This webinar hosted by Scintica Instrumentation will discuss the benefits of ultrasound imaging in cancer research. Ms. Tonya Coulthard will be the presenter of this webinar, she will touch on several topics throughout the presentation as they pertain to cancer biology and preclinical imaging. First off a brief overview of the different types of tumor models, followed by a look at the differences between optical and ultrasound imaging in cancer research, and finally a brief thought on image guided injections for placement of tumor cells in various models. During the last half of the webinar Tonya will walk participants through some of the recent publications using high frequency ultrasound imaging to monitor tumor growth, study therapeutic effect on progression, as well as some publications using a variety of contrast agents. The work will highlight researchers using the Prospect T1 system manufactured by S-Sharp. Ultrasound imaging is a non-invasive imaging technique using sound waves to produce images of a variety of internal structures, including tumors. Ultrasound is used clinically, as well as preclinically to detect and assess tumor location and size, as well as therapeutic response. Many tumor models use mice and/or rats, necessitating high frequency ultrasound with image resolution down to 30µm. Topics discussed in this webinar will include: Different types of tumor models Ultrasound vs. optical imaging in cancer research Image guided injections of cancer cells Publication overview

1. Tonya Coulthard, MSc.
Manager, Imaging Division
Scintica Instrumentation
Phone: +1 (519) 914 5495
tcoulthard@scintica.com
Cancer Research in Small
Animals: A Review of
Recent Publications Using
High Frequency
Ultrasound

2. • Preclinical Cancer Research
• Imaging in Preclinical Cancer Research
• Recent Publications
Topics of Discussion

3. Preclinical Cancer Research
• Preclinical Solid Tumor Model Overview
• Cell Line Derived / Transplantable Tumor Models
• Genetically Engineered Mouse Tumor Models
• Patient Derived Xenograft Tumor Models
• Preclinical Metastatic Tumor Models

4. • Preclinical cancer research focuses on the following:
• identify biological pathways and mechanisms of
tumorgenesis, growth and metastasis
• screen potential therapeutic compounds or
approaches for their efficacy
• The focus is on the translational aspects of bringing
novel therapeutic approaches to the clinic
Begley et al. Nature (2012) 483(7391): 531-533.
Preclinical Cancer Research

5. • A number of different types of solid tumor models are used
commonly in preclinical research:
• Cell line-derived models
• Patient Derived Xenograft (PDX) models
• Environmentally induced models
• Genetically Engineered Mouse (GEM) models
Gengenbacher et al. Nature Reviews Cancer (2017) 17:751-765.
Preclinical Solid Tumor Models

6. • A review of publications from
2016 found that most work was
being done using a single type
of model, on a single tumor
type
• Breast, lung, colorectal,
melanoma, pancreas, brain and
liver were the top solid tumor
types being studied
• There is a clear preference for
cell line derived models, with
varying levels of PDX, GEM, and
environmental models depending
on the tumor type
Gengenbacher et al. Nature Reviews Cancer (2017) 17:751-765.
Preclinical Solid Tumor Models

7. • Findings may not correlate to clinical outcomes – cell lines represent only a single population of cancer patients
Dranoff, Glenn Nature Reviews Immunology (2012) 12:61-66.
Cell Line Derived / Transplantable Tumor Models
• Work horse in cancer research – propagated
easily
• Subcutaneous injection
• Orthotopic injection – relevant
microenvironment may be present
• IV injection for metastasis
• May be genetically engineered to contain
luciferase or other compounds to allow them to be
imaged – i.e. bioluminescence

8. Dranoff, Glenn Nature Reviews Immunology (2012) 12:61-66.
Genetically Engineered Mouse Tumor Models
• Mice with genetically engineered/humanized immune
systems
• used to study specific parts of the immune system
• may cause enhanced tumor susceptibility
• Tissue-specific and/or temporally controlled
expression of oncogenes or loss of tumor
suppressor genes
• spontaneous tumor development
• more closely mimic the human disease
• Tumor formation is variable and takes longer to
develop
• Model the multiple stages in the development of cancer, and importance of the interaction between neoplastic cells
and the tissue microenvironment

9. Lai et al. Journal of Hematology & Oncology (2017) 10:106-.
Patient Derived Xenograft Tumor Models
• Resected tumor tissue is implanted into
immunodeficient mice; may be placed
subcutaneously or orthotopically
• Can be expanded in vivo through limited
passages in mice
• Only model system that allows for the vast
inter-patient and intra-tumor
heterogeneity that is inherent to human
cancer
• PDX models can be used to identify and test efficacy of therapeutic compounds in vivo and in vitro
• The actual patient may enroll in a specific trial, simultaneously avatar mice can be used in parallel to explore
multiple therapeutic options

10. • When used, primary tumor may be engrafted
ectopically or orthotopically, and in some studies
both methods were employed
• Metastatic studies make up only about 25% of the
cancer research being performed
• Metastasis can be induced in two ways
• IV injection of tumor cells either in the tail
vein, intracardiac injection, splenic vein
injection or intraperitoneal injection
• By spontaneous metastasis from a
primary tumor – at times the primary tumor
is resected to ensure survival of the animal
Gengenbacher et al. Nature Reviews Cancer (2017) 17:751-765.
Preclinical Metastatic Tumor Models

11. Audience Poll #1

12. Imaging in Preclinical Cancer Research
• Imaging Modality Review
• High Frequency Ultrasound – Prospect T1 Imaging Capabilities
• Tumor detection/monitoring and surrounding tissue imaging
• Injections for orthotopic placement or metastatic tumor models
• Tumor vasculature and biomarker detection
• Advanced techniques for drug delivery and early lesion detection

13. Li et al. Cancers (2019) 11:1800.
Imaging in Cancer Research
• Each modality has specific strengths/weaknesses as they
relate to cancer research
• CT provides good resolution of bones and lung, while
exposing the imaging subject to radiation
• PET requires the use of radioactive tracers to visualize
the tumors, and should be coregistered with another
imaging modality to provide anatomical context, but is
highly sensitive to metastasis and early pathological
changes
• Bioluminescence requires the tumor that express
luciferase; tumor volume measurements are dependent
on expression of the luciferase enzyme and delivery of
the luciferin substrate to the tumor cells, which may
change throughout disease progression or regression

14. Li et al. Cancers (2019) 11:1800.
Imaging in Cancer Research
• MRI provides excellent soft tissue resolution to
visualize normal and abnormal tissue structures, including
tumors; acquisition times vary from 2-5 minutes, but
provides good resolution (150-300µm) images of the
whole body. Contrast agents can be used to study
perfusion and specific biomarkers.
• Ultrasound (high frequency) provides high-resolution
real-time images with a strong soft tissue contrast to
visualize the tumor; contrast agents may be used to
study tumor perfusion or molecular expression of specific
targets

15. • Ultrasound is a non-invasive imaging technique which does not required the use of ionizing radiation,
instead it uses sound waves
• The transducer both sends and receives the ultrasound waves and the computer interprets the returned signal
into an image
Note – these images were not taken
with the Prospect T1 system
Ultrasound Imaging

16. • High-frequency ultrasound waves are necessary to resolve the small anatomical targets in preclinical
research
• Compromise is shorter penetration depth
Ultrasound Imaging

17. Prospect T1
• High-frequency ultrasound system specifically small animal imaging
• Up to 30µm resolution - small tumors, early pathological changes
• B-mode imaging to identify tumors and investigate surrounding
tissue
• 3D imaging to allow for tumor volume quantification
• Image guided needle injections for orthotopic tumor models
• Power Doppler imaging to study tumor vasculature
• Contrast agent imaging for tumor perfusion and biomarker detection
• Integrated sonoporation for drug delivery and cell membrane
permeability applications
• Shear wave elastography for early lesion detection

18. Tumor Detection and Monitoring
• Tumors of all types are visible, whether
subcutaneous or orthotopic
• Standard 2D B-mode imaging is used to
provide a greyscale image, tumors often
show up as a different echogenicity than
the surrounding tissue
• 2D measurements can be done to
measure linear or area measurements of
tumor size

19. Surrounding Tissue Imaging
• Normal structures have a characteristic look
for that specific tissue
• Abnormal changes can be identified – for
example cysts located in the ovaries
• It may be important to look at tissues such as
the lymph node and spleen to identify
systemic changes
Lymph Node
Skeletal Muscle Liver
Spleen
Cyst
Ovary

20. 3D B-Mode Imaging
• The 3D motor expands the capabilities of the Prospect T1
to acquire 3D B-mode images
• Add-on includes the software analysis package to view
the 3D images and perform volume calculations

21. 3D B-Mode Imaging
• Multiple slices are acquired
to cover the entire tumor
volume
• Two areas are drawn
perpendicular to one
another
• Software performs edge
detection and creates a
volume
• 39.0mm3

22. Image Guided Needle Injection
• The image guided needle injection mount integrates with probe
• Injections may be performed with a regular syringe and steel
needle, or pulled glass capillary needle
• Injections may be made into developing embryos, adult
myocardium, or any number of abdominal organ/muscle targets
Adult mouse
myocardium

23. Power Doppler – Tumor Vasculature
• Power Doppler is used to visualize the tumor
vasculature
• Larger vasculature is detected and displayed as a
color overlay on the B-mode image

24. Contrast Imaging – Tumor Microvasculature
• Contrast agents used to detect the
microvasculature – microbubbles are 2-3µm
• Imaging can be done using reference
subtraction or subharmonic imaging
techniques
• Reference subtraction imaging provides a
color overlay to highlight the inflowing
microbubbles
• Multiple regions of interest can be drawn to

25. Contrast Imaging – Tumor Microvasculature
• Subharmonic imaging
isolates the signal coming only
from the microbubbles –
listening only for the 1st
harmonic signal, tissue signal
is removed
• Multiple regions of interest
can be drawn to create time
vs. intensity curves

26. Integrated Sonoporation
• Sonoporation is the controlled cavitation or
bursting of microbubbles with the intention of
increasing the permeability of the cell
membrane, to open to blood brain barrier, or to
facilitate drug delivery
• Sonoporation is performed by a secondary, non-
imaging, probe directed at the anatomical target
• Software integration and control of the
sonoporation probe is included with this add-on

27. Shear Wave Elastography
• Shear wave elastography is used to quantify mechanical/elastic properties of
tissues
• The acoustic radiation force is generated by a push probe mounted on the side of
the imaging probe
• The software analysis generates a colored elastogram which is overlaid on a B-
mode image

28. Audience Poll #2

29. Recent Publications

30. • Targeted tumor theranostic agent was created with selective
tumor accumulation with diagnostic capabilities, as well as
increased retention, and drug release to effect tumor growth
• Trimodal imaging capabilities were included in the design,
with folate on the surface for binding to tumor cells, along
with 5-fluorouracil included as the therapeutic agent which
blocks DNA synthesis
• Fluorescence
• IR-780 in the
nanoparticle shell
• MRI
• Gd conjugated to
the 5-FU in shell
• Ultrasound
• gas filled core
Li et al. NPG Asia Materials (2018) 10: 1046-1060

31. • In vitro – nanobubbles were clearly visible in a gel mold
• In vivo – clear accumulation of the targeted nanobubbles was observed using
all three imaging modalities
• Ultrasound - red arrows below show accumulation within the tumor at the
same location over time
Li et al. NPG Asia Materials (2018) 10: 1046-1060

32. • 5-FU was released from the nanobubble
surface in an acidic pH environment and with
laser irradiation
• Various combined therapies were applied to the
mice; only the nanobubbles with laser resulted
in a decrease in tumor volumes. Other
therapies slowed the growth, but tumors remained
Li et al. NPG Asia Materials (2018) 10: 1046-1060

33. • Hepatocellular carcinoma (HCC) is one of the most
common malignant tumors, causing significant mortality
• Synergistic effect of chemotherapy and gene therapy
• Oxaliplatin – mechanism is not fully understood, but
believed to act on DNA synthesis pathways
• Recombinant human adenovirus Aspp2 (apoptosis
stimulating protein of p53-2) – Aspp2 binds to p53 and
inhibits cell growth and induces apoptosis
• in vivo and in vitro studies on HCC cell lines and xenograft
models in nude mice
Liu et al. International Journal of Oncology (2017) 51:1291-1299.

34. • Relative tumor volume decreased with all treatments,
however, was only significant when both chemotherapy and
gene therapy were combined
• This group was also able to use Power Doppler to assess
the small vessels within the tumors – they noted that vessel
quantity decreased in all three treatment groups (data not
shown)
Liu et al. International Journal of Oncology (2017) 51:1291-1299.

35. Want to see the system in action? Join us for a virtual demo
• Images were acquired by implanting a small portion of a blackberry into clear gelatin. The inner structure of the flesh of
the berry as well as the seeds are clearly visible through the clear gelatin mold

36. Audience Poll #3

37. • Targeted ultrasound contrast agents combine the advantages of
ultrasound imaging with molecular imaging to visualize molecular
biomarkers with high sensitivity and specificity in vivo
• Gas filled nanobubbles may be functionalized with specific cell
targets
• VEGFR2 (vascular endothelial grown factor receptor 2) –
expressed by endothelial cells of newly formed blood vessels
• HER2 (human epidermal growth factor 2) – has been identified as
a key target to improve detection and diagnosis of breast cancer
• Traditional microbubbles are typically blood pool agents as they are
too large to fit through the leaky tumor vasculature limiting their targets
to endothelial cell surface markers (VEGFR2); nanobubbles however
are able to extravasate from the vasculature and bind to tumor cell
surface markers (HER2)
Du et al. Scientific Reports (2018) 8:3887.

38. • Both in vitro and in vivo studies showed enhanced binding of dual-targeted (VEGFR2 & HER2) nanobubbles compared
to single targeted (VEGFR2 or HER2) nanobubbles, or untargeted (no antibody) nanobubbles alone
• Sustained increased greyscale intensity was seen as far out as 6-minute post injection, showing retention of the
nanobubbles within the tumor
• Fluorescent in vivo imaging and other histological techniques were used to confirm these results
Du et al. Scientific Reports (2018) 8:3887.

39. • Theranostic agents combine imaging capabilities with therapeutic
function into a single agent
• Multiwalled carbon nanotubes targeted to prostate stem cell
antigen (PSCA) are used to both detect tumors but also to deliver a
therapeutic compound – doxorubicin
• PSCA is only expressed at very low levels in normal prostate tissue,
but is highly expressed in all forms of prostate cancer, and even
more so in all metastatic prostate tumors
• Subcutaneous prostate tumors were used to study the
characteristics of this contrast agent
Wu et al. Biomaterials (2014) 35:5369-5380.

40. • Targeted carbon nanotubes were found to
accumulate in the tumors over time; non-
targeted and antibody blocked nanotubes
did not show the same level of binding
within the tumor
• Further studies showed the therapeutic
efficacy of the drug delivery component of
the nanotubes, as well as the reduced
overall toxicity of doxorubicin when
delivered in a targeted manner
Wu et al. Biomaterials (2014) 35:5369-5380.

41. • Preclinical Cancer Research
• Imaging in Preclinical Cancer Research
• Recent Publications
Topics of Discussion

42. Tonya Coulthard, MSc.
Manager, Imaging Division
Scintica Instrumentation
Phone: +1 (519) 914 5495
tcoulthard@scintica.com
Q&A
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43. • Molecular imaging, using targeted ultrasound contrast agents, allows
for subtle changes to be detected non-invasively, prior to any
phenotypical changes which may be observed without a contrast agent
• Ultrasound imaging is advantageous for frequent monitoring as there is
no ionizing radiation
• Dual targeted gold nanoshelled poly(lactic-co-glycolic acid) [PLGA]
nanocapsules were created targeting both VEGFR2 (vascular
endothelial growth factor receptor 2) and and p53
Xu et al. International Journal of Nanomedicine (2018): 13:1791-1807.
• Orthotopic breast tumors
were used to study
diagnostic capabilities of
this contrast agent

44. • Ultrasound results showed that dual-targeted nanocapsules
showed higher signal enhancement then either of the single
targeted agents (VEGFR2 or p53), and that non-targeted and
dual-targeted nanocapsules that were pre-treated with
antibodies before injection showed the lowest signal intensity,
similar to that seen in the hind limb adductor muscle
Xu et al. International Journal of Nanomedicine (2018): 13:1791-1807.

45. • Hepatocellular carcinoma is characterized by chronic inflammation and
an immune-suppressive tumor microenvironment
• Immuno-oncology is a new area of drug development focused on
enhancing the host’s effective anti-cancer immune response
• Orthotopic liver tumors expressing programmed death ligand 1 (PD-L1)
to explore the growth characteristics, regulation of the immune
microenvironment, and potential association with anti-PD1 therapy
• Tumor perfusion was assessed using an intravenous microbubble
injection, measured using time-intensity curves as well as vascular
index curves
Ou et al. Liver Cancer (2019) 8:155-171.

46. • Expression of PD-L1 in the tumor cells did not effect
• Survival
• Tumor growth
• Tumor perfusion
• Anti-PD1 antibodies, as immunotherapy re-sensitized the PD-L1 expressing
tumors to sorafenib (conventional chemotherapy)
• Anti-CD8+ antibodies were used to deplete the CD8+ T cells from the host
 neither anti-PD1 nor sorafenib were effective treatments when these T
cells were removed from hosts immune system response
Ou et al. Liver Cancer (2019) 8:155-171.

47. • Exosomes are created by a number of different cell types; natural
killer (NK) cells play a central role in the immune response against
cancer
• Exosomes from NK cells could be used to exploit the antitumor
properties of NK cells
• Melanoma is an aggressive type of skin cancer with a poor prognosis,
and is often resistant to multimodal treatment approaches (resection,
chemo, radiation)
• Ultrasound and bioluminescence was used to assess tumor volumes
in this study
Zhu et al. Theranostics (2017) 7(10):2732-45.

48. • Bioluminescence and ultrasound both showed the effectiveness of the
NK derived exosomes and controlling tumor growth; these results
correlated well with the final tumor weights taken at the end of the
study
Zhu et al. Theranostics (2017) 7(10):2732-45.