TWI521207B - Method and for the determination of colon cancer and application therefor - Google Patents

Description

Detection method of colorectal cancer and its application

The present invention mainly discloses the use of an antibody against human chemokine (C-C motif) ligand 5 (CCL5-antibody), particularly in the detection and treatment of colorectal cancer.

The microenvironment of the tumor includes stromal cells, immune cells (including lymphocytes, macrophages, and dendritic cells), composition of extracellular matrices, growth factors, cytokines/chemical hormones, and metabolites (Peltekova et al., 2014), and It plays an important role in the development of tumors and the chemical resistance of cancer drugs (Katoh et al., 2013).

It is currently known that the microenvironment of tumors is closely related to the generation and progression of cancer. Peripheral cells provide a basic scaffold or facilitator for tumor progression.

Human chemical hormone (CC motif) receptor 5 (CCL5), known to regulate the activation and expression of normal T cells (RANTES), is also considered to be an important promoter of a variety of chronic inflammatory diseases, and this degree of malignancy can be attributed To recruit inflammatory cells (Mackay, 2001). CCL5 is thought to be overexpressed in a variety of cancers and is involved in several important steps in the process of cancer spread, including the activation of CC chemokine receptors type 1, 3 and 5, followed by Regeneration, migration, invasion, vascular proliferation and metastasis.

In addition to cancer cell growth and development, CCL5 is also associated with resistance to traditional chemotherapy drugs such as cisplatin and tamoxifen. High levels of CCL5 have been considered as cancer organisms Indicators and can guide prognosis and treatment guidelines.

The transformation of epithelial cells into mesenchymal cells (EMT) is an important factor in the development of cancer, as it enhances the ability of tumor cells to metastasize.

Snail is a basic helix-loop-helix transcription factor that regulates EMT by reducing the expression of attached connexins, such as E-cadherin. Increasing evidence suggests that Snail activity is elevated in multiple human tumor tissues and that its performance is associated with invasion, metastasis, and poor prognosis.

Long non-coding RNAs (over 200 nucleotide transcripts) are thought to be involved in the regulation of gene expression and genome composition, or a structural backbone specific to functional regions in the nucleus.

Metastasis-associated lung adenocarcinoma transcription factor 1 (MALAT-1) is a long-length non-coding RNA that is considered a regulator of metastasis and activity. The message delivery profile of MALAT-1 is highly correlated with the metastasis of a variety of human solid tumors, including lung cancer, liver cancer, prostate cancer, bladder cancer, and colorectal cancer. MALAT-1 is also involved in some steps in cancer metastasis, including cell viability and EMT, and can encourage the transformation of epithelial cells into mesenchymal phenotype cells by increasing the expression of the EMT transcription factors ZEB and Slug.

However, it has not yet been fully clarified which molecules are involved in the interaction between colorectal cancer and tumor-associated dendritic cells.

The main object of the present invention is to provide a method for detecting cancer by measuring one of the information ribonucleic acid (mRNA) and protein expression of colorectal cancer cells.

An embodiment of the invention provides a method for evaluating the efficacy of an antibody for colorectal cancer treatment. The method wherein the anti-system is resistant to a human chemokine (C-C motif) ligand 5 (CCL5) secreted by a tumor-associated dendritic cell (TADC) infiltrated by a large intestine cancer cell. The method comprises the steps of: mixing the antibody with the colorectal cancer cell; and assessing the performance of a message transmission of the colorectal cancer cell, wherein the message comprises metastasis-associated lung adenocarcinoma transcription factor 1 (MALAT-1) and alkaline One of the helix-loop-helix transcription factors (Snail).

Another embodiment of the present invention is to propose an antibody against human chemokine (C-C motif) ligand 5 (CCL5) which can be used to slow cell migration of colorectal cancer cells.

Another embodiment of the present invention is to provide an antibody against human chemokine (C-C motif) ligand 5 (CCL5) which can be used to alleviate the invasion of colorectal cancer cells.

Another embodiment of the present invention is to provide an antibody against human chemokine (C-C motif) ligand 5 (CCL5) which can be used to slow the transformation of epithelial cells of a colorectal cancer cell into a mesenchymal cell.

Another embodiment of the present invention provides a method for detecting colorectal cancer. The detection method comprises the steps of: secreting a human chemokine (CC motif) ligand 5 (CCL5) by a tumor-associated dendritic cell (TADC) infiltrated by a large intestine cancer cell; and detecting the expression amount of the CCL5 To determine the degree of malignancy of the colorectal cancer cells.

Another embodiment of the present invention is to provide a pharmaceutical composition for treating colorectal cancer. The pharmaceutical composition includes an antibody against human chemokine (C-C motif) ligand 5 (CCL5).

mdDC-CM‧‧‧ culture medium for dendritic cell environment derived from monocytes

TADC-CM‧‧‧ Medium for tumor-associated dendritic cell environment

SW480 SW620‧‧‧ Human colorectal cancer cell line

N-cadherin‧‧‧Calcene N

E-cadherin‧‧‧Calcene E

SMA‧‧‧smooth muscle α-actin

Vimentin‧‧‧Vimentin

Claudin-3‧‧‧Claudin 3

PAPDH‧‧‧Glyceraldehyde 3-phosphate dehydrogenase

SW480-TADC-CM‧‧‧ Medium for tumor-associated dendritic cell environment of human colorectal cancer cell line SW480

SW620-TADC-CM‧‧‧Mechanism of tumor-associated dendritic cell environment of human colorectal cancer cell line SW620

SW620-TADC-CM+IgG‧‧‧The medium of tumor-associated dendritic cell environment of human colorectal cancer cell line SW620 plus immunoglobulin G

SW620-TADC-CM+CCL5 ab.‧‧‧The medium of tumor-associated dendritic cell environment of human colorectal cancer cell line SW620 plus anti-CCL5 antibody

Figure 1A shows the experimental results of the wound healing assay; Figures 1B-1 and 1B-2 show the experimental results of the permeability nesting system; Figures 1C-1 and 1C-2 show the experimental results of cell invasion; Figure 1D shows Experimental results of transformation of epithelial cells into mesenchymal cells; Figure 2A shows the expression of CCL5 mRNA in tumor-associated dendritic cells; Figure 2B shows the expression of CCL5 protein in tumor-associated dendritic cells; Figure 2C shows the detection of colorectal cancer cells by wound healing Experimental results; Figures 2D-1 and 2D-2 show experimental results of a colony system for colorectal cancer cells; Figure 2E-1 and Figure 2E-2 show the transformation of colorectal cancer cells into mesenchymal cells via epithelial cells. Experimental results; Figure 2F shows the effect of CCL5 concentration changes on epithelial cell markers and fibroblasts; Figure 3A-1 and Figure 3A-2 show experimental results of cell migration of colorectal cancer cells after antibody blocking CCL5; 3B-1 and FIG. 3B-2 show experimental results of cell invasion of colorectal cancer cells after antibody blocking CCL5; FIG. 3C shows experimental results of transformation of epithelial cells of colon cancer cells into interstitial cells after antibody blocking CCL5 ; Figure 4A, Figure 4B and FIG. 4C show the amount of Snail expression of colorectal cancer cells; FIGS. 4D-1, 4D-2, 4D-3, and 4D-4 show experimental results of cell migration of colorectal cancer cells after inhibiting the expression of Snail; Figure 4E-1, Figure 4E-2, Figure 4E-3, and Figure 4E-4 show experimental results of cell invasion of colorectal cancer cells after inhibition of Snail performance; Figures 4F-1 and 4F-2 show inhibition of Snail performance After that, the epithelial cells of colorectal cancer cells were transformed into interstitial cells; FIG. 5A shows the microarray expression of MALAT-1 of colorectal cancer cells; FIG. 5B-1 and FIG. 5B-2 show MALAT-1 of colorectal cancer cells. Immediate polymerase chain reaction performance; Figure 5C-1 and Figure 5C-2 show the amount of MALAT-1 expression in colorectal cancer cells; Figures 5D-1 and 5D-2 show inhibition of CCL5 expression, colorectal cancer cells Figure IA shows the efficiency of action of MALAT-1 small interfering RNA; Figure 6B-1, Figure 6B-2, Figure 6B-3 and Figure 6B-4 show inhibition of MALAT-1 expression after the large intestine Experimental results of cell migration of cancer cells; Figures 6C-1, 6C-2, 6C-3, and 6C-4 show experimental results of cell invasion of colorectal cancer cells after inhibition of MALAT-1 expression; Figure 6D- 1 and FIG. 6D-2 show the expression of Snail and E-cadherin in colorectal cancer cells after inhibiting the expression of MALAT-1; FIG. 7A shows the distribution of CCL5 in CD11c ⁺ dendritic cells of colorectal cancer patients; FIG. 7B shows the large intestine. The distribution of CCL5 in the serum of cancer patients; FIG. 8 shows an embodiment of a method for evaluating the efficacy of an antibody for colorectal cancer; and FIG. 9 shows an embodiment of a method for detecting colorectal cancer.

[Embodiment 1]

Please refer to FIGS. 1A, 1B-1, 1B-2, 1C-1, 1C-2 and 1D. Figure 1A shows the injury Experimental results of the mouth healing test. Figures 1B-1 and 1B-2 show experimental results of a permeability nesting system. Figures 1C-1 and 1C-2 show experimental results of cell invasion. Figure 1D shows the results of an experiment in which epithelial cells are transformed into mesenchymal cells. This example is intended to demonstrate whether tumor-associated dendritic cells promote the development of colorectal cancer. After invented by the wound healing assay, the inventors found that the culture medium of the tumor-associated dendritic cell environment promoted the migration of colorectal cancer cells (see Figure 1A); in addition, in the permeability transwell system (see Figure 1B- 1 and Figure 1B-2), invasion (see Figure 1C-1 and Figure 1C-2) and the process of transformation of epithelial cells into mesenchymal cells can have the same result; that is, indicators of epithelial cells, such as cadherin E (E) -cadherin) and Claudin-3; and markers of fibroblasts such as N-cadherin, Vimentin, and α-smooth muscle actin , SMA) different performance (see Figure 1D).

In the present example, SW480 cells were treated with a tumor-associated dendritic cell environment medium (SW480-TADC-CM) of human colon cancer cell line SW480 at a specific time, and a tumor-associated tree of human colon cancer cell line SW620 was used. The cell culture medium (SW620-TADC-CM) was used to treat SW620 cells, and after 72 hours of treatment, the wound healing test was performed, and after 24 hours, the epithelial cells were transformed into a mesenchymal cell process assay.

The ability of SW480 and SW620 cells to migrate and invade was observed by QCM ^TM 24 small cell migration and invasion assay.

In the 48-hour migration and invasion experiments of cancer cells, SW620 cells were treated with SW480 cells (SW480-TADC) and SW620 tumor-associated dendritic cell environment in a medium of SW480 tumor-associated dendritic cell environment containing 20% by volume ( SW620-TADC) and monocyte-derived dendritic cell environment medium (mdDC-CM), as a control group, the above medium is used as a source of chemical hormones.

Each value is taken after 3 experiments and the average is added or subtracted from the standard error. After analysis by Student's t test, the asterisk represents a significant difference in both test groups (P value less than 0.05).

[Embodiment 2]

Please refer to Figures 2A, 2B, 2C, 2D-1, 2D-2, 2E-1, 2E-2 and 2F. Figure 2A shows the expression of CCL5 mRNA in tumor-associated dendritic cells. Figure 2B shows the expression of CCL5 protein in tumor-associated dendritic cells. Figure 2C shows the results of an experimental study of wound healing in colorectal cancer cells. Figures 2D-1 and 2D-2 show experimental results of a colorectal cancer cell through a permeability nesting system. Figure 2E-1 and Figure 2E-2 show the results of an experiment in which colorectal cancer cells are transformed into mesenchymal cells by epithelial cells. This example is intended to confirm whether CCL5 is involved in the malignant development of cancer regulated by tumor-associated dendritic cells. Colorectal cancer cells increase the expression of CCL5 mRNA (see Figure 2A) and protein (see Figure 2B) in tumor-associated dendritic cells. CCL5 increases cell migration, which can be seen in wound healing assays (see Figure 2C) and permeability nesting systems (see Figure 2B). CCL5 increases the invasion of SW480 and SW620 cells (see Figure 2D-1 and Figure 2D-2) and the process by which epithelial cells transform into mesenchymal cells (see Figure 2E-1 and Figure 2E-2). Figure 2F shows the effect of changes in CCL5 concentration on the following indicators: E-cadherin, Claudin-3, N-cadherin, Vimentin, and Smooth muscle α-actin (SMA).

Using CD14 ⁺ monocyte culture medium (20% by volume) in SW480 or SW620 environment, then add macrophage colony-stimulating factor (GM-CSF) (20 ng/ml) and interleukin-4 (IL-4). (20ng/ml) can produce monocyte-derived dendritic cells, SW480 and SW620 tumor-associated dendritic cells in 5 days. The mRNA and protein expression levels were assessed by real-time polymerase chain reaction (qRT-PCR) and enzyme immunoassay (ELISA). The migration ability of SW480 and SW620 cancer cells was assessed by wound healing assay. The invasion and migration ability of SW480 and SW620 cells was evaluated by the above method.

CCL5 (0.1 and 1 ng/ml) acts as a chemical hormone in cancer cell migration and invasion (48 hours) experiments. Each value is taken after 3 experiments and the average is added or subtracted from the standard error. After analysis by Student's t test, the asterisk representative was significantly different after comparison with the control group (P value less than 0.05).

[Embodiment 3]

Please refer to Figures 3A-1, 3A-2, 3B-1, 3B-2 and 3C. 3A-1 and 3A-2 show experimental results of cell migration of colorectal cancer cells after antibody blocking CCL5. Figure 3B-1 and Figure 3B-2 show the results of experimental cell invasion of colorectal cancer cells after antibody blocking CCL5; Figure 3C shows the transformation of epithelial cells of colorectal cancer cells into interstitial cells after antibody blocking CCL5 result. This example uses a specific antibody to neutralize CCL5, which can reduce the development of cancer promoted by the culture medium of the tumor-associated dendritic cell environment. Blocking CCL5 with commercially available anti-CCL5 antibodies (Abcam/UK; see the sequence listing) can reduce the mediation of tumor-associated dendritic cell environment for cell migration (see Figure 3A-1 and Figure 3A-2), invasion (see Figure 3A-1 and Figure 3A-2) See Figure 3B-1 and Figure 3B-2) and the effect of epithelial cell transformation into a mesenchymal cell process (see Figure 3C).

Mononuclear cell-derived dendritic cell environment medium (mdDC-CM) or tumor-associated dendritic cell environment medium (TADC-CM) plus anti-CCL5 antibody (CCL5 ab.) or immunoglobulin G (IgG) ) to treat SW480 and SW620 cells. The colon cancer cells were seeded in the upper tank and the anti-CCL5 antibody (2 μg/ml) or immunoglobulin G (2 μg/ml) was treated, and the culture medium (volume percent concentration 20) of the tumor-associated dendritic cell environment was added. The bottom plate is used as an attraction factor for 48 hours. Cells with migration and invasion are measured by the above method It.

Each value is taken after 3 experiments and the average is added or subtracted from the standard error. After analysis by Student's t test, the asterisk represents a significant difference in both test groups (P value less than 0.05).

[Embodiment 4]

Please refer to FIGS. 4A, 4B, 4C, 4D-1, 4D-2, 4D-3, 4D-4, 4E-1, 4E-2, 4E-3, and 4E-4. 4A, 4B, and 4C show the amount of Snail expression of colorectal cancer cells. 4D-1, 4D-2, 4D-3, and 4D-4 show experimental results of cell migration of colorectal cancer cells after inhibiting the expression of Snail. 4E-1, 4E-2, 4E-3, and 4E-4 show experimental results of cell invasion of colorectal cancer cells after inhibiting the expression of Snail. Figure 4F-1 and Figure 4F-2 show the results of experiments in which epithelial cells of colorectal cancer cells are transformed into mesenchymal cells after inhibiting the expression of Snail. This example is intended to demonstrate that Snail is an important regulator in the progression of cancer-associated dendritic cells to promote cancer. The culture medium of the tumor-associated dendritic cell environment increases the amount of Snail expression (see Figure 4A). CCL5 will increase the amount of Snail's performance (see Figure 4B). Inhibition of CCL5 expression can block the expression of Snail caused by the culture medium of the tumor-associated dendritic cell environment (see Figure 4C). Inhibition of Snail performance can reduce the mediation of tumor-associated dendritic cell environment and cell migration promoted by CCL5 (see Figure 4D-1, Figure 4D-2, Figure 4D-3, and Figure 4D-4), invasion (see Figure 4E-1, Fig. 4E-2, Fig. 4E-3, and Fig. 4E-4)) and the process of transforming epithelial cells into interstitial cells (see Fig. 4F-1 and Fig. 4F-2).

SW480 and SW620 cells were treated with CCL5, monocyte-derived dendritic cell environment medium or tumor-associated dendritic cell environment medium and anti-CCL5 antibody (2 μg/ml) or immunoglobulin G (2 μg/ml) for 24 hours. Then use the immunodot ink method to evaluate Snail's Performance. The cells are transfected into the control group or Snail's small interfering message ribonucleic acid (Snail siRNA), followed by the addition of a monocyte-derived dendritic cell environment medium or a tumor-associated dendritic cell environment medium. The migration and invasion ability of colorectal cancer cells is measured by the above method.

Each value is taken after 3 experiments and the average is added or subtracted from the standard error. After analysis by Student's t test, the asterisk represents a significant difference in both test groups (P value less than 0.05).

[Embodiment 5]

Please refer to Figures 5A, 5B-1, 5B-2, 5C-1, 5C-2, 5D-1 and 5D-2. Figure 5A shows the microarray performance of MALAT-1 of colorectal cancer cells. Figures 5B-1 and 5B-2 show the instantaneous polymerase chain reaction performance of MALAT-1 of colorectal cancer cells. Figures 5C-1 and 5C-2 show the amount of MALAT-1 expression in colorectal cancer cells. Figures 5D-1 and 5D-2 show the amount of MALAT-1 expression in colorectal cancer cells after inhibition of CCL5 expression. This example mainly illustrates that CCL5 secreted by tumor-associated dendritic cells increases the expression of long-length non-coding RNA MALAT-1 in colorectal cancer cells. The amount of CCL5 that enhances the performance of MALAT-1 can be measured by a microarray (see Figure 5A) and an instant polymerase chain reaction (see Figures 5B-1 and 5B-2). The culture medium of SW480 and SW620 tumor-associated dendritic cell environment increased the expression of MALAT-1 in SW480 and SW620 cells (see Figure 5C-1 and Figure 5C-2). Inhibition of CCL5 expression can block the expression of MALAT-1 by the culture medium of the tumor-associated dendritic cell environment (see Figures 5D-1 and 5D-2).

Cells were treated with media for tumor-associated dendritic cell environment or CCL5 and anti-CCL5 antibodies (2 μg/ml) or immunoglobulin G (2 μg/ml) for 6 hours, followed by measurement of MALAT-1 expression by real-time polymerase chain reaction .

Each value is taken after three experiments and the average is added or subtracted from the standard error. After analysis by Student's t test, the asterisk represents a significant difference in both test groups (P value less than 0.05).

[Embodiment 6]

Please refer to FIGS. 6A, 6B-1, 6B-2, 6B-3, 6B-4, 6C-1, 6C-2, 6C-3, 6C-4, 6D-1 and 6D-2. Figure 6A shows the efficiency of action of MALAT-1 small interfering RNA. 6B-1, 6B-2, 6B-3, and 6B-4 show experimental results of cell migration of colorectal cancer cells after inhibiting the expression of MALAT-1. 6C-1, 6C-2, 6C-3, and 6C-4 show experimental results of cell invasion of colorectal cancer cells after inhibiting the expression of MALAT-1. Figures 6D-1 and 6D-2 show the expression of Snail and E-cadherin in colorectal cancer cells after inhibition of the expression of MALAT-1. This example explores how MALAT-1 regulates the performance of Snail. The efficiency of MALAT-1 small interfering RNA (see Figure 6A). Inhibition of MALAT-1 expression reduces the mediation of tumor-associated dendritic cell environment and cell migration promoted by CCL5 (see Figure 6B), invasion (see Figure 6C-1, Figure 6C-2, Figure 6C-3 and Figure) 6C-4), and reduce the amount of Snail and E-cadherin in colorectal cancer cells (see Figure 6D-1 and Figure 6D-2).

The cells were transfected with small interfering RNA (MALAT-1 siRNA) of MALAT-1, and the biological activity of the cells and the expression of E-cadherin were measured by the above method.

Each value is taken after three experiments and the average is added or subtracted from the standard error. After analysis by Student's t test, the asterisk represents a significant difference in both test groups (P value less than 0.05).

[Embodiment 7]

Please refer to Figure 7A and. Figure 7A shows the distribution of CCL5 in CD11c ⁺ dendritic cells in colorectal cancer patients. Figure 7B shows the distribution of CCL5 in the serum of patients with colorectal cancer. This example demonstrates that the content of CCL5 in CD11c ⁺ dendritic cells of patients with colorectal cancer is very high. There are many CD11c ⁺ dendritic cells that display large amounts of CCL5 in tumor sections (see Figure 7A). Tumor and non-tumor areas (10 samples) were excised and stained and analyzed using a conjugated focus microscope (10x and 40x). The serum of patients with colorectal cancer was used to assess the content of CCL5 (see Figure 7B).

Enzyme immunoassay was used to measure the level of CCL5 in serum of patients with colorectal cancer (60 samples) and healthy donors (15 samples).

[Embodiment 8]

Please refer to Figure 8, which shows an embodiment of a method for assessing the efficacy of an antibody in the treatment of colorectal cancer. In step S801, the anti-system is defined to be a human chemokine (CC motif) ligand 5 (CCL5) secreted by a tumor-associated dendritic cell (TADC) infiltrated by a large intestine cancer cell; The antibody is mixed with the colorectal cancer cell; and in step S803, the expression of a message transmission of the colorectal cancer cell is evaluated, wherein the message transmission includes a metastasis-associated lung adenocarcinoma transcription factor 1 (MALAT-1) And one of the basic helix-loop-helix transcription factors (Snail).

[Embodiment 9]

Please refer to FIG. 9, which shows an embodiment of a method for detecting colorectal cancer. In step S901, a tumor-associated dendritic cell (TADC) infiltrated by a large intestine cancer cell secretes a human chemokine (CC motif) ligand 5 (CCL5); and in step S902, the detection is performed One of CCL5 is expressed in order to determine the degree of malignancy of the colorectal cancer cells.

【Results and discussion】

Tumor-associated dendritic cells promote the migration, invasion and transformation of epithelial cells into interstitial cells

In order to understand the importance of tumor-associated dendritic cells for the development of colorectal cancer, in one embodiment, the inventors used a medium of tumor-associated dendritic cell environment to explore migration, invasion, and transformation of epithelial cells into mesenchymal cells. What is the impact of the process. After the wound healing test, it was found that the culture medium (20%) of the tumor-associated dendritic cell environment of SW480 and SW620 increased the migration of colorectal cancer cells SW480 and SW620. In addition, the culture medium of the tumor-associated dendritic cell environment of SW480 and SW620 also enhances the invasion ability of SW480 and SW620 cells. Furthermore, the culture medium of the tumor-associated dendritic cell environment of SW480 and SW620 causes SW480 and SW620 cells to transform into epithelial cells into interstitial cells, which includes indicators of epithelial cells (E-cadherin and The expression of Claudin-3 is decreased, and the indicators of fibroblasts (N-cadherin, Vimentin, and α-smooth muscle actin, SMA)) The amount of performance increased.

CCL5 plays an important role in the development of cancer regulated by tumor-associated dendritic cells

In order to find out which factor is involved in the process of tumor-associated dendritic cells regulating the development of colorectal cancer, in one embodiment, a microarray is used to evaluate the gene profile of tumor-associated dendritic cells of SW480. The results showed that the expression of several soluble factors in tumor-associated dendritic cells of SW480 increased compared to monocyte-derived dendritic cells. Among the genes with increased expression, the results of real-time polymerase chain reaction showed that the expression levels of allergic cytokines in tumor-associated dendritic cells of SW480 and SW620 increased by 8.18 and 21.02 times, respectively. The results of enzyme-linked immunosorbent assay showed that the protein expression of CCL5 also increased in tumor-associated dendritic cells of SW480 and SW620.

Next, evaluate the migration and entry of CCL5 protein for SW480 and SW620 cells. Invasion of epithelial cells into the process of interstitial cell processes. CCL5 will enhance the ability of SW480 and SW620 cells to migrate and invade. In addition, CCL5 reduced the performance of E-cadherin and claudin-3 and enhanced the performance of N-cadherin and α-smooth muscle actin. The results indicated that CCL5 promoted the transformation of epithelial cells into interstitial cells in SW480 and SW620 cells.

In order to clarify the role of CCL5, in one embodiment, the inventors used specific neutralizing antibodies to block the effects of CCL5, and then evaluated the culture of tumor-associated dendritic cell environment for SW480 and SW620 cancer development. Impact. The results of the experiment indicated that the effect of the culture medium of tumor-associated dendritic cell environment of SW480 and SW620 on the migration of colorectal cancer cells SW480 and SW620 was neutralized under the treatment of anti-CCL5 antibody.

Similarly, neutralizing anti-CCL5 antibodies also reduced the effects of SW480 and SW620 tumor-associated dendritic cell culture media on invasion of colorectal cancer cells SW480 and SW620 and transformation of epithelial cells into interstitial cells.

Snail participates in the process of cancer development regulated by CCL5 derived from tumor-associated dendritic cells

Since Snail is a very important regulator in the evolution of cancer, the inventors speculated that Snail would be involved in the development of mediators in tumor-associated dendritic cells and in the development of colorectal cancer regulated by CCL5. The experimental results indicated that the culture medium and CCL5 of tumor-associated dendritic cell environment increased the expression of Snail in SW480 and SW620 cells.

In addition, the use of neutralizing antibodies to block CCL5 also reduced Snail with increased expression, suggesting that CCL5 plays an important role in the increase in the amount of Snail regulated by tumor-associated dendritic cells.

To investigate the migration of Snail in tumor-associated dendritic cells and CCL5-promoted cells Whether it is a very important regulator during migration and invasion, in one embodiment, transfection techniques using small interfering RNA (siRNA) are used to inhibit the performance of Snail. The efficiency of inhibition of Snail performance was assessed by real-time polymerase chain reaction and Western blotting.

The results showed that Snail can be efficiently inhibited in SW480 and SW620 cells, and can also reduce the migration and invasion of tumor-associated dendritic cells and CCL5-promoted cells after Snail is inhibited. After Snail is inhibited, it can also restore the expression of E-cadherin in the culture medium of tumor-associated dendritic cells and CCL5-treated colorectal cancer cells.

The effects of CCL5 in colorectal cancer include an increase in the performance of MALAT-1

To clarify the molecular mechanisms by which CCL5 regulates cancer development, in one embodiment, the inventors used microarrays to evaluate gene profiles of SW480 cells under treatment with CCL5. The experimental results indicated that the MALAT-1 expression of SW480 cells increased by 3.53 times under CCL5 treatment. The results of real-time polymerase chain reaction experiments showed that the expression of MALAT-1 increased under CCL5 treatment, which is similar to the treatment of large intestine cells with a medium of tumor-associated dendritic cell environment.

Similar results were also observed in SW480 and SW620 cells treated with SW480 and SW620 tumor-associated dendritic cell culture media. The use of neutralizing antibodies to inhibit the expression of CCL5 also reduced MALAT-1, which increased the amount of expression, indicating that CCL5 plays an important role in the increase in the amount of MALAT-1 regulated by tumor-associated dendritic cells.

MALAT-1 is the upstream regulator of Snail

In order to understand the effect of MALAT-1 on CCL5 derived from tumor-associated dendritic cells in colorectal cancer, in one embodiment, the inventors used small interfering RNA transfection techniques to reduce the performance of Snail, and then Medium for evaluating tumor-associated dendritic cell environment Or what effect CCL5 will have on the development of cancer. The small interfering RNA of MALAT-1 was transfected into SW480 and SW620 cells by transfection technique to reduce the expression of MALAT-1. Inhibition of MALAT-1 expression in SW480 and SW620 cells reduced the media and CCL5 regulated migration and invasion of tumor-associated dendritic cell environment. In addition, SW480 and SW620 cells transfected with MALAT-1 small interfering RNA completely abolished the performance of Snail caused by SW480, SW620 tumor-associated dendritic cell environment and CCL5.

Similar results were also observed in SW480 and SW620 cells expressing MALAT-1 small interfering RNA, which prevents E-cadherin caused by the media of tumor-associated dendritic cell environment and the action of CCL5. The amount of performance has decreased.

Tumor infiltrating dendritic cells enhance the metastasis and invasion of colorectal cancer cells

Due to paracrine CCL5, TADC increases the migration, invasion and EMT of colorectal cancer cells. This suggests that CCL5 derived from TADC may promote the development of colorectal cancer.

Experiments have shown that elevated levels of CCL5 in patients are not only derived from cancer cells, but also from tumor-associated dendritic cells (TADC). TADC secretes high levels of CCL5 and communicates with cancer cells, which in turn increases the migration, invasion and EMT performance of cancer cells. Furthermore, neutralizing CCL5 reduces the activity of the TADC promoter in cancer progression, including cell migration, invasion, and EMT.

CCL5 can be used as another therapeutic target to prevent the progression of colorectal cancer

Experimental data show that CD11c ⁺ dendritic cells in human colorectal cancer tissue also produce large amounts of CCL5 compared to non-tumor regions. Both experimental cell and clinical patient sections strongly suggest that CCL5 is an important agent that increases the development of colorectal cancer through TADC. In this case, the inventors suggest that both SW480 and SW620 cells treated with TADC-CM or CCL5 exhibited a dramatic increase in Snail performance. The evoked effect of TADC-CM or CCL5 is derived from the inhibition of the expression of the phenotypic marker E-cadherin.

Furthermore, the selective inhibition of Snail by siRNA can also reduce the effect of TADC-CM or CCL5 on cell migration and invasion, suggesting that the colorectal cancer phenotype transformation triggered by TADC involves mediation by CCL5, for Snail Up-regulation and subsequent E-cadherin down regulation.

The inventors found that SW480 and SW620 cells treated with TADC-CM or CCL5 would result in increased MALAT-1 and Snail performance. Inhibition of MALAT-1 expression by specific siRNAs suggests that MALAT-1 is an upstream molecule of Snail.

Furthermore, knockdown of MALAT-1 reduced the effects of TADC-CM and CCL5 on cell migration, invasion and EMT of colorectal cancer cells SW480 and SW620, suggesting that activation of MALAT-1 is involved in colorectal cancer growth and metastasis. The importance of it.

TADC enhances colorectal cancer progression, in which CCL5 derived from TADC causes phenotypic transformation and promotes cancer progression. CCL5 promotes the performance of MALAT-1 and subsequently increases the performance of Snail, which promotes the progression of colorectal cancer. There is an interaction between dendritic cells and cancer. Using anti-CCL5 strategy (such as anti-human chemokine (CC motif) ligand 5 (CCL5) antibody and other pharmaceutical composition) to lock invasive colorectal cancer, to inhibit CCL5 or CCL5-related message transmission is expected to become a new Targets for the treatment of colorectal cancer patients.

In summary, the inventors of the present invention investigated that allergic cytokines secreted by tumor-associated dendritic cells promote the migration, invasion, and transformation of epithelial cells into interstitial cells. Human chemical hormone (C-C motif) receptor 5 (CCL5), which is abundantly expressed in colorectal cancer tumor-associated dendritic cells, is used to mimic colorectal cancer cells in a medium associated with tumor-associated dendritic cell environment. Migration, invasion, and transformation of epithelial cells into interstitial cells.

In addition, tumor-infiltrating CD11c ⁺ dendritic cells were found to secrete CCL5 in human colorectal cancer sections. CCL5 secreted by tumor-associated dendritic cells not only promotes the development of colorectal cancer, but also promotes the expression of non-coding RNA-associated lung adenocarcinoma transcription factor 1 (MALAT-1), and then MALAT-1 will increase downstream. Performance.

Therefore, blocking MALAT-1, thereby significantly reducing the performance of Snail, reducing the culture and CCL5-mediated migration and invasion of tumor-associated dendritic cell environment. This suggests that the mechanism of MALAT-1/Snail plays an important role in the development of cancer regulated by tumor-associated dendritic cells.

【references】

Katoh H, Wang D, Daikoku T, Sun H, Dey SK, Dubois RN. 2013. CXCR2-expressing myeloid-derived suppressor cells are essential to promote colitis-associated tumororigenesis. Cancer Cell 24(5):631-644.

Mackay CR. Chemokines: immunology's high impact factors. 2001. Nat Immunol 2(2): 95-101.

Peltekova VD, Lemire M, Qazi AM, Zaidi SH, Trinh QM, Bielecki R, Rogers M, Hodgson L, Wang M, D'Souza DJ, Zandi S, Chong T, Kwan JY, Kozak K, De Borja R, Timms L , Rangrej J, Volar M, Chan-Seng-Yue M, Beck T, Ash C, Lee S, Wang J, Boutros PC, Stein LD, Dick JE, Gryfe R, McPherson JD, Zanke BW, Pollett A, Gallinger S, Hudson TJ. Identification of genes expressed by immune cells of the colon that are regulated by colorectal cancer-associated variants. 2014. Int J Cancer 134(10):2330-2341.

<110> Kaohsiung Medical University

<120> Detection method of colorectal cancer and its application

<211> 91

<212> PRT

<213> Human

<223> Antigen of CCL5 antibody

<400> 1

Claims (6)

A method for evaluating the efficacy of an antibody for treating colorectal cancer, wherein the anti-system is resistant to a human chemokine (CC motif) ligand 5 (CCL5) secreted by a tumor-associated dendritic cell (TADC) infiltrated by a large intestine cancer cell. ), comprising the steps of: mixing the antibody with the colorectal cancer cell; and assessing the performance of a message transmission of the colorectal cancer cell, wherein the message comprises metastasis-associated lung adenocarcinoma transcription factor 1 (MALAT-1) and alkaline At least one of a helix-loop-helix transcription factor (Snail), and the antibody blocks the increase in the expression of MALAT-1 and Snail. The method of claim 1, further comprising the step of assessing the performance of cell migration of the colorectal cancer cell. The method of claim 2, further comprising the step of assessing the performance of the invasion of the colorectal cancer cells. The method of claim 3, further comprising the step of assessing the transformation of the epithelial cells of the colorectal cancer cells into interstitial cells. A method for detecting colorectal cancer, comprising the steps of: secreting a human chemokine (CC motif) ligand 5 (CCL5) by a tumor-associated dendritic cell (TADC) infiltrated by a large intestine cancer cell; and detecting the CCL5 One of the expression amounts to determine the degree of malignancy of the colorectal cancer cell, wherein the amount of expression of the CCL5 is proportional to the degree of malignancy of the colorectal cancer cell. The method of detecting according to claim 5, wherein the amount of expression is selected from one of a message ribonucleic acid (mRNA) and a protein expression amount of the CCL5.