Gary V. Martinez

Imaging pH and metastasis

Metastasis is a multistep process that culminates in the spread of cells from a primary tumor to a distant site or organs. For tumor cells to be able to metastasize, they have to locally invade through basement membrane into the lymphatic and the blood vasculatures. Eventually they extravasate from the blood and colonize in the secondary organ. This process involves multiple interactions between the tumor cells and their microenvironments. The microenvironment surrounding tumors has a significant impact on tumor development and progression. A key factor in the microenvironment is an acidic pH. The extracellular pH of solid tumors is more acidic in comparison to normal tissue as a consequence of high glycolysis and poor perfusion. It plays an important role in almost all steps of metastasis. The past decades have seen development of technologies to non‐invasively measure intra‐ and/or extracellular pH. Most successful measurements are MR‐based, and sensitivity and accuracy have dramatically improved. Quantitatively imaging the distribution of acidity helps us understand the role of the tumor microenvironment in cancer progression. The present review discusses different MR methods in measuring tumor pH along with emphasizing the importance of extracelluar tumor low pH on different steps of metastasis; more specifically focusing on epithelial‐to‐mesenchymal transition (EMT), and anti cancer immunity. Copyright

High resolution pHe imaging of rat glioma using pH-dependent relaxivity

Previous studies using MR spectroscopy have shown that the extracellular pH (pHe) of tumors is acidic compared to normal tissues. This has a number of important sequelae that favor the emergence of more aggressive and therapy‐resistant tumors. New MRI methods based on pH‐sensitive T1 relaxivity are an attractive alternative to previous spectroscopic methods, as they allow improvements in spatial and temporal resolution. Recently, pH‐dependent GdDOTA‐4AmP5‐ and a pH‐independent analog, GdDOTP5‐, were used to image renal pH in mice. The current study has used a similar approach to image pHe in rat gliomas. Significant differences were observed compared to the renal study. First, the relaxivity of GdDOTP5‐ was found to be affected by the higher extracellular protein content of tumors. Second, the pixel‐by‐pixel analysis of the GdDOTP5‐ and GdDOTA‐4AmP5‐ pharmacokinetics showed significant dispersion, likely due to the temporal fluctuations in tumor perfusion. However, there was a robust correlation between the maximal enhancements produced by the two boluses. Therefore, to account for the local time‐courses differences, pHe maps were calculated at the time of maximal enhancement in each pixel. Finally, the comparison of the pHe and the time to maximal intensity maps revealed an inverse relationship between pHe and tumor perfusion. Magn Reson Med, 2006.

Exploiting evolutionary principles to prolong tumor control in preclinical models of breast cancer

Using evolutionary principles to guide drug administration, monotherapy with paclitaxel can maintain prolonged stability of breast cancer in preclinical models. Evolution of cancer therapy The standard approach to treating cancer is giving patients the maximum tolerated amount of chemotherapy with the goal of doing the maximum possible damage to the tumor without killing the patient. This method is relatively effective, but it also causes major toxicities. Now, Enriquez-Navas et al. have demonstrated a different approach for ensuring efficacy of chemotherapy and minimizing toxicity. The authors used an evolutionary approach, where the dose of chemotherapy is guided by the tumor’s response to the previous dose, allowing a gradual withdrawal of the drug if the tumor continues to respond. This method proved quite effective for paclitaxel treatment in two different mouse models and warrants further evaluation in additional models as well as human trials. Conventional cancer treatment strategies assume that maximum patient benefit is achieved through maximum killing of tumor cells. However, by eliminating the therapy-sensitive population, this strategy accelerates emergence of resistant clones that proliferate unopposed by competitors—an evolutionary phenomenon termed “competitive release.” We present an evolution-guided treatment strategy designed to maintain a stable population of chemosensitive cells that limit proliferation of resistant clones by exploiting the fitness cost of the resistant phenotype. We treated MDA-MB-231/luc triple-negative and MCF7 estrogen receptor–positive (ER+) breast cancers growing orthotopically in a mouse mammary fat pad with paclitaxel, using algorithms linked to tumor response monitored by magnetic resonance imaging. We found that initial control required more intensive therapy with regular application of drug to deflect the exponential tumor growth curve onto a plateau. Dose-skipping algorithms during this phase were less successful than variable dosing algorithms. However, once initial tumor control was achieved, it was maintained with progressively smaller drug doses. In 60 to 80% of animals, continued decline in tumor size permitted intervals as long as several weeks in which no treatment was necessary. Magnetic resonance images and histological analysis of tumors controlled by adaptive therapy demonstrated increased vascular density and less necrosis, suggesting that vascular normalization resulting from enforced stabilization of tumor volume may contribute to ongoing tumor control with lower drug doses. Our study demonstrates that an evolution-based therapeutic strategy using an available chemotherapeutic drug and conventional clinical imaging can prolong the progression-free survival in different preclinical models of breast cancer.

HDAC inhibitors enhance T-cell chemokine expression and augment response to PD-1 immunotherapy in lung adenocarcinoma

Purpose: A significant limitation of checkpoint blockade immunotherapy is the relatively low response rate (e.g., ∼20% with PD-1 blockade in lung cancer). In this study, we tested whether strategies that increase T-cell infiltration to tumors can be efficacious in enhancing immunotherapy response. Experimental Design: We performed an unbiased screen to identify FDA-approved oncology agents with an ability to enhance T-cell chemokine expression with the goal of identifying agents capable of augmenting immunotherapy response. Identified agents were tested in multiple lung tumor models as single agents and in combination with PD-1 blockade. Additional molecular and cellular analysis of tumors was used to define underlying mechanisms. Results: We found that histone deacetylase (HDAC) inhibitors (HDACi) increased expression of multiple T-cell chemokines in cancer cells, macrophages, and T cells. Using the HDACi romidepsin in vivo, we observed increased chemokine expression, enhanced T-cell infiltration, and T-cell–dependent tumor regression. Importantly, romidepsin significantly enhanced the response to PD-1 blockade immunotherapy in multiple lung tumor models, including nearly complete rejection in two models. Combined romidepsin and PD-1 blockade also significantly enhanced activation of tumor-infiltrating T cells. Conclusions: These results provide evidence for a novel role of HDACs in modulating T-cell chemokine expression in multiple cell types. In addition, our findings indicate that pharmacologic induction of T-cell chemokine expression represents a conceptually novel approach for enhancing immunotherapy response. Finally, these results suggest that combination of HDAC inhibitors with PD-1 blockade represents a promising strategy for lung cancer treatment. Clin Cancer Res; 22(16); 4119–32. ©2016 AACR.

Imaging the extracellular pH of tumors by MRI after injection of a single cocktail of T 1 and T 2 contrast agents

The extracellular pH (pHe) of solid tumors is acidic, and there is evidence that an acidic pHe is related to invasiveness. Herein, we describe an MRI single‐infusion method to measure pHe in gliomas using a cocktail of contrast agents (CAs). The cocktail contained gadolinium–1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraaminophosphonate (GdDOTA‐4AmP) and dysprosium–1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetrakis(methylenephosphonic acid) (DyDOTP), whose effects on relaxation are sensitive and insensitive to pH, respectively. The Gd‐CA dominated the spin–lattice relaxivity ΔR1, whereas the Dy‐CA dominated the spin–spin relaxivity ΔR2*. The ΔR2* effects were used to determine the pixel‐wise concentration of [Dy] which, in turn, was used to calculate a value for [Gd] concentration. This value was used to convert ΔR1 values to the molar relaxivity Δr1 and, hence, pHe maps. The development of the method involved in vivo calibration and measurements in a rat brain glioma model. The calibration phase consisted of determining a quantitative relationship between ΔR1 and ΔR2* induced by the two pH‐independent CAs, gadolinium–diethylenetriaminepentaacetic acid (GdDTPA) and DyDOTP, using echo planar spectroscopic imaging (EPSI) and T1‐weighted images. The intensities and linewidths of the water peaks in EPSI images were affected by CA and were used to follow the pharmacokinetics. These data showed a linear relationship between inner‐ and outer‐sphere relaxation rate constants that were used for CA concentration determination. Nonlinearity in the slope of the relationship was observed and ascribed to variations in vascular permeability. In the pHe measurement phase, GdDOTA‐4AmP was infused instead of GdDTPA, and relaxivities were obtained through the combination of interleaved T1‐weighted images (R1) and EPSI for ΔR2*. The resulting r1 values yielded pHe maps with high spatial resolution. Copyright

Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302

BackgroundHypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models.ResultsThree human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors.ConclusionsUsing metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Diffusion MRI and Novel Texture Analysis in Osteosarcoma Xenotransplants Predicts Response to Anti-Checkpoint Therapy

Combinations of targeted drugs have been employed to treat sarcomas, however, response rates have not improved notably, therefore emphasizing the need for novel treatments. In addition, imaging approaches to assess therapeutic response is lacking, as currently measurable indices, such as volume and/or diameter, do not accurately correlate with changes in tumor biology. In this study, quantitative and profound analyses of magnetic resonance imaging (MRI) were developed to evaluate these as imaging biomarkers for MK1775 and Gem in an osteosarcoma xenotransplant model at early time-points following treatment. Notably, we showed that Gem and Gem+MK1775 groups had significantly inhibited tumor growth by day 4, which was presaged by elevations in mean ADC by 24 hours post treatment. Significant differences were also observed at later time points for the Gem+MK1775 combination and MK1775 therapy. ADC distribution and entropy (randomness of ADC values) were also elevated by 24 hours following therapy. Immunohistochemistry demonstrated that these treatment-related increases in ADC correlated with apoptosis and observed cell condensations (dense- and exploded bodies). These findings underline the role of ADC as a quantitative imaging biomarker for therapy-induced response and show promising clinical relevance in the sarcoma patient population.

Evaluation of LDH-A and Glutaminase Inhibition In Vivo by Hyperpolarized 13C-Pyruvate Magnetic Resonance Spectroscopy of Tumors

Hyperpolarized (13)C magnetic resonance spectroscopy provides a unique opportunity to detect real-time metabolic fluxes as a means to measure metabolic treatment responses in vivo. Here, we show that pharmacologic inhibition of lactate dehydrogenase-A suppressed the conversion of hyperpolarized (13)C-pyruvate to lactate in murine xenografts of P493 human lymphoma. In contrast, a glutaminase inhibitor reduced conversion of (13)C-pyruvate to alanine without affecting conversion of pyruvate to lactate. These results illustrate the ability to monitor biomarkers for responses to antimetabolic therapy in real-time, paving the way for clinical development of imaging biomarkers to monitor metabolic pharmacodynamics.

Development and in vivo quantitative magnetic resonance imaging of polymer micelles targeted to the melanocortin 1 receptor.

Recent emphasis has focused on the development of rationally designed polymer-based micelle carriers for drug delivery. The current work tests the hypothesis that target specificity can be enhanced by micelles with cancer-specific ligands. In particular, we describe the synthesis and characterization of a new gadolinium texaphyrin (Gd-Tx) complex encapsulated in an IVECT micellar system, stabilized through Fe(III) cross-linking and targeted with multiple copies of a specific ligand for the melanocortin 1 receptor (MC1R), which has been evaluated as a cell-surface marker for melanoma. On the basis of comparative MRI experiments, we have been able to demonstrate that these Gd-Tx micelles are able to target MC1R-expressing xenograft tumors in vitro and in vivo more effectively than various control systems, including untargeted or un-cross-linked Gd-Tx micelles. Taken in concert, the findings reported herein support the conclusion that appropriately designed micelles are able to deliver contrast agent payloads to tumors expressing the MC1R.