Cathepsin K in lymphangioleiomyomatosis: LAM cell-fibroblast Interactions enhance protease activity by extracellular acidification

Lymphangioleiomyomatosis (LAM) is a rare disease in which clonal ‘LAM’ cells infiltrate the lungs and lymphatics. In association with recruited fibroblasts, LAM cells form nodules adjacent to lung cysts. It is assumed LAM nodule derived proteases lead to cyst formation although, this is uncertain. We profiled protease gene expression in whole lung tissue and observed cathepsin K was 40 fold over-expressed in LAM compared with control lungs (p≤0.0001). Immunohistochemistry confirmed cathepsin K protein in LAM nodules but not control lungs. Cathepsin K gene expression, protein and protease activity was detected in LAM associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immune reactivity was predominantly co-localised with LAM associated fibroblasts. In vitro, extra-cellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced in fibroblast cultures at pH 7 and 6. 621-101 cells reduced extracellular pH by 0.5 units over 24 hours. Acidification was dependent upon 621-101 cell mTOR activity and net hydrogen ion transporters, particularly sodium/bicarbonate co-transporters and carbonic anhydrases which were also expressed in LAM lung tissue. In LAM cell/fibroblast co-cultures, acidification paralleled cathepsin K activity and both were inhibited by sodium bicarbonate co-transporter (p≤0.0001) and carbonic anhydrase inhibitors (p=0.0021). Our findings suggest cathepsin K activity is dependent on LAM cell/fibroblast interactions and inhibitors of extracellular acidification may be potential therapies for LAM

Interrogating Tumor Metabolism with AcidoCEST MRI

Tumor metabolism is a highly dysregulated process that is identified as a unique target for therapy. Current philosophy proposes that tumor metabolism is a plastic and flexible process which sustains proliferative and survival advantages. Tumors employ an anaerobic glycolytic pathway resulting in the overproduction of lactate. Additional thinking suggests that the conversion of pyruvate to lactate regenerates the NAD+ pool in the cell, maintaining a sustainable oxidative environment. Regardless of the reasons for lactate overproduction, its excretion and build up in the microenvironment results in acidic tumor microenvironments. Tumor acidosis has been measured with several different methods, but consistently averages from pH 6.6 to 7.0. Tumor acidity can thus be measured as a biomarker for tumor metabolism. This work examines the commonly explored energy pathways available to the cancer cell and a non-invasive MRI method to measure the efficacy of the tumor metabolism targeting agent. Appendix A is an introduction to tumor metabolism pathways and the large list of candidate therapies in interfering with energy production. Glucose, fatty acid, and glutamine metabolisms are all discussed along with PI3K/AKT/mTOR and HIF growth signals and ion transport. Magnetic resonance imaging and positron emission tomography are examined as imaging methods for non-invasively interrogating tumor acidosis. Appendix B presents the findings in a study where tumor metabolism was targeted with an mTOR inhibitor, where tumor growth rate was initially decreased and accompanied by an early, acute increase in tumor extracellular pH with acidoCEST MRI. Chapter 2 discusses the combination of a lactate dehydrogenase inhibitor in conjunction with doxorubicin in a breast cancer model. Tumor extracellular pH was shown to increase when measured with acidoCEST MRI, and an increase in cell death was measured. Chapter 4 discusses the studies and experimental designs that can be done in the near future

Interrogating tumor energy metabolism with acidocest MRI

Tumor metabolism is a highly dysregulated process that is identified as a unique target for therapy. Current philosophy proposes that tumor metabolism is a plastic and flexible process which sustains proliferative and survival advantages. Tumors employ an anaerobic glycolytic pathway resulting in the overproduction of lactate. Additional thinking suggests that the conversion of pyruvate to lactate regenerates the NAD+ pool in the cell, maintaining a sustainable oxidative environment. Regardless of the reasons for lactate overproduction, its excretion and build up in the microenvironment results in acidic tumor microenvironments. Tumor acidosis has been measured with several different methods, but consistently averages from pH 6.6 to 7.0. Tumor acidity can thus be measured as a biomarker for tumor metabolism. This work examines the commonly explored energy pathways available to the cancer cell and a non-invasive MRI method to measure the efficacy of the tumor metabolism targeting agent. Appendix A is an introduction to tumor metabolism pathways and the large list of candidate therapies in interfering with energy production. Glucose, fatty acid, and glutamine metabolisms are all discussed along with PI3K/AKT/mTOR and HIF growth signals and ion transport. Magnetic resonance imaging and positron emission tomography are examined as imaging methods for non-invasively interrogating tumor acidosis. Appendix B presents the findings in a study where tumor metabolism was targeted with an mTOR inhibitor, where tumor growth rate was initially decreased and accompanied by an early, acute increase in tumor extracellular pH with acidoCEST MRI. Chapter 2 discusses the combination of a lactate dehydrogenase inhibitor in conjunction with doxorubicin in a breast cancer model. Tumor extracellular pH was shown to increase when measured with acidoCEST MRI, and an increase in cell death was measured. Chapter 4 discusses the studies and experimental designs that can be done in the near future

Abstract LB-161: Monitoring early therapeutic response by measuring extracellular pH in a tumor model with acidoCEST MRI

Abstract Introduction: The purpose of the present study is to examine whether tumor extracellular pH (pHe) is a sensitive and specific biomarker for measuring immediate, short term drug response to an mTOR inhibiting agent (RAD001, everolimus) by a novel, non-invasive MRI-based pH imaging method (acidoCEST). Methods: Two groups of SCID mice with Granta 519 human mantle cell lymphoma flank xenograft models were used in the study. First, to investigate if RAD001 has anti-tumor activity in the Granta 519 model, mice were injected IP with 5 mg/kg per day with RAD001 or vehicle (DMSO) (N=12 per group) every day until tumors reached 2000 mm3. Survival analysis was conducted using GraphPad software. In a separate correlative imaging study mice with tumors averaging 400 mm3 (N=8) were scanned with acidoCEST MRI pretreatment, one day post initial treatment, and one week after continuous treatment. To prepare for each acidoCEST MRI scan session, a mouse was anesthetized with 1.5% isoflurane, respiration rate and body temperature were monitored, and body temperature was maintained at 37.0᠑C with warm air. A bolus of 200 μL of 370 mgI/mL iopamidol was injected IV prior to scanning, followed by continuous infusion of iopamidol at 150 uL/hour during scanning. A CEST-FISP MRI protocol was performed with a Bruker Biospec 7T MRI scanner with a 72 mm volume coil using previously published procedures. The results were analyzed using Matlab, following previously published procedures. The average pHe was determined for each tumor. Pixels which registered a pHe of 6.00 to 6.99 were grouped together as the “acidic fraction,” and those pixels with pH ≥7 were collectively grouped together as the “neutral fraction.” The percent uptake of the contrast agent in the tumor area was measured to assess vascular perfusion in the tumor microenvironment. Results: RAD001 treatment results in a significant survival advantage in Granta 519 flank xenograft tumors. AcidoCEST imaging of tumors showed pretreatment an average pHe value of 6.82, demonstrating that the tumor model was moderately acidic. The standard deviation of pixelwise pHe values was 0.24, indicating moderate heterogeneity of pHe in the measured tumor area. Post initial treatment, the pHe had a statistically significant increase to 6.92 with a standard deviation of 0.14, indicating a decrease in average acidosis and a decrease in heterogeneity of acidosis. After continuous treatment, the pHe dropped to 6.74 with a standard deviation of 0.26, suggesting a return to a more normal metabolic rate. In keeping with the trend, the acidic fraction of the tumor decreased from 33.1% to 19.6%, before increasing to 49% during this study. The tumor growth study showed a temporary growth delay before returning to a more normal growth rate, which paralleled the acidoCEST MRI results. Conclusion: Tumor pHe measured with acidoCEST MRI can detect early response to RAD001. Additional pre-clinical studies are warranted to determine if this biomarker is a robust measure of early therapeutic response. In addition, translation of acidoCEST MRI to the clinic should be pursued to provide this biomarker for clinical studies. Citation Format: Paul J. Akhenblit, Christine M. Howison, Scott W. Malm, Liu Qi Chen, Amanda F. Baker, Mark D. Pagel. Monitoring early therapeutic response by measuring extracellular pH in a tumor model with acidoCEST MRI. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-161. doi:10.1158/1538-7445.AM2014-LB-161</jats:p

Assessing Metabolic Changes in Response to mTOR Inhibition in a Mantle Cell Lymphoma Xenograft Model Using AcidoCEST MRI

AcidoCEST magnetic resonance imaging (MRI) has previously been shown to measure tumor extracellular pH (pHe) with excellent accuracy and precision. This study investigated the ability of acidoCEST MRI to monitor changes in tumor pHe in response to therapy. To perform this study, we used the Granta 519 human mantle cell lymphoma cell line, which is an aggressive B-cell malignancy that demonstrates activation of the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin ( mTOR) pathway. We performed in vitro and in vivo studies using the Granta 519 cell line to investigate the efficacy and associated changes induced by the mTOR inhibitor, everolimus (RAD001). AcidoCEST MRI studies showed a statistically significant increase in tumor pHe of 0.10 pH unit within 1 day of initiating treatment, which foreshadowed a decrease in tumor growth of the Granta 519 xenograft model. AcidoCEST MRI then measured a decrease in tumor pHe 7 days after initiating treatment, which foreshadowed a return to normal tumor growth rate. Therefore, this study is a strong example that acidoCEST MRI can be used to measure tumor pHe that may serve as a marker for therapeutic efficacy of anticancer therapies.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]