RIF-1 Tumors Research Articles

Comparison of the accumulation manner of a macromolecular drug between two mouse tumour models: study with magnetic resonance imaging and the model macromolecular drug, gadolinium-conjugated dextran

A knowledge of the difference of spatio-temporal behaviour of nanomedicine in different type of tumour models is important to develop well-targeted nanomedicine for tumour. In this study, intratumoral accumulation of the model nanomedicine, gadolinium-conjugated dextran (Gd-Dex), was examined with magnetic resonance imaging in two tumour models; mouse sarcoma S180 and radiation-induced mouse fibrosarcoma RIF-1. From time-course of the distribution images, the plasma-to-tumour interstitial tissue transfer constant (Ktrans ) and fractional plasma volume (Vp ) were calculated and mapped. Gd-Dex preferentially distributed to the marginal region of S180 tumours immediately after its injection, and then started to accumulate in some parts of the central region. Ktrans and Vp values were large in the marginal region, while only Ktrans was large in some parts of the central region. In contrast, the distribution of Gd-Dex in RIF-1 tumours was fairly homogeneous, and may have resulted from the homogeneous distributions of Ktrans and Vp . The amounts of Gd-Dex that accumulated in entire tumours in both tumour models correlated with the volume of tumours; however, accumulation in large S180 tumours deviated from the correlation in the early phase. The differences in the manner and pharmacokinetics of nanomedicine among tumour models may affect the accumulation of the medicine.

Temporal variation in the response of tumors to hyperoxia with breathing carbogen and oxygen.

The effect of hyperoxygenation with carbogen (95% O2 + 5% CO2) and 100% oxygen inhalation on partial pressure of oxygen (pO2) of radiation-induced fibrosarcoma (RIF-1) tumor was investigated. RIF-1 tumors were innoculated in C3H mice, and aggregates of oximetry probe, lithium phthalocyanine (LiPc), was implanted in each tumor. A baseline tumor pO2 was measured by electron paramagnetic resonance (EPR) oximetry for 20 minutes in anesthetized mice breathing 30% O2 and then the gas was switched to carbogen or 100 % oxygen for 60 minutes. These experiments were repeated for 10 days. RIF-1 tumors were hypoxic with a baseline tissue pO2 of 6.2–8.3 mmHg in mice breathing 30% O2. Carbogen and 100% oxygen significantly increased tumor pO2 on days 1 to 5, with a maximal increase at approximately 32–45 minutes on each day. However, the extent of increase in pO2 from the baseline declined significantly on day 5 and day 10. The results provide quantitative information on the effect of hyperoxic gas inhalation on tumor pO2 over the course of 10 days. EPR oximetry can be effectively used to repeatedly monitor tumor pO2 and test hyperoxic methods for potential clinical applications.

EPR Oximetry for Investigation of Hyperbaric O2 Pre-treatment for Tumor Radiosensitization.

A number of studies have reported benefits associated with the application of hyperbaric oxygen treatment (HBO) delivered immediately prior to radiation therapy. While these studies provide evidence that pre-treatment with HBO may be beneficial, no measurements of intratumoral pO2 were carried out and they do not directly link the apparent benefits to decreased hypoxic fractions at the time of radiation therapy. While there is empirical evidence and some theoretical basis for HBO to enhance radiation therapy, without direct and repeated measurements of its effects on pO2, it is unlikely that the use of HBO can be understood and optimized for clinical applications. In vivo EPR oximetry is a technique uniquely capable of providing repeated direct measurements of pO2 through a non-invasive procedure in both animal models and human patients. In order to evaluate the ability of pretreatment with HBO to elevate tumor pO2, a novel small animal hyperbaric chamber system was constructed that allows simultaneous in vivo EPR oximetry. This chamber can be placed within the EPR magnet and is equipped with a variety of ports for multiplace gas delivery, thermoregulation, delivery of anesthesia, physiologic monitoring, and EPR detection. Initial measurements were performed in a subcutaneous RIF-1 tumor model in C3H/HeJ mice. The mean baseline pO2 value was 6.0 ± 1.2 mmHg (N = 7) and responses to two atmospheres absolute pressure HBO varied considerably across subjects, within tumors, and over time. When an increase in pO2 was observed, the effect was transient in all but one case, with durations lasting from 5 min to over 20 min, and returned to baseline levels during HBO administration. These results indicate that without direct measurements of pO2 in the tissue of interest, it is likely to be difficult to know the effects of HBO on actual tissue pO2.

Tumour T1 changes in vivo are highly predictive of response to chemotherapy and reflect the number of viable tumour cells – a preclinical MR study in mice

BackgroundEffective chemotherapy rapidly reduces the spin–lattice relaxation of water protons (T1) in solid tumours and this change (ΔT1) often precedes and strongly correlates with the eventual change in tumour volume (TVol). To understand the biological nature of ΔT1, we have performed studies in vivo and ex vivo with the allosteric mTOR inhibitor, everolimus.MethodsMice bearing RIF-1 tumours were studied by magnetic resonance imaging (MRI) to determine TVol and T1, and MR spectroscopy (MRS) to determine levels of the proliferation marker choline and levels of lipid apoptosis markers, prior to and 5 days (endpoint) after daily treatment with vehicle or everolimus (10 mg/kg). At the endpoint, tumours were ablated and an entire section analysed for cellular and necrotic quantification and staining for the proliferation antigen Ki67 and cleaved-caspase-3 as a measure of apoptosis. The number of blood-vessels (BV) was evaluated by CD31 staining. Mice bearing B16/BL6 melanoma tumours were studied by MRI to determine T1 under similar everolimus treatment. At the endpoint, cell bioluminescence of the tumours was measured ex vivo.ResultsEverolimus blocked RIF-1 tumour growth and significantly reduced tumour T1 and total choline (Cho) levels, and increased polyunsaturated fatty-acids which are markers of apoptosis. Immunohistochemistry showed that everolimus reduced the %Ki67+ cells but did not affect caspase-3 apoptosis, necrosis, BV-number or cell density. The change in T1 (ΔT1) correlated strongly with the changes in TVol and Cho and %Ki67+. In B16/BL6 tumours, everolimus also decreased T1 and this correlated with cell bioluminescence; another marker of cell viability. Receiver-operating-characteristic curves (ROC) for everolimus on RIF-1 tumours showed that ΔT1 had very high levels of sensitivity and specificity (ROCAUC = 0.84) and this was confirmed for the cytotoxic patupilone in the same tumour model (ROCAUC = 0.97).ConclusionThese studies suggest that ΔT1 is not a measure of cell density but reflects the decreased number of remaining viable and proliferating tumour cells due to perhaps cell and tissue destruction releasing proteins and/or metals that cause T1 relaxation. ΔT1 is a highly sensitive and specific predictor of response. This MRI method provides the opportunity to stratify a patient population during tumour therapy in the clinic.

Monitoring vascular changes induced by photodynamic therapy using contrast-enhanced micro-computed tomography

The aim of this study was to determine whether contrast-enhanced micro-computed tomography can be used for non-invasive imaging of the early-stage changes in the vasculature of tumours that have been treated with photodynamic therapy (PDT). The subjects used were C3H mice with an RIF-1 tumour implanted subcutaneously and allowed to grow for 3 weeks prior to treatment. The experimental groups were PDT-treated (150 J/cm2 and 50 J/cm2) and control (150 J/cm2 light-only and untreated). The laser light exposure was performed at 15 - 30 minutes after the administration of the photosensitizer (BPD-MA). The contrast-enhanced micro-computed tomography imaging procedure consisted of eight-second scans taking place before treatment and up to 24 hours after treatment. The 150 J/cm2 PDT group showed a significant increase in the ratio of blood volume to tumour volume at 2, 8 and 24 hours after treatment when compared to pre-treatment measurements (p 2 PDT group at 24 hours after treatment. This preliminary study indicates that micro-CT can detect compromised vasculature in tumours treated with high-fluence photodynamic therapy as early as 2 hours post treatment.

Radioiodinated Phenylalkyl Malonic Acid Derivatives as pH-Sensitive SPECT Tracers

Introduction In vivo pH imaging has been a field of interest for molecular imaging for many years. This is especially important for determining tumor acidity, an important driving force of tumor invasion and metastasis formation, but also in the process of apoptosis.Methods2-(4-[123I]iodophenethyl)-2-methylmalonic acid (IPMM), 2-(4-[123I]iodophenethyl)-malonic acid (IPM), 2-(4-[123I]iodobenzyl)-malonic acid (IBMM) and 4-[123I]iodophthalic acid (IP) were radiolabeled via the Cu+ isotopic nucleophilic exchange method. All tracers were tested in vitro in buffer systems to assess pH driven cell uptake. In vivo biodistribution of [123I]IPMM and [123I]IPM was determined in healthy mice and the pH targeting efficacy in vivo of [123I]IPM was evaluated in an anti-Fas monoclonal antibody (mAb) apoptosis model. In addition a mouse RIF-1 tumor model was explored in which tumor pH was decreased from 7.0 to 6.5 by means of induction of hyperglycemia in combination with administration of meta-iodobenzylguanidine.ResultsRadiosynthesis resulted in 15–20% for iodo-bromo exchange and 50–60% yield for iodo-iodo exchange while in vitro experiments showed a pH-sensitive uptake for all tracers. Shelf-life stability and in vivo stability was excellent for all tracers. [123I]IPMM and [123I]IPM showed a moderately fast predominantly biliary clearance while a high retention was observed in blood. The biodistribution profile of [123I]IPM was found to be most favorable in view of pH-specific imaging. [123I]IPM showed a clear pH-related uptake pattern in the RIF-1 tumor model.ConclusionIodine-123 labeled malonic acid derivates such as [123I]IPM show a clearly pH dependent uptake in tumor cells both in vitro and in vivo which allows to visualize regional acidosis. However, these compounds are not suitable for detection of apoptosis due to a poor acidosis effect.

Radiolabeled iodohypericin as tumor necrosis avid tracer: diagnostic and therapeutic potential

It is estimated that 30-80% of solid tumor mass represents necrotic tissue that consists out of a significant number of dead and dying cells. The fact that these necrotic zones are restricted to dysplastic and malignant tissue and are rarely present in normal tissue makes necrosis an interesting target both for cancer diagnosis and therapy. In this study, the avidity of hypericin, [(123) I]iodohypericin and [(131) I]iodohypericin to tumor necrosis was explored for both diagnosis and therapy of experimental malignancies. The intratumoral distribution in RIF-1 tumors was investigated by means of fluorescence microscopy (hypericin) and autoradiography ([(123) I]iodohypericin). Results show high uptake of the tracers in necrosis at 24 hr, lasting for up to 72 hr p.i. Ratios of activity of [(123) I]iodohypericin in necrotic tissue over viable tumor reached up to 19.63 ± 4.66, correlating with 9.20% ID/g in necrosis. Nude mice bearing RIF-1 tumors that received three injections of 300 μCi over a 3-week treatment period showed stabilization in tumor growth for 5 days, as measured by caliper and micro-positron emission tomography using [(18) F]fluorodeoxyglucose. Based on these results, we suggest the potentials of radiolabeled hypericin (1) in diagnostic aspects including prognosis or staging assessment of bulky necrotic cancers, monitoring of treatments and therapeutic follow-up; and (2) in cancer treatment based on tumor necrosis. In conclusion, we showed that hypericin radiolabeled with iodine is a necrosis avid tracer that can be used both as a tumor diagnostic and therapeutic.

Pretargeting of necrotic tumors with biotinylated hypericin using 123I-labeled avidin: evaluation of a two-step strategy

As an alternative to directly targeting of necrotic tissue using hypericin, we synthesized a conjugate of hypericin to biotin for use in a pretargeting approach. With this conjugate, we explored the possibility of a two-step pretargeting strategy using (123)I-labeled avidin as effector molecule directed against necrotic RIF-1 tumors. Hypericin was conjugated to biotin-ethylenediamine in a straightforward coupling method using n-hydroxysuccinimide and dicyclohexylcarbodiimide. The necrosis avidity of the conjugate was first confirmed in necrotic liver tissue by means of fluorescence microscopy. Using autoradiography imaging and whole body-biodistribution, the accumulation of (123)I-avidin in necrotic tumor tissue was evaluated 24 h after administration and 48 h after pretargeting with hypericin-biotin. Analysis of autoradiography images show a higher accumulation of (123)I-avidin in pretargeted compared to nontargeted tissue. However, absolute accumulation of (123)I-avidin in necrotic tumors was low as shown by biodistribution experiments. Direct injection of hypericin-biotin or biotin-fluorescein did not substantially improve (123)I-avidin accumulation after pretargeting, pointing towards a poor penetration of avidin in necrotic tissue. Our results show the feasibility of a pretargeting technique using a small molecule as targeting agent. However, for a more efficient accumulation of the effector molecule in necrotic tissue, other pretargeting strategies need to be investigated.

Influence of the vascular damaging agents DMXAA and ZD6126 on hypericin distribution and accumulation in RIF-1 tumors

We investigated the influence of two types of vascular damaging agents (VDAs) (DMXAA vs. ZD6126) and sequence of administration (VDA 24 h before HYP vs. HYP 1 h before VDA) to evaluate the effect on hypericin (HYP) accumulation and distribution in necrotic tumors. Frozen sections of dorsally inoculated RIF-1 tumors were analyzed by fluorescence microscopy and H&E stained for histological evaluation. The localization of HYP was assessed both qualitatively and semi-quantitatively in necrotic tumor, viable tumor, or nontarget host tissue. Whereas the type of VDA did not influence HYP accumulation and distribution, a clear advantage could be seen when administering VDA 24 h before HYP compared to HYP 1 h before VDA, pointing toward the absence of a "trapping" mechanism. In DMXAA-treated and not in ZD6126-treated tumors, spotty fluorescence was observed which is likely to be a consequence of neutrophil phagocytosis. Dexamethasone treatment neither did influence this phenomenon nor did change HYP uptake in necrotic tumor. We conclude that HYP accumulation is optimal when it is administered after VDA injection. We also found that HYP accumulation in necrosis is not changed when using VDAs with different working mechanisms. This insight provides a rationale for tumor necrosis therapy (TNT) using iodine-131-labeled hypericin ([(131)I]-HYP) in combination with VDAs.

Thymidine Kinase Gene Modified Bone Marrow Mesenchymal Stem Cells as Vehicles for Antitumor Therapy

Bone marrow mesenchymal stem cells (BMSCs) represent an important source of cells for tissue repair. The tropism of these cells to the sites of injury and tumors has been well established. Their tumor-homing properties make BMSCs good candidates as antitumor agent delivery vehicles. In this study, we showed that BMSCs have the ability to migrate toward various cancer cells, including prostate cancer cells in vitro and in vivo and incorporating into the tumor mass. When infected with herpes simplex virus thymidine kinase (TK) gene by lentiviral transduction, TK-BMSCs maintained their tumor tropism capabilities and significantly inhibited the growth of subcutaneous PC3 prostate cancer xenografts in nude mice, in the presence of prodrug ganciclovir (GCV). Xenogenic TK-BMSCs also survived and exerted a significant antitumor effect in an animal model bearing metastastic RIF-1 (fibrosarcoma) tumor in the presence of prodrug GCV. The present study demonstrated that overexpression of TK in BMSCs did not affect their multidifferentiation potentials and their specific homing capacities toward the tumor mass, and the TK-BMSCs alone did not cause any harmful side effects in vivo. The use of TK-BMSCs as tumor-specific delivery vehicles together with controlled prodrug treatment may be a safe and novel anticancer therapy approach.

Synergistic Combination of Hyperoxygenation and Radiotherapy by Repeated Assessments of Tumor pO2 with EPR Oximetry

The effect of hyperoxygenation with carbogen (95% O(2) + 5% CO(2)) inhalation on RIF-1 tumor pO(2 )and its consequence on growth inhibition with fractionated radiotherapy is reported. The temporal changes in the tumor pO(2) were assessed by in vivo Electron Paramagnetic Resonance (EPR) oximetry in mice breathing 30% O(2) or carbogen and the tumors were irradiated with 4 Gy/day for 5 consecutive days; a protocol that emulates the clinical application of carbogen. The RIF-1 tumors were hypoxic with a tissue pO(2) of 5-9 mmHg. Carbogen (CB) breathing significantly increased tumor pO(2), with a maximum increase at 22.9-31.2 min on days 1-5, however, the magnitude of increase in pO(2) declined on day 5. Radiotherapy during carbogen inhalation (CB/RT) resulted in a significant tumor growth inhibition from day 3 to day 6 as compared to 30%O(2)/RT and carbogen (CB/Sham RT) groups. The results provide unambiguous quantitative information on the effect of carbogen inhalation on tumor pO(2) over the course of 5 days. Tumor growth inhibition in the CB/RT group confirms that the tumor oxygenation with carbogen was radiobiologically significant. Repeated tumor pO(2) measurements by EPR oximetry can provide temporal information that could be used to improve therapeutic outcomes by scheduling doses at times of improved tumor oxygenation.

The role of nitric oxide in the treatment of tumours with aminolaevulinic acid-induced photodynamic therapy

Photodynamic therapy (PDT) is a local cancer treatment which induces cell death by the interaction of light with a photosensitizing drug. Previous studies indicate that nitric oxide (NO) plays a role in Photofrin-PDT, but this has not been investigated in aminolaevulinic acid (ALA)-PDT. The current study determines whether inhibition of nitric oxide synthase (NOS) activity modulates treatment responses to ALA-PDT, in tumours displaying differential levels of NO. Murine tumours with low (EMT6) or high (RIF-1) NO levels were implanted into the cremaster muscle of BALB/c or C3H/HeN mice respectively. Animals were prepared for in vivo microscopy 7–14 days later. Mice received oral ALA (200 mg/kg) 4 h before PDT. l-NAME, l-NNA or 1400 W (10 mg/kg) were administered via the jugular vein 5 min before PDT. NOS inhibition ( l-NAME or l-NNA) combined with ALA-PDT in RIF-1 tumours demonstrated enhanced damage to both the tumour and normal microvasculature, with increased macromolecular leak and reduction in vessel diameter, whereas ALA-PDT alone had no effect. In contrast, EMT6 tumours responded to ALA-PDT alone but sensitivity was not enhanced in the presence of NOS inhibition. 1400 W combined with ALA-PDT induced similar microvascular effects to l-NAME in both tumours, but were less pronounced. The data demonstrates that NO has an important role in events likely to be critical for treatment response, sensitivity and therapeutic outcome of ALA-PDT.

Effect of a Topical Vasodilator on Tumor Hypoxia and Tumor Oxygen Guided Radiotherapy using EPR Oximetry

We sought to reduce tumor hypoxia by topical application of a vasodilator, benzyl nicotinate (BN), and investigated its effect on the growth of tumors irradiated at times when tumor pO(2) increased. EPR oximetry was used to follow the changes in the tissue pO(2) of subcutaneous radiation-induced fibrosarcoma (RIF-1) tumors during topical applications of 1.25-8% BN formulations for 5 consecutive days. The RIF-1 tumors were hypoxic with a tissue pO(2) of 4.6-7.0 mmHg. A significant increase in tumor pO(2) occurred 10-30 min after BN application. The formulation with the minimal BN concentration that produced a significant increase in tumor pO(2) was used for the radiation study. The tumors were irradiated (4 Gy x 5) at the time of the maximum increase in pO(2) observed with the 2.5% BN formulation. The tumors with an increase in pO(2) of greater than 2 mmHg from the baseline after application of BN on day 1 had a significant growth inhibition compared to the tumors with an increase in pO(2) of less than 2 mmHg. The results indicate that the irradiation of tumors at the time of an increase in pO(2) after the topical application of the 2.5% BN formulation led to a significant growth inhibition. EPR oximetry provided dynamic information on the changes in tumor pO(2), which could be used to identify responders and non-responders and schedule therapy during the experiments.

Abstract 1660: The antitumor action of ASA404 (vadimezan; DMXAA); potential involvement of vascular endothelial growth factor (VEGF)

Abstract ASA404, a Tumor-Vascular Disrupting Agent developed at the Auckland Cancer Society Research Centre, is currently undergoing two Phase III clinical trials (Novartis) against non-small cell lung cancer. Its early effects on tumor tissue include increased tumor vascular permeability, increased tumor endothelial apoptosis, decreased tumor blood flow and increased tumor hypoxia. The basis for the selective effect of ASA404 on tumor versus normal vasculature is not clear. We hypothesized that VEGF, produced in tumor tissue in responses to hypoxia and other stresses, played a possible role in this selectivity. We compared the actions of ASA404 and VEGF in vitro and also examined the potential for induction of tumor VEGF by ASA404 treatment in vivo. We grew human umbilical vein endothelial cells (HUVEC) on Matrigel and compared in vitro responses to ASA404 and human VEGF using time-lapse microscopy and image analysis. Both ASA404 (30 and 300 µM; 9.9 and 99 µg/ml) and VEGF (50 ng/ml) stimulated the initial formation of 2-dimensional tubular networks on Matrigel. Both ASA404 and VEGF induced actin stress fiber formation in cultured HUVEC's, consistent with increased migration. To determine if there was an in vivo relationship between ASA404 and VEGF expression, mice with Rif-1 tumors (7-9 mm diameter) were treated i.p. with ASA404 (70 µmol/kg; 21 mg/kg) and mRNA was extracted from tumor tissue 4 hours later. VEGF-A mRNA abundance was measured by co-amplification with endogenous 18S mRNA as standard. VEGF-A relative abundance rose from 0.6 ± 0.1 to 1.3 ± 0.1 (p < 0.01). VEGF-B and VEGF-D concentrations also increased but the changes were not significant. The results are consistent with the following model: hypoxia and other stresses in tumor tissue lead to synthesis by both tumor and host cells of VEGF, which increases vascular permeability and partially compromises tumor blood flow. The administration of ASA404 leads to increased tumor vascular permeability and vascular endothelial apoptosis, possibly by a pathway involving p38 kinase, ceramide synthesis and actin stress fiber formation. The resultant decrease in tumor blood flow further increases hypoxia, potentially providing a positive feedback loop by stimulating further VEGF production. These effects, in combination with other cytokines such as tumor necrosis factor that are induced by ASA404, lead to tumor vascular failure. Thus, endogenous VEGF production by tumor tissue may contribute to the antitumor selectivity of ASA404. Further studies addressing the modulatory role of VEGF in the action of ASA404 are warranted. 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 1660.

Abstract 4337: Tumor T1 is a non-invasive imaging marker of proliferation and response to the mTOR inhibitor everolimus

Abstract Everolimus (RAD001) is an oral mTOR inhibitor recently approved for the treatment of 2nd-line renal cell carcinoma. Further development of everolimus will be aided by identification of suitable biomarkers for stratification and response. We have recently shown that a non-invasive imaging marker (T1) is a rapid and robust marker of tumor response for five compounds with different mechanisms of action (Clinical Cancer Res, in the press). Here we have correlated everolimus-induced changes in T1 (ΔT1) with other biomarkers in a murine tumor model. Cultured RIF-1 tumor cells (5 × 106) were injected s.c. in the flank of C3H mice (n=14) and after 2 weeks were treated daily with everolimus (10 mg/kg p.o.) or vehicle. Magnetic resonance imaging and 1H-spectroscopy (MRI and MRS) were performed at baseline (day-0) and after 5 days treatment on a 2 mm central slice of the tumor. MRI used an IR-TrueFISP method to quantify the spin-lattice relaxation time of water protons (T1) and MRS the total levels of choline as the Cho/H20 ratio. On day-5, the tumors were ablated, paraffin-embedded and stained for the percentage of cells positive for cleaved caspase-3 (apoptosis) and the proliferation marker Ki67 using immunohistochemistry (IHC). Effects are summarized as mean T/C (ratio of treated divided by vehicle-control); relationships between variables were assessed using Pearson's correlation, with significance set at p<0.05. Everolimus strongly inhibited tumor growth (T/C=0.05, p<0.001) without affecting body-weight. MRS signals for the proliferation marker choline were clearly detectable in all tumors at both baseline and day-5, and in 6/7 tumors everolimus reduced choline (T/C=0.73, p=0.1). Basal T1 was 2.3 sec and this was always reduced by everolimus (T/C=0.89, p<0.01). The ΔT1 correlated with changes in tumor volume, dTVol (r=0.81, p<0.001), and choline (r=0.59, p<0.05). Basal T1 did not correlate with dTVol, i.e. did not predict response to everolimus, but choline did (r= −0.81, p<0.05), so that higher levels of choline predicted a better response. IHC showed that the %-caspase and %-necrosis were unchanged by everolimus but the %Ki67 positive cells was reduced (T/C=0.55, p<0.001) and there was a significant correlation between %Ki67 and ΔT1 (r=0.61, p<0.05). These results show that everolimus-induced changes in T1 correlate with known markers of proliferation. The speed and robustness of the method should facilitate its use in clinical trials as an early-response marker. In this model, basal levels of choline were a stratifier for response to everolimus. 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 4337.

Quantified Tumor T1 Is a Generic Early-Response Imaging Biomarker for Chemotherapy Reflecting Cell Viability

Identification of a generic response biomarker by comparison of chemotherapeutics with different action mechanisms on several noninvasive biomarkers in experimental tumor models. The spin-lattice relaxation time of water protons (T(1)) was quantified using an inversion recovery-TrueFISP magnetic resonance imaging method in eight different experimental tumor models before and after treatment at several different time points with five different chemotherapeutics. Effects on T(1) were compared with other minimally invasive biomarkers including vascular parameters, apparent diffusion coefficient, and interstitial fluid pressure, and were correlated with efficacy at the endpoint and histologic parameters. In all cases, successful chemotherapy significantly lowered tumor T(1) compared with vehicle and the fractional change in T(1) (DeltaT(1)) correlated with the eventual change in tumor size (range: r(2) = 0.21, P < 0.05 to r(2) = 0.73, P < 0.0001), except for models specifically resistant to that drug. In RIF-1 tumors, interstitial fluid pressure was decreased, but apparent diffusion coefficient and permeability increased in response to the microtubule stabilizer patupilone and 5-fluorouracil. Although DeltaT(1) was small (maximum of -20%), the variability was very low (5%) compared with other magnetic resonance imaging methods (24-48%). Analyses ex vivo showed unchanged necrosis, increased apoptosis, and decreased %Ki67 and total choline, but only Ki67 and choline correlated with DeltaT(1). Correlation of Ki67 and DeltaT(1) were observed in other models using patupilone, paclitaxel, a VEGF-R inhibitor, and the mammalian target of rapamycin inhibitor everolimus. These results suggest that a decrease in tumor T(1) reflects hypocellularity and is a generic marker of response. The speed and robustness of the method should facilitate its use in clinical trials.

Radiotherapy in Conjunction with 7-Hydroxystaurosporine: A Multimodal Approach with Tumor pO2as a Potential Marker of Therapeutic Response

Checkpoint inhibitors potentially could be used to enhance cell killing by DNA-targeted therapeutic modalities such as radiotherapy. UCN-01 (7-hydroxystaurosporine) inhibits S and G2 checkpoint arrest in the cells of various malignant cell lines and has been investigated in combination with chemotherapy. However, little is known about its potential use in combination with radiotherapy. We report the effect of 20 Gy radiation given in conjunction with UCN-01 on the pO2 and growth of subcutaneous RIF-1 tumors. Multisite EPR oximetry was used for repeated, non-invasive tumor pO2 measurements. The effect of UCN-01 and/or 20 Gy on tumor pO2 and tumor volume was investigated to determine therapeutic outcomes. Untreated RIF-1 tumors were hypoxic with a tissue pO2 of 5-7 mmHg. Treatment with 20 Gy or UCN-01 significantly reduced tumor growth, and a modest increase in tumor pO2 was observed in tumors treated with 20 Gy. However, irradiation with 20 Gy 12 h after UCN-01 treatment resulted in a significant inhibition of tumor growth and a significant increase in tumor pO2 to 16-28 mmHg from day 1 onward compared to the control, UCN-01 or 20-Gy groups. Treatment with UCN-01 12 h after 20 Gy also led to a similar growth inhibition of the tumors and a similar increase in tumor pO2. The changes in tumor pO2 observed after the treatment correlated inversely with the tumor volume in the groups receiving UCN-01 with 20 Gy. This multimodal approach could be used to enhance the outcome of radiotherapy. Furthermore, tumor pO2 could be a potential marker of therapeutic response.

Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine N3 DNA minor groove alkylators

Hypoxia represents an important therapeutic target in tumors because of the resistance of hypoxic cells to radiotherapy and chemotherapy and because it is more severe in many tumors than in normal tissues. Here, we describe a class of prodrugs, nitro-chloromethylindolines, which undergo hypoxia-selective activation by endogenous nitroreductases in tumor cells to form the corresponding amino compounds. The latter are chemically related to the cyclopropylindoline antitumor antibiotics and they share the same properties of sequence-selective DNA minor groove alkylation and high cytotoxic potency. Of three alkylating subunits investigated, the chloromethylbenzindoline (CBI) structure provided the most favorable prodrug properties: aerobic cytotoxic potency of the amines was approximately 90- to 3,000-fold higher than the corresponding nitro compounds, and the nitro compounds showed air/anoxia potency differentials of up to 300-fold. Selective alkylation of adenine N3 in calf thymus DNA by an amino-CBI was shown by characterization of the thermal depurination product; the same adduct was shown in hypoxic RIF-1 cells exposed to the corresponding nitro-CBI prodrug under hypoxic (but not oxic) conditions. The amino metabolite generated from a nitro-CBI by cells expressing Escherichia coli nfsB nitroreductase in multicellular layer cultures was shown to elicit bystander killing of surrounding cells. Nitro-CBI prodrugs were >500-fold less toxic to mice than amino-CBIs by i.p. administration and provided selective killing of hypoxic cells in RIF-1 tumors (although only at maximally tolerated doses). Nitro-CBIs are novel lead hypoxia-activated prodrugs that represent the first examples of hypoxia-selective generation of potent DNA minor groove alkylating agents.

1249 Combination of platinum standard first front line chemotherapy and anti-idiotype 1E10/aluminum vaccine in patients with advanced non- small- cell lung cancer (NSCLC)

Background and purpose: Drug toxicities are often a limiting factor in long term treatment regimes used in conjunction with radiotherapy. If the drug could be localized to the tumor site and released slowly, then optimal, intra-tumoral drug concentrations could be achieved without the cumulative toxicity associated with repeated systemic drug dosage. In this paper we describe the use of a biodegradable polymer implant for sustained intra-tumoral release of high concentrations of drugs targeting hypoxic cells.Materials and methods: The RIF-1 tumor was implanted subcutaneously or rntramuscularly in C3H mice and irradiated with 60Co gamma rays. The drug delivery device was the co-polymer CPP-SA;20:80 into which the drug was homogeneously incorporated. The hypoxic radiosensitizer Etanidazole or the bioreductive drug Tirapazamine were delivered intra-tumorally by means of implanted polymer rods containing the drugs. Tumor growth delay (TGD) was used as the end point in these experiments.Results: Both Etanidazole and Tirapazamine potentiated the effects of acute and fractionated radiation in the intra-muscular tumors but neither drug was effective in sub-cutaneous tumors. Since both drugs target hypoxic cells we hypothesized that the lack of effect in the subcutaneous tumor was attributable to the smaller size of the hypoxic fraction in this tumor model. This was confirmed using the hypoxia marker EF5.Conclusions: These results indicate that the biodegradable polymer implant is an effective vehicle for the intra-tumoral delivery of Etanidazole and Tirapazamine and that, in conjunction with radiation, this approach could improve treatment outcome in tumors which contain a sub-population of hypoxic, radioresistant cells.

1250 Phase I study of intra-tumoral injection of a newly developed enzyme-targeting radiosensitizer (KORTUC) containing hydrogen peroxide & sodium hyaluronate for unresectable and/or recurrent malignant neoplasms

Background and purpose: Drug toxicities are often a limiting factor in long term treatment regimes used in conjunction with radiotherapy. If the drug could be localized to the tumor site and released slowly, then optimal, intra-tumoral drug concentrations could be achieved without the cumulative toxicity associated with repeated systemic drug dosage. In this paper we describe the use of a biodegradable polymer implant for sustained intra-tumoral release of high concentrations of drugs targeting hypoxic cells.Materials and methods: The RIF-1 tumor was implanted subcutaneously or rntramuscularly in C3H mice and irradiated with 60Co gamma rays. The drug delivery device was the co-polymer CPP-SA;20:80 into which the drug was homogeneously incorporated. The hypoxic radiosensitizer Etanidazole or the bioreductive drug Tirapazamine were delivered intra-tumorally by means of implanted polymer rods containing the drugs. Tumor growth delay (TGD) was used as the end point in these experiments.Results: Both Etanidazole and Tirapazamine potentiated the effects of acute and fractionated radiation in the intra-muscular tumors but neither drug was effective in sub-cutaneous tumors. Since both drugs target hypoxic cells we hypothesized that the lack of effect in the subcutaneous tumor was attributable to the smaller size of the hypoxic fraction in this tumor model. This was confirmed using the hypoxia marker EF5.Conclusions: These results indicate that the biodegradable polymer implant is an effective vehicle for the intra-tumoral delivery of Etanidazole and Tirapazamine and that, in conjunction with radiation, this approach could improve treatment outcome in tumors which contain a sub-population of hypoxic, radioresistant cells.