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Autologous Cancer Stem Cell Transplants to Alter Cancer Cell

Reproduction
Emily Scott
July 29th, 2016

Introduction
Delivering high doses of ionizing radiation to tissue has been proven to be an effective
method of cellular destruction. DNA is damaged under ionizing radiation and can lead to direct
death of the cell itself or damage to the mechanisms that allow the cells to proliferate. While
ionizing radiation is very effective in destroying normal, healthy cells, it is not always as
effective when treating tumor cells. Radioresistance, the defense of tumor cells against radiation,
is a challenge in the field of radiation oncology. Malignant cells can protect themselves against
damaging intrusions, making it very difficult to clinically treat and control cancer. The study of
the human genome, stem cells, gene expression, and cancer therapies, among countless other
topics, has provided massive amounts of insight on how to more effectively treat malignancy, but
radioresistance continues to be an obstacle.
Previous and ongoing work on this topic is vast. Researchers have introduced
chemotherapy agents, enzymes, and other genetic and chemical materials to tumor cells in the
hopes of reducing their resistance to ionizing radiation. The addition of some of these agents has
led to a greater understanding of radioresistance and radiosensitivity, but the ability for the
medical community to control all cancer cells is still unattainable. Some of these materials have
shown to be effective in targeting and weakening certain types of cancer cells, including the
introduction of thalidomide, survivin, and a CHK1 inhibitor. One of the common themes in this
research is trying to decrease cell division by damaging the mechanisms that allow division to
occur. The study of cancer stem cells has started to become the focus of targeting cancer so that it
may be effectively treated. According to Skvortsova and associates, cancer stem cells are
defined as a small subpopulation of carcinoma cells that can renew itself and can also generate
the heterogeneous cell lineages comprising the tumor.1 Cancer stem cells are similar to the
normal, healthy stem cells in the human body in their purpose and general mechanisms of
function, but research has shown that cancer stem cells have different genetic markers that we
may be able to identify and use as targets. They are generally more chemoresistant,
radioresistant, and have a greater ability to metastasis than normal cancer cells.2 If researchers
are able to pinpoint a specific agent or combination of agents to stop malignant cell reproduction
via these markers, the destruction and elimination of tumor cells may be accomplished.

Hypothesis

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Targeting cancer cells ability to reproduce is vitally important when considering how to
eradicate them from a human body. If the cells are unable to reproduce, there is no chance of
metastasis or growth and they will eventually die off. We can speed the process of eradication by
using radiation therapy to kill any living cancer cells, thereby making the process of curing a
patient more efficient. I propose utilizing a combination of total body irradiation (TBI),
autologous cancer stem cell (CSC) transplant, introduction of a combination of enzymes and
proteins to disrupt the replication process, and radiation therapy to stop cancer cells ability to
reproduce. My hypothesis for this experiment is that the altered stem cells will integrate
themselves into the body, causing a genetic change in the malignant cells, which will prevent
them from reproducing. Once the tumor has adopted the altered DNA, we can then irradiate them
with high-energy photons to destroy the cancer cells quickly. Once the radiation therapy is
complete, the patient will be able to heal and replenish his or her normal, healthy cells without
malignant cells taking over. The critical prediction of this proposal is that this process will
prevent metastasis, recurrence, and secondary malignancies.

Literature Review
There is a plethora of past and ongoing research regarding radiosensitivity of cancer cells
as well as trying to find effective ways of causing cellular destruction by introducing various
materials to the tissue. Cancer stem cell research is still relatively new in the realm of radiation
oncology. Khan and his colleagues discuss some of the biomarkers found on cancer stem cells
and some of the current therapies being evaluated as potential mechanisms to destroy these stem
cells. Some of the possible interventions this group discusses include targeting certain gene
pathways such as the Notch, Hedgehog, and Wnt pathways, targeting biochemical processes
within the stem cells, or blocking surface markers so that essential functions of the stem cells
cannot go forward. A study by Takebe and associates also investigated cancer stem cells by
targeting the Notch, Hedgehog, and Wnt pathways, all three of which have distinct cellular
functions.3 Figure 1, pulled from Khan's study, lists several drugs being investigated for their
various effects on therapeutic targets on cancer stem cells. Khan's study summarizes many
different research topics in the field, but does not draw any significant conclusions in the paper.4
Another study by Greve and associates tested their theory of targeting survivin, a protein
that is highly expressed in the majority of human cancers. Some of the roles of survivin include

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cell cycle division, cell repair, and cell communication, among others. The researchers
manipulated Ewings sarcoma cells so that there was less survivin present, then irradiated the
cells to determine whether or not a decreased amount of survivin led to more cancer cell death.
The researchers concluded that lower levels of survivin, termed survivin knockdown, plus
ionizing radiation led to more apoptosis of sarcoma cells. For example, ionizing radiation alone
resulted in 10.8% apoptosis of tumor cells, while combining radiation with survivin knockdown
resulted in 24.7% apoptosis. They also conclude that survivin knockdown cells combined with
ionizing radiation resulted in decreased proliferation and colony formation. This made Ewings
sarcoma cells far more radiosensitive. This study is one good example of an agent that can be
applied to cancerous cells to reduce their radioresistance and make treating with ionizing
radiation more successful.5
A study conducted by Yu et al at the Soochow University in China investigated the
response of esophageal carcinoma cells after the introduction of thalidomide. Thalidomide is a
pharmaceutical that was originally used as a sedative in the mid-1900s, but was found to be a
teratogen in pregnant women and caused severe birth defects. There has since been evidence that
thalidomide may be useful in conditions in which the production of blood vessels needs to be
slowed to prevent further destruction, such as AIDS, multiple myeloma, and other cancers. Yus
study investigated the effects of combining treatment of esophageal carcinoma with thalidomide
with radiation therapy. The researchers determined that this combination did result in an increase
in cell death as long as the cells were exposed to the thalidomide for longer than 24 hours. As the
length of exposure increased, as well as the dose of radiation, more esophageal carcinoma cells
were destroyed. This study is another example of an agent being introduced to cancerous cells
and having a positive effect on destroying cancerous tissue.6
In another study by Zhang et al, radioresistant breast cancer cells were treated with an
agent called AZD7762 which inhibits a particular molecule found within human cells. This
molecule, CHK1, promotes replication and transformation of cells and plays a role in protection
against DNA damage. The researchers found that introducing AZD7762 inhibited the
functionality of CHK1, resulting in suppressed breast cancer cell growth, decreased replication
fork speed, and increased cytotoxicity of the cells. The researchers did note that despite these
positive outcomes of introducing AZD7762, the agent did not make the cells more sensitive to

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ionizing radiation. Zhang et al also only tested the impact of AZD7762 on breast cancer cells and
no other types of cancer.7

Methods
Several groups of patients will need to be utilized for this study. I will choose three
different types of cancer, including breast, liver, and lung, and all of these patients will need to be
either stage I or stage II in order to participate. These three cancer sites were chosen due to ease
of surgical access and high probability of disease progression. Each patient must not have any
other known malignancies and the cancer being examined must be their only primary diagnosis
in their medical history. Fifty patients with each disease will be utilized for this experiment,
twenty of which will be part of the control group while the other thirty will receive stem cell
treatment (see Figure 2). A control group is necessary in this experiment so that we may compare
the results between patients receiving stem cell treatment and patients not receiving treatment.
Initial positron emission tomography (PET) and computed tomography (CT) scans will be taken
of each patient to serve as a baseline. The control group of twenty patients per disease site will
not receive stem cell treatment, but will receive standard radiation therapy. The treatment group
will have biopsies taken of their tumors, which will allow us to extract cancer stem cells. These
stem cells will be treated in a laboratory with agent X, a combination of enzymes and proteins,
which will cause disruption to the replicating mechanisms of the cells. The cells will be given
two weeks to adopt the new sequencing.
While the stem cells are being altered in the laboratory, the patients in the treatment
group will undergo a round of total body irradiation (TBI). The purpose of TBI is to suppress the
patients immune system so that the stem cells that are reintroduced after alteration are not
rejected. Each patient will receive 1200 cGy in six fractions, 200 cGy per fraction, three fractions
per week (Monday, Wednesday, and Friday). This will be done on a 6 MV linear accelerator with
the patient at an extended distance from the source in a reclined position so that the beams hit the
patient on each lateral side (see Figure 3).8
After the sixth fraction of TBI, the cancer stem cells will be reintroduced into the patients
via an injection directly into the tumor vasculature. Depending upon where the tumor is located,
this may be done under local anesthesia or under general anesthesia. These patients will remain
in the hospital under isolation for three days to ensure they do not contract any illnesses from the

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environment at home. The reintroduced stem cells will be given two additional weeks to
integrate themselves into the tumor in the hopes that they will transmit the altered sequencing to
other cancer stem and non-stem cells in the tissue. After the two weeks, patients in both the
control and treatment groups will undergo standard radiation therapy for their disease sites.
All patients will have both PET and CT scans at three-months, six-months, twelve-
months, and five-years after treatment is complete. Tumor volumes, lymph node involvement,
and metastasis will be evaluated at each set of scans and the results will then be compiled after
the five-year mark.

Possible Results
If my hypothesis is true, I would expect to see complete remission in the treatment group.
The altered stem cells will have incorporated themselves into the malignant tissue, preventing
further proliferation of the malignant cells. Since the cells will have properties that prevent
replication, and not necessarily properties that instigate apoptosis, the earlier scans following
treatment (three and six-month scans) may show some residual tumor volume since the cells will
not have completely died off yet. The one-year and five-year scans will hopefully lack in any
tumor volume since the cells were never able to reproduce and proliferate, eventually dying off
from the effects of radiation. The control group will show similar results to what the medical
community has seen for each disease site at follow up appointments. Five-year survival rates for
stage I-II breast cancer patients are between 88% to 74%.9 Five-year survival rates for stage I-II
lung cancer patients are between 12.3% to 65.1%.10 Finally, five-year survival rates for stage I-II
liver cancer patients is approximately 31%.11 See Figure 4 for a graphical representation of these
expected results.
If my hypothesis fails, I expect to see similar results between the control group and
treatment group. There may be a slight increase in survival and tumor suppression in the
treatment group when compared to the control group because of the addition of TBI, which may
have eliminated some of the cancer cells prior to reintroduction of the altered stem cells.
However, there may also be a decrease in survival and tumor suppression, and even an increase
in secondary malignancies, in the treatment group after TBI and stem cell transplant due to the
additional ionizing radiation and altered genetic material introduced to the patient. See Figure 5
for a graphical representation of these potential results.

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Figures

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Figure 1: This table, pulled from Khans study, shows some of the current agents in clinical trials
that are being tested against cancer stem cells.4

Breakdown of Participants

Breast Treatment
20 Breast Control
30
Liver Treatment
Liver Control
30
20 Lung Treatment
Lung Control
20 30

Figure 2: This chart shows the breakdown of the participants for my proposed study. There are
three different cancer types, which are further broken down into treatment and control groups.
There are 20 participants in each control group and 30 participants in each treatment group.

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Figure 3: This image shows the patient positioning for the TBI procedure.8

Results if Hypothesis is True


1

0.8

0.6
Fraction of patients showing disease
0.4

0.2

0
0.0 3.0 6.0 12.0 60.0

Time (months)

Treatment group Control group

Figure 4: This graph shows potential results in the case that my hypothesis is true.

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Results if Hypothesis is False
1

0.8

0.6
Fraction of patients with disease
0.4

0.2

0
0.0 3.0 6.0 12.0 60.0

Time (months)

Treatment group Control group

Figure 5: This graph shows potential results in the case that my hypothesis is true.

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References Cited
1
Skvortsova I, Debbage P, Kumar V, Skvortsov S. Radiation resistance: Cancer stem cells (CSCs)
and their enigmatic pro-survival signaling. Seminars in Cancer Biology. 2015;35:39-44.
doi:10.1016/j.semcancer.2015.09.009.
2
MacDonagh L, Gray S, Breen E et al. Lung cancer stem cells: The root of resistance. Cancer
Letters. 2016;372(2):147-156. doi:10.1016/j.canlet.2016.01.012.
3
Takebe N, Miele L, Harris P et al. Targeting Notch, Hedgehog, and Wnt pathways in cancer
stem cells: clinical update. Nature Reviews Clinical Oncology. 2015;12(8):445-464.
doi:10.1038/nrclinonc.2015.61.
4
Khan I, Al-Karim S, Bora R, Chaudhary A, Saini K. Cancer stem cells: a challenging paradigm
for designing targeted drug therapies. Drug Discovery Today. 2015;20(10):1205-1216.
doi:10.1016/j.drudis.2015.06.013.
5
Greve B, Sheikh-Mounessi F, Kemper B, Ernst I, Gtte M, Eich H. Survivin, a target to
modulate the radiosensitivity of Ewings sarcoma. Strahlenther Onkol. 2012;188(11):1038-1047.
doi:10.1007/s00066-012-0223-z.
6
Yu J, Liu F, Sun Z, Sun M, Sun S. The Enhancement of Radiosensitivity in Human Esophageal
Carcinoma Cells by Thalidomide and Its Potential Mechanism. Cancer Biotherapy &
Radiopharmaceuticals. 2011;26(2):219-227. doi:10.1089/cbr.2010.0897.
7
Zhang Y, Lai J, Du Z et al. Targeting radioresistant breast cancer cells by single agent CHK1
inhibitor via enhancing replication stress. Oncotarget. 2014. doi:10.18632/oncotarget.9156.
8
Hackworth R. Skin Cancer. 2016.
9
Hackworth R. Breast Treatment. 2016.
10
Hackworth R. Respiratory Tract. 2015.
11
Liver Cancer: Statistics | Cancer.Net. CancerNet. 2012. Available at:
http://www.cancer.net/cancer-types/liver-cancer/statistics. Accessed July 16, 2016.