Journal club

Persent by: Li Feifei ( 李菲菲） , M.D. &Ph.D, Associate prof.

Department of pathophysiology,
Anhui Medical University
Email ： 349711832@qq.com
 Cancer stem cells
Rare cells within tumors with the ability to self-renew
and give rise to the phenotypically diverse tumor cell
population to drive tumorigenesis

Seed: tumor cell with growth and metastatic potential

heterogeneity
Cancer stem cells theory
 Normal stem cells
Rare cells within organs with the ability to self-renew
and give rise to all types of cells within the organ to
drive organogenesis (differentiation)

Cancer stem cells

Rare cells within tumors with the ability to self-renew

and give rise to the phenotypically diverse tumor cell
population to drive tumorigenesis
Normal stem cell and cancer stem cell characteristic
Cancer stem cell characteristic
 Cancer stem cell and Tumor
• leukemia stem cells
breast cancer stem cells
glioma stem cells
…
…

• Breast cancer stem cell Marker ：

CD44+ CD24low Lin- B38.1+ ESA+
ALDH1+ ：Aldehyde dehydrogenase 1

Glioma stem cells marker

CD133+

Liver cancer stem cells marker

CD45+
CD90+

Tannishtha , Sean JM , Michael FC,et al. Nature,2001,414:105

 Isolation of cancer stem cells

• ① CSC markers
cell surface markers→e.g flow cytometry CD44, CD133…
cytoplasma markers → Aldefluor assay to identify cells with ALDH activity

• ② CSC colony formation

Tumor cell were cultured on soft agar with growth factors without serum.CSC can
still maitein strong ability on self-renew and differentiation and form colony, while
non-CSC will die after several generations.
 Isolation of cancer stem cells
• ⑤Mammosphere formation
CSC can grow very well on media without serum for a very
long time and form spheres. But non-CSC grow slowly and
eventually die. By collecting spheres we could enrich CSC.

• ④Tumor formation
Normal tumor cell need very high concentration(>10 6/ml)
to form tumor in NOD/SCID mice. But CSC only need very
few numbers to let tumor formation. Therefore, we can
test different subtypes of tumor cell and find out the CSC
cluster which have the most highest tumor formation
ability with lowest concentration. This is the golden rule
of identity of CSC
。
 Resistance to Chemotherapy and Radiation therapy

Despite current advances in cancer therapy, tumor recurrence and metastasis remain
clinically challenge. Cancer stem cells (CSCs) are a highly tumorigenic subset of the total
cancer cell population. While chemotherapy and radiation have been shown to affect
the majority of tumor cells, CSCs may be highly resistant to these therapies, resulting in
tumor recurrence and metastasis.
Moreover, conventional chemotherapy and radiation therapy may induce stemness in
non-stem cancer cells.
Mechanism of CSC involved resistance to Chemotherapyt and Radiation therapy
 Triple negative breast cancers
(TNBCs)
Triple negative breast cancers (TNBCs) are
defined by the lack of
estrogen receptor (ER),
progesterone receptor (PR),
and human
epidermal growth factor receptor 2 expression.

Usually treated with cytotoxic chemotherapy

such as paclitaxel or gemcitabine, with a durable
response rate of less than 20%.

TNBCs are enriched for the basal subtype gene

expression profile and the presence of
breast cancer stem cells, which are endowed with
self-renewing and tumor-initiating properties and
resistance to chemotherapy.

In vitro cell model: Sum149, sum 159, MAD-231

 ？ Hypoxia-inducible factors
CSC (HIFs) and their target gene
products
are highly active in TNBCs
Paclitaxel Induces HIF Expression and Transcriptional Activity in TNBC Cells.

HIF Inhibitors
D ： digoxin
A ： acriflavine

CA9 ： cabonic anhydrase

ENO1 ： enolase 1 （ 2-
pospho-D-glycerate hydro-
lyase ）
RPL13A:ribosomal preotein
13a, catelyze protein synthesis
Paclitaxel Treatment Increases the Percentage of
BCSCs.

Detection for BCSCs:

markers mammospheres
 Mechansim?

Interleukin 6 (IL-6) and IL-8 have been shown to regulate BCSC

survival and self-renewal

? Is Paclitaxel-Induced Interleukin Expression Blocked by HIF Inhibitors

Paclitaxel-Induced Interleukin Expression Is Blocked by HIF Inhibitors.

HIF Inhibitors
D ： digoxin
A ： acriflavine
HIF-1α and HIF-2α Are Required for Paclitaxel-Induced BCSC Enrichment.

NTC: MDA-MB-231stably
transfected with a lentiviral expression
vector encoding a nontargeting
control (NTC) short hairpin RNA (shRNA)
DKD: MDA-MB-231stably
transfected with shRNAs targeting HIF-
1α and HIF-2α
JMJD1A/3:the histone demethylase,
which is the product of a HIF target
gene, binds to the IL8 promoter and
stimulates IL-8 mRNA expression

paclitaxel
digoxin
HIFs acriflavine

JMJD1A and JMJD3

IL8 and IL6

BCSC enrichment
 What signals mediate induction of HIF-1α and enrichment of BCSCs ？

X Paclitaxel-Induced SMAD2 and STAT3 Activity Is Insufficient to Induce BCSC Enrichment

X TLR-4 and NF-κB Do Not Mediate Chemotherapy-Induced BCSC Enrichment.

Dose Increase ROS Levels Mediate Induction of HIF-1α and Enrichment of BCSCs ？
Increased ROS Levels Mediate Induction of HIF-1α and Enrichment of BCSCs.

MitoSOX Red Staining is

rapidly and selectively targeted to
mitochondria and produces
fluorescence when oxidized by superoxide
radicals, thereby
serving as an indicator of ROS in live cells

MnTMPyP ： a cell-permeable
superoxide scavenger

paclitaxel

ROS ↑

HIFs

BCSC enrichment
 paclitaxel

ROS ↑

Genes ？ MDR ？

HIFs

BCSC enrichment
Paclitaxel-Induced MDR1 Expression Is Blocked by HIF Inhibitors

Paclitaxel

HIF↑

BCSC enrichment
digoxin
IL8 and IL6 acriflavine

MDR-1

Tumor growth
Digoxin Blocks Gemcitabine-Induced IL-6 Expression and BCSC Enrichment.

Gemcitabine

HIF-1a↑

BCSC enrichment
digoxin
IL8 and IL6 acriflavine

MDR-1

Tumor growth
In vivo study

Coadministration of Digoxin with Gemcitabine Prevents Tumor Relapse.

MDA-MB-231 cells were implanted into the mammary fat pad of

female Scid mice. When a tumor was palpable (day 1), the mice were
randomized to three groups, which were treated with i.p. injections of:
saline, gemcitabine [20 mg/kg on days 5, 10, 15, 20, and 25 (arrows)], or
gemcitabine and digoxin (2 mg/kg on days 1–25). Tumor volumes were
determined every 2–3 d
 HIF Signature Predicts Mortality of Patients Treated with Chemotherapy

HIF-1 signature composed of 16 genes (PLOD1, VEGFA, LOX, P4HA2, NDRG1, SLC2A1,
ERO1L, ADM, LDHA, PGK1, ANGPTL4, SLC2A3, CA9, HIF1A, IL6, and IL8),
PAM50 signature, which consists of 50 genes, the expression of which divides human breast
cancer specimens into five groups, designated basal, HER2 enriched, luminal B, luminal A,
and normal
Summary