Main description:

This volume includes comprehensive methods and protocols on autophagic pathways in cancer disorders. Chapters cover basic principles of autophagy methodology in cancer cells, translational approaches, systematic computational analyses of TCGA (The Cancer Genome Atlas) cohorts, CCLE (The Cancer Cell Line Encyclopedia) analysis, CRISPR genetics, metabolomics, biochemistry, and immunohistochemistry methodologies. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Autophagy and Cancer: Methods and Protocols aims to be a useful practical guide to researches to help further their study in this field.

Contents:

Part I: OVERVIEW OF THE FIELD

1. A balance between autophagy and other cell death modalities in cancer

Anna S. Gorbunova, Gelina S. Kopeina and Boris D. Zhivotovsky

Part II: CHAPERONE-MEDIATED AUTOPHAGY (CMA)

2. Isolation of Autophagy competent Lysosomes from Cancer cells by Differential Large-scale Multi-layered Density Gradient Centrifugations

Merve Kacal and Helin Vakifahmetoglu-Norberg.

3. CRISPR-Cas9 Gene Editing to create Isoform specific LAMP-2A Knockout in Human Cancer Cells

Ting-ting Yu, Xun Zhou and Helin Vakifahmetoglu-Norberg

Part III: MACROAUTOPHAGY

4. Modified LC3 dot quantification method

Tahira Anwar and Eeva-Liisa Eskelinen.

5. A quantitative flow cytometry-based method for autophagy detection across the cell cycle

Vitaly O. Kaminsky.

6. A dual HiBiT-GFP-LC3 lentiviral reporter for autophagy flux assessment

Rainer Will, Katja Bauer, Matthias Kudla, Jetsy Montero-Vergara, Stefan Wiemann, Verena Jendrossek, Samuel Pena-Llopis and Silvia Vega-Rubin-de-Celis.

7. Measuring autophagic cargo flux with Keima-based probes

Nikolai Engedal, Tonje Sonstevold, Carsten J. Beese, Sarvini Selladurai, Thea Melcher, Julia E. Simensen, Lisa B. Frankel, Alfonso Urbanucci and Maria L. Torgersen.

8. Detection of nuclear biomarkers for chromosomal instability

Carles Pons, Eugenia Almacellas, Albert Tauler and Caroline Mauvezin.

9. Analysis of autophagic vesicles in mitotic cells

Eugenia Almacellas, Marta Garcia-Cajide, Albert Tauler and Caroline Mauvezin.

10. Approaches and protocols to analyze autophagy and its role in death of apoptosis-resistant senescent tumor cells

Elena Y. Kochetkova, Valery A. Pospelov and Tatiana V. Pospelova.

11. Concepts and design of introducing synthetic microRNAs into mammalian cells

Per Hydbring.

12. Studying autophagy using a TMT-based quantitative proteomics approach

Kiran Bala Sharma, Suruchi Aggarwal, Amit Kumar Yadav, Sudhanshu Vrati and Manjula Kalia.

Part IV: MITOPHAGY

13. Immunofluorescence-based measurement of autophagosome formation during mitophagy

Benjamin S. Padman and Michael Lazarou.

14. Induction and detection of mitophagy

Maria A. Yapryntseva, Boris Zhivotovsky and Vladimir Gogvadze.

Part V: AUTOPHAGY ANALYSIS In vivo

15. Analysis of pancreatic acinar protein solubility in autophagy-deficient mice

Matthew Smith, Carla Salomo-Coll, Morwenna Muir and Simon Wilkinson.

16. Studying autophagy in vivo in the mammary gland and in xenograft samples

Zhongju Zou, Alvaro Fernandez Fernandez, Verena Jendrossek and Silvia Vega-Rubin-de-Celis.

Part VI: COMPUTATIONAL APPROACHES

17. A method to Identify Potential Prognostic Markers Across Distinct Tumor Types

Boxi Zhang, Elena Kochetkova and Erik Norberg.

18. A method for Co-Expression Analysis

Boxi Zhang and Erik Norberg.

Part VII: METABOLISM

19. Methodological approaches for assessing metabolomic changes in glioblastomas -MS

Trang T. T. Nguyen, Enyuan Shang, Mike-Andrew Westhoff, Georg Karpel-Massler and Markus D. Siegelin

20. Extraction of Metabolites from Cancer Cells

Elena Kochetkova, Tina Becirovic and Erik Norberg

21. Measuring glucose consumption and gluconeogenesis in 3D human tissue cultures with nanoliter input volumes

Aurino M. Kemas and Volker M. Lauschke