Multiscale Measurements Distinguish Cellular and Interstitial Hindrances to Diffusion In Vivo

Molecular cancer therapy relies on interstitial diffusion for drug distribution in solid tumors. A mechanistic understanding of how tumor components affect diffusion is necessary to advance cancer drug development. Yet, because of limitations in current techniques, it is unclear how individual tissue components hinder diffusion. We developed multiscale fluorescence recovery after photobleaching (MS-FRAP) to address this deficiency. Diffusion measurements facilitated by MS-FRAP distinguish the diffusive hindrance of the interstitial versus cellular constituents in living tissue. Using multiscale diffusion measurements in vivo, we resolved the contributions of these two major tissue components toward impeding diffusive transport in solid tumors and subcutaneous tissue in mice. We further used MS-FRAP in interstitial matrix-mimetic gels and in vivo to show the influence of physical interactions between collagen and hyaluronan on diffusive hindrance through the interstitium. Through these studies, we show that interstitial hyaluronan paradoxically improves diffusion and that reducing cellularity enhances diffusive macromolecular transport in solid tumors.     

Diffusion and convection in collagen gels: implications for transport in the tumor interstitium

Diffusion coefficients of tracer molecules in collagen type I gels prepared from 0-4.5% w/v solutions were measured by fluorescence recovery after photobleaching. When adjusted to account for in vivo tortuosity, diffusion coefficients in gels matched previous measurements in four human tumor xenografts with equivalent collagen concentrations. In contrast, hyaluronan solutions hindered diffusion to a lesser extent when prepared at concentrations equivalent to those reported in these tumors. Collagen permeability, determined from flow through gels under hydrostatic pressure, was compared with predictions obtained from application of the Brinkman effective medium model to diffusion data. Permeability predictions matched experimental results at low concentrations, but underestimated measured values at high concentrations. Permeability measurements in gels did not match previous measurements in tumors. Visualization of gels by transmission electron microscopy and light microscopy revealed networks of long collagen fibers at lower concentrations along with shorter fibers at high concentrations. Negligible assembly was detected in collagen solutions pregelation. However, diffusion was similarly hindered in pre and postgelation samples. Comparison of diffusion and convection data in these gels and tumors suggests that collagen may obstruct diffusion more than convection in tumors. These findings have significant implications for drug delivery in tumors and for tissue engineering applications.     

In vivo measurement of tumor redox environment using EPR spectroscopy

Solid tumors are characterized by a number of physiological properties such as occurrence of significant hypoxia, large amounts of cellular reducing equivalents, compromised blood-flow and low pH, all of which are distinctly different from normal tissues. Tumor therapeutic regimens such as radiation or chemotherapy attempt to exploit these physiological differences between normal and malignant tissue. Thus, methods that can detect these subtle differences would greatly aid in devising appropriate treatment strategies. Low-frequency in vivo electron paramagnetic resonance (EPR) spectroscopy is capable of providing non-invasive measurements of these parameters in tumors. This requires the use of appropriate exogenously injected free radical reporter molecules (probes), such as nitroxides. In the present study we performed measurements of nitroxide metabolism in RIF-1 murine tumors, in vivo, and demonstrated that the rate of nitroxide decay correlated with the tumor redox environment. The results showed the existence of significantly higher reducing environment in the tumor tissue compared to normal tissue. The dependence of the tumor redox status on the intracellular GSH levels and tissue oxygenation was investigated. The measurement of redox status and its manipulation may have important implications in the understanding of tumor growth and therapy.     

Gas exchange measurements, what can they tell us about the underlying limitations to photosynthesis? Procedures and sources of error

The principles, equipment and procedures for measuring leaf and canopy gas exchange have been described previously as has chlorophyll fluorescence. Simultaneous measurement of the responses of leaf gas exchange and modulated chlorophyll fluorescence to light and CO2 concentration now provide a means to determine a wide range of key biochemical and biophysical limitations on photo synthesis in vivo. Here the mathematical frameworks and practical procedures for determining these parameters in vivo are consolidated. Leaf CO2 uptake (A) versus intercellular CO2 concentration (Ci) curves may now be routinely obtained from commercial gas exchange systems. The potential pitfalls, and means to avoid these, are examined. Calculation of in vivo maximum rates of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) carboxylation (Vc,max), electron transport driving regeneration of RuBP (Jmax), and triose-phosphate utilization (VTPU) are explained; these three parameters are now widely assumed to represent the major limitations to light-saturated photosynthesis. Precision in determining these in intact leaves is improved by the simultaneous measurement of electron transport via modulated chlorophyll fluorescence. The A/Ci response also provides a simple practical method for quantifying the limitation that stomata impose on CO2 assimilation. Determining the rate of photorespiratory release of oxygen (Rl) has previously only been possible by isotopic methods, now, by combining gas exchange and fluorescence measurements, Rl may be determined simply and routinely in the field. The physical diffusion of CO2 from the intercellular air space to the site of Rubisco in C3 leaves has long been suspected of being a limitation on photosynthesis, but it has commonly been ignored because of the lack of a practical method for its determination. Again combining gas exchange and fluorescence provides a means to determine mesophyll conductance. This method is described and provides insights into the magnitude and basis of this limitation.     

Flux through the de novo pyrimidine pathway in vivo. Effect of N-phosphonacetyl-L-aspartate, a potent inhibitor of aspartate transcarbamylase

The activity of the de novo pyrimidine biosynthetic pathway has been measured in resistant and sensitive murine tumors in vivo following a single intraperitoneal dose of N-phosphonacetyl-L-aspartate (PALA) (400 mg/kg). For these studies, we utilized a gas chromatograph-mass spectrometric technique which enabled measurement of 13C incorporation from 13CO2 into the uracil nucleotide pool (sigma uracil) of tumors in situ. Flux through the de novo pathway was 75-85% inhibited 1 h after PALA treatment in both sensitive (Lewis lung carcinoma) and the resistant (L1210) tumors, but there was a lag time before this inhibition was reflected in reduced sigma uracil pools. The activity of the pathway in the Lewis lung carcinoma tumors remained maximally depressed (5-15% of control activity) for up to 48 h after the dose of PALA. In contrast, flux through the pathway of L1210 tumors remained 80% inhibited for up to 4 h following PALA administration, but recovered to 70% of control activity between 4 and 12 h after PALA treatment. Recovery of the remaining 30% of control activity in the L1210 tumor was at a much slower rate requiring between 12 and 48 h after PALA treatment to regain full activity of the pathway. This recovery of flux through the de novo pyrimidine biosynthetic pathway did not correlate with the measurement of recovery of aspartate transcarbamylase activity in similarly treated tumors. These data argue strongly in favor of the importance of the de novo biosynthetic pathway, rather than salvage mechanisms, for determining in vivo sensitivity or resistance of these tumors to PALA treatment.     

Diffusion-Weighted MRI Is Insensitive to Changes in the Tumor Microenvironment Induced by Antiangiogenic Therapy

Antiangiogenic treatment (AAT) used in combination with radiation therapy or chemotherapy is a promising strategy for the treatment of several cancer diseases. The vascularity and oxygenation of tumors may be changed significantly by AAT, and consequently, a noninvasive method for monitoring AAT-induced changes in these microenvironmental parameters is needed. The purpose of this study was to evaluate the potential usefulness of diffusion-weighted magnetic resonance imaging (DW-MRI). DW-MRI was conducted with a Bruker Biospec 7.05-T scanner using four diffusion weightings and diffusion sensitization gradients in three orthogonal directions. Maps of the apparent diffusion coefficient (ADC) were calculated by using a monoexponential diffusion model. Two cervical carcinoma xenograft models (BK-12, HL-16) were treated with bevacizumab, and two pancreatic carcinoma xenograft models (BxPC-3, Panc-1) were treated with sunitinib. Pimonidazole and CD31 were used as markers of hypoxia and blood vessels, respectively, and fraction of hypoxic tissue (HF_Pim) and microvascular density (MVD) were quantified by analyzing immunohistochemical preparations. MVD decreased significantly after AAT in BK-12, HL-16, and BxPC-3 tumors, and this decrease was sufficiently large to cause a significant increase in HF_Pim in BK-12 and BxPC-3 tumors. The ADC maps of treated tumors and untreated control tumors were not significantly different in any of these three tumor models, suggesting that the AAT-induced microenvironmental changes were not detectable by DW-MRI. DW-MRI is insensitive to changes in tumor vascularity and oxygenation induced by bevacizumab or sunitinib treatment.     

Explicit Separation of Growth and Motility in a New Tumor Cord Model

We investigate a new model of tumor growth in which cell motility is considered an explicitly separate process from growth. Bulk tumor expansion is modeled by individual cell motility in a density-dependent diffusion process. This model is implemented in the context of an in vivo system, the tumor cord. We investigate numerically microscale density distributions of different cell classes and macroscale whole tumor growth rates as functions of the strength of transitions between classes. Our results indicate that the total tumor growth follows a classical von Bertalanffy growth profile, as many in vivo tumors are observed to do. This provides a quick validation for the model hypotheses. The microscale and macroscale properties are both sensitive to fluctuations in the transition parameters, and grossly adopt one of two phenotypic profiles based on their parameter regime. We analyze these profiles and use the observations to classify parameter regimes by their phenotypes. This classification yields a novel hypothesis for the early evolutionary selection of the metastatic phenotype by selecting against less motile cells which grow to higher densities and may therefore induce local collapse of the vascular network.     

Magnetic Resonance Assessment of Response to Therapy: Tumor Change Measurement,Truth Data and Error Sources

This article describes methods and issues that are specific to the assessment of change in tumor characteristics as measured using quantitative magnetic resonance (MR) techniques and how this relates to the establishment of quantitative MR imaging (MRI) biomarkers of patient response to therapy. The initial focus is on the various sources of bias and variance in the measurement of microvascular parameters and diffusion parameters as such parameters are being used relatively commonly as secondary or exploratory end points in current phase 1/2 clinical trails of conventional and targeted therapies. Several ongoing initiatives that seek to identify the magnitude of some of the sources of measurement variations are then discussed. Finally, resources being made available through the National Cancer Institute Reference Image Database to Evaluate Response (RIDER) project that might be of use in investigations of quantitative MRI biomarker change analysis are described. These resources include 1) data from phantom-based assessment of system response, including short-term (1 hour) and moderate-term (1 week) contrast response and relaxation time measurement, 2) data obtained from repeated dynamic contrast agent-enhanced MRI studies in intracranial tumors, and 3) data obtained from repeated diffusion MRI studies in both breast and brain. A concluding section briefly discusses issues that must be addressed to allow the transition of MR-based imaging biomarker measures from their current role as secondary/exploratory end points in clinical trials to primary/surrogate markers of response and, ultimately, in clinical application.     

Retinoblastoma cell cycling

Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material. Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesis-phase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48). DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.     

The universal dynamics of tumor growth

Scaling techniques were used to analyze the fractal nature of colonies of 15 cell lines growing in vitro as well as of 16 types of tumor developing in vivo. All cell colonies were found to exhibit exactly the same growth dynamics-which correspond to the molecular beam epitaxy (MBE) universality class. MBE dynamics are characterized by 1), a linear growth rate, 2), the constraint of cell proliferation to the colony/tumor border, and 3), surface diffusion of cells at the growing edge. These characteristics were experimentally verified in the studied colonies. That these should show MBE dynamics is in strong contrast with the currently established concept of tumor growth: the kinetics of this type of proliferation rules out exponential or Gompertzian growth. Rather, a clear linear growth regime is followed. The importance of new cell movements-cell diffusion at the tumor border-lies in the fact that tumor growth must be conceived as a competition for space between the tumor and the host, and not for nutrients or other factors. Strong experimental evidence is presented for 16 types of tumor, the growth of which cell surface diffusion may be the main mechanism responsible in vivo. These results explain most of the clinical and biological features of colonies and tumors, offer new theoretical frameworks, and challenge the wisdom of some current clinical strategies.     

The in vivo differentiation of murine neuroblastoma

C1300 neuroblastoma was implanted with regenerating skeletal muscle to study the role of tissue interactions during tumor cell differentiation. Combined tumor-muscle implants, placed subcutaneously or within diffusion chambers were compared with control tumors implanted without muscle. Neuroblastoma implanted with injured muscle undergoes a partial neuronal differentiation. The tumor cells lose their normal round cell configuration and develop numerous cytoplasmic processes. Accompanying these outward changes are an increased content of microtubules in the neuritic processes; the appearance of glial-like processes containing abundant microfilaments; and the occurrence of growth vesicles identical to those of the growth cones of normal neurites. Although the implants usually contain large numbers of regenerated myofibers, tumor cell differentiation is not dependent upon the presence of these newly formed fibers. Tumor differentiation occurs equally well on the surfaces of degenerating muscle fragments, fibrin deposits and on the membrane surfaces of the diffusion chambers. These observations suggest that non-specific cell surface phenomena, rather than neuromuscular interactions were primarily responsible for the tumor cell differentiation in vivo.     

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn²⁺ and is used in vivo.The mean lifetime of water inside human erythrocytes was found to be 17 ms at 24°C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

Simultaneous in vivo optical quantification of key metabolic and vascular endpoints reveals tumor metabolic diversity in murine breast tumor models

Therapeutically exploiting vascular and metabolic endpoints becomes critical to translational cancer studies because altered vascularity and deregulated metabolism are two important cancer hallmarks. The metabolic and vascular phenotypes of three sibling breast tumor lines with different metastatic potential are investigated in vivo with a newly developed quantitative spectroscopy system. All tumor lines have different metabolic and vascular characteristics compared to normal tissues, and there are strong positive correlations between metabolic (glucose uptake and mitochondrial membrane potential) and vascular (oxygen saturations and hemoglobin concentrations) parameters for metastatic (4T1) tumors but not for micrometastatic (4T07) and nonmetastatic (67NR) tumors. A longitudinal study shows that both vascular and metabolic endpoints of 4T1 tumors increased up to a specific tumor size threshold beyond which these parameters decreased. The synchronous changes between metabolic and vascular parameters, along with the strong positive correlations between these endpoints suggest that 4T1 tumors rely on strong oxidative phosphorylation in addition to glycolysis. This study illustrates the great potential of our optical technique to provide valuable dynamic information about the interplay between the metabolic and vascular status of tumors, with important implications for translational cancer investigations.      

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique.

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn2+, and is used in vivo. The mean lifetime of water insed human erythrocytes was found to be 17 ms at 24 degrees C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

Simple and Reliable Determination of Intravoxel Incoherent Motion Parameters for the Differential Diagnosis of Head and Neck Tumors

Intravoxel incoherent motion (IVIM) imaging can characterize diffusion and perfusion of normal and diseased tissues, and IVIM parameters are authentically determined by using cumbersome least-squares method. We evaluated a simple technique for the determination of IVIM parameters using geometric analysis of the multiexponential signal decay curve as an alternative to the least-squares method for the diagnosis of head and neck tumors. Pure diffusion coefficients (D), microvascular volume fraction (f), perfusion-related incoherent microcirculation (D*), and perfusion parameter that is heavily weighted towards extravascular space (P) were determined geometrically (Geo D, Geo f, and Geo P) or by least-squares method (Fit D, Fit f, and Fit D*) in normal structures and 105 head and neck tumors. The IVIM parameters were compared for their levels and diagnostic abilities between the 2 techniques. The IVIM parameters were not able to determine in 14 tumors with the least-squares method alone and in 4 tumors with the geometric and least-squares methods. The geometric IVIM values were significantly different (p<0.001) from Fit values (+2±4% and −7±24% for D and f values, respectively). Geo D and Fit D differentiated between lymphomas and SCCs with similar efficacy (78% and 80% accuracy, respectively). Stepwise approaches using combinations of Geo D and Geo P, Geo D and Geo f, or Fit D and Fit D* differentiated between pleomorphic adenomas, Warthin tumors, and malignant salivary gland tumors with the same efficacy (91% accuracy = 21/23). However, a stepwise differentiation using Fit D and Fit f was less effective (83% accuracy = 19/23). Considering cumbersome procedures with the least squares method compared with the geometric method, we concluded that the geometric determination of IVIM parameters can be an alternative to least-squares method in the diagnosis of head and neck tumors.     

Fluorescence correlation spectroscopy and quantitative cell biology

Fluorescence correlation spectroscopy (FCS) analyzes fluctuations in fluorescence within a small observation volume. Autocorrelation analysis of FCS fluctuation data can be used to measure concentrations, diffusion properties, and kinetic constants for individual fluorescent molecules. Photon count histogram analysis of fluorescence fluctuation data can be used to study oligomerization of individual fluorescent molecules. If the FCS observation volume is positioned inside a living cell, these parameters can be measured in vivo. FCS can provide the requisite quantitative data for analysis of molecular interaction networks underlying complex cell biological processes.     

Engineering tumors with 3D scaffolds

Microenvironmental conditions control tumorigenesis and biomimetic culture systems that allow for in vitro and in vivo tumor modeling may greatly aid studies of cancer cells' dependency on these conditions. We engineered three-dimensional (3D) human tumor models using carcinoma cells in polymeric scaffolds that recreated microenvironmental characteristics representative of tumors in vivo. Strikingly, the angiogenic characteristics of tumor cells were dramatically altered upon 3D culture within this system, and corresponded much more closely to tumors formed in vivo. Cells in this model were also less sensitive to chemotherapy and yielded tumors with enhanced malignant potential. We assessed the broad relevance of these findings with 3D culture of other tumor cell lines in this same model, comparison with standard 3D Matrigel culture and in vivo experiments. This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.     

Improved Model of Fluorescence Recovery Expands the Application of Multiphoton Fluorescence Recovery after Photobleaching in Vivo

Multiphoton fluorescence recovery after photobleaching is a well-established microscopy technique used to measure the diffusion of macromolecules in biological systems. We have developed an improved model of the fluorescence recovery that includes the effects of convective flows within a system. We demonstrate the validity of this two-component diffusion-convection model through in vitro experimentation in systems with known diffusion coefficients and known flow speeds, and show that the diffusion-convection model broadens the applicability of the multiphoton fluorescence recovery after photobleaching technique by enabling accurate determination of the diffusion coefficient, even when significant flows are present. Additionally, we find that this model allows for simultaneous measurement of the flow speed in certain regimes. Finally, we demonstrate the effectiveness of the diffusion-convection model in vivo by measuring the diffusion coefficient and flow speed within tumor vessels of 4T1 murine mammary adenocarcinomas implanted in the dorsal skinfold chamber.