23 days to those of 50 days. Only this range was modelled in part I because it covered the fewest and the most fractions, and the longest overall times that will probably be used in practice. Most UK schedules, typically using five fractions a week, yield tumour effective doses about 10% less than the best schedules in other developed countries. The present paper covers a much wider range of fraction numbers from one to 115, and from 1 to 80 days. Some numerical errors in the Tables in part I are also corrected in the present appendix. These made no difference to the main conclusions just described. Materials and methods Standard linear quadratic modelling was used, assuming at first α/β = 10 Gy, α = 0.35 ln/Gy, Tk = 21 days, Tp = 3 days for tumours, but with Tk = 7 days, Tp = 2.5 Gy for acute mucosal reactions, as before. A late complications constraint of 70 Gy was accepted, and an acute constraint of 51 Gy (both at 2 Gy fractions). Alternative values of more rapid or slower repopulation were also explored (Tp = 2 days or Tp = 5 days, respectively). Results Optimal values were shown at 22–32 days for one fraction/day five times a week, and at 42–49 days for two fractions/day at 10 fractions/week. Repopulation caused a rapid fall in tumour dose after 30 days with one fraction/day, but not until after 50 days with two fractions/day, and so was not seen in part I with its too-practical end time. Conclusions Biological modelling can extrapolate calculations outside the borders of published treatment schedules to clarify borderline situations. Optimum schedules in radiotherapy can reliably give more tumour control if two fractions/day are used. The potential gains are equivalent to about two fractions of 2 Gy as given by this modelling. However, the late complications will be less with some nearly tumour equivalent shorter schedules if optimally designed.

CpG oligodeoxynucleotides are potent enhancers of radio- and chemoresponses of murine tumors

Background and purpose Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess anti-tumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074–7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361–9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC). Materials and methods CpG ODN 1826 (100 μg) was given sc three times: when leg tumors were 6 mm, when they grew to 8 mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10 Gy) were given when tumors were 8 mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter. Results Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. Conclusion Overall results show that CpG ODN 1826 can markedly improve tumor response to radiation and chemotherapy (DOC), suggesting that CpG ODNs have potential to be beneficial when used singly or in combination with other standard treatment modalities such as taxane chemotherapy, radiotherapy or both.

Characterization of the tumor-specific activity of WT1 specific T cells generated in vitro from normal individuals by sensitization with WT1-peptide loaded autologous EBV transformed B cells

The Wilms’ tumor protein WT1 is over-expressed in most acute and chronic leukemias. Isolated T-cell clones have been reported to recognize WT1 peptides binding by HLA-A0201 and HLA-A2402 and lyse WT1+ leukemic lines. To test WT1 peptide immunogenicity in normal individuals, we sensitized T-cells from 9 HLA-A0201+ and 4 HLA-A2402+ donors with autologous EBV transformed B cells (EBV-BLCL) or cytokine-activated monocytes (CAM), loaded with HLA-A0201-binding WT1 peptides RMFPNAPYL or SLGEQQYSV or the HLA- A2402- binding WT1 peptide RVPGVAPTL. WT1-peptide specific cytotoxic and interferon-gamma producing T-cells were generated from each donor. T-cells sensitized with peptide-loaded EBV-BLCL generated higher numbers of WT1 specific T-cells than peptide loaded CAM. The frequencies of WT1 peptide-specific T-cells measured by binding to peptide-HLA tetramers were similar to those generated against individual immunogenic HLA-A0201 binding EBV peptides. These T-cells specifically lysed WT1+ leukemias (n = 16) and solid tumors (n = 12) in an HLA-restricted manner, but neither lysed autologous or HLA-sharing normal CD34+ hematopoietic progenitor cells (n = 12) nor affected their yield of CFU-GM, BFU-C or CFU-MIX. Following adoptive transfer into SCID mice bearing subcutaneous xenografts of WT1+ and WT1−, HLA-A0201+ leukemias, WT1-peptide-specific T-cells preferentially accumulated in and induce regressions only of WT1+ leukemias . The long-term repeated in vivo scintigraphic imaging of such T cells, transduced to express an HSV-TK/GFP fusion gene, following intravenous infusions of the HSV-TK substrate [131I]-FIAU demonstrated progressive accumulation of the T cells over a period of 8 days in tumors, expressing targeted antigen and the HLA restricting allele. These studies demonstrate that T cells specific to the self antigen WT1 can be regularly generated from normal individuals and that these cells exhibit significant anti-tumor activity both in vitro and in vivo. Such cells may prove useful for adoptive therapy of leukemias in man.

Inhibition of spontaneous metastases formation by amifostine.

Amifostine was investigated for its ability to inhibit spontaneous metastases formation using the well-characterized murine sarcoma, Sa-NH. Amifostine was administered intraperitoneally at a dose of 50 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8-8.5 mm in diameter. Amifostine was again administered immediately after surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Twenty-one days later, animals were evaluated for the presence of spontaneously developed pulmonary metastases. Nontumor-bearing control animals were sham treated using the same dosing and surgery schedules. Treatment with amifostine appeared to slightly delay tumor growth, that is, 13 vs. 12 days for tumors to reach an average diameter of 8 mm. Amifostine reduced both the incidence of pulmonary metastases formed in experimental animals from 77% to 57% (p < 0.05), and their average number per animal from 12.8 +/- 5.4 (SEM) to 2.9 +/- 1.1 (SEM). The effect of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin was also determined using Western blot analysis. Consistent with the antimetastatic effect, exposure of animals to 50 mg/kg of amifostine resulted in a 4-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in tumor-bearing and nontumor-bearing animals. The effects of amifostine on matrix metalloproteinase (MMP) enzymatic activity was also determined using gelatin zymography. Conditioned growth medium collected from Sa-NH cells grown to confluency was exposed to various concentrations of SH, i.e., 2-[(aminopropyl)amino]ethane-thiol (WR-1065), the active thiol form of amifostine, for either 30 min or 18 hr. WR-1065, as a function of increasing dose and time, inhibited the enzymatic activities of MMP-2 and MMP-9. At a concentration and time of exposure likely to be achieved in vivo, that is, 40 microM and 30 min, MMP-2 and MMP-9 activities were reduced to between 30% and 40% of control values. Consistent with these affects, WR-1065 was also found to be effective in inhibiting the ability of Sa-NH cells to migrate through Matrigel membranes. After an 18-hr exposure under in vitro conditions, WR-1065 at concentrations of 4, 40 and 400 microM, and 4 mM, inhibited Sa-NH migration to 11%, 44%, 81% and 97% of control values, respectively. The abilities of amifostine and its active thiol WR-1065 to stimulate angiostatin production in mice, and to inhibit the MMP enzymatic activities and invasion ability of Sa-NH cells under in vitro conditions, are consistent with the observed antimetastatic effects exhibited against Sa-NH tumors growing in vivo.