(December 2, 2021) The Bench to Bedside Series: Preclinical Cancer Research with Scintica

Overview:

The goal of this webinar will be to provide a high-level overview of the various stages of preclinical cancer research and discuss the role that innovative instrumentation can play in moving science forward.

To better understand how to treat and control cancer, researchers start by investigating the basics – the cells, molecules, and genes that make up the human body. This type of study, which is often referred to as basic or discovery research, aims to understand the underlying mechanisms contributing to cancer growth and spread. This knowledge is an essential starting point for developing future diagnostic tests and treatment strategies.

After finding an innovative idea that works in cells, researchers need to take their studies to the next level by employing animal models that have similar biology to humans. Animal models have helped scientists make some of the most important cancer discoveries over the years. Furthermore, preclinical imaging technologies allow researchers to perform longitudinal animal studies that are noninvasive leaving the underlying biology intact so that one can track changes throughout the entire disease process.

It was previously thought that the journey from bench to bedside was unidirectional, starting with discovery research and moving towards clinical trials. However, in the last decade, it has become crucial for basic scientists and clinicians to work together towards finding innovative solutions that will positively impact patient care.

After attending this webinar, we hope you will have a better understanding of the preclinical workflow needed to push an idea from bench to bedside as well as some of the key equipment that is needed along the way.

This webinar series will be hosted by Drs. Katie Parkins and Tyler Lalonde, both of which have extensive experience in translational research areas including oncology, neuroscience, molecular imaging, and drug development.

In this webinar we will discuss the following topics:

• Introduction To Cancer Research
• What does “Bench to Bedside” mean?
• In vitro characterization
• Rapid throughput screening
• Quantitative tools
• Moving towards translation

1. The Bench to Bedside Series: Preclinical Cancer Research with Scintica Katie Parkins, PhD Scientific Product Manager [email protected] Tyler Lalonde, PhD Scientific Product Manager [email protected]

2. Topics of Discussion 2 Introduction to Cancer Research What Does Bench to Bedside Mean? In Vitro C haracterization Rapid Throughput vs. Quantitative Tools Moving Towards Translation

3. What is Cancer? 3 M ost common cancers (new cases 2020): breast (2.26 million) lung (2.21 million) colon and rectum (1.93 million) prostate (1.41 million) skin (non-melanoma) (1.20 million) stomach (1.09 million) Most common causes of cancer deaths (2020): lung (1.80 million) colon and rectum (935 000) liver (830 000) stomach (769 000) breast (685 000) Cancer accounts for nearly 10 million deaths in 2020 World Health Organization

4. Introduction to Cancer Research 4 All stages of cancer journey are important and can be studied This research happens at many levels and in many different settings The goal is to move basic science discoveries more quickly and efficiently into practice

5. What does “Bench to Bedside” mean? 5 No longer a unidirectional process

6. Transition into Preclinical Models 6 In Vivo Clinical Billions of Drug Candidates In Vitro C haracterization (High Throughput)

7. Why Is This Important? We need to find more efficient and translational tools to help us get from development phase to clinic faster and more reliably 7

8. Plan Your Study 8 Define clear research objectives Search the literature and understand the road map that already exists What are the gaps and how can we fill them? Outline experiments and hypotheses Set reasonable timelines /milestones Look for answers in the data NOT in your hypotheses or past literature

9. Starting with Cells Control for Researchers Test different conditions at once High throughput Inexpensive & easy to implement (resources, equipment costs, personnel, animal facilities)

10. cBioPortal – Great Starting Point

11. Start with Something that is High T hroughput 11 Start with a 2D Immortalized L ine Simple to culture Well known Allows for initial high-throughput evaluation with therapies or intervention

12. What is Live Cell Imaging? Cells need to be monitored in order to study the fundamentals of cell growth dynamics Removing cells to take measurements and images prevent accurate measurements under physiological conditions Live cell monitoring can be done with compact brightfield microscopes that fit within incubators Basic brightfield and fluorescent imaging Routine cell culture processes i.e. tracking cell confluency 12

13. What is Live Cell Imaging? 13 Lux2 Lux3 FL OMNI Bench Bedside

14. Live Cell Imaging- Oncology Example 14 Measuring proliferation rates Assessing transfection/transduction efficiency Monitoring co-culture i.e. cancer cells + immune cells Effect of drugs on cells Bench Bedside

15. Transition into Preclinical Models 15 In Vivo Clinical Billions of Drug Candidates In Vitro C haracterization (High Throughput)

16. Entire subject Non-invasive Dynamic In Vitro Intact biology In Vivo Transition into Preclinical Models

17. The Newton 7.0 – Optical Imaging 17 TdTomato FLI Luciferase BLI Bench Bedside Multiple animals or samples to be imaged simultaneously Full spectrum tunability (400-800nm) User friendly & easy to adapt Little operation costs Whole-body imaging No r adiation Bioluminescence, Fluorescence, 3D Tomography

18. Optical Imaging: Oncology Example 18 Huizi Keiko Li et al., 2017 Xiang Ao et al., 2019 Bench Bedside

19. Audience Poll

20. IVIM - IntraVital Microscopy (IVM) 20 Designed and optimized for longitudinal imaging of live animal models in vivo All-in-one confocal/two-photon microscopy Bench Bedside

21. IVIM – Oncology Example 21 Bench Bedside

22. IVIM – Oncology Example 22 Bench Bedside

23. IVIM – Oncology Example 23 Bench Bedside

24. Immune Cells Infiltration in Various Tissue and Organ Models 24 Lymph Node (High Endothelial Venule) T-cell B-cell FRC KARS Granulocyte S kin ( Inflammatory response) Ultra-high speed in vivo imaging (up to 100fps @ 512×512 pixels) 

25. Prospect T1 - High Frequency Ultrasound 25 Bench Bedside Tumor detection 3D volume measurements Surrounding tissue investigation Blood flow monitoring – tumor vascularization High-Frequency Ultrasound

26. Cancer Research – Preclinical Solid Tumor Models Solid tumor models commonly used in preclinical research: Cell line-derived models Patient Derived Xenograft (PDX) models Environmentally induced models Genetically Engineered Mouse (GEM) models 26 Figure from Gengenbacher et al. Nature Reviews Cancer (2017) 17:751-765.

27. High-frequency Ultrasound - Oncology Example 27 Subcutaneous tumor model Transgenic liver tumor model Tumor Volume Bench Bedside

28. Orthotopic versus IP Injected Tumors 28 Orthotopic Mammary Fat Pad Tumor (MDA-MB-231) IP injection of ovarian tumor cells (SKOV-3) Tumor Bench Bedside

29. High-frequency Ultrasound – Oncology Example Complex tumor models can be investigated using ultrasound Normal tissues are identified, followed by identification of abnormal tissues Changes in nearby tissues could also be investigated 29 Kidney Splenic Vein Tumour Intestine Bench Bedside

30. The SuperArgus - PET/CT 30 Bench Bedside Cell proliferation Apoptosis Angiogenesis Metastasis Gene Expression Receptor-ligand interactions Substrate transportation Metabolism of nutrients PET Tracers developed to study: Orthotopic Transgenic/spontaneous Xenografts Metastatic PET imaging for the following tumor model types: High sensitivity High spatial resolution Quantifiable Why PET Imaging for Oncology:

31. PET/CT - Oncology Example 31 Courtesy of Dr. M. Desco & J.J. Vaquero, UMCE Hospital Gregorio Marañón HGUGM (Madrid, Spain) Bench Bedside 168g Wistar Male Rat with large subcutaneous tumor on hind limb Dose: 1.15 mCi (42.55 MBq ) of 18F-FDG Incubation period: 49 min PET Acq . Time: 45 min; 3 FOV; 400-700 keV; 8 slices overlap CT Acq : 150 μ A; 45 kVp ; 360 deg; 8 shots; 200 μ m resolution; 10 min acq .

32. Heterogeneity of a Tumor Using PET 32 47g nude mouse; Pancreatic subcutaneous tumor Dose: 590 μ Ci (21.83 MBq ) 18F-FDG; Incubation period: 47 min CT contrast: Iopamiro ; 0.2 mL; ip CT Acq : 350 μ A; 45 kVp ; 360 projections; 8 shots; 200 μ m resolution PET Acq : Static; 40 min; 400-700 keV

33. PET/CT - Oncology Example 33 Bench Bedside Minn , Il, et al. "Imaging CAR T cell therapy with PSMA-targeted positron emission tomography." Science advances 5.7 (2019): eaaw5096.

34. PET/CT - Oncology Example 34 Bench Bedside Kumar, Dhiraj, et al. "Peptide-based PET quantifies target engagement of PD-L1 therapeutics." The Journal of clinical investigation 129.2 (2019): 616-630.

35. The Aspect M Series- MRI 35 Bench Bedside Mouse 1 Mouse 2 Day 10 Day 23 Day 10 Day 23 Mouse 1 4.9 mm 3 248 mm 3 Mouse 2 10 mm 3 106 mm 3 Anatomy and Morphology Neurology Cancer Biology Cardiovascular Biology Multi-Modal Imaging Ex Vivo Imaging Contrast or no contrast imaging Preclinical MRI

36. Preclinical MRI - Oncology Example 36 Day 4 – normal anatomical structures are visible Day 15 – tumor is visible, spread throughout the brain, enlarged ventricles Tumor volume = 20mm 3 4 days post injection 15 days post injection T2 weighted: FSE (TE/TR=73.8/3100, FOV=40x20mm, Matrix=256x128, NEX=20, ETL=16, Res. 156um, Acq. Time 10 min) Bench Bedside

37. Preclinical MRI - Oncology Example 37 Bench Bedside Therapeutic effect can be monitored over time using the same animal as its own control Control (n=4) Treated (n=30) 5.5 weeks 179±46 mm 3 93±8.5 mm 3 7 weeks 227±64 mm 3 134±11 mm 3 Control Treated T2 weighted: FSE (TE/TR=52.7/3500, FOV=80x30mm, Matrix=256x96, NEX=8, ETL=16, Res. 312um, Acq Time 4:40 min) T1 weighted: SE (TE/TR=9.8/500, FOV=80x30mm, Matrix=256x96, NEX=3, Res. 312um, Acq Time 2:42min:sec) Model Courtesy of Drs. Naz Chaudary , Richard Hill & Shawn Stapleton, Princess Margaret Cancer Center

38. Cancer Biology: IP Injected Ovarian Tumor Cells 38 SKOV3 cell line, injected IP 250um in plane resolution; 4.5 min acquisition time T2 Weighted T1 Weighted T1 Weighted T2 Weighted Region Of Interest Color Volume (mm³) Upper Tumor red 144 Mid Tumor green 6 Lower Tumor blue 9 Lower Tumor #2 cyan 7 Mid Tumor #2 magenta 64

39. SimPET - PET insert 39 Bench Bedside True simultaneous PET/MRI Combines the benefits of the M-Series MR imaging with PET imaging Further image details and analysis Simultaneous PET/MR Imaging

40. Simultaneous PET/MR - Oncology Example Tumor showed increase metabolism on FDG-PET compared to contralateral muscle (ratio = 2.7) Central region of tumor showed decreased PET signal, T2 weighted MR image indicates increased fluid content – possibly a necrotic core CT images may provide additional anatomical context 40 PET PET + CT + MRI MRI – T1w CT MRI – T2w Necrotic Core Tumor Tumor volume is best measured on MRI = 410 mm 3 Bench Bedside Model courtesy of Dr. R. DeSouza , STTARR (UHN)

41. Complimentary Nature of Imaging Modalities -Example Bioluminescence helps to confirm viability of the tumor cells, as they express luciferase, approximate volumes may be possible from the BLI signal; anatomical images help to confirm tumor volume - ultrasound (263mm 3 ) or MRI (273mm 3 ) 41 Orthotopic Mammary Fat Pad Tumor (MDA-MB-231) Optical Imaging - BLI Ultrasound MRI

42. Transition into Preclinical Models 42 In Vivo Clinical Billions of Drug Candidates In Vitro C haracterization (High Throughput)

43. NGB-R - 4D Bioprinting 43 Bench Bedside Multimodal, 4D bioprinting Print from cells to spheroids using a large number of biomaterials and hydrogels Bridge the translational gap from animal to human Bio-printed autologous tissues for personalized cancer therapies Tissue Engineering

44. 4D Bioprinting – Tissue Printed Example 44 Bench Bedside 40 cm 2 Stratified epidermis 500-700 µm thickness 21 Days availability

45. Bioprinting - Oncology Example 45 Bioprinting was evolved to overcome the limitations of conventional 2D cell culture to create functional tissues, organoids, tumors, and organ-on-a-chip models Provides more clinically relevant 3D cancer models for chemotherapeutic screening R. Augustine, S.N. Kalva, R. Ahmad et al. Translational Oncology, 2021. Bench Bedside

46. Bioprinting - Oncology Example 46 Bench Bedside Wang, Ying, et al. "3D bioprinting of breast cancer models for drug resistance study." ACS Biomaterials Science & Engineering 4.12 (2018): 4401-4411.

47. Hypoxia Chambers & Workstations 47 Bench Bedside

48. Hypoxia chambers – Oncology Example 48 Used to grow and maintain cancer cell populations within the relevant environment Test therapeutics i.e. T-cells behave differently in hypoxic conditions Use hypoxia inducible systems i.e. gene expression driven by hypoxia Bench Bedside

49. Using Tools & Systems Together 49 NK cells maintained under hypoxic conditions and characterized in vitro Co-culture experiments: Cancer cells expressing RFP and NK cells expressing GFP Tumor monitoring (RFP imaging); NK cell tracking (GFP imaging) Tumor volume (MRI); Tumor metabolism (PET)

50. In Summary 50 Cancer research is multidisciplinary and requires many areas of expertise to come together to answer important questions about underlying mechanisms, treatment response, causes of recurrence. Overall Goal: Encourage scientific collaboration and conduct multicenter preclinical trials to be more efficient and effective in studying the progression of cancer. Considerations: Infrastructure Resources Instrumentation Expertise

51. Audience Poll

52. Q&A WWW.SCINTICA.COM [email protected] Please enter your questions in the Q&A section.

53. Thank You WWW.SCINTICA.COM [email protected]