Activating IGFLR1 to Promote Melanoma Cell Apoptosis

Kehan Ren

College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China

Keywords: Melanoma, IGFLR1, IFN-γ, IGFL1, IGFL3, CD4+ T Cell.

Abstract: Purpose: Melanoma is one of the most common types of cancer with a high incidence rate. Surgical

resection therapy is difficult to be applied to patients with extensive metastasis. Previous studies have

reported that IGFL3 promotes the activation of CD4+ T cell and secretion of IFN-γ through the

transmembrane protein IGFLR1, and IFN-γ inhibits tumor growth by inducing apoptosis of cancer cells.

Activating IGFLR1 will upregulate the release of IFN-γ and promote cancer cell apoptosis. This paper

investigates the effect of activation of IGFLR1 using IGFL1 recombinant protein on anti-melanoma

treatment in both in vitro and in vivo conditions. In this work, eight possible outcomes of the experiments

are discussed respectively. Different results will indicate whether or not IGFLR1 activation has a

therapeutic effect on the anti-cancer treatment and provide critical information for the future clinical trial of

IGFLR1 activation therapy.

INTRODUCTION

Melanoma is the most dangerous type of skin cancer

(World Cancer Report. World Health Organization.

2014). According to the American Cancer Society

(ACS), it affects about 100 thousand people per year

in the United States, and it has a long-term survival

rate of less than 30% (American Cancer Society,

2021). Compared with other types of cancer cells,

melanoma cell is more likely to spread to other parts

of the body. Additionally, since some of the

melanomas do not make melanin, people may miss

prime therapy time.

Surgical resection is currently the primary

treatment for malignant melanoma at the early stage,

but it is not suitable for patients with extensive

metastasis (Niederhuber, 2019). Immunotherapy is a

treatment method that is widely applied to advanced

cancer (Abbott, M., and Y. Ustoyev, 2019). It aims

to stimulate or suppress certain type of the immune

cells through certain target in order to help the

body’s immune system attack melanoma cells more

effectively. Immunotherapy has revolutionized

anti-melanoma therapy through anti-programmed

cell death protein 1 (anti-PD-1) (Onitilo, 2019).

However, there are still over 60% of the patients

who do not respond or develop resistance to these

treatments (Hugo, 2016). Therefore, a more suitable

treatment for melanoma and new drug target on

immune cells to enhance the anti-melanoma

immmune response are still needed to be developed.

CD4+ T cell is an immune cell in the body's

immune system that plays a vital role in fighting

cancer (Luckheeram, 2012). CD4+ T cells would not

kill the cancer cells directly. However, it can

produce and secrete IFN-γ, which is a glycosylated

protein (Ngai. 2007). IFN-γ inhibits tumor growth

by inducing apoptosis and dormancy of tumor cells

(Takeda, 2017). IFN-γ induces the expression of

IRF1, a tumor suppressor gene (Yan Zhou, Crystal

M. Weyman, 2008). It also participates in the

IFN-γ/STAT1 pathway, which leads to the

dormancy of tumor cells (Kortylewski, 2004).

Figure 1: IGFLR1 Pathway. This figure shows the position

and activities of IGFLR1.

Insulin growth factor-like receptor 1 (IGFLR1) is

a transmembrane protein, and it is encoded by the

Ren, K.

Activating IGFLR1 to Promote Melanoma Cell Apoptosis.

DOI: 10.5220/0011312600003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 883-888

ISBN: 978-989-758-595-1

883

IGFLR1 gene located on chromosome 19

(Fagerberg, 2014). IGFLR1 is highly expressed in

CD4+ T cells (Zhang, 2018). Insulin growth

factor-like family member 1 (IGFL1) is known as

the ligand of IGFLR1, and it is induced in

inflammatory skin conditions (Song, 2020); (Lobito,

2011). IGFL3 is another ligand of IGFLR1, showing

high-affinity interactions with IGFLR1 (Zhang,

2018). IGFLR1 pathway (See Figure 1) could be a

potential drug therapy target. IGFL3 binds with

IGFLR1, promoting CD4+ T cell activation and

IFN-γ secretion (Zhang, 2018) . However, the effect

of IGFL1 binding with IGFLR1 in immunotherapy

has not be studied.

Therefore, in order to test the therapeutic effect of

activating IGFLR1 in preclinical conditions, a

comparative study should be designed. This paper

investigates the effect of activating IGFLR1 using

IGFL1 recombinant protein on anti-melanoma

treatment in both in vitro and in vivo conditions. If

IGFLR1 is activated in T cells through binding

IGFL1 recombinant protein produced in e coli, then

the function of T cell will be enhanced, and

melanoma progression will be suppressed, because

IGFL3 promotes CD4+ cell activation and IFN-γ

release through IGFLR1, and IFN-γ plays an

important role in killing cancer cells.

2 METHODS

2.1 Materials

This experiment will use a human melanoma cell

line (A375), a murine melanoma cell line (B16-F1),

human and murine CD4+ T cells. Cell line A375 and

B16 are obtained from the China Center for Type

Culture Collection. Human CD4+ T cells are

isolated from healthy donor PBMCs, and murine

CD4+ T cells are isolated from the murine spleen.

For all the experiments, cells are randomly allocated

to different experimental groups.

C57BL/6 mice will be used in vivo study.

Four-week old C57BL/6 female and male mice are

purchased from the Center of Medical Experimental

Animals of Hubei Province (Wuhan, China). The

mice are randomly assigned to the control group or

the treated group. The mice are housed under

specific pathogen-free conditions. Animals will be

euthanized immediately if they display excessive

discomfort.

IGFL1 recombinant protein is produced in e coli

and IGFL3 is ordered from CUSABIO for both in

vitro and in vivo experiments.

2.2 In Vitro Cell Culture

Co-culture system will be established for in vitro

study. A375, B16 cells are cultured on RPMI-1640

supplemented with 10% fetal bovine serum and 1%

penicillin and streptomycin at 37 °C with 5% CO2

(Ye, 2017). The cells are passaged every 2 days

using TrypLE. Isolated T cells are cultured in

RPMI-1640 supplemented with 10%

heat-inactivated FBS, 100 U/ml penicillin and

streptomycin and 2-mercaptoethanol (Zhang, 2018).

T cells are primarily activated with anti-CD3

(UCHT1) and anti-CD28 (CD28.2) (Zhang, 2018).

Melanoma cells are labeled with CFDA-SE.

CFDA-SE-labeled melanoma cells and T cells are

added into the 12-well plate, shaken evenly, and

placed in a cell culture box for co-culture.

Each co-culture cells will be divided into six

groups: (1) negative control: PBS; (2) positive

control: 100ng/ml IGFL3 solution; (3) 10 ng/ml

IGFL1 solution; (4) 50 ng/ml IGFL1 solution; (5)

100 ng/ml IGFL1 solution; and (6) 200 ng/ml

IGFL1 solution. IGFL1 is given and incubated in the

cell culture box for 2 hours. in vitro experiment will

be repeated three times.

2.3 Flow Cytometry

The cell apoptosis will be measured through the

Flow Cytometry system offered by ThermoFisher

Scientific. The co-cultured cells will be collected

and measured every 24 hours after treatment. The PI

staining solution in the apoptosis PI staining kit is

used for staining. The therapeutic effect of the in

vivo experiment will be evaluated after one course

of treatment (30 days), with three courses of

treatment in total. Each experiment is repeated three

times. Flow cytometry analysis showed that the ratio

of CFDA-SE+/PI+ cells was the ratio of tumor cells

killed after IGFL1 stimulated CD4+ T cells to exert

an antitumor immune effect. Each experiment is

repeated for five times.

2.4 ELISA

IFN-γ expression level will be measured via

sandwich ELISA. The sandwich is formed by adding

the samples, then the second antibody, which has a

measurable signal on it. Specific anti-murine IFN-γ

antibodies and anti-human IFN-γ antibodies are

precoated in the wells of the microplate. Samples

and controls are then added into these wells and bind

to the immobilized antibody. Then the second

antibodies is added and bind to the samples. After

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

884

incubation, the intensity of signal is measured.

2.5 Western Blotting

Whole-cell lysates will be prepared with FLAG-IP

lysis buffer (50 mM tris, 150 mM NaCl, 1 mM

EDTA, 0.5% NP-40, and 10% glycerol), with

protease inhibitor tablets. Protein concentration will

then be determined with DC Protein Assay from

Bio-Rad Laboratory. Proteins will be separated

using SDS Page gel electrophoresis and

wet-transferred to polyvinylidene difluoride

membranes. Blots will be visualized with

SuperSignal West Pico Chemiluminescent®

substrate from Thermo Fisher Scientific. IGFL1

Antibody and IGFLR1 Antibody from ThermoFisher

Scientific will be used for Western blotting.

2.6 Animal Model

C57BL/6 mice aged from 4 to 6 weeks will be used.

B16 cells are injected subcutaneously to form a

model of Subcutaneous melanoma. As a minimum

of eight mice per group is required for having a

statistical power, each group has ten mice. Each

C57BL/6 mouse is injected with B16 cells

subcutaneously at the left hind leg on day 0. The

experimentalists are blinded from the expected

outcome of the treatment.

Then the mice are randomly divided into three

groups: (1) negative control: PBS; (2) positive

control: 100ng/mL IGFL3 solution; (3) IGFL1

treatment, with the optimum blood concentration

determined by the cell experiment. For the positive

control group, there is no experimental data to

confirm that IGFL3 would function in vivo

experiments. However, IGFL3 is the possible

molecule that may activate IGFLR1 in vivo

experiments. in vivo experiment will be repeated

three times.

2.7 In Vivo IGFL1 Delivery

IGFL1 will be given on day 8. Hydrodynamic tail

vein injection will be performed. The growth of B16

tumors is monitored by measuring tumor size every

other day. On day 30, all the mice will be

euthanized. The IGFL1 level in the blood will be

determined by western blot.

2.8 Histologic Evaluation and

Immunohistochemistry

Tissue of mice in experimental groups is collected

for histologic evaluation in order to determine tissue

toxicity. Tumor tissue, lung, liver, spleen and

stomach of mice are fixed by 4% paraformaldehyde

and then embedded in paraffin and cut to ~4 µm

thick sections by Thermo FINESSE 325. Organ

sections are stained by H&E and slides are evaluated

for tumor formation by a veterinary (Chen, 2018).

2.9 Statistical Analysis

The statistical significance of all numerical data

gathered through ELISA, Western Blot, and Flow

Cytometry will be analyzed using the student's

T-Test on GraphPad Prism® at (p <0.05).

3 RESULTS

Possible Results on Melanoma Cell Apoptosis (The

overview of six possible results is also shown in

Table 1.)

Table 1: Possible Results on Melanoma Cell Apoptosis.

Cell Lines

Result 1 Result 2 Result 3 Result 4 Result 5

Result 6

Killing of xenograft cells

+ - + - - -

Killing of Human A375 Cell Lines

+ + - - + -

Activating IGFLR1 to Promote Melanoma Cell Apoptosis

885

Killing of Murine B16 Cell Lines

+ + + + - -

Note. “+” represents a significant increase in melanoma cell apoptosis. “-” represent not significantly different from negative

control.

Possible Result 1: Applying IGFL1 promotes

melanoma cell apoptosis in determined human

and murine melanoma cell lines and the cell line

from the in vivo animal models.

IGFL1 activates IGFLR1 in all in vitro CD4+ T

cell samples, increasing the expression of IFN-γ.

The apoptosis of melanoma cell samples is

promoted significantly. The animal experiments

display that IGFL1 activation of IGFLR1 has a

therapeutic effect on melanoma.

Possible Result 2: Applying IGFL1 promotes

melanoma cell apoptosis in determined human

and murine melanoma cell lines, but not the cell

line from the in vivo animal models.

IGFL1 activates IGFLR1 in all in vitro CD4+ T

cell samples, increasing the expression of IFN-γ.

The apoptosis of both human and murine melanoma

cell samples is promoted significantly. However,

IGFL1 does not successfully increase in vivo IFN-γ

expression, or the animal experiments do not display

a significant therapeutic effect of IGFL1 activation

of IGFLR1 on melanoma.

Possible Result 3: Applying IGFL1 promotes

melanoma cell apoptosis in the determined

murine melanoma cell line in vitro and in vivo

animal models, but not the human melanoma cell

line.

IGFL1 activates IGFLR1 in murine CD4+ T

cells, increasing the expression of IFN-γ. The

apoptosis of B16 cell samples is promoted

significantly. However, the IGFL1 does not

successfully increase in vitro IFN-γ expression or

the apoptosis of A375 cell samples. The animal

experiments display that IGFL1 activation of

IGFLR1 inhibits the growth of B16 tumors since

this model uses the same melanoma cell line as in

vitro murine cell samples.

Possible Result 4: Applying IGFL1 only

promotes melanoma cell apoptosis in determined

murine melanoma cell line.

IGFL1 activates IGFLR1 in murine CD4+ T

cells. The apoptosis of B16 cell samples is promoted

significantly. However, applying IGFL1 to the

co-culture of human CD4+ T cells and A375 cells

does not successfully increase in vitro IFN-γ

expression or the apoptosis of A375 cell samples.

Furthermore, IGFL1 does not successfully increase

in vivo IFN-γ expression, or the animal experiments

do not display a significant therapeutic effect of

IGFL1 activation of IGFLR1 on melanoma.

Possible Result 5: Applying IGFL1 only

promotes melanoma cell apoptosis in determined

human melanoma cell line.

IGFL1 activates IGFLR1 in human CD4+ T

cells. The apoptosis of A375 cell samples is

promoted significantly. However, applying IGFL1

to the co-culture of murine CD4+ T cells and B16

cells does not successfully increase in vitro IFN-γ

expression or the apoptosis of B16 cell samples. The

animal experiment will not be successfully

conducted in this scenario.

Possible Results 6: Applying IGFL1 does not

promote melanoma cell apoptosis.

Applying IGFL1 does not successfully stimulate

CD4+ T cells. The expression level of IFN-γ does

not change significantly. Moreover, there is not a

significant increase in the ratio of melanoma cell

apoptosis.

Possible Result 7: Applying IGFL1 increases

IFN-γ expression but does not promote

melanoma cell apoptosis in any cell lines.

The IFN-γ level in the blood is upregulated.

However, there is not a significant increase in the

ratio of melanoma cell apoptosis after IGFL1

activation.

Possible Result 8: Applying IGFL1 promote

melanoma cell apoptosis, but does not increases

IFN-γ expression in any cell lines.

Melanoma cell apoptosis is prompted. However,

there is not a significant increase in the IFN-γ

level in the blood.

4 DISCUSSION

Previous studies report that activation of IGFLR1

will increase the secretion of IFN-γ in CD4+ T cells,

which promotes cell apoptosis in many known

cancer cell lines. In order to test the preclinical

therapeutic effect of activating IGFLR1 in

melanoma cell samples and animal models by using

agonist, this study induces potential agonist IGFL1

to one well-studied melanoma cell line from humans

and one from mice, as well as an in vivo animal

melanoma model.

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Possible Results 1 fully support the hypothesis.

Compared with the negative group, the expression

level of IFN-γ is upregulated and the melanoma cell

apoptosis is promoted, which is consistent with the

positive group. The result is consistent with previous

studies investigating IGFLR1's effect on CD4+ T

cells and IFN-γ’s effect on cancer cells. It indicates

that IGFL1 activating IGFLR1 therapy has potential

value in anti-melanoma treatment. Further studies

investigating the specific gene regulation

mechanism of IGFLR1 should be done for a

thorough understanding of its structures and

functions. The relation between IGFLR1 and IGFL1

should also be investigated to investigate the more

specific IGFLR1 pathway. Preclinical testing on

more complex and representative animal models

such as rabbits and monkeys should also be done

before the transition to clinical testing of IGFLR1

activation therapy. In order to improve this

therapeutic method, better delivery platforms like

small molecule carriers or mechanisms involving

controlled-release should be applied as well.

Possible Results 2 partially support the

hypothesis. In Possible Result 2, IGFL1 activation

fails in vivo experiment. In mice experiment,

compared with positive group, the ELISA result will

indicate a low expression level of IFN-γ. And

IGFL1 level is almost no higher than the negative

group. The failure of the in vivo experiment is most

likely to be caused by the unsuccessful delivery of

IGFL1 in vivo: IGFL1 does not reach CD4+ T cells

through vein injection or does not maintain in the

body long enough for its functions. To improve the

experiment, a highly efficient and dependable

delivery method should be developed. The safety

level of activating the IGFLR1 pathway to enhance

antitumor immune responses should be improved

before clinical trials. Furthermore, new agonists

activating IGFLR1 could be developed in place of

IGFL1.

Possible Results 3 and 4 both partially support

the hypothesis. Both of the results indicate that

IGFLR1 activation cannot promote the cell

apoptosis in human melanoma cell lines. Compared

with negative group, the ELISA result will indicate

almost no difference in the expression level of

IFN-γ. Only if the IGFLR1 activation promotes the

cell apoptosis in human melanoma cell samples, the

IGFL1 activating IGFLR1 treatment will potentially

have therapeutic effects and should be carried on to

clinical trial. Possible Results 3 and 4 indicate that

the IGFLR1 activation is not qualified to be a

universal treatment for melanoma either because the

A375 cell line does not have an IFN-γ receptor or

has a different type of IFN-γ mechanism. This will

require future studies to re-evaluate the relationship

between IFN-γ, IGFLR1, and general types of

melanoma.

Possible Results 5 and 6 indicate potential errors

in the experimental designs. Possible Results 5

partially support the hypothesis. The expression

level of IFN-γ and cancer cell apoptosis rate in

murine cell line does not change significantly. It

indicates that IGFL1 cannot be applied to mice.

Searching for new agonists will be required for

future studies on animal cell lines. At the same time,

IGFL1 activating IGFLR1 treatment should be

applied to other types of human melanoma cell lines

to investigate the therapeutic effect. Possible Results

6 contradicts the hypothesis. In result 6, IGFLR1

activation is unsuccessful in all cell samples.

Compared with the negative group, the expression

level of IFN-γ is not considerably changed. It

indicates that IGFL1 does not activate IGFLR1,

which means bioinformatic data deviates from the

actual situation. This result may be caused by an

inappropriate cell incubation method or IGFL1

concentration. In this case, changing experimental

designs is required. This result may also be caused

by IGFLR1 participating in multiple pathways. This

will require further studies on IGFLR1 pathways

and the development of new agonists aiming at

IGFLR1.

The Possible Result 7 and 8 partially contradicts

the hypothesis. In Result 7, The IFN-gamma level in

the blood is upregulated, which is consistent with

positive group. However, there is not a significant

increase in the ratio of melanoma cell apoptosis after

IGFL1 activation. This result indicates that CD4+ T

cells is successfully activated, however, some

oncogenic mutations may be present in the

melanoma cell line, which means that future clinical

melanoma therapies through activating IGFLR1

could not be applied for all kinds of melanoma types

since each cell line can have such a mutation.

However, this result is unlikely to happen on the

well-studied melanoma cell lines since the IFN-γ

pathways are relatively well studied in these cell

lines. In Possible Result 8, melanoma cell apoptosis

is prompted, which is consistent with positive group.

However, there is not a significant increase in the

IFN-gamma level in the blood. It indicates that an

alternative IGFL1 pathway may be crucial for CD4+

T cell stimulation. Future studies should be focused

on investigating the expression level of other

cytokines to verify the effects of IGFL1 activation

on CD4+ T cells.

Activating IGFLR1 to Promote Melanoma Cell Apoptosis

887

5 CONCLUSION

As a newly identified potential drug target, IGFLR1

has not been thoroughly studied in melanoma

treatment. This study explores the therapeutic effect

of IGFLR1 activation in human and murine

melanoma cell lines, as well as Xenograft murine

models. The result of this study will indicate

whether or not IGFLR1 activation has a therapeutic

effect in preclinical conditions, preparing the basis

for the transition to clinical trials. The feasibility of

using IGFL1 as the agonist of IGFLR1 will also be

tested. Future studies should focus on improving in

vivo IGFL1 delivery methods, including active

targeting delivery and small molecule carriers, as

well as developing IGFLR1 agonists such as

monoclonal antibodies (mAbs). And we could pay

attention to potential IGFLR1 functions in different

kinds of T cells. Further studies on IGFLR1

pathways may find new targets for enhancing T cell

function in immunotherapy of melanoma.

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