(December 2, 2021) The Bench to Bedside Series: Preclinical Cancer Research with Scintica
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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
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(December 2, 2021) The Bench to Bedside Series: Preclinical Cancer Research with Scintica
- 1. The Bench to Bedside Series: Preclinical Cancer Research with Scintica Katie Parkins, PhD Scientific Product Manager kparkins@scintica.com Tyler Lalonde, PhD Scientific Product Manager tlalonde@scintica.com
- 2. WWW.SCINTICA.COM Topics of Discussion 2 Introduction to Cancer Research What Does Bench to Bedside Mean? In Vitro Characterization Rapid Throughput vs. Quantitative Tools Moving Towards Translation
- 3. WWW.SCINTICA.COM What is Cancer? 3 Most 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM What does “Bench to Bedside” mean? 5 No longer a unidirectional process 1.Discovery Translational/Pre clinical Clinical
- 6. WWW.SCINTICA.COM Transition into Preclinical Models 6 In Vivo Clinical Billions of Drug Candidates In Vitro Characterization (High Throughput)
- 7. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM Starting with Cells Control for Researchers Test different conditions at once High throughput Inexpensive & easy to implement (resources, equipment costs, personnel, animal facilities)
- 10. WWW.SCINTICA.COM cBioPortal – Great Starting Point
- 11. WWW.SCINTICA.COM Start with Something that is High Throughput 11 Start with a 2D Immortalized Line • Simple to culture • Well known • Allows for initial high-throughput evaluation with therapies or intervention
- 12. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM What is Live Cell Imaging? 13 Lux2 Lux3 FL OMNI Bench Bedside
- 14. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM Transition into Preclinical Models 15 In Vivo Clinical Billions of Drug Candidates In Vitro Characterization (High Throughput)
- 16. WWW.SCINTICA.COM Entire subject Non-invasive Dynamic In Vitro Intact biology In Vivo Transition into Preclinical Models
- 17. WWW.SCINTICA.COM 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 radiation Bioluminescence, Fluorescence, 3D Tomography
- 18. WWW.SCINTICA.COM Optical Imaging: Oncology Example 18 Huizi Keiko Li et al., 2017 Xiang Ao et al., 2019 Bench Bedside
- 19. Audience Poll
- 20. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM IVIM – Oncology Example 21 Bench Bedside
- 22. WWW.SCINTICA.COM IVIM – Oncology Example 22 Bench Bedside
- 23. WWW.SCINTICA.COM IVIM – Oncology Example 23 Bench Bedside
- 24. WWW.SCINTICA.COM Immune Cells Infiltration in Various Tissue and Organ Models 24 Lymph Node (High Endothelial Venule) T-cell B-cell FRC KARS Granulocyte Skin (Inflammatory response) Ultra-high speed in vivo imaging (up to 100fps @ 512×512 pixels)
- 25. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM High-frequency Ultrasound - Oncology Example 27 Subcutaneous tumor model Transgenic liver tumor model Tumor Volume Bench Bedside
- 28. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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 μA; 45 kVp; 360 deg; 8 shots; 200 μm resolution; 10 min acq.
- 32. WWW.SCINTICA.COM Heterogeneity of a Tumor Using PET 32 • 47g nude mouse; Pancreatic subcutaneous tumor • Dose: 590 μCi (21.83 MBq) 18F-FDG; • Incubation period: 47 min • CT contrast: Iopamiro; 0.2 mL; ip • CT Acq: 350 μA; 45 kVp; 360 projections; 8 shots; 200 μm resolution • PET Acq: Static; 40 min; 400-700 keV
- 33. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM The Aspect M Series-MRI 35 Bench Bedside Mouse 1 Mouse 2 Day 10 Day 23 Day 10 Day 23 Mouse 1 4.9 mm3 248 mm3 Mouse 2 10 mm3 106 mm3 • Anatomy and Morphology • Neurology • Cancer Biology • Cardiovascular Biology • Multi-Modal Imaging • Ex Vivo Imaging • Contrast or no contrast imaging Preclinical MRI
- 36. WWW.SCINTICA.COM 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 = 20mm3 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. WWW.SCINTICA.COM Preclinical MRI - Oncology Example 37 Bench Bedside Therapeutic effect can be monitored over time using the same animal as its own control 0 50 100 150 200 250 300 350 5.5 Weeks 7 Weeks Tumor Volume (mm 3 ) Control Treated Control (n=4) Treated (n=30) (n=30) 5.5 weeks 179±46 mm3 93±8.5 mm3 7 weeks 227±64 mm3 134±11 mm3 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. WWW.SCINTICA.COM 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) (mm³) Upper Tumor red 144 Mid Tumor green 6 Lower Tumor blue 9 Lower Tumor #2 cyan 7 Mid Tumor #2 magenta 64
- 39. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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 mm3 Bench Bedside Model courtesy of Dr. R. DeSouza, STTARR (UHN)
- 41. WWW.SCINTICA.COM 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 (263mm3) or MRI (273mm3) 41 Orthotopic Mammary Fat Pad Tumor (MDA-MB-231) Optical Imaging - BLI Ultrasound MRI
- 42. WWW.SCINTICA.COM Transition into Preclinical Models 42 In Vivo Clinical Billions of Drug Candidates In Vitro Characterization (High Throughput)
- 43. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 4D Bioprinting – Tissue Printed Example 44 Bench Bedside 40 cm2 Stratified epidermis 500-700 µm thickness 21 Days availability
- 45. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM Hypoxia Chambers & Workstations 47 Bench Bedside
- 48. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM 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. WWW.SCINTICA.COM Q&A WWW.SCINTICA.COM INFO@SCINTICA.COM Please enter your questions in the Q&A section.
Editor's Notes
- Cancer refers to any one of a large number of diseases characterized by the development of abnormal cells that divide uncontrollably and have the ability to infiltrate and destroy normal body tissue. Cancer often has the ability to spread to other parts of the body through blood or lymphatic vessels and this is known as metastatsis. Cancer is the second-leading cause of death in the world accounting for nearly 10 million deaths in 2020. However survival rates are improving for many types of cancer, thanks to improvements in cancer screening, treatment and prevention which is largely a result of efforts in cancer research..
- Cancer research is a careful, step-by-step process. Researchers across the globe study every stage of the cancer journey, from prevention and screening to diagnosis, treatment, life after cancer and end-of-life care. What we know about cancer – how to prevent it, how it develops, how to treat it, how to help people cope with it – depends on different kinds of research. This research happens at many levels and in many diverse settings.
- It was previously thought that the journey from bench to bedside was unidirectional, starting with discovery research, moving towards preclinical or translational studies and ending with 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 and this starts early on during the research question stage as clinicians have a better understanding of the needs and/or gaps that exist in the clinic and where the focus needs to lie.
- Why is this important? Well if we look at this funnel representing the standard workflow of bench to bedside we know there may be billions of potential drug candidates, many will appear promising in their design and targeted approach and will move on to in vitro studies. From here it’s important to characterize the drug, particle, cell etc. to better understand it’s potential as a therapeutic so investigating things like efficacy, potency and toxicity and narrowing it down to a more limited list of candidates to assess in vivo. And then based on our findings from animal studies, very few will check all the boxes necessary to move on to clinical trials. And even with all of this screening (which typically takes years of research), many of the candidates that make it to the clinical phase, will not show to have the same effects in patients as was seen in the preclinical studies.
- As seen here in this chart published back in 2018, if we focus in on oncology studies only, of the 57% of studies that successfully make it from phase 1 to 2 during clinical trials, only 3.4% of those will end up getting approved. Its really important that as scientists we look for more efficient and translational tools to help us get from the development phase to clinic faster and more reliably.
- 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
- To understand how to treat and control cancer, researchers start by studying the basics – the cells, molecules and genes that are the building blocks of life. First, researchers work to understand how healthy cells grow. Then, they look for differences in cancer cells. This type of laboratory research, which is sometimes called basic research, aims to understand how cancer starts, grows and spreads. This knowledge is an essential starting point for developing future tests and treatments. Studying individual cells or tissues, rather than whole organisms, allows researchers to control and test many different factors. They can turn specific genes off or on, or expose cells to a certain substance, condition or possible treatment, and measure the effects.
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- Start with a 2D immortalized line It is simple to culture It is well known Allows for initial high-throughput evaluation with therapies or intervention
- Both CO2 and tri-gas (hypoxia) incubators are designed to generate and maintain a defined environment. They do a great job of imitating physiological conditions so that cells can grow in their ideal environment. The problem lies in the frequency the door of these chambers are opened and cells are removed from this ideal environment every time a measurement is to be made or an image is to be taken (usually under an external microscope). What this means is most scientists never actually measure cells under physiological conditions and the cells they are looking at are generally in some state of shock. Live-cell imaging itself has become a desired and often necessary analytical tool in many cell biology laboratories that operate in the field of neurobiology, developmental biology, pharmacology. Currently live-cell imaging is often difficult, because it required large costly high-end devices that are difficult to operate. The CytoSMART products are highly compact, easy to use, and affordable microscopes for bright-field and fluorescence live cell imaging so it can be used in every biological laboratory. While it has functionality for basic imaging, it also has the capability to be used in routine cell culture processes like tracking confluency over time.
- The field of in vivo animal studies grew out of the enormous wealth of information provided by in vitro diagnostics. These assays include histological assays, protein assays, microarrays, etc all giving information regarding the changes that occur during the pathogenesis of a disease. However, these assays have their limitations. Importantly, they only sample a part of the subject or diseased tissue, they give static information at a single time point during the disease process, they are invasive, and many times they are destructive which limits the amount of information we can get out of that sample. These limitations led people to envision if it’s possible to perform these types of assays in living subjects which we now know as in vivo research The advantages here are you can assay the entire subject, you get dynamic information since you can image the same subject longitudinally over time, and for the most part these assays are non-invasive leaving the underlying biology intact so that one can track changes in the disease process.
- Bioluminescence, fluorescence, 3D tomography Allows for multiple animals or samples to be imaged simultaneously Full spectrum tunability (400-800nm) User friendly modality that is easy to adapt, with little operation costs Whole-body imaging without any radiation
- All-in-one confocal/two-photon microscopy system designed and optimized for longitudinal imaging of live animal models in vivo
- SQ tumor model. To look and progression and regression of the tumor over time and response to treatment at cellular resolutions. Here you are looking at the tissue through a window chamber, tumor cells have been labelled in green and blood vessels have been labelled with intravenous injection of a red Fluorescent dye. The platforms allows you to look at the structure of the vessels in 3D down to capillary level. it has long been known that tumour blood vessels are heterogeneous with regard to organisation, function and structure. Whereas the normal vasculature is arranged in a hierarchy of evenly spaced, well-differentiated arteries, arterioles, capillaries, venules and veins, the tumour vasculature is unevenly distributed and chaotic.
- Longitudinal imaging of the tumor cells and vascular allows analyzing the drug’s anti-angiogenic effects on tumor tissue. We could find that in the drug treated group, the tumor vessels are less dilated compared to the normal group. How do angiogenesis inhibitors work? Angiogenesis inhibitors are unique cancer-fighting agents because they block the growth of blood vessels that support tumor growth rather than blocking the growth of tumor cells themselves. Angiogenesis inhibitors interfere in several ways with various steps in blood vessel growth. Some are monoclonal antibodies that specifically recognize and bind to VEGF. When VEGF is attached to these drugs, it is unable to activate the VEGF receptor. Other angiogenesis inhibitors bind to VEGF and/or its receptor as well as to other receptors on the surface of endothelial cells or to other proteins in the downstream signaling pathways, blocking their activities. Some angiogenesis inhibitors are immunomodulatory drugs—agents that stimulate or suppress the immune system—that also have antiangiogenic properties. In some cancers, angiogenesis inhibitors appear to be most effective when combined with additional therapies. Because angiogenesis inhibitors work by slowing or stopping tumor growth without killing cancer cells, they are given over a long period. What angiogenesis inhibitors are being used to treat cancer in humans? The U.S. Food and Drug Administration (FDA) has approved a number of angiogenesis inhibitors to treat cancer. Most of these are targeted therapies that were developed specifically to target VEGF, its receptor, or other specific molecules involved in angiogenesis. Approved angiogenesis inhibitors include: Axitinib (Inlyta®) Bevacizumab (Avastin®) Cabozantinib (Cometriq®) Everolimus (Afinitor®) Lenalidomide (Revlimid®) Lenvatinib mesylate (Lenvima®) Pazopanib (Votrient®) Ramucirumab (Cyramza®) Regorafenib (Stivarga®) Sorafenib (Nexavar®) Sunitinib (Sutent®) Thalidomide (Synovir, Thalomid®) Vandetanib (Caprelsa®) Ziv-aflibercept (Zaltrap®)
- After treating the drugs, vessel morphology become more normalized compared to no treatment model.
- High endothelial venules (HEVs) in a lymph node in the hind leg of the mouse, immune cell infiltrating Neutrophils infiltration in skin in Ear skin model They have injected KARS protein which is known to cause inflammation in cancer models. The KARS gene encodes lysyl-tRNA synthetase, which catalyzes the aminoacylation of tRNA-lys in both the cytoplasm and mitochondria. Fibroblastic reticular cells (FRCs) in the T cell zone of lymph nodes (LNs) are pivotal for T cell survival, mobility, and peripheral tolerance.
- Tumor detection 3D volume measurements Surrounding tissue investigation Blood flow monitoring – tumor vascularization
- PET Tracers have been developed to study: Cell proliferation Apoptosis Angiogenesis Metastasis Gene Expression Receptor-ligand interactions Substrate transportation Metabolism of nutrients PET may be used on the following tumor model types: Orthotopic Transgenic/spontaneous Xenografts Metastatic Oncology with PET: High sensitivity High spatial resolution Quantifiable
- True simultaneous PET/MRI system Combines the benefits of the M-Series MR imaging with that of the added PET image for further image details and analysis
- Autologous approach Multimodal, 4D bioprinting system 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
- Autologous approach
- Various in vitro cancer models used in chemotherapeutic screening. Evolution of cell-culture models from simple 2D to complex 3D bio-printed models. Conventional 2D monolayer culture, monolayer co-culture, cells grown over floating membranes, and cell monolayer sandwiched between membranes, are the commonly used 2D cancer models in research and drug screening. Cancer cells cultured in hydrogels, spheroid monoculture, spheroid co-culture, cancer/stromal cells cultured in porous 3D scaffolds, and advanced bioprinted constructs are amongst the available 3D cancer models.
- The bioprinted constructs with bottom and top ADMSC layers were cultured for 21 d and then treated with different concentrations of DOX. The constructs with 21PT alone served as controls. We evaluated the 21PT apoptosis ratio by staining them with cleaved Caspase-3. As shown in Figure 3A, the percentage of cleaved Caspase-3 positive cells increased with increasing concentrations of DOX. For 3D bioprinted 21PT and ADMSC constructs, DOX can penetrate through the ADMSC layer into the cancer region, but at a lower DOX dose (i.e., 1 and 10 μg/mL), the cleaved Caspase-3 positive cell percentage was significantly lower than that for the cancer cell alone constructs (Figure 3B). A high DOX dose (i.e., 100 μg/mL) resulted in a high apoptosis rate for both 21PT alone and 21PT and ADMSC constructs. Apoptosis and LOX expression of the bioprinted constructs in response to various DOX doses. (A) Representative IF staining for vimentin (red), cleaved Caspase-3 (green), and nuclei (blue) of 21PT and ADMSC within bioprinted constructs after 21 d culture and with the addition of DOX for another 3 d culture; scale bar = 250 μm. (B) Percentage of cells that were stained positive to cleaved Caspase-3 based on IF staining analysis; n = 3− 5, **p < 0.01, ***p < 0.001. (C) Entrapped DOX concentration within different 3D bioprinted constructs after DOX treatment. The amount of DOX was measured by HPLC and normalized to the wet weight of the hydrogels. n = 3, *p < 0.05. (D) Representative IF staining for αSMA (red), LOX (green), and nuclei (blue) of 21PT and ADMSC within bioprinted constructs after 21 d culture and with the addition of DOX for another 3 d culture; scale bar = 250 μm. (E) LOX activity at day 21, with or without ADMSC; LOX secretion in the media at different time points (n = 3). The data are normalized to the average of bioprinted constructs with 21PT only, **p < 0.05.
- 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 etc. Considerations: Infrastructure Resources Instrumentation Expertise