ISCO2023

New treatment strategies for intractable cancers brought
about by the fusion of different fields of science

Hideyuki Saya, MD, PhD
Division of Gene Regulation, Cancer Center
Fujita Health University, Aichi, Japan
Keio University School of Medicine, Tokyo, Japan

Malignant brain cancer : Glioblastoma
surgery
Radiation therapy
Chemotherapy

Resistance and RecurrenceCancer
A phenomenon in which normal cells change due togene mutations, break the rules, proliferate
abnormally, and survive for a long time.

Cancer cells invade surrounding organs or metastasize
to distant organs, resulting in impairment of theirfunctions.
Without treatment, various organ disorders eventuallyoccur, leading to death.

How does cancer arise?

Skin cancer

Normal skin structure

http://www.kumc.edu/instruction/medicine/anatomy/histoweb/skin/skin.htm

Normal tissue organization
Regulated cell proliferation, rapid turnoverdeath
senescencedifferentiation

Progenitor cell（rapid growth）

Stem cell（slow growth）

28 daysCancer cells are derived from stem-likecancer cells (cancer stem cells)

 Continuous growth Prolonged survival

Critical mutation(s) ina stem cell or a progenitor cellCancer cells are derived from stem-likecancer cells (cancer stem cells)
Character of cell isdetermined bywhich genes areexpressed

Cancer stemcells (CSC)

The combinationof 23,000 gene
expressions
determines thenature of each cellCancer cells are derived from stem-likecancer cells (cancer stem cells)

Cancer cells are derived from stem-likecancer cells (cancer stem cells)Continuous
growth andlonger survival

Cance Stem Cells

Structure of skin cancer

Cancer stem cells (CSCs)
CSC is resistant toconventional therapies

Self-renewed
CSC

Non-CSCTherapeutic predictions of cancer stem cell model
Conventional cancer therapy
Chemotherapy
Radiotherapy

Tumor
grows back

CSC survived

Anti-cancer stem cell therapy
Kill CSC

Chemotherapy
Radiotherapy

What do we need for cancer eradication?

Targets

Modalities(cell, gene, protein(small molecule,nucleic acid, antibody,peptide etc)

mRNA, signal etc)

 Novel therapeutic strategies for eliminationof cancer stem cells
 Detection of cancer stem cells in whole body Monitoring the cancer stem cells in patientstreated by various therapeutic interventions

Therapeutic resistant mechanisms of cancer stem cells• Resistant cells require high energy production (highATP production) for survival and growth.
• High-energy-producing cells produce large amounts ofreactive oxygen species (ROS).
• Resistant cells have anti-oxidant system for survival.
Anti-oxidant
mechanisms

Reactiveoxygenspecies(ROS)

Cell
Death

Ferroptosis

Blocking the anti-oxidant system may induce cell
death in the resistant cells
Ishimoto et al., Cancer Cell 2011

Yae et al. Nat Commun, 2012

Drugs Induce Ferroptosis in Cancer Stem Cells
Two primary targets:xCT and ALDH both collaborate to block oxidative stresscancer stem cell
① xCT
② ALDHROS

Cancer cell survive
by blocking
oxidative stress
Okazaki et al., Oncotarget 2019
Otsuki et al., Cancer Sci 2020

Drugs Induce Ferroptosis in Cancer Stem Cells
Two primary targets:xCT and ALDH both collaborate to block oxidative stresscancer stem cell
① xCT
② ALDHROS
Ferroptosis

Inhibition of
xCT+ALDH
induces Ferroptosis
Okazaki et al., Oncotarget 2019
Otsuki et al., Cancer Sci 2020

Drugs Induce Ferroptosis in Cancer Stem Cells
Two primary targets:xCT and ALDH both collaborate to block oxidative stressIn vivo Mouse Model (HCT116 colon cancer cell line)

Phase I clinical trial in 2024

Otsuki et al., Cancer Sci 2020

What do we need for cancer eradication?

Targets

Modalities(cell, gene, protein(small molecule,nucleic acid, antibody,peptide etc)

mRNA, signal etc)

 Novel therapeutic strategies for eliminationof cancer stem cells
 Detection of cancer stem cells in whole body Monitoring the cancer stem cells in patientstreated by various therapeutic interventions

OIST Mini Symposium "New Medical Imaging and Advanced Cancer Therapy(BNCT) Instrumentation" May 14-16, 2015
Organizer: Hirotaka Sugawara, Advanced Medical Instrumentation Unit, OISTKavli IPMU-JAXA-Keio collaboration project
Whole body imaging of cancer stem cells andtherapeutic resistant cells by applying thetechnologies of particle physics and space science
Technologies for space telescope
are applied to microscope

March 26,2018

Application of the most advanced space
exploration sensors to medicine
Space exploration sensors
may allow the detectionof RI-labeled compounds
incorporated in cancer
stem cells.

xCT inhibitor
RI

Theranostics = Therpeutic +Diagnostic(Alphatherapy, BNCT etc.)
225 Ac211 AtAnimal models which pathologically andclinically recapitulate human cancers

Induced cancer stem cell︓︓ iCSCXY
ShortperiodiCSC

differentiation

100%
Normal cells(progenitor cells/stem cells)

Self-renewal

Organoid
cultureInoculation tosyngeneic miceexcessive growthheterogeneity

Invasion/metastasis

Drugdevelopment

Mouse cancer models established by using iCSC
tumorcells-of-origin

transgenes

lymphoma

HSCs/progenitors• N-myc• c-mycosteosarcoma

INK4a/Arf KOBMSCs• c-myc

pathology

references••

Pre-B LBL
Mature B-cell
lymphoma

Oncogene (2011)
Blood (2017)
Cancer Res (2020)•Osteosarcoma

JEM (2009)
Oncogene (2010)
Cancer Res (2019)brain tumors

INK4a/Arf KO
neural stem cells• RasV12• c-myc••

Glioblastoma
PNET

Neoplasia (2011)
Stem Cells (2013)
Neuro-Oncol (2014)
Neuro-Oncol (2017)
Nat Cell Biol (2019)
Commun Biol (2020)
JCI (2021)

breast cancer

INK4a/Arf KO
mammary stem cells• RasV12•

Basal type

Oncogene (2013)ovarian cancer

Normal ovarian
Premature epithelialcells• p53siRNA+RasV12+c-myc•

Serousadenocarcinoma

Carcinogenesis (2011)

lung cancer

INK4a/Arf KO
EpCAM-positive lungcells• KRasV12• ELM4-ALK••

Adenocarcinoma
Adenocarcinoma

Cancers (2021)

cholangiocarcinoma

INK4a/Arf KO
EpCAM-positive bile
duct cells• KRasV12•

Adenocarcinoma

Cancer Sci (2021)

iCSC-based mouse tumor models

Glioblastoma multiforme
Sampetrean et al., Neoplasia (2011)

Ovarian serous adenocarcinoma

Lung adenocarcinoma
Semba et al., Cancers (2021)

Motohara et al., Carcinogenesis (2011)

Osteosarcoma

Pre-B lymphoblastic leukemia

Cholangiocarcinoma

Shimizu et al., Oncogene (2010)

Sugihara et al., Oncogene (2011)

Kasuga et al., Cancer Sci (2021)

Genetically engineered glioma model
Isolation of adultbrain cells

NeurospherecultureRetroviral
infection

Neural stemcells
Ink4a/Arf C57BL/6

7days:
1-2x106 cells

5-10 days

Orthotopic
implantation

iGSCs

1x105 cells
C57BL/6

Retroviral constructs
5’-LTRΨ

5’-LTRΨ

5’-LTR

Ψ

IRES

EGFP

3’-LTR

H-RAS V12IRESEGFP

3’-LTR

H-RAS V12IRES

dsRed

3’-LTR

Sampetrean et.al., Neoplasia 2011

Glioma stem cell (GSC)-based brain tumor model
RL

Humanglioblastoma

T2WIL

R

T1WIGd100% penetrance, 5-week median survival
Sampetrean et.al., Neoplasia 2011

Self-renewal and differentiation abilities of GSCs
GFP

Nestin

GFAP

GFP

CD44

TUJ-1

O4

Sampetrean et.al., Neoplasia 2011

In vitro/ex vivo/in vivo
evaluation
In vitro: efficacy towards
iGSCsiGSC

Ex vivo: efficacy towards
iGIC/GSCs in native
microenvironment, toxicity

In vivo: efficacy, toxicity,side effects

drugdrugdrug

survivaltumor size
side effects

Cell cycle
visualization
5’-LTR

NSC

5’-LTRΨ mCherry

Ψ

H-RAS
V12

“FUCCI”: fluorescentubiquitination-based cell
cycle indicator

Hygromycin B

3’-LTR

GSC

GIC

hCdt1

SV40

P2A

EGFP

hGeminin

IRES

Blasticidine

3’-LTR

A genetic marker forvisualization of cell cycle
Sakaue-Sawano, Cell, 2008
RL Mort, Cell Cycle , 2014

Cell cycle
visualization
Check:☑ mitotic spindle☑proper chr alignment

MG2

Check:☑ proper DNA synthesis☑ no DNA damage

G1Check:☑ cell size
☑ enough nutrients☑ enough growth factors☑ no DNA damage

SGoStopRadiation-induced changes in the cell cycle- GSCs Medium change
Day2

Day4

Day7

IR 5GyIR 0GyDay0Regrowth after 5Gy seen after day 4, in anutrient/oxygen-dependent manner.

In vitro/ex vivo/in vivo
evaluation
In vitro: efficacy towards
iGSCs

iGIC/iGSC

Ex vivo: efficacy towards
iGIC/GSCs in native
microenvironment, toxicity

In vivo: efficacy, toxicity,side effectsdrugdrug

drug

survivaltumor size
side effects

Brain slice culture for visualizing iCSC in brain
Isolation of adult
SVZ cells

Ink4a/Arf C57BL/6

Live brain
extraction

2-14days
3% agarose gel

NeurospherecultureRetroviral
infection

7days:
1-2x106 cells

5-10 days

Orthotopic
implantation

1x105 cells
C57BL/6

Coronal slice
preparation

Culture and video
acquisitionWeek 1:

External capsule

GFP stainingIn vitro/ex vivo/in vivo
evaluation
In vitro: efficacy towards
iGIC/iGSCs

iGIC/iGSC

Ex vivo: efficacy towards
iGIC/GSCs in native
microenvironment, toxicity

In vivo: efficacy, toxicity,side effects

drugdrugdrug

survivaltumor size
side effects

High invasion ability of glioma iCSC

However, there is no way to detect a
specific cancer cell population fromoutside the body without invasive
procedures.
PET can detect approximately 1cm tumor whichcontains 109 cells.

Team Takahashi in Kavli IPMU

Takahashi

Katsuragawa

TakedaOrita

Yagishita

Umeda

Sampetrean

Imaging of cancer stem cells
Clinically recognized cancer

cancer stem
cellSpheroid/organoidCSCs
neovessels
immune cells
mesenchymalcells

10〜40 μm

100〜400 μm
Initial phase tumor
metastatic lesion

Separation of cancer stem cells from other cells requires
high spatial resolution and multi-probe detection

SPECT imaging of cancer stem cell organoid
Organoids of NIS (natrium iodide symporter)
expressing CSCs which can incorporate 125I

control

NIS
inhibitor

control

NIS
inhibitor

multi-nuclide in vivo imaging
Spatial resolution

Yagishita, Takeda et al., Nat Biomed Eng 2022

multi-nuclide in vivo imaging

Yagishita, Takeda et al., Nat Biomed Eng 2022

Conclusions• Collaborative research to detect heterogeneous cellgroups in tumor tissue with 3D and multi-probe
using state-of-the-art space observation sensor
technology has started.
• We intend to build a system that can detect cancer
cells with different properties in the living body at
the single cell level and rapidly evaluate thetherapeutic effect.
• We intend to developed a new THERANOSTICS byloading RI into the specific probe.