Delta radiomics analysis of Magnetic Resonance guided radiotherapy imaging data can enable treatment response prediction in pancreatic cancer.

BACKGROUND: Magnetic Resonance Image guided Stereotactic body radiotherapy (MRgRT) is an emerging technology that is increasingly used in treatment of visceral cancers, such as pancreatic adenocarcinoma (PDAC). Given the variable response rates and short progression times of PDAC, there is an unmet clinical need for a method to assess early RT response that may allow better prescription personalization. We hypothesize that quantitative image feature analysis (radiomics) of the longitudinal MR scans acquired before and during MRgRT may be used to extract information related to early treatment response. METHODS: Histogram and texture radiomic features (n = 73) were extracted from the Gross Tumor Volume (GTV) in 0.35T MRgRT scans of 26 locally advanced and borderline resectable PDAC patients treated with 50 Gy RT in 5 fractions. Feature ratios between first (F1) and last (F5) fraction scan were correlated with progression free survival (PFS). Feature stability was assessed through region of interest (ROI) perturbation. RESULTS: Linear normalization of image intensity to median kidney value showed improved reproducibility of feature quantification. Histogram skewness change during treatment showed significant association with PFS (p = 0.005, HR = 2.75), offering a potential predictive biomarker of RT response. Stability analyses revealed a wide distribution of feature sensitivities to ROI delineation and was able to identify features that were robust to variability in contouring. CONCLUSIONS: This study presents a proof-of-concept for the use of quantitative image analysis in MRgRT for treatment response prediction and providing an analysis pipeline that can be utilized in future MRgRT radiomic studies.

Green does not always mean go: A sulfated galactan from Codium isthmocladum green seaweed reduces melanoma metastasis through direct regulation of malignancy features.

Melanoma is the most lethal form of skin cancer, with a worldwide increase in incidence. Despite the increased overall survival of metastatic melanoma patients given recent advances in targeted and immunotherapy, it still has a poor prognosis and available treatment options carry diverse severe side effects. Polysaccharides from seaweed have been shown to exert antitumor activities. Here we show in vitro and in vivo antitumor activities of a sulfated homogalactan (named 3G4S) from Codium isthmocladum seaweed in the B16-F10 murine melanoma cell line. 3G4S did not induce cytotoxicity or proliferation changes; however, it was able to reduce solid tumor growth and metastasis, while not inducing side effects in mice. B16-F10 cells traits related to the metastatic cascade were also impaired by 3G4S, reducing cell invasion, colony-forming capacity and membrane glycoconjugates. Therefore, 3G4S shows promising antitumor activities without the commonly associated drawbacks of cancer treatments and can be further explored.

Targeting ion transport in cancer.

The metabolism of cancer cells differs substantially from normal cells, including ion transport. Although this phenomenon has been long recognized, ion transporters have not been viewed as suitable therapeutic targets. However, the acidic pH values present in tumours which are well outside of normal limits are now becoming recognized as an important therapeutic target. Carbonic anhydrase IX (CAIX) is fundamental to tumour pH regulation. CAIX is commonly expressed in cancer, but lowly expressed in normal tissues and that presents an attractive target. Here, we discuss the possibilities of exploiting the acidic, hypoxic tumour environment as possible target for therapy. Additionally, clinical experience with CAIX targeting in cancer patients is discussed.

Predicting the safety and efficacy of buffer therapy to raise tumour pHe: an integrative modelling study.

BACKGROUND: Clinical positron emission tomography imaging has demonstrated the vast majority of human cancers exhibit significantly increased glucose metabolism when compared with adjacent normal tissue, resulting in an acidic tumour microenvironment. Recent studies demonstrated reducing this acidity through systemic buffers significantly inhibits development and growth of metastases in mouse xenografts. METHODS: We apply and extend a previously developed mathematical model of blood and tumour buffering to examine the impact of oral administration of bicarbonate buffer in mice, and the potential impact in humans. We recapitulate the experimentally observed tumour pHe effect of buffer therapy, testing a model prediction in vivo in mice. We parameterise the model to humans to determine the translational safety and efficacy, and predict patient subgroups who could have enhanced treatment response, and the most promising combination or alternative buffer therapies. RESULTS: The model predicts a previously unseen potentially dangerous elevation in blood pHe resulting from bicarbonate therapy in mice, which is confirmed by our in vivo experiments. Simulations predict limited efficacy of bicarbonate, especially in humans with more aggressive cancers. We predict buffer therapy would be most effectual: in elderly patients or individuals with renal impairments; in combination with proton production inhibitors (such as dichloroacetate), renal glomular filtration rate inhibitors (such as non-steroidal anti-inflammatory drugs and angiotensin-converting enzyme inhibitors), or with an alternative buffer reagent possessing an optimal pK of 7.1-7.2. CONCLUSION: Our mathematical model confirms bicarbonate acts as an effective agent to raise tumour pHe, but potentially induces metabolic alkalosis at the high doses necessary for tumour pHe normalisation. We predict use in elderly patients or in combination with proton production inhibitors or buffers with a pK of 7.1-7.2 is most promising.

The biology underlying molecular imaging in oncology: from genome to anatome and back again.

Cancers are complex, evolving, multiscale ecosystems that are characterized by profound spatial and temporal heterogeneity. The interactions in cancer are non-linear in that small changes in one variable can have large changes on another. These multiple interacting phenotypes and spatial scales can best be understood with appropriate mathematical and computational models. Imaging is central to this investigation because it can non-destructively and longitudinally characterize spatial variations in the tumour phenotype and environment so that the system dynamics over time can be captured quantitatively.

Cellular adaptations to hypoxia and acidosis during somatic evolution of breast cancer.

Conceptual models of carcinogenesis typically consist of an evolutionary sequence of heritable changes in genes controlling proliferation, apoptosis, and senescence. We propose that these steps are necessary but not sufficient to produce invasive breast cancer because intraductal tumour growth is also constrained by hypoxia and acidosis that develop as cells proliferate into the lumen and away from the underlying vessels. This requires evolution of glycolytic and acid-resistant phenotypes that, we hypothesise, is critical for emergence of invasive cancer. Mathematical models demonstrate severe hypoxia and acidosis in regions of intraductal tumours more than 100 microm from the basement membrane. Subsequent evolution of glycolytic and acid-resistant phenotypes leads to invasive proliferation. Multicellular spheroids recapitulating ductal carcinoma in situ (DCIS) microenvironmental conditions demonstrate upregulated glucose transporter 1 (GLUT1) as adaptation to hypoxia followed by growth into normoxic regions in qualitative agreement with model predictions. Clinical specimens of DCIS exhibit periluminal distribution of GLUT-1 and Na(+)/H(+) exchanger (NHE) indicating transcriptional activation by hypoxia and clusters of the same phenotype in the peripheral, presumably normoxic regions similar to the pattern predicted by the models and observed in spheroids. Upregulated GLUT-1 and NHE-1 were observed in microinvasive foci and adjacent intraductal cells. Adaptation to hypoxia and acidosis may represent key events in transition from in situ to invasive cancer.

Microenvironmental and cellular consequences of altered blood flow in tumours.

Tumour angiogenesis is triggered by various signals characteristic of the tumour microenvironment, including low oxygen tension, low extracellular pH and low glucose concentration. Tumour microvasculature is chaotic, producing perfusion heterogeneities which can be visualized by MRI and other modalities. Inefficient perfusion in tumours produces regions of transient and chronic hypoxia. Tumour hypoxia is associated with adverse clinical outcomes and reduced patient survival. Hypoxia may be a factor in activation of extracellular matrix-degrading proteases, and some studies have correlated primary tumour hypoxia with likelihood of tumour cell dissemination. Exposure to hypoxia either induces or selects for cells that are hyperglycolytic, and this in turn produces local acidosis which is also a common feature of solid tumours. Increased glucose uptake in hyperglycolyzing tumour cells is the basis of lesion-visualization in positron emission tomography using 18F-fluorodeoxyglucose. Tumour acidity can reduce the effectiveness of weak-base drugs, but can be exploited to increase the anti-tumour activity of weak-acid chemotherapeutics. Evidence linking tumour acidity with increased activity of several extracellular matrix-degrading enzyme systems is examined. High levels of lactate, another end-product of glycolysis, in primary lesions have been correlated with increased likelihood of metastasis. In the numerous studies correlating hypoxia, acidity and lactate with metastasis, the direction of the causality has not been adequately established. We hypothesize that adoption of a hyperglycolytic phenotype is a necessary feature of carcinogenesis itself, and confers a survival and proliferative advantage to tumour cells over surrounding normal cells. Empirical evidence supporting this "acid-mediated tumour invasion" model is discussed.

Combined vascular and extracellular pH imaging of solid tumors.

The unique physiological environment of solid tumors, frequently characterized by areas of poor flow, hypoxia, high lactate and low extracellular pH (pHe), influences vascularization, invasion and metastasis. Thus, vascularization and the physiological and metabolic environment play permissive (and conversely preventive) roles in invasion and metastasis. By using a multi-parametric approach of combined vascular and spectroscopic imaging, we can begin to evaluate which combinations of vascular, metabolic and physiological regions in a solid tumor represent the highest 'metastatic threat'. Here, we present measurements of pHe, vascular volume and permeability from co-localized regions within a solid tumor. These studies were performed for a group of metastatic (MDA-MB-231) and non-metastatic (MCF-7) human breast cancer xenografts. In this study, we have demonstrated the feasibility of such an approach, and presented methods of analyses to detect differences in patterns of combined parameters obtained from spatially co-registered regions in a solid tumor.

In vivo molecular imaging.

The relatively young field of molecular imaging is focused on the visualization of molecular phenotypes in whole organisms. This is achieved using imaging systems based on radionuclides, nuclear magnetic resonance, ultrasound, or the visible-IR region of the optical spectrum. Molecularly defined contrast in these modalities is generated by exogenous probes of the endogenous proteome, or through transgenes. Examples of exogenous probes include those that are transported and trapped (glucose, nucleoside analogs), those directed against extracellular receptors (somatostatin, opioid, melanotropin), and those activated by extracellular proteases. Transgenes that have been used in molecular imaging include the above receptors, non-mammalian enzymes that trap pro-drugs (HSV-tk, yeast CD), and optical reporter proteins (luciferase, fluorescent proteins). Cutting edge technologies in this field include in vivo assays for protein-protein interactions, and in vivo assays for mRNA expression patterns. The number of degrees of freedom in designing new agents is daunting, and advancements in this field will require a significant participation from molecular and cellular biochemists.

pH and chemotherapy.

In vivo pH measurements by magnetic resonance spectroscopy reveal the presence of large regions of acidic extracellular pH in tumours, with the intracellular pH being maintained in the neutral-to-alkaline range. This acid-outside plasmalemmal pH gradient acts to exclude weak base drugs such as the anthracyclines and vinca alkaloids, a behaviour that is predicted by the decrease in octanol-water partition coefficients of mitoxantrone and doxorubicin with decreasing solution pH. This pH gradient can be reduced or eliminated in mouse models of breast cancer by systemic treatment with sodium bicarbonate. We have demonstrated tumour alkalinization following chronic ad libitum administration of NaHCO3 and acute intraperitoneal administration of NaHCO3 to tumour-bearing mice. Chronic treatment of tumour-bearing SCID mice with NaHCO3 results in an enhancement in MCF-7 tumour xenograft response to doxorubicin. Intraperitoneal administration of NaHCO3 to tumour-bearing C3H/Hen mice prior to treatment with mitoxantrone results in a greater-than 4.5-fold increase in cell-kill in the syngeneic C3H mammary tumour model. Most combination chemotherapy regimens include at least one weak base drug. Our results suggest that agents such as sodium bicarbonate, Carbicarb and the diuretic furosemide--which are known to induce metabolic alkalosis in humans--may be useful in enhancing the efficacy of these treatment regimens in humans.

The physiological environment in cancer vascularization, invasion and metastasis.

One of the most lethal aspects of cancer arises from its ability to invade and metastasize. Determining the factors that promote cancer cell invasion and metastasis is therefore critically important in treating this disease. The tumour physiological environment is uniquely different from normal tissue, and exhibits hypoxia, acidic extracellular pH and high levels of lactate. This environment, dictated largely by abnormal tumour vasculature and metabolism, in turn also promotes angiogenesis. The physiological environment, tumour metabolism, angiogenesis and vascularization are therefore inextricably linked. We have developed and applied non-invasive magnetic resonance (MR) imaging (I) and spectroscopy (S) techniques to understand the role of vascular, physiological and metabolic properties in cancer invasion and metastasis. These MR studies are performed with human breast and prostate cancer cells maintained in culture or grown as solid tumours in immune-suppressed mice. We have detected significant differences in vascular, physiological and metabolic characteristics of metastatic and non-metastatic human breast and prostate cancer models with MRI and MRS. Using a combined MRI/MRS approach we are currently acquiring metabolic, extracellular pH and vascular images from the same localized regions within a solid tumour to further understand the dynamics between these parameters and their role in cancer invasion and metastasis.

Frontiers in the measurement of cell and tissue pH.

Investigation into the causes and consequences of aberrant pH regulation is entirely dependent on the ability to measure this parameter with specificity and sensitivity. Since their beginnings in the 1950s, techniques for measuring cell and tissue pH have undergone a number of significant advances. Following each of these advances, new investigators have been brought into the field, and new discoveries have been made. From microelectrode and dye distribution studies, measurement of pH underwent a revolution with the advent of pH-sensitive dyes that could be loaded into the cytosol. A further significant advance came from the measurement of cell and tissue pH in whole organisms by magnetic resonance spectroscopy (MRS). Frontiers in pH measurement exist in the development of pH-sensitive proteins and pH-sensitive MR contrast agents. These are predicted to bring even more people into this fascinating field, and generate more important discoveries.

Acute metabolic alkalosis enhances response of C3H mouse mammary tumors to the weak base mitoxantrone.

Uptake of weak acid and weak base chemotherapeutic drugs by tumors is greatly influenced by the tumor extracellular/interstitial pH (pH(e)), the intracellular pH (pH(i)) maintained by the tumor cells, and by the ionization properties of the drug itself. The acid-outside plasmalemmal pH gradient in tumors acts to exclude weak base drugs like the anthracyclines, anthraquinones, and vinca alkaloids from the cells, leading to a substantial degree of "physiological drug resistance" in tumors. We have induced acute metabolic alkalosis in C3H tumor-bearing C3H/hen mice, by gavage and by intraperitoneal (i.p.) administration of NaHCO(3). (31)P magnetic resonance spectroscopic measurements of 3-aminopropylphosphonate show increases of up to 0.6 pH units in tumor pH(e), and 0.2 to 0.3 pH units in hind leg tissue pH(e), within 2 hours of i.p. administration of NaHCO(3). Theoretical calculations of mitoxantrone uptake into tumor and normal (hind leg) tissue at the measured pH(e) and pH(i) values indicate that a gain in therapeutic index of up to 3.3-fold is possible with NaHCO(3) pretreatment. Treatment of C3H tumor-bearing mice with 12 mg/kg mitoxantrone resulted in a tumor growth delay of 9 days, whereas combined NaHCO(3)--mitoxantrone therapy resulted in an enhancement of the TGD to 16 days.

Applications of magnetic resonance in model systems: tumor biology and physiology.

A solid tumor presents a unique challenge as a system in which the dynamics of the relationship between vascularization, the physiological environment and metabolism are continually changing with growth and following treatment. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) studies have demonstrated quantifiable linkages between the physiological environment, angiogenesis, vascularization and metabolism of tumors. The dynamics between these parameters continually change with tumor aggressiveness, tumor growth and during therapy and each of these can be monitored longitudinally, quantitatively and non-invasively with MRI and MRS. An important aspect of MRI and MRS studies is that techniques and findings are easily translated between systems. Hence, pre-clinical studies using cultured cells or experimental animals have a high connectivity to potential clinical utility. In the following review, leaders in the field of MR studies of basic tumor physiology using pre-clinical models have contributed individual sections according to their expertise and outlook. The following review is a cogent and timely overview of the current capabilities and state-of-the-art of MRI and MRS as applied to experimental cancers. A companion review deals with the application of MR methods to anticancer therapy.

Applications of magnetic resonance in model systems: cancer therapeutics.

The lack of information regarding the metabolism and pathophysiology of individual tumors limits, in part, both the development of new anti-cancer therapies and the optimal implementation of currently available treatments. Magnetic resonance [MR, including magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and electron paramagnetic resonance (EPR)] provides a powerful tool to assess many aspects of tumor metabolism and pathophysiology. Moreover, since this information can be obtained nondestructively, pre-clinical results from cellular or animal models are often easily translated into the clinic. This review presents selected examples of how MR has been used to identify metabolic changes associated with apoptosis, detect therapeutic response prior to a change in tumor volume, optimize the combination of metabolic inhibitors with chemotherapy and/or radiation, characterize and exploit the influence of tumor pH on the effectiveness of chemotherapy, characterize tumor reoxygenation and the effects of modifiers of tumor oxygenation in individual tumors, image transgene expression and assess the efficacy of gene therapy. These examples provide an overview of several of the areas in which cellular and animal model studies using MR have contributed to our understanding of the effects of treatment on tumor metabolism and pathophysiology and the importance of tumor metabolism and pathophysiology as determinants of therapeutic response.

Imaging prostate cancer invasion with multi-nuclear magnetic resonance methods: the Metabolic Boyden Chamber.

The physiological milieu within solid tumors can influence invasion and metastasis. To determine the impact of the physiological environment and cellular metabolism on cancer cell invasion, it is necessary to measure invasion during well-controlled modulation of the physiological environment. Recently, we demonstrated that magnetic resonance imaging can be used to monitor cancer cell invasion into a Matrigel layer [Artemov D, Pilatus U, Chou S, Mori N, Nelson JB, and Bhujwalla ZM (1999). Dynamics of prostate cancer cell invasion studied in vitro by NMR microscopy. Mag Res Med 42, 277-282.]. Here we have developed an invasion assay ("Metabolic Boyden Chamber") that combines this capability with the properties of our isolated cell perfusion system. Long-term experiments can be performed to determine invasion as well as cellular metabolism under controlled environmental conditions. To characterize the assay, we performed experiments with prostate cancer cell lines preselected for different invasive characteristics. The results showed invasion into, and degradation of the Matrigel layer, by the highly invasive/metastatic line (MatLyLu), whereas no significant changes were observed for the less invasive/metastatic cell line (DU-145). With this assay, invasion and metabolism was measured dynamically, together with oxygen tensions within the cellular environment and within the Matrigel layer. Such a system can be used to identify physiological and metabolic characteristics that promote invasion, and evaluate therapeutic interventions to inhibit invasion.

Enhancement of chemotherapy by manipulation of tumour pH.

The extracellular (interstitial) pH (pHe) of solid tumours is significantly more acidic compared to normal tissues. In-vitro, low pH reduces the uptake of weakly basic chemotherapeutic drugs and, hence, reduces their cytotoxicity. This phenomenon has been postulated to contribute to a 'physiological' resistance to weakly basic drugs in vivo. Doxorubicin is a weak base chemotherapeutic agent that is commonly used in combination chemotherapy to clinically treat breast cancers. This report demonstrates that MCF-7 human breast cancer cells in vitro are more susceptible to doxorubicin toxicity at pH 7.4, compared to pH 6.8. Furthermore 31P-magnetic resonance spectroscopy (MRS) has shown that the pHe of MCF-7 human breast cancer xenografts can be effectively and significantly raised with sodium bicarbonate in drinking water. The bicarbonate-induced extracellular alkalinization leads to significant improvements in the therapeutic effectiveness of doxorubicin against MCF-7 xenografts in vivo. Although physiological resistance to weakly basic chemotherapeutics is well-documented in vitro and in theory, these data represent the first in vivo demonstration of this important phenomenon.

In vivo imaging of extracellular pH using 1H MRSI.

Tumor pH is physiologically important since it influences a number of processes relevant to tumorigenesis and therapy. Hence, knowledge of localized pH within tumors would contribute to understanding these processes. The destructiveness, poor spatial resolution, and poor signal-to-noise ratio (SNR) of current technologies (e.g., microelectrodes, 31P magnetic resonance spectroscopy) have limited such studies. An extrinsic chemical extracellular pH (pHe) probe is described that is used in combination with 1H magnetic resonance spectroscopic imaging to yield pHe maps with a spatial resolution of 1 x 1 x 4 mm3. The principle of the technique is demonstrated on a phantom. Further data are shown to demonstrate its application in vivo, and results agree with previously reported pH values. The accuracy of the reported pH measurements is <0.1 pH units, as derived from a detailed analysis of the errors associated with the technique, the description of which is included.