WO2023090976A1 - Outer membrane vesicles and composition for cancer treatment or prevention including same - Google Patents

Abstract

The present invention provides bacterial outer membrane vesicles that are homogenous in size and components, and a composition for cancer treatment or prevention including same. More particularly, the present invention provides bacterial outer membrane vesicles having higher anticancer activity and more suitable properties for commercial development as pharmaceuticals.

Description

Extracellular vesicles and compositions for treating or preventing cancer containing the same

The present invention relates to bacterial extracellular vesicles having a homogeneous size and composition, and to a composition for treating or preventing cancer comprising the same. Specifically, the present invention provides bacterial extracellular vesicles with higher anticancer activity and properties more suitable for commercial development as pharmaceuticals.

Bacterial extracellular vesicles (EVs) are double-lipid membrane structured nanoparticles naturally secreted by bacteria to communicate with the host or other bacteria. In particular, extracellular vesicles secreted by Gram-negative bacteria are also called OMVs (outer membrane vesicles), and are known to have an average diameter of about 20 nm to 200 nm [JH Kim, et al., "Gram-negative and Gram-positive bacterial extracellular vesicles", Semin. Cell. Dev. Biol. 40, 97-104, (2015)].

According to recent research results, various bacterial extracellular vesicles not only have direct therapeutic efficacy for various diseases including cancer, but can also be used as drug delivery systems for treating these diseases. And it is being used or developed clinically as a vaccine delivery system to prevent or treat various diseases such as meningitis.

Bacterial extracellular vesicles contain various immune-inducing substances that can activate the host immune system when injected into an animal host having an immune system, including humans. The extracellular endoplasmic reticulum, which is a double lipid membrane structure, has various components such as proteins, lipids, and nucleic acids in the double lipid membrane and lumen. It was presumed to trigger an anti-cancer effect, and this was first revealed in a previous study by the present research team. It has been reported that when a variety of purified bacterial extracellular vesicles are intravenously injected into mice with cancer, they effectively remove cancer tissue and induce long-term anticancer immune responses [OY Kim, et al., "Bacterial outer membrane vesicles suppress tumor by interferon-γ-mediated antitumor response", Nature Communications . 8:626, (2017)].

Furthermore, bacterial extracellular vesicles are specifically accumulated in cancer tissues with enhanced permeability and retention effect (EPR effect) due to their nano-sized characteristics, and anticancer cytokines from natural killer cells and T lymphocytes. It induces a strong anticancer immune response through a mechanism of action that promotes the secretion of IFN-γ (Ibid.). According to the preceding study, the particle size of Escherichia coli extracellular vesicles measured by DLS (dynamic light scattering) corresponds to about 20-200 nm, and the mixture nature of particles having various sizes within this range is mixed. have Bacterial extracellular vesicles vary greatly in composition as well as in size, and this diversity is particularly prominent in extracellular vesicles derived from Gram-negative bacteria composed of outer and inner membranes. The extracellular vesicles naturally secreted by Gram-negative bacteria contain a mixture of inner membrane-derived extracellular vesicles and nucleic acid particles in addition to the cell outer membrane-derived extracellular vesicles.

The cell membrane-derived extracellular vesicles have lipopolysaccharides (LPS) in the outer cell membrane on the membrane surface, and contain the cell membrane proteins and periplasm proteins. On the other hand, intracellular membrane-derived extracellular vesicles contain intracellular membranes, intracellular proteins, and cytoplasmic proteins. The outer and inner membrane-derived extracellular vesicles are secreted out of the cells and mixed in the culture medium. It is very difficult to separate and purify the outer membrane-derived extracellular vesicles from the inner membrane-derived extracellular vesicles, so the purified Gram-negative bacteria used in previous studies The extracellular cell body included extracellular vesicles derived from the outer and inner membranes of the cell.

As such, the fact that the sizes and components of bacterial extracellular vesicles are diverse and that the intracellular membrane-derived extracellular vesicles are mixed is consistent with the development of bacterial extracellular vesicle-based drugs, CMC (Chemistry, Manufacturing, Control) , and acts as a major stumbling block in terms of efficacy.

Therefore, it is necessary to ensure consistent quality and efficacy in the commercial development of bacterial extracellular vesicles as immunocancer agents. To this end, it would be a very useful technique to identify the characteristics of various types of extracellular vesicles secreted from Gram-negative bacteria.

According to many studies accumulated so far, it is commonly understood that extracellular vesicles have various sizes as well as components, even if they are derived from a single cell. Therefore, it has been accepted as a natural phenomenon that extracellular vesicles purified by conventional separation or purification methods of naturally secreted extracellular vesicles have various components and size distributions.

However, contrary to this general understanding, the present inventors, through further studies on the properties of bacterial extracellular vesicles purified by conventional methods, found that they contained two distinct groups of extracellular vesicles, and could not further isolate or purify them. discovered that it can be

As a result of isolating and purifying the above two distinct groups of extracellular vesicles and confirming their characteristics, it was found that they were composed of relatively large extracellular vesicles and relatively small and homogeneous extracellular vesicles, respectively. Relatively large extracellular vesicles were designated as P1, and small and homogeneous extracellular vesicles were designated as P2. The size range of the extracellular vesicles of P1 is about 30-200 nm, whereas the size of the extracellular vesicles of P2 is about 20-30 nm.

The present inventors confirmed that they are clearly distinguished from each other in terms of spectroscopic characteristics and constituent proteins as well as size through identification work on each of P1 and P2.

P1 is relatively large in size and heterogeneous, and has spectroscopic characteristics with an absorbance value at 280 nm smaller than that at 260 nm (A ₂₈₀ /A ₂₆₀ <1), and is derived from endomembrane-derived proteins and cytoplasm. Contains a lot of protein.

On the other hand, P2 was shown to have a relatively small and homogeneous size, spectroscopic characteristics with an absorbance value at 280 nm greater than that at 260 nm (A ₂₈₀ /A ₂₆₀ >1), and a large amount of cell membrane-derived proteins. included.

Based on Dynamic Light Scattering (DLS) analysis, the average particle size of P2 is in the range of about 20-30 nm, and the polydispersity index is about 0.35 or less, or about 0.3 or less, or about 0.25 or less. It has very homogeneous properties. As confirmed through electron microscopic analysis, P2 is 90% or more, preferably 95% or more, more preferably 97% or more of the total particles of extracellular vesicles in the above range. Their zeta potential value is about -14 (±3) mV, and they are negatively charged.

In addition, the anticancer efficacy of P1 and P2 was compared in an animal model, and it was confirmed that P2 had higher anticancer efficacy than P1 at the same dose. Therefore, it was confirmed that the main anticancer effect of bacterial extracellular vesicles was derived from P2.

The bacterial extracellular vesicles according to the present invention are superior in the treatment or prevention of cancer compared to extracellular vesicles naturally secreted and purified from bacteria and having heterogeneous characteristics. In addition, the bacterial extracellular vesicles according to the present invention have relatively homogeneous physicochemical properties, so quality control (QC) is easy in terms of drug development, which is more advantageous for the development of anticancer drugs based on bacterial extracellular vesicles.

1 is an absorption chromatogram according to elution time obtained by fractionating extracellular vesicles purified by a conventional method using size-exclusion chromatography (SEC, size-exclusion chromatography) (FIG. 1a) and the SEC elution time (retention time) It is the transmission electron microscope observation result of each fraction (FIG. 1b).

Figure 2 is a result of separating the extracellular vesicles purified by a conventional method into P1 and P2 through centrifugation, and performing proteomics analysis for each. The content of proteins identified in P1 and P2 (Fig. 2a), the top 5% proteins in the content ranking (Fig. 2b), the number of proteins according to subcellular localizations (Fig. 2c) and the content (Fig. 2d) is the result showing

Figure 3 is an animal experiment method and design for comparing the anticancer activity of P1 and P2 according to the dose.

Figure 4 shows the results of animal experiments comparing the anticancer activity of P1 (a) and P2 (b) according to the dose.

As used herein, the term "about" means to include a commonly accepted error range (standard deviation) depending on a device, instrument, or method used to measure or determine a numerical value defined by the term. For example, the size of extracellular vesicles measured by dynamic light scattering (DLS) may have an error range of ±5 nm.

As used herein, the term "size" means "average diameter" unless otherwise indicated.

As used herein, the terms "separation" or "purification" are used interchangeably without any significant difference in meaning. The term is not limited to the act of fractionating only the desired component excluding other components, but may mean any action for selectively enriching the content of the desired component.

As used herein, the term "extracellular vesicles purified by a conventional method" or "extracellular vesicles purified using a conventional separation or purification method" refers to components other than extracellular vesicles obtained from bacterial cell culture prior to the present invention. It refers to extracellular vesicles obtained after a series of processes to remove various biologically active substances derived from bacteria, such as phosphoproteins, lipids, genetic materials (DNA, RNA), and virulence factors. Therefore, it should be understood that the extracellular endoplasmic reticulum has not been subjected to a special separation, purification or concentration process for P1 or P2 discovered in the present invention.

As used herein, the term "P2" or "extracellular vesicles that are relatively small and homogeneous in size" has an average particle size in the range of about 20-30 nm and is less than or equal to about 0.35, or less than or equal to about 0.3, as identified herein. , or polydispersity of about 0.25 or less, meaning extracellular vesicles with highly homogeneous properties.

The present invention has revealed that extracellular vesicles (P2) having a very homogeneous size distribution exist among the population of naturally secreted extracellular vesicles or extracellular vesicles purified therefrom by conventional methods.

Accordingly, the present invention provides extracellular vesicles having a relatively small size and a high content of homogeneous extracellular vesicles (P2) compared to naturally secreted extracellular vesicles.

The content of P2 in the extracellular vesicles according to the present invention is 90% or more, preferably 95% or more, more preferably 97% or more of the total particles.

As known to those skilled in the art, the extracellular vesicles (P2) according to the present invention are cultured with bacteria, removed by centrifugation, and then used in addition to chromatographic methods such as size exclusion chromatography and ion exchange chromatography. It can be separated by various methods known in the art, such as centrifugation, filtration, dialysis, ultrafiltration, immunoaffinity separation, microfluidic separation, aqueous two-phase system, and polymer-based precipitation. Specific conditions and experimental protocols according to each method for selectively purifying the extracellular vesicles (P2) according to the present invention are appropriately selected by those skilled in the art who understand the composition and characteristics of the extracellular vesicles disclosed in the present invention can be used

The bacteria include, but are not limited to, Gram-negative bacteria.

The gram-negative bacteria are Escherichia genus, Helicobacter genus, Hemophilus genus, Neisseria genus, Cyanobacterium genus, Klebsiella genus, Aceto Genus Acetobacter , Genus Acinetobacter , Genus Enterobacter , Genus Chlamydia , Genus Vibrio , Genus Pseudomonas , Genus Salmonella , Genus Thiobacter Genus , Borrelia , Burkholderia , Serratia, Treponema , Rikenella , Alistipes , Marinillabili Sub ( Marinilabilia ), Proteus ( Proteus ), Enhydrobacter ( Enhydrobacter ) genus, Methylobacterium ( Methylobacterium ) genus, Morganella ( Morganella ) genus, Cupriavidus ( Cupriavidus ) genus, Yersinia ( Yersinia ) ) genus, Shigella genus, Legionella genus, Stenotrophomonas genus, and Moraxella genus bacteria, etc., but is not limited thereto. .

As confirmed by the present inventors, it was difficult to confirm the presence of P2 when extracellular vesicles were isolated from a bacterial culture medium using centrifugation and size exclusion chromatography techniques used for conventional separation and purification of extracellular vesicles. For example, in the centrifugation method mainly used in the step of treating a substance inducing precipitation of extracellular vesicles and recovering the precipitated extracellular vesicles, centrifugation must be performed for a longer period of time than is sufficient to separate normal extracellular vesicles. It is difficult to confirm the presence of P2 enough to be mistaken for nucleic acid particles and relatively large proteins because it can be separated, or it is eluted later than the point at which extracellular vesicles are eluted even in a purification method using size fractionation technology through size exclusion chromatography. circumstances existed.

Therefore, confirmation of the existence of P2 according to the present invention was an unexpected finding. The inventors of the present technology thus deviate from the separation or purification parameters used in conventional purification methods of extracellular vesicles and purify the corresponding substances (P2) having unique properties, so that they not only have relatively homogeneous particulate properties, It was confirmed to have a clear protein-lipid bilayer structure, which is a key feature of the extracellular endoplasmic reticulum.

If the characteristics of P2 disclosed in the present invention are understood, P2 can be easily isolated or purified from the naturally secreted extracellular vesicle population or extracellular vesicles purified therefrom by conventional methods.

For example, the extracellular vesicles (P2) according to the present invention are subjected to centrifugation for a longer period of time than sufficient to separate normal extracellular vesicles, or in the size-specific fractionation method through size exclusion chromatography, the P2 particles disclosed herein It can be separated with reference to size and elution time, or purified through a filter method and ultrafiltration method having a pore size of 0.1 μm to 0.05 μm. In addition, in the precipitation step after concentration of the cell culture solution, differential centrifugation is used to first remove large particles at a low centrifugation speed, and then the supernatant can be separated by centrifugation at high speed for a long time.

The extracellular vesicles (P2) according to the present invention have a size range of about 20-30 nm, which is smaller than the size range of about 20-200 nm of the extracellular vesicles separated by conventional methods and is homogeneous. In addition, when proteomics quantitative analysis was performed, the content ranking of proteins selected from the group consisting of OmpF (Outer membrane protein F), LamB (Maltoporin), Lpp (Major outer membrane lipoprotien Lpp), and OmpA (Outer membrane protein A) It is within the top 5%.

As a result of comparative experiments on anticancer efficacy in animal models, it was confirmed that P2 has higher anticancer efficacy than P1 at the same dose. Previously, in the case of mammalian-derived extracellular vesicles, larger ones were more effective in terms of pharmacological activity, and smaller ones were considered impurities. . This hypothesis was expected to be common to bacterial extracellular vesicles, but in the present invention, contrary to this expectation, it was confirmed that small-sized extracellular vesicles exhibited higher anticancer efficacy.

Although not bound by the theory described later, these results suggest that P2 has a relatively large number of outer membrane-derived factors that have been shown to have anticancer effects through stimulation of the immune system, and is small in size, so it is internal (luminal) compared to large extracellular vesicles. ) is expected to be because it is unlikely to contain various substances that do not specifically contribute to immune activation.

Therefore, according to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the extracellular vesicles (P2) according to the present invention as an active ingredient.

The pharmaceutical composition according to the present invention can be used for the treatment and prevention of cancer.

In the present invention, the cancer is liver cancer, thyroid cancer, testicular cancer, bone cancer, glioblastoma, oral cancer, ovarian cancer, brain tumor, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, head and neck cancer, lymphoma, bladder cancer, leukemia, esophageal cancer, kidney cancer, and stomach cancer. , breast cancer, cervical cancer, prostate cancer, rectal cancer, spinal cord tumor, pancreatic cancer, salivary gland cancer, lung cancer, skin cancer, laryngeal cancer, melanoma, etc., but may be selected from the group, but is not limited thereto.

The administration route of the pharmaceutical composition according to the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration may be administered, but is not limited thereto.

The pharmaceutical composition according to the present invention may be formulated in a suitable form together with a pharmaceutically acceptable carrier commonly used for anticancer treatment. 'Pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness, or similar reaction when administered to humans. Pharmaceutically acceptable carriers include, for example, parenteral administration carriers such as sterile water, physiological saline, suitable oil, aqueous glucose and glycol, and the like, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, sucrose, albumin, and the like. Suitable preservatives include dimethylsulfoxide (DMSO), glycerol, ethylene glycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, etc. there is

According to another aspect of the present invention, there is provided a cancer prevention or treatment method comprising the step of administering to a subject the cell therapy composition for anticancer comprising the extracellular vesicles according to the present invention. The subject means a mammal, preferably a human.

Hereinafter, examples will be described in detail to explain the present invention in detail. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto in any sense.

[Example]

Example 1. Preparation of bacterial extracellular vesicles and size-exclusion chromatography (SEC) analysis

1. Strain culture and extracellular vesicle purification

After culturing E. coli BL21(DE3) ΔmsbB in aerobic conditions for 16 hours using a fermentor system (75 L scale), a culture solution from which cells were removed was prepared by centrifugation at 6,000 × g for 20 minutes. After filtering the culture solution with a 0.2 μm pore-sized filter, a 10-fold concentrated culture solution was produced through tangential flow filtration (TFF) using a 100 kDa molecular weight cut-off (MWCO) membrane filter. Proteolytic enzymes were inactivated by adding 50 μM phenylmethylsulfonyl fluoride (PMSF) to the concentrated culture solution and shaking for 30 minutes. Thereafter, a buffer solution (pH 7.2) containing 25 mM CaCl ₂ was added to the concentrated culture medium, and the reaction was refrigerated and shaken for 1 hour to precipitate the extracellular vesicles selectively. After recovering the precipitate by centrifugation, it was dissolved in a buffer solution, and 1 mM MgSO ₄ and Benzonase (2.5 U/mL) were added, followed by shaking at room temperature for 1 hour to remove nucleic acid impurities.

Thereafter, in order to remove non-particulate contaminants having a molecular weight smaller than that of bacterial extracellular vesicles, the sample was placed in a 100 kDa MWCO dialysis membrane, and the dialysis solution was changed 5 times for a total of 18 hours at 4° C. to purify the bacterial extracellular vesicles.

2. Results of SEC fractionation of purified extracellular vesicles

The extracellular vesicles purified by the method in 1 above were injected into a column (10 x 200 mm) filled with S500, developed with HEPES buffer at a flow rate of 1.0 mL/min, and the absorbance at 260 nm, 280 nm, and 450 nm was measured. measured. Specific SEC fractionation conditions are as follows:

Column: S500 (10 × 200 mm)

Mobile phase: 20 mM HEPES, 500 mM NaCl (pH 7.2)

Flow Rate: 1.0 mL/min

Sample volume injected: 0.1 mL in 20 mM HEPES, 1 M NaCl (pH 7.2)

As shown in FIG. 1a, two distinct peaks were found in the absorption chromatograms at 260 nm, 280 nm, and 450 nm. As a result of observation using transmission electron microscopy (TEM) of each fraction according to the retention time, it was confirmed that the size of the observed particles was clearly distinguished according to the fraction (Fig. 1b, P1 and P2). ).

Example 2. P1 and P2 isolation and proteomics analysis

The extracellular vesicles purified by the method of Example 1-1 were centrifuged at 13,000 × g for 40 minutes. After centrifugation, the pellet was referred to as P1 and the supernatant as P2. After extracting proteins from P1 and P2, they were digested into peptides by treatment with trypsin, and proteomics analysis was performed by LC-ESI-MS/MS (Fig. 2 and Table 1).

[Table 1]

As shown in Table 1 above, 567 and 506 proteins were identified in P1 and P2, respectively. There were 357 P1-enriched proteins in P1 compared to P2, and among them, 198 proteins were found only in P1 and 159 proteins were more than 1.5 times more abundant than P2. There were 297 P2-enriched proteins in P2 compared to P1, and among them, 137 proteins were found only in P2 and 160 proteins were more than 1.5 times more abundant than P1. On the other hand, 50 proteins were found in both P1 and P2 and differed by less than 1.5 times in content.

As shown in FIG. 2a and Table 1, among the P1-enriched proteins, cytoplasmic proteins were high in content, and representatively, there were Tuf (Elongation factor Tu) and RplB (50S ribosomal protein L2). Among the P2-enriched proteins, outer membrane proteins with a high content were mostly outer membrane proteins. Representatively, OmpF (Outer membrane protein F), LamB (Maltoporin), Lpp (Major outer membrane lipoprotein Lpp), OmpA (Outer membrane protein) A) was

Figure 2b shows the protein types belonging to the top 5% of the content ranking among the proteins of P1 and P2.

On the other hand, in the case of P1, both the number and content of cytoplasmic proteins were higher than those of P2. In the case of P2, both the number and content of outer membrane proteins were higher than those of P1, and the number of periplasmic proteins was higher than that of P1 (FIGS. 2c and 2d).

Considering that outer membrane-derived proteins are enriched and cytoplasmic proteins are de-enriched in the extracellular vesicles of Gram-negative bacteria, P2 can be said to be closer to the extracellular vesicles of Gram-negative bacteria.

Example 3. P1 and P2 in vivo Confirmation of anticancer activity

The fraction (P1 - pellet, large extracellular vesicles; P2 - supernatants, small extracellular vesicles) were compared according to the dose. The animal experiment method and design were performed as shown in FIG. 3 .

The mouse colorectal cancer cell line CT26 (1×10 ⁶ cells/head) was injected into the right dorsal side of BALB/c female mice, and after 1 week, cancer tissues were identified and the experimental groups were divided. A buffer solution was injected into the control group, and 2, 5, or 10 μg/head of P1 and P2 were injected into the cancer tissues at 8, 11, 14, and 17 days after colon cancer cell line injection, depending on the experimental group. Direct injection (intratumoral administration) was performed. In addition, the size of cancer tissue was measured up to 22 days after colon cancer cell line injection.

Both P1 and P2 increased the anticancer effect in a dose-dependent manner, but it was confirmed that the anticancer effect of P2 was greater than that of P1 at the same dose (FIG. 4).

That is, the extracellular vesicles of Gram-negative bacteria separated by a conventional method could be further purified to obtain extracellular vesicles that were relatively small and homogeneous in size and had improved anticancer efficacy.

Claims (9)

1. Bacterial extracellular vesicles with a high content of extracellular vesicles with an average diameter of about 20 to 30 nm compared to naturally secreted extracellular vesicles.
2. 2. The bacterial extracellular vesicles of claim 1, wherein the content of extracellular vesicles having an average diameter of about 20 to 30 nm is greater than 90%, or greater than 95%, or greater than 97%.
3. 3. The bacterial extracellular vesicle of claim 2, wherein the polydispersity index of the mean diameter of the extracellular vesicle is less than or equal to about 0.35, or less than or equal to about 0.3, or less than or equal to about 0.25.
4. 4. The bacterial extracellular vesicle according to claim 3, characterized in that the absorbance value at 280 nm of the extracellular vesicle is greater than the absorbance value at 260 nm (A ₂₈₀ /A ₂₆₀ >1).
5. The bacterial extracellular vesicle according to claim 1 , wherein the bacteria are gram negative bacteria.
6. The method of claim 5, wherein the gram-negative bacteria are Escherichia , Helicobacter , Hemophilus , Neisseria , Cyanobacterium , Krebsiella ( Klebsiella ), Acetobacter genus, Acinetobacter genus, Enterobacter genus, Enterobacter genus, Chlamydia genus, Vibrio genus, Pseudomonas genus ( Pseudomonas ) genus, Salmonella genus , Thiobacter genus, Borrelia genus, Burkholderia genus, Burkholderia genus, Serratia genus, Treponema genus, Rikenella genus, Alistipes ) Genus, Marinilabilia Genus, Proteus Genus, Enhydrobacter Genus, Methylobacterium Genus, Morganella Genus, Cupriavidus Genus , a bacterial cell selected from the group consisting of bacteria of the genus Yersinia , Shigella , Legionella , Stenotrophomonas , and Moraxella . outer endoplasmic reticulum.
7. The method according to claim 6, wherein the content ranking of the protein selected from the group consisting of OmpF (Outer membrane protein F), LamB (Maltoporin), Lpp (Major outer membrane lipoprotein Lpp) and OmpA (Outer membrane protein A) is within the top 5% phosphorus bacterial extracellular vesicles.
8. A pharmaceutical composition for treating or preventing cancer, comprising the bacterial extracellular vesicles according to any one of claims 1 to 7 as an active ingredient.
9. The method of claim 8, wherein the cancer is liver cancer, thyroid cancer, testicular cancer, bone cancer, glioblastoma, oral cancer, ovarian cancer, brain tumor, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, head and neck cancer, lymphoma, bladder cancer, leukemia, esophageal cancer, kidney A pharmaceutical composition for the treatment or prevention of cancer, which is selected from the group consisting of cancer, stomach cancer, breast cancer, cervical cancer, prostate cancer, rectal cancer, spinal cord tumor, pancreatic cancer, salivary gland cancer, lung cancer, skin cancer, laryngeal cancer, melanoma, and the like .

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