Radiosensitizing oxygenation changes in murine tumors treated with VEGF-ablation therapy are measurable using oxygen enhanced-MRI (OE-MRI).

PURPOSE: There is a significant need for a widely available, translatable, sensitive and non-invasive imaging biomarker for tumor hypoxia in radiation oncology. Treatment-induced changes in tumor tissue oxygenation can alter the sensitivity of cancer tissues to radiation, but the relative difficulty in monitoring the tumor microenvironment results in scarce clinical and research data. Oxygen-Enhanced MRI (OE-MRI) uses inhaled oxygen as a contrast agent to measure tissue oxygenation. Here we investigate the utility of dOE-MRI, a previously validated imaging approach employing a cycling gas challenge and independent component analysis (ICA), to detect VEGF-ablation treatment-induced changes in tumor oxygenation that result in radiosensitization. METHODS: Murine squamous cell carcinoma (SCCVII) tumor-bearing mice were treated with 5 mg/kg anti-VEGF murine antibody B20 (B20-4.1.1, Genentech) 2-7 days prior to radiation treatment, tissue collection or MR imaging using a 7 T scanner. dOE-MRI scans were acquired for a total of three repeated cycles of air (2 min) and 100% oxygen (2 min) with responding voxels indicating tissue oxygenation. DCE-MRI scans were acquired using a high molecular weight (MW) contrast agent (Gd-DOTA based hyperbranched polygylcerol; HPG-GdF, 500 kDa) to obtain fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters derived from the MR concentration-time curves. Changes to the tumor microenvironment were evaluated histologically, with cryosections stained and imaged for hypoxia, DNA damage, vasculature and perfusion. Radiosensitizing effects of B20-mediated increases in oxygenation were evaluated by clonogenic survival assays and by staining for DNA damage marker γH2AX. RESULTS: Tumors from mice treated with B20 exhibit changes to their vasculature that are consistent with a vascular normalization response, and result in a temporary period of reduced hypoxia. DCE-MRI using injectable contrast agent HPG-GDF measured decreased vessel permeability in treated tumors, while dOE-MRI using inhaled oxygen as a contrast agent showed greater tissue oxygenation. These treatment-induced changes to the tumor microenvironment result in significantly increased radiation sensitivity, illustrating the utility of dOE-MRI as a non-invasive biomarker of treatment response and tumor sensitivity during cancer interventions. CONCLUSIONS: VEGF-ablation therapy-mediated changes to tumor vascular function measurable using DCE-MRI techniques may be monitored using the less invasive approach of dOE-MRI, an effective biomarker of tissue oxygenation that can monitor treatment response and predict radiation sensitivity.

Fast and sensitive dynamic oxygen-enhanced MRI with a cycling gas challenge and independent component analysis.

PURPOSE: There is a critical need for non-invasive imaging biomarkers of tumor oxygenation to assist in patient stratification and development of hypoxia targeting therapies. Using a cycling gas challenge and independent component analysis (ICA), we sought to improve the sensitivity and speed of existing oxygen enhanced MRI (OE-MRI) techniques to detect changes in oxygenation with dynamically acquired T1 W signal intensity images (dOE-MRI). METHODS: Mice were implanted with SCCVII, HCT-116, BT-474, or SKOV3 tumors in the dorsal subcutaneous region and imaged at 7T. T1 W images were acquired during a respiratory challenge with alternating 2-minute periods of air and 100% oxygen for three cycles. Data were analyzed with ICA and oxygenation maps were generated and compared to corresponding histology sections stained for hypoxia (pimonidazole) and blood vessels (CD31). RESULTS: Cycling air-oxygen-air gas challenges were well tolerated and ICA permitted extraction of the oxygen-enhancing component in all imaged tumors from four different models. Comparison with synthetic response functions showed that dOE-MRI does not require any a-priori knowledge of the physiological response. The fraction of O2 -negative dOE-MRI voxels that correlate inversely with the ICA gas-cycling component correspond well with the histological hypoxic fraction in SCCVII tumors (r = 0.91, p = 0.0016) but did not correlate in HCT-116 tumors (r = 0.13, p = 0.81). CONCLUSIONS: Using ICA and adding a cycling gas challenge extends the sensitivity of OE-MRI and allows the oxygenation status of tumors to be assessed in as little as six minutes. These findings support further development of OE-MRI as a biomarker of tumor oxygenation.

Heterogeneous distribution of trastuzumab in HER2-positive xenografts and metastases: role of the tumor microenvironment.

Most HER2-positive metastatic breast cancer patients continue to relapse. Incomplete access to all target HER2-positive cells in metastases and tumor tissues is a potential mechanism of resistance to trastuzumab. The location of locally bound trastuzumab was evaluated in HER2-positive tissues in vivo and as in vivo xenografts or metastases models in mice. Microenvironmental elements of tumors were related to bound trastuzumab using immunohistochemical staining and include tight junctions, vasculature, vascular maturity, vessel patency, hypoxia and HER2 to look for correlations. Trastuzumab was evaluated alone and in combination with bevacizumab. Dynamic contrast-enhanced magnetic resonance imaging parameters of overall vascular function, perfusion and apparent permeability were compared with matched histological images of trastuzumab distribution and vascular patency. Trastuzumab distribution is highly heterogeneous in all models examined, including avascular micrometastases of the brain and lung. Trastuzumab distributes well through the extravascular compartment even in conditions of high HER2 expression and poor convective flow in vivo. Microregional patterns of trastuzumab distribution in vivo do not consistently correlate with vascular density, patency, function or maturity; areas of poor trastuzumab access are not necessarily those with poor vascular supply. The number of vessels with perivascular trastuzumab increases with time and higher doses and dramatically decreases when pre-treated with bevacizumab. Areas of HER2-positive tissue without bound trastuzumab persist in all conditions. These data directly demonstrate tissue- and vessel-level barriers to trastuzumab distribution in vivo that can effectively limit access of the drug to target cells in brain metastases and elsewhere.

Multi-modal magnetic resonance imaging and histology of vascular function in xenografts using macromolecular contrast agent hyperbranched polyglycerol (HPG-GdF).

Macromolecular gadolinium (Gd)-based contrast agents are in development as blood pool markers for MRI. HPG-GdF is a 583 kDa hyperbranched polyglycerol doubly tagged with Gd and Alexa 647 nm dye, making it both MR and histologically visible. In this study we examined the location of HPG-GdF in whole-tumor xenograft sections matched to in vivo DCE-MR images of both HPG-GdF and Gadovist. Despite its large size, we have shown that HPG-GdF extravasates from some tumor vessels and accumulates over time, but does not distribute beyond a few cell diameters from vessels. Fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters were derived from the MR concentration-time curves of HPG-GdF. Non-viable necrotic tumor tissue was excluded from the analysis by applying a novel bolus arrival time (BAT) algorithm to all voxels. aPS derived from HPG-GdF was the only MR parameter to identify a difference in vascular function between HCT116 and HT29 colorectal tumors. This study is the first to relate low and high molecular weight contrast agents with matched whole-tumor histological sections. These detailed comparisons identified tumor regions that appear distinct from each other using the HPG-GdF biomarkers related to perfusion and vessel leakiness, while Gadovist-imaged parameter measures in the same regions were unable to detect variation in vascular function. We have established HPG-GdF as a biocompatible multi-modal high molecular weight contrast agent with application for examining vascular function in both MR and histological modalities.

Mapping of amide, amine, and aliphatic peaks in the CEST spectra of murine xenografts at 7 T.

PURPOSE: To evaluate the performance of endogenous chemical exchange saturation transfer (CEST) spectra and derived maps in a longitudinal study of tumor xenografts to ascertain the role of CEST parameters in describing tumor progression and in distinguishing between tumor, muscle, and necrosis. METHODS: CEST spectra of 24 mice with tumor xenografts (20 LLC and 4 MDA) were acquired at three time-points. We employed a novel method of decomposing the CEST spectrum into a sum of four Lorentzian shapes, each with a corresponding measured amplitude, width and frequency offset. This semi-quantitative method is an improvement over techniques which simply assess the asymmetry in the spectrum for the presence of CEST, due to the fact that it is not confounded by CEST peaks on opposing sides of the direct effect. The CEST images were compared to several other commonly employed contrast mechanisms: T1 relaxation, T2 relaxation, diffusion (ADC), and magnetization transfer (MT). RESULTS: Tumor spectra had distinct CEST peaks corresponding to the presence of hydrogen exchange between free water and amide, amine, and aliphatic groups. All three CEST peaks (amide, amine, and aliphatic) were larger in the tumor tissue as compared with the adjacent healthy muscle. CONCLUSIONS: CEST contrast (particularly the amine peak amplitude) performed especially well in distinguishing areas of apoptosis and/or necrosis from actively progressing tumor, as validated by histology.

Mapping water exchange rates in rat tumor xenografts using the late-stage uptake following bolus injections of contrast agent.

PURPOSE: To map the intra-to-extracellular water exchange rate constant in rat xenografts using a two-compartment model of relaxation with water exchange and a range of contrast agent concentrations and compare with histology. METHODS: MDA-MB-231 cells were xenografted into six nude rats. Three bolus injections of gadodiamide were administered. When uptake in the tumor demonstrated a steady-state, T1 data were acquired by spoiled gradient recalled acquisitions at four flip angles. A global fit of data to a two-compartment model incorporating exchange was performed, assuming a distribution volume of 20% of the rat. RESULTS: Voxels that did not reach steady-state and were excluded from parametric maps tended to be in large necrotic areas. TUNEL-negative (nonapoptotic) regions tended to have well-defined error bounds, with an average intra-to-extracellular exchange rate constant of 0.6 s(-1) . Apoptotic regions had higher exchange, but poorly determined upper bounds, with goodness of fit similar to that for a model assuming infinitely fast exchange. A lower bound of >3 s(-1) was used to establish voxels where the exchange rate constant was fast despite a large upper bound. CONCLUSION: Water exchange rates were higher in apoptotic regions, but examination of statistical errors was an important step in the mapping process.