BM 599

Optical Methods for Detection and

Treatment of Cancer
Week 5: Introduction to Fluorescence-guided Resection
 Summary and Midterm

Hallmarks of Cancer The Jablonski Diagram

Open-book exam:
• Doesn’t mean that you don’t have to study. You should be able to quickly find relevant information and apply knowledge.
• Can’t copy paste
Aim of today’s course: Fluorescence Guided Resection (FGR)

• Why is fluorescence guidance needed?

• Desirable characteristics of FGR probes
• FDA approval of ALA-mediated FGR for glioma
Current Cancer Treatment Methods

Surgery
Chemotherapy
Immunotherapy
External beam radiation therapy
The primary treatment modality for most solid tumors is surgery. If all the cancer cells are removed by
surgery, the patient is cured of that cancer.

Ideal Case The problem

Fi gure: Mondal et a l, “Cha pter Fi ve - Rea l-Time Fl uorescence Image-Guided Oncol ogic Surgery”, Adva nces i n ca ncer research 2014
Which information helps surgeons during a tumor resection surgery?
- Palpation
- Visual examination
- Images taken before surgery

Examples of surgeries that have high local recurrence (at the site of the initial
surgery) or positive margin rates are resections of:

• Malignant gliomas (80%)1

• Conservation surgery for breast cancer (20–40%)1
• Ovarian cancer (35%)2

Therefore, Real-time intraoperative guidance is essential during oncological

surgery for complete and safe tumour resection.

1 Nguyen et al., “Fluorescence-guided surgery with live molecular navigation – a new cutting edge”, Nature Reviews Cancer, 2013
2 Orosco et al,. “Positive Surgical Margins in the 10 Most Common Solid Cancers” Scientific Reports, 2017
Fluorescence guided resection
Fluorescently labelled, tumor targeted probe would provide real-time, intra-operative
distinction of the molecular edge between cancer and adjacent normal tissue.
Fluorescence guided resection

Fluorescence imaging uses a

specialized camera to capture
light emitted by a fluorescent
contrast agent after
excitation with an
appropriate light source.

All imaging equipment can be

integrated into a compact
open-field device or within
laparoscopic or robotic
instruments for contact-free
and real-time interrogation

Sophie Hernot et al. ”Latest developments in molecular tracers for fluorescence image-guided cancer surgery”
Benefits of FGR

• Decreased positive margins.

• Decreased surgical duration.

• Minimize costs to the overall health-care system.

Important Parameters in FGR
We want to make the tumor tissue fluorescent. Which parameters are important?

1. Dosing

2. Wavelengths and dyes

3. Instrumentation

4. Targeting

Nguyen et al., “Fluorescence-guided surgery with live molecular navigation – a new cutting edge”, Nature Reviews Cancer, 2013
 Important Parameters in FGR

Dosing: Fluorescence imaging agents for a given surgical procedure should ideally be administered topically during the
surgery itself, or intravenously 0.5–2 hours before the start of the operation. Contrast should ideally last the duration of
the surgery or at least during the period of tumor resection and margin evaluation (typically 1–4 hours); if contrast is lost,
re-administration of the agent may be necessary.

Topical Intravenous
(Systemic)
• Minimizes the overall dose and systemic side effects • Fluorescence from all lesions and whole tumor, even
if the tumor is underlying other tissue.
• Limited probe penetration
• Surgeon can skip lesions that are separated from the • Higher doses / risk of systemic side effects
main tumor. • Longer time for contrast to develop and for
nonspecific fluorescence to wash out.

Pros ?
Cons ? Nguyen et al., “Fluorescence-guided surgery with live molecular navigation – a new cutting edge”, Nature Reviews Cancer, 2013
Important Parameters in FGR

2) Wavelengths and dyes:

• In mammals, there is a strong scattering and strong absorbance below 600 nm from hemoproteins such as
hemoglobin.

• Also, wavelengths below 600 nm encounter high autofluorescence due to many endogenous fluorophores

• Therefore, longer excitation and emission wavelengths give less autofluorescence, deeper tissue penetration.

Fi gure: Keereweer et a l. “Opti cal Image-Guided Ca ncer Surgery: Cha l lenges and Li mitations “American As sociation for Ca ncer Research, 2013
Important Parameters in FGR

3) Instrumentation

• Ideally, the instrument should provide multiple excitation wavelengths

for accommodating wide variety of molecularly targeted probes being
developed.

• Both white light and fluorescence view should have independent

control of intensity to adapt variations of lighting and probe uptake.

• The device should go through premarket approval (PMA), which is the

FDA process of scientific and regulatory review to evaluate the safety
and effectiveness of class III medical devices.
Important Parameters in FGR: Targeting

Non-tumor-specific dyes can be used to assist intra-operative dissection. These cannot highlight tumors, but
they can provide an improved visualization in the resection area

• Sodium fluorescein
• ICG

White light ICG Fluorescence

EPR Effect
Enhanced retention and permeability effect is
a passive targeting mechanism.

Molecules tend to accumulate

in cancer tissue because of:

• Poorer lymphatic drainage,

• Angiogenesis leads to high vascular density and large gaps between endothelial cells (Leaky
vasculature).
• Or more specific interactions between the photosensitizers and cells due to the charge, size, structure
of the photosensitizer etc.
Normal tissue Tumor tissue

Drain
Drain
ICG for Fluorescence Guided Surgery
https://www.youtube.com/watch?v=TPMT1JW_5KI
Important Parameters in FGR

4) Targeting: Preferentially, fluorescent molecules should specifically localize to target tissues.

Tumor-specific Membrane-associated Cancer-activated

molecules enzymes metabolism pathways
➢ EGFR antibody
➢ EPCAM antibody ➢ Activatable cell-
➢ PSMA antibody penetrating peptides
➢ CEA antibody
2-deoxyglucose probes Heme biosynthesis pathway
➢ CA19-9 antibody (ALA-induced PpIX)
➢ Folate receptor-α antibody
EGFR signaling pathways
Simplified representation of the two main EGFR signaling
pathways: PI3K/Akt and MEK/ERK pathways.

EGF
TGF-alpha
EGFR antibody mediated targeting

EGFR is overexpressed in many cancer types.

EGFR antibodies are used to inhibit EGFR signaling pathways to

treat cancer.

EGFR antibodies can also be used to label cancer cells with

fluorescent molecules.

FDA approved EGFR antibodies: cetuximab, panitumumab,

nimotuzumab, and necitumumab
EpCAM antibody
Epithelial cell adhesion molecule (EpCAM) is
a transmembrane glycoprotein mediating cell–cell adhesion.

EpCAM is overexpressed in human epithelial-derived neoplasms, including cancer of

the tongue, thyroid, prostate, esophagus, liver, colon, breast, ovary, pancreas,
bladder, lung, stomach and kidney.
 PSMA, CEA, CA19-9 antibody

Prostate-specific membrane antigen (PSMA),

Carcinoembryonic antigen (CEA)
Carbohydrate antigen 19-9 (CA19-9)

are markers for pancreatic or hepatobiliary (liver, bile ducts, pancreas and gall bladder) cancers.

• For instance, CEA is expressed in up to 98% of pancreatic cancers.*

*Tumor-Specific Fluorescent Antibody Imaging Enables Accurate Staging Laparoscopy in an Orthotopic Model of Pancreatic Cancer
Hop S Tran Cao
Folate receptor-α (FR-α)
This receptor is responsible for binding to folate which
is used for metabolic processes such as DNA replication
and cell division.

FR-α is overexpressed in different types of cancer

including ovarian, breast, renal, lung, colorectal, and
brain cancer. For example, it has been shown that 72%
of primary and 81% of recurrent epithelial ovarian
carcinomas express FR-α.

Therefore, folate receptor can be used to target active

cancer cells.

Utilizing the folate receptor for active targeting of cancer nanotherapeutics

Zwicke et al.
Important Parameters in FGR

4) Targeting: Preferentially, fluorescent molecules should specifically localize to target tissues.

Tumor-specific Membrane-associated Cancer-activated

molecules enzymes metabolism pathways
➢ EGFR antibody
➢ EPCAM antibody ➢ Activatable cell-
➢ PSMA antibody penetrating peptides
➢ CEA antibody
2-deoxyglucose probes Heme biosynthesis pathway
➢ CA19-9 antibody (ALA-induced PpIX)
➢ Folate receptor-α antibody

Tumor-specific molecules in or on cancer cells are usually the most specific. However, they have
the drawback of usually being applicable only to a particular type of cancer.
Important Parameters in FGR

4) Targeting: Preferentially, fluorescent molecules should specifically localize to target tissues.

Tumor-specific Membrane-associated Cancer-activated

molecules enzymes metabolism pathways
➢ EGFR antibody
➢ EPCAM antibody ➢ Activatable cell-
➢ PSMA antibody penetrating peptides
➢ CEA antibody
2-deoxyglucose probes Heme biosynthesis pathway
➢ CA19-9 antibody (ALA-induced PpIX)
➢ Folate receptor-α antibody
Activatable cell-penetrating peptides (ACCPs)
In most types of solid tumors, MMP-2 and MMP-9 are

They degrade the extracellular matrix

and activate growth factors and angiogenesis.

ACCPs can be cleaved by MMP-2

Membrane-associated or excreted enzymes such as MMPs are second best in

terms of specificity but may have the advantage of being generalizable to all
cancers.
In vivo characterization of activatable cell penetrating peptides for targeting protease activity in cancer
Emilia S. Olson, Todd A. Aguilera, Tao Jiang, Lesley G. Ellies, Quyen T. Nguyen, Edmund Wong, Larry Gross, and Roger Y. Tsien
Important Parameters in FGR

4) Targeting: Preferentially, fluorescent molecules should specifically localize to target tissues.

Tumor-specific Membrane-associated Cancer-activated

molecules proteins/enzymes metabolism pathways
➢ EGFR antibody
➢ EPCAM antibody ➢ Activatable cell-
➢ PSMA antibody penetrating peptides
➢ CEA antibody
2-deoxyglucose probes Heme biosynthesis pathway
➢ CA19-9 antibody (ALA-induced PpIX)
➢ Folate receptor-α antibody
 2-Deoxy-d-glucose (2-DG) Probes
• 2-Deoxy-d-glucose is similar to glucose molecule. Therefore, it is taken
up by cancer cells, which prefers glycolysis.

• In 2-DG, the 2-hydroxyl group replaced by hydrogen, so that it cannot

undergo further glycolysis.

• 2-DG can be used to treat cancer because it inhibits glycolysis.

• 2-DG can also be used to target cancer cells as a delivery component.

Aminolevulinic Acid (ALA) White light PpIX Fluorescence

• ALA-induced PpIX fluorescence is approved for fluorescence-

guided tumor resection of glioma.1,2

• ALA-PDT is approved for non-melanoma skin cancers, actinic

keratosis and acne treatment.

1Pottier R.H. ve ark,. “Non-Invasive Technique for Obtaining Fluorescence Excitation and Emission Spectra In Vivo” Photochemistry and Photobiology, 1986.
2Hadjipanayis ve ark. “5-ALA and FDA approval for glioma surgery” Journal of Neuro-oncology 2019
Surgery Figure: Van Dam et al,. “Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results”, 2011
 Aminolevulinic acid (ALA, 5-ALA) is a naturally occurring precursor for PpIX and heme in
the heme biosynthesis pathway.

Heme biosynthesis pathway1-3 1Shemin,

2Shemin
3Shemin,
D. and Rittenberg, D., “The utilization of glycine for the synthesis of a porphyrin,” J. Biol. Chem. 159, 567–568 (1945).
D. and Kumin, S., “The mechanism of porphyrin formation; the formation of a succinyl intermediate from succinate.,” J. Biol. Chem. (1952) .
D. and Russell, C., “Aminolevulinic acid, its role in the biosythesis of porphyins and purines,” J. Am. Chem. Soc. 75, 4873–4873 (1953).
 Aminolevulinic acid (ALA, 5-ALA) is a naturally occurring precursor for PpIX and heme in
the heme biosynthesis pathway.

ALA

Protoporphyrin IX
HEME +
Fe2+

Mitochondrion

Heme biosynthesis pathway1-3 1Shemin,

2Shemin
3Shemin,
D. and Rittenberg, D., “The utilization of glycine for the synthesis of a porphyrin,” J. Biol. Chem. 159, 567–568 (1945).
D. and Kumin, S., “The mechanism of porphyrin formation; the formation of a succinyl intermediate from succinate.,” J. Biol. Chem. (1952) .
D. and Russell, C., “Aminolevulinic acid, its role in the biosythesis of porphyins and purines,” J. Am. Chem. Soc. 75, 4873–4873 (1953).
 Heme: An iron ion in the middle of a porphyrin ring.

Protoporphyrin IX
(PpIX)

PpIX is highly
fluorescent!

Heme forms many hemoproteins, including hemoglobin and cytochromes.

Heme and PpIX levels in normal cells

Because PpIX and heme can create oxidative stress and cell damage, cells maintain a
low level of free porphyrins.
How?

Heme biosynthesis is negatively regulated by the Excess PpIX, heme and other
intracellular level of heme. porphyrins are taken out of the
cells by various transporters
(mainly by ABCG2 protein).
High heme level in cells Excess amount of heme
impose negative stimulates heme
feedback inhibition on degradation by
ALAS. promoting the heme
degradation enzyme
heme oxygenase.
 What if we increase ALA?

Heme biosynthesis pathway

Production of PpIX can be increased by the administration of excess ALA.

et al. Fluorescence-guided surgery with live molecular navigation – a new cutting edge, Nature Reviews Cancer, 2013
wska et al,. “Aminolevulinic acid as a prodrug in photodynamic therapy of cancer”, 2011

1. Wachowska et al,. “Aminolevulinic acid as a prodrug in photodynamic therapy of cancer”, 2011

ALA-mediated PpIX production in tumors

• Exogenous administration of ALA bypasses the feedback control, which leads to increased
production of PpIX in mitochondria.

• Production of PpIX is typically higher in tumors than normal tissue because:

o FECH (Ferrochelatase) gene expression or activity has often been found reduced in
a variety of tumor cells/tissues.1
o Evidence of increased activity of heme biosynthesis pathway enzymes such as
PBGD and UROD.1
o Limited availability of iron in tumor cells.2

1 Nguyen et al. Fluorescence-guided surgery with live molecular navigation – a new cutting edge, Nature Reviews Cancer, 2013
2 Wachowska et al,. “Aminolevulinic acid as a prodrug in photodynamic therapy of cancer”, 2011
Key elements in heme biosynthesis pathway

• Protoporphyrin (PpIX) is a precursor of heme with good fluorescence and

photosensitizing activity.

• Aminolevulinic acid (ALA) is the first metabolite in the heme biosynthesis

pathway. It is formed in the mitochondria from Glycine and Succinyl CoA by ALA
synthase (ALAS).

• FECH (Ferrochelatase) catalyzes the insertion of iron into the PpIX to form heme.
FECH should be inhibited to increase PpIX level.

• CPOX (Coproporphyrinogen-III oxidase) expression can be increased by using

certain chemicals to increase PpIX level.
Challenges in ALA-mediated PpIX production in tumors

• The application is limited by inadequate and heterogeneous PpIX production in

tumor cells.1

• PpIX production may also be heterogeneous between patients.2

1 Nguyen et al., “Fluorescence-guided surgery with live molecular navigation – a new cutting edge”, Nature Reviews Cancer, 2013
2 Yang et al., “Aminolevulinic acid-based tumor detection and therapy: Molecular mechanisms and strategies for enhancement” International journal of Molecular Sciences, 2015
Current approaches to handle the problem

• Differentiation agents

• Transporter Inhibitors

• Iron chelators
Current approaches to handle the problem
Differentiation Agents Transporter Inhibitors Iron Chelators

Vitamin D: CPOX Fumitremorgin C Ethylenediamine tetraacetic

acid (EDTA)

Induction of keratinocyte Kol43 Deferoxamine (DFO)

differentiation by increasing
calcium concentration in
medium ( ALA uptake, CPOX,
expression, PpIX efflux)
Calcitriol CPOX, FECH Tyrosine kinase inhibitors CP94
such as imatinib mesylate
and gefitinib
“Here we describe the FDA process for 5-ALA approval in the US and some of the
challenges faced during this journey.”
Dr. Hadjipanayis talks about ALA
https://www.youtube.com/watch?v=UP3CphWlD8s
Peritoneal carcinomatosis is the formation of wide-spread metastases throughout the
abdominal cavity. It occurs frequently in patients with advanced stage gastrointestinal
and gynecological cancers.

In the review, authors present an overview of relevant clinical trials published since
2010 and discuss their major findings.
They mention that “Almost all trials used ALA, given 2–4 h preoperatively with an oral
dosage ranging from 10 to 20 mg/kg”
Latest developments in molecular tracers for fluorescence image-guided cancer surgery

Sophie Hernot et. al. ”Latest developments in molecular tracers for fluorescence image-guided cancer surgery”
Sophie Hernot et. al. ”Latest developments in molecular tracers for fluorescence image-guided cancer surgery”
Sophie Hernot et. al. ”Latest developments in molecular tracers for fluorescence image-guided cancer surgery”
Traditional cancer detection tools
(mainly used for surgical planning. Not to provide real-time intraoperative guidance.)

• MRI
• CT (x-ray)
• PET
• PET-CT
Magnetic resonance imaging (MRI)

• MRI can be used:

• To learn the size and location of the
tumor.
• To plan cancer treatments, such as
surgery or radiation therapy.
• To see how well treatment is working.
Magnetic resonance imaging (MRI)
Computed tomography (CT)
• Similar images with MRI but different working principles.
• In CT, a rotating X-ray tube and a row of detectors
measure X-ray attenuations by different tissues inside the
body.
• Faster scan than MRI
• Ionizing radiation (x-ray)
Positron emission tomography (PET, usually PET-CT)

Before the scan, a radioactive sugar (tracer)

called fluorodeoxyglucose-18 is injected to the
patient.

PET CT PET-CT